Patent application title: COMPOSITIONS AND RELATED METHODS FOR AGRICULTURE
Inventors:
Ignacio Martinez (Lexington, MA, US)
Zachary Garo Armen (Boston, MA, US)
Jonathan Friedlander (Cambridge, MA, US)
Christine Cezar (Sammamish, WA, US)
Barry Andrew Martin (Boston, MA, US)
Maier Steve Avendano Amado (Cambridge, MA, US)
IPC8 Class: AA23K20153FI
USPC Class:
1 1
Class name:
Publication date: 2021-07-01
Patent application number: 20210195917
Abstract:
Provided herein are agents, compositions, and methods for agricultural
use, e.g., for altering the level, activity, or metabolism of one or more
microorganisms resident in a host nematode or arthropod (e.g., honeybee
or silkworm), the alteration resulting in an increase in the fitness of
the host. The invention features a composition that includes an agent
(e.g., phage, peptide, small molecule, antibiotic, or combinations
thereof) that can alter the host's microbiota in a manner that is
beneficial to the host. By promoting favorable microbial levels,
microbial activity, microbial metabolism, and/or microbial diversity, the
agents described herein may be used to increase the fitness of a variety
of beneficial nematodes or arthropods, such as bees and silkworms,
utilized in agriculture and commerce.Claims:
1. A method for increasing fitness of an insect, the method comprising:
administering to the insect a composition comprising an effective amount
of amino-acid-producing bacteria formulated with an insect comestible
carrier.
2. The method of claim 1, wherein the administration comprises delivering the composition to at least one habitat where the insect grows, lives, reproduces, or feeds.
3. The method of claim 1, wherein the composition is a liquid, a solid, an aerosol, a paste, a gel, or a gas.
4. The method of claim 1, wherein the amino acid is glutamate or methionine.
5. The method of claim 1, wherein the carrier is a seed coating.
6. (canceled)
7. A composition comprising an effective amount of amino-acid-producing bacteria formulated with an insect comestible carrier as a liquid, a solid, an aerosol, a paste, a gel, or a gas.
8. The composition of claim 7, wherein the amino-acid-producing bacteria produce glutamate or methionine.
9. The composition of claim 7, wherein the carrier is a seed coating.
10. The composition of claim 7, wherein the amino-acid-producing bacteria are at a concentration of at least 100,000 cells/ml.
11. A method for increasing the developmental rate of an insect, the method comprising: administering to the insect a composition comprising an amino-acid-producing bacteria formulated in an insect comestible carrier.
12. The method of claim 11, wherein the developmental rate of the insect is increased relative to an insect to which the composition has not been administered.
13. The method of claim 11, wherein the amino-acid-producing bacteria was identified or selected, and wherein the bacteria secretes an amino acid at a concentration of at least 0.1 mg/ml in a culture.
14. The method of claim 11, wherein the bacteria is administered at a concentration of at least 150,000 cells/ml.
15. The method of claim 11, wherein the amino acid is methionine or glutamate.
16. The method of claim 11, wherein the insect is honeybee, silkworm, or Drosophila melanogaster.
17. The method of claim 11, wherein administering the composition further increases insect biomass of the insect relative to an insect to which the composition has not been administered.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 62/450,017, filed on Jan. 24, 2017, and U.S. Provisional Application No. 62/583,736, filed on Nov. 9, 2017, the contents of which are hereby incorporated herein by reference in their entireties.
BACKGROUND
[0002] Certain invertebrates, such as nematodes and arthropods (e.g., insects, e.g., European honey bees (Apis melliferia) or silkworms (Bombyx mori)), are utilized in agriculture for pollination efforts and pest control as well as in commerce for the production of commercial products, such as honey or silk. To cultivate beneficial nematodes and arthropods for use in agricultural or commercial industries, there is a need in the art for ways to promote the growth and fitness of beneficial invertebrates.
SUMMARY OF THE INVENTION
[0003] Disclosed herein are compositions and methods for modulating the fitness of invertebrates for agriculture or commerce. The composition includes an agent that alters a level, activity, or metabolism of one or more microorganisms resident in a host, the alteration resulting in a modulation in the host's fitness.
[0004] In one aspect, provided herein is a method for increasing the fitness of a honeybee, the method including administering to the honeybee a composition including an effective amount of an organophosphorus insecticide-metabolizing bacteria formulated with an insect comestible carrier.
[0005] In some embodiments, the administration involves delivering the composition to a honeybee hive or at least one habitat where the honeybee grows, lives, reproduces, or feeds.
[0006] In some embodiments, the composition may be a liquid, a solid, an aerosol, a paste, a gel, or a gas.
[0007] In some embodiments, the organophosphorus insecticide may be fenitrothion.
[0008] In some embodiments, the carrier may be a seed coating.
[0009] In some embodiments, the honeybee may be in a honeybee colony.
[0010] In another aspect, provided herein is a composition including an effective amount of an organophosphorus insecticide-metabolizing bacteria formulated with an insect comestible carrier as a liquid, a solid, an aerosol, a paste, a gel, or a gas.
[0011] In some embodiments of the second aspect, the organophosphorus insecticide-metabolizing bacteria metabolize fenitrothion.
[0012] In some embodiments of the second aspect, the carrier is a seed coating.
[0013] In some embodiments of the second aspect, the organophosphorus insecticide-metabolizing bacteria are at a concentration of at least 100,000 cells/ml (e.g., at least about 100,000 cells/ml, at least about 150,000 cells/ml, at least about 200,000 cells/ml, at least about 250,000 cells/ml, at least about 300,000 cells/ml, at least about 350,000 cells/ml, at least about 400,000 cells/ml, at least about 450,000 cells/ml, or at least about 500,000 cells/ml).
[0014] In yet another aspect, the composition includes an agent that alters a level, activity, or metabolism of one or more microorganisms resident in an insect host, the alteration resulting in an increase in the insect host's fitness.
[0015] In another instance, the composition includes an agent that alters a level, activity, or metabolism of one or more microorganisms resident in a nematode host, the alteration resulting in an increase in the nematode host's fitness.
[0016] In some embodiments of any of the above compositions, the one or more microorganisms may be a bacterium or fungus resident in the host. In some embodiments, the bacterium resident in the host is at least one selected from the group consisting of Candidatus spp, Buchenera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacter spp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, and Escherichia spp. In some embodiments, the fungus resident in the host is at least one selected from the group consisting of Candida, Metschnikowia, Debaromyces, Starmerella, Pichia, Cryptococcus, Pseudozyma, Symbiotaphrina bucneri, Symbiotaphrina kochii, Scheffersomyces shehatae, Scheffersomyces stipites, Cryptococcus, Trichosporon, Amylostereum areolatum, Epichloe spp, Pichia pinus, Hansenula capsulate, Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamyces bromatificus.
[0017] In any of the above compositions, the agent, which hereinafter may also be referred to as a modulating agent, may alter the growth, division, viability, metabolism, and/or longevity of the microorganism resident in the host. In any of the above embodiments, the modulating agent may decrease the viability of the one or more microorganisms resident in the host. In some embodiments, the modulating agent increases growth or viability of the one or more microorganisms resident in the host.
[0018] In any of the above embodiments, the modulating agent is a phage, a polypeptide, a small molecule, an antibiotic, a bacterium, or any combination thereof.
[0019] In some embodiments, the phage binds a cell surface protein on a bacterium resident in the host. In some embodiments, the phage is virulent to a bacterium resident in the host. In some embodiments, the phage is at least one selected from the group consisting of Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and Tectiviridae.
[0020] In some embodiments, the polypeptide is at least one of a bacteriocin, R-type bacteriocin, nodule C-rich peptide, antimicrobial peptide, lysin, or bacteriocyte regulatory peptide.
[0021] In some embodiments, the small molecule is a metabolite.
[0022] In some embodiments, the antibiotic is a broad-spectrum antibiotic.
[0023] In some embodiments, the modulating agent is a naturally occurring bacteria. In some embodiments, the bacteria is at least one selected from the group consisting of Bartonella apis, Parasaccharibacter apium, Frischella perrara, Snodgrassella alvi, Gilliamela apicola, Bifidobacterium spp, and Lactobacillus spp. In some embodiments, the bacterium is at least one selected from the group consisting of Candidatus spp, Buchenera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacter spp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, and Escherichia spp. In certain instances, the bacterium is a naturally occurring bacterium that is capable of degrading pesticides such as organophosphorus insecticides (e.g., phosphorothioate, e.g., fenitrothion).
[0024] In any of the above compositions, host fitness may be measured by survival, reproduction, or metabolism of the host. In some embodiments, the modulating agent modulates the host's fitness by decreasing pesticidal susceptibility of the host (e.g., susceptibility to a pesticide listed in Table 12). In some embodiments, the pesticidal susceptibility is bactericidal or fungicidal susceptibility. In some embodiments, the pesticidal susceptibility is insecticidal or nematicidal susceptibility.
[0025] In any of the above compositions, the composition may include a plurality of different modulating agents. In some embodiments, the composition includes a modulating agent and a pesticidal agent (e.g., a pesticide listed in Table 12). In some embodiments, the pesticidal agent is a bactericidal or fungicidal agent. In some embodiments, the pesticidal agent is an insecticidal or nematicidal agent.
[0026] In any of the above compositions, the composition may include a modulating agent and an agent that increases crop growth.
[0027] In any of the above compositions, modulating agent may be linked to a second moiety. In some embodiments, the second moiety is a modulating agent.
[0028] In any of the above compositions, the modulating agent may be linked to a targeting domain. In some embodiments, the targeting domain targets the modulating agent to a target site in the host. In some embodiments, the targeting domain targets the modulating agent to the one or more microorganisms resident in the host.
[0029] In any of the above compositions, the modulating agent may include an inactivating pre- or pro-sequence, thereby forming a precursor modulating agent. In some embodiments, the precursor modulating agent is converted to an active form in the host.
[0030] In any of the above compositions, the modulating agent may include a linker. In some embodiments, the linker is a cleavable linker.
[0031] In any of the above compositions, the composition may further include a carrier. In some instances, the carrier may be an agriculturally acceptable carrier.
[0032] In any of the above compositions, the composition may further include a host bait, a sticky agent, or a combination thereof. In some embodiments, the host bait is a comestible agent and/or a chemoattractant.
[0033] In any of the above compositions, the composition may be at a dose effective to modulate host fitness.
[0034] In any of the above compositions, the composition may be formulated for delivery to a microorganism inhabiting the gut of the host.
[0035] In any of the above compositions, the composition may be formulated for delivery to a microorganism inhabiting a bacteriocyte of the host and/or the gut of the host. In some embodiments, the composition may be formulated for delivery to a plant. In some embodiments, the composition may be formulated for use in a host feeding station.
[0036] In any of the above compositions, the composition may be formulated as a liquid, a powder, granules, or nanoparticles. In some embodiments, the composition is formulated as one selected from the group consisting of a liposome, polymer, bacteria secreting peptide, and synthetic nanocapsule. In some embodiments, the synthetic nanocapsule delivers the composition to a target site in the host. In some embodiments, the target site is the gut of the host. In some embodiments, the target site is a bacteriocyte in the host.
[0037] In a further aspect, also provided herein are hosts that include any of the above compositions. In some embodiments, the host is an insect. In some embodiments, the insect aids in pest control, pollination, generation of a commercial product, or a combination thereof. In some embodiments, the insect is a species belonging to Coccinellidae, Carabidae, Mantidae, Syrphidae, Lampyridae, Myrmeliontidase, Chrysopidae, Hemerobiidae, Brachonidae, Ichneumonidae, or Odonata. In some embodiments, the insect is a species belonging to Andrenidae, Apidae, Colletidae, Halicitdae, or Megahlidae. In some embodiments, the insect is a species belonging to Bombycidae or Saturniidae. In certain embodiments, the insect is a honey bee or silkworm.
[0038] In some embodiments, the host is a nematode. In some embodiments, the nematode is a species belonging to Heterorhabditis or Steinernema.
[0039] In yet a further aspect, also provided herein is a system for modulating a host's fitness comprising a modulating agent that targets a microorganism that is required for a host's fitness, wherein the system is effective to modulate the host's fitness, and wherein the host is an insect or nematode. The modulating agent may include any of the compositions described herein. In some embodiments, the modulating agent is formulated as a powder. In some embodiments, the modulating agent is formulated as a solvent. In some embodiments, the modulating agent is formulated as a concentrate. In some embodiments, the modulating agent is formulated as a diluent. In some embodiments, the modulating agent is prepared for delivery by combining any of the previous compositions with a carrier.
[0040] In another aspect, also provided herein are methods for modulating the fitness of an insect or nematode using any of the compositions described herein. In one instance, the method of modulating the fitness of an insect or nematode host includes delivering the composition of any one of the previous claims to the host, wherein the modulating agent targets the one or more microorganisms resident in the host, and thereby modulates the host's fitness. In another instance, the method of modulating microbial diversity in an insect or nematode host includes delivering the composition of any one of the previous claims to the host, wherein the modulating agent targets the one or more microorganisms resident in the host, and thereby modulates microbial diversity in the host.
[0041] In some embodiments of any of the above methods, the modulating agent may alter the levels of the one or more microorganisms resident in the host. In some embodiments of any of the above methods, the modulating agent may alter the function of the one or more microorganisms resident in the host. In some embodiments, the one or more microorganisms may be a bacterium and/or fungus. In some embodiments, the one or more microorganisms are required for host fitness. In some embodiments, the one or more microorganisms are required for host survival.
[0042] In some embodiments of any of the above methods, the delivering step may include providing the modulating agent at a dose and time sufficient to effect the one or more microorganisms, thereby modulating microbial diversity in the host. In some embodiments, the delivering step includes topical application of any of the previous compositions to a plant. In some embodiments, the delivering step includes providing the modulating agent through a genetically engineered plant. In some embodiments, the delivering step includes providing the modulating agent to the host as a comestible. In some embodiments, the delivering step includes providing a host carrying the modulating agent. In some embodiments the host carrying the modulating agent can transmit the modulating agent to one or more additional hosts.
[0043] In some embodiments of any of the above methods, the composition is effective to increase health and/or survival of the host. In some embodiments, the composition is effective to increase host fitness, increase host lifespan, increase effective pollination, increase generation of a host product, increase host reproduction, or a combination thereof. In some embodiments, the composition is effective to decrease the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12). In certain embodiments, the pesticidal agent is a neonicotinoid (e.g., imidacloprid). In certain embodiments, the pesticidal agent is an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion). In some embodiments, the composition is effective to increase the host's resistance to an allelochemical agent produced by a plant. In some embodiments, the allelochemical agent is toxic to the host prior to delivery of the composition. In some embodiments, the allelochemical agent is caffeine, soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds.
[0044] In some embodiments of any of the above methods, the host is an insect. In some embodiments, the insect aids in pest control, pollination, generation of a commercial product, waste degradation, or a combination thereof. In some embodiments, the insect is a species belonging to Coccinellidae, Carabidae, Mantidae, Syrphidae, Lampyridae, Myrmeliontidase, Chrysopidae, Hemerobiidae, Brachonidae, Ichneumonidae, or Odonata. In some embodiments, the insect is a species belonging to Andrenidae, Apidae, Colletidae, Halicitdae, or Megahlidae. In some embodiments, the insect is a species belonging to Bombycidae or Saturniidae. In certain embodiments, the insect is a honey bee or silkworm.
[0045] In some embodiments, the host is a nematode. In some embodiments, the nematode is a species belonging to Heterorhabditis or Steinernema.
[0046] In some embodiments of any of the above methods, the delivering step includes delivering any of the previous compositions to a plant. In some embodiments, the plant is an agricultural crop. In some embodiments, the crop is an unharvested crop at the time of delivery. In some embodiments, the crop is a harvested crop at the time of delivery. The some embodiments, the crop comprises harvested fruits or vegetables. In some embodiments, the composition is delivered in an amount and for a duration effective to increase growth of the crop. In some embodiments, the crop includes corn, soybean, or wheat plants.
[0047] In another aspect, also provided herein are screening assays to identify modulating agent that modulate the fitness of a host. In one instance, the screening assay to identify a modulating agent that modulates the fitness of a host, includes the steps of (a) exposing a microorganism that can be resident in the host to one or more candidate modulating agents and (b) identifying a modulating agent that increases the fitness of the host.
[0048] In some embodiments of the screening assay, the modulating agent is a microorganism resident in the host. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium, when resident in the host, increases host fitness. In some embodiments, the bacterium degrades a pesticide (e.g., a pesticide listed in Table 12). In certain embodiments, the pesticide is a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion). In some embodiments, the bacterium secretes an amino acid. In certain embodiments, wherein the amino acid is methionine.
[0049] In some embodiments of the screening assay, the modulating agent affects an allelochemical-degrading microorganism. In some embodiments, the modulating agent is a phage, an antibiotic, or a test compound. In certain embodiments, the antibiotic is timentin or azithromycin.
[0050] In some embodiments of the screening assay, the host may be an invertebrate. In some embodiments, the invertebrate is an insect or a nematode. In certain embodiments, the insect is a honey bee. In other particular embodiments, the insect is a silkworm.
[0051] In any of the above embodiments of the screening assay, host fitness may be modulated by altering the host microbiota.
Definitions
[0052] As used herein, the term "bacteriocin" refers to a peptide or polypeptide that possesses anti-microbial properties. Naturally occurring bacteriocins are produced by certain prokaryotes and act against organisms related to the producer strain, but not against the producer strain itself. Bacteriocins contemplated herein include, but are not limited to, naturally occurring bacteriocins, such as bacteriocins produced by bacteria, or derivatives thereof, such as engineered bacteriocins, recombinantly expressed bacteriocins, or chemically synthesized bacteriocins. In some instances, the bacteriocin is a functionally active variant of the bacteriocins described herein. In some instances, the variant of the bacteriocin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a bacteriocin described herein or a naturally occurring bacteriocin.
[0053] As used herein, the term "bacteriocyte" refers to a specialized cell found in certain insects where intracellular bacteria reside with symbiotic bacterial properties.
[0054] As used herein, the term "effective amount" refers to an amount of a modulating agent (e.g., a phage, lysin, bacteriocin, small molecule, or antibiotic) or composition including said agent sufficient to effect the recited result, e.g., to increase or promote the fitness of a host organism (e.g., insect); to reach a target level (e.g., a predetermined or threshold level) of a modulating agent concentration inside a target host; to reach a target level (e.g., a predetermined or threshold level) of a modulating agent concentration inside a target host gut; to reach a target level (e.g., a predetermined or threshold level) of a modulating agent concentration inside a target host bacteriocyte; to modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host.
[0055] As used herein, the term "fitness" refers to the ability of a host organism to survive, grow, and/or to produce surviving offspring. Fitness of an organism may be measured by one or more parameters, including, but not limited to, life span, reproductive rate, mobility, body weight, and/or metabolic rate. Fitness may additionally be measured based on measures of activity (e.g., pollination) or product output (e.g., honey or silk).
[0056] As used herein, the term "gut" refers to any portion of a host's gut, including, the foregut, midgut, or hindgut of the host.
[0057] As used herein, the term "host" refers to an organism (e.g., insect) carrying resident microorganisms (e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g., primary or secondary endosymbionts), commensal organisms, and/or pathogenic microorganisms).
[0058] As used herein "increasing host fitness" or "promoting host fitness" refers to any favorable alteration in host physiology, or any activity carried out by said host, as a consequence of administration of a modulating agent, including, but not limited to, any one or more of the following desired effects: (1) increasing a population of a host by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) increasing the reproductive rate of a host (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (3) increasing the mobility of a host (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increasing the body weight of a host (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5) increasing the metabolic rate or activity of a host (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (6) increasing pollination (e.g., number of plants pollinated in a given amount of time) by a host (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) increasing production of host (e.g., insect, e.g., bee or silkworm) byproducts (e.g., honey from a honeybee or silk from a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8) increasing nutrient content of the host (e.g., insect) (e.g., protein, fatty acids, or amino acids) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (9) increasing host resistance to pesticides (e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. An increase in host fitness can be determined in comparison to a host organism to which the modulating agent has not been administered.
[0059] The term "insect" includes any organism belonging to the phylum Arthropoda and to the class Insecta or the class Arachnida, in any stage of development, i.e., immature or adult insects.
[0060] As used herein, "lysin" also known as endolysin, autolysin, murein hydrolase, peptidoglycan hydrolase, or cell wall hydrolase refers to a hydrolytic enzyme that can lyse a bacterium by cleaving peptidoglycan in the cell wall of the bacterium. Lysins contemplated herein include, but are not limited to, naturally occurring lysins, such as lysins produced by phages, lysins produced by bacteria, or derivatives thereof, such as engineered lysins, recombinantly expressed lysins, or chemically synthesized lysins. A functionally active variant of the bacteriocin may have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a synthetic, recombinant, or naturally derived bacteriocin, including any described herein.
[0061] As used herein, the term "microorganism" refers to bacteria or fungi. Microorganisms may refer to microorganisms resident in a host organism (e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g., primary or secondary endosymbionts)) or microorganisms exogenous to the host, including those that may act as modulating agents. As used herein, the term "target microorganism" refers to a microorganism that is resident in the host and impacted by a modulating agent, either directly or indirectly.
[0062] As used herein, the term "agent" or "modulating agent" refers to an agent that is capable of altering the levels and/or functioning of microorganisms resident in a host organism (e.g., insect), and thereby modulate (e.g., increase) the fitness of the host organism (e.g., insect).
[0063] As used herein, the term "pesticide" or "pesticidal agent" refers to a substance that can be used in the control of agricultural, environmental, or domestic/household pests, such as insects, fungi, bacteria, or viruses. The term "pesticide" is understood to encompass naturally occurring or synthetic insecticides (larvicides or adulticides), insect growth regulators, acaricides (miticides), nematicides, ectoparasiticides, bactericides, fungicides, or herbicides (substance which can be used in agriculture to control or modify plant growth). Further examples of pesticides or pesticidal agents are listed in Table 12. In some instances, the pesticide is an allelochemical. As used herein, "allelochemical" or "allelochemical agent" is a substance produced by an organism that can effect a physiological function (e.g., the germination, growth, survival, or reproduction) of another organism (e.g., a host insect or nematode).
[0064] As used herein, the term "peptide," "protein," or "polypeptide" encompasses any chain of naturally or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or more amino acids), the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation), or the presence of, e.g., one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to the peptide, and includes, for example, natural proteins, synthetic, or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics.
[0065] As used herein, "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, which is described in Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
[0066] As used herein, the term "phage" or "bacteriophage" refers to a virus that infects and replicates in bacteria. Bacteriophages replicate within bacteria following the injection of their genome into the cytoplasm and do so using either a lytic cycle, which results in bacterial cell lysis, or a lysogenic (non-lytic) cycle, which leaves the bacterial cell intact. The phage may be a naturally occurring phage isolate, or an engineered phage, including vectors, or nucleic acids that encode either a partial phage genome (e.g., including at least all essential genes necessary to carry out the life cycle of the phage inside a host bacterium) or the full phage genome.
[0067] As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, or microspores. Plant parts include differentiated or undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, or callus tissue). The plant tissue may be in a plant or in a plant organ, tissue, or cell culture. In addition, a plant may be genetically engineered to produce a heterologous protein or RNA, for example, of any of the modulating agents in the methods or compositions described herein.
[0068] The terms "obtainable by", "producible by" or the like are used to indicate that a claim or embodiment refers to compound, composition, product, etc. per se, i. e. that the compound, composition, product, etc. can be obtained or produced by a method which is described for manufacture of the compound, composition, product, etc., but that the compound, composition, product, etc. may be obtained or produced by other methods than the described one as well. The terms "obtained by," "produced by," or the like indicate that the compound, composition, product, is obtained or produced by a recited specific method. It is to be understood that the terms "obtainable by," "producible by" and the like also disclose the terms "obtained by", "produced by" and the like as a preferred embodiment of "obtainable by", "producible by" and the like.
[0069] Other features and advantages of the invention will be apparent from the following Detailed Description and the Claims.
BRIEF DESCRIPTION OF THE FIGURES
[0070] The figures are meant to be illustrative of one or more features, aspects, or embodiments of the invention and are not intended to be limiting.
[0071] FIG. 1 is a graph showing the time to reach adulthood from embryos in Drosophila melanogaster. Embryos of Drosophila melanogaster were either raised on diet seeded with Corynebacterium glutamicum (a strain that produces glutamate--C. glutamicum Glu) or on axenic diet without any bacteria. The percentage of adults emerging from their pupa was measured every 12 hours from the time of the emergence of the first adult. The organisms raised on bacteria supplemented diet reach adulthood faster than their bacteria free counterparts.
[0072] FIG. 2A is a graph showing the effects of male gender on the developmental rate differences in Drosophila melanogaster. The adults emerging from FIG. 1 were sexed and their rate of emergence was plotted.
[0073] FIG. 2B is a graph showing the effects of female gender on the developmental rate differences in Drosophila melanogaster. The adults emerging from FIG. 1 were sexed and their rate of emergence was plotted. The enhancement in the rate of development in the females due to the presence of bacteria in the diet is significantly more than in their male counterparts. The benefits of the presence of bacteria in the fly diet are higher in the females compared to the males.
[0074] FIG. 3 is a graph showing C. glutamicum strains promoted larval biomass. Larvae raised on diet supplemented with C. glutamicum strains either producing glutamate or methionine are bigger than those raised on sterile diet or diet supplemented with Escherichia coli. The areas of the larvae are measured as the number of pixels in the images of the larvae. The medians and the 95% confidence intervals are shown as lines on the graph.
[0075] FIG. 4 is a panel of graphs showing the results of a Seahorse flux assay for bacterial respiration. Bacteria were grown to logarithmic phase and loaded into Seahorse XFe96 plates for temporal measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as described in methods. Treatments were injected into the wells after approximately 20 minutes and bacteria were monitored to detect changes in growth. Rifampicin=100 .mu.g/mL; Chloramphenicol=25 .mu.g/mL; Phages (T7 for E. coli and .PHI.SmVL-C1 for Serratia marcescens) were lysates diluted either 1:2 or 1:100 in SM Buffer. The markers on each line are solely provided as indicators of the condition to which each line corresponds, and are not indicative of data points
[0076] FIG. 5 is a graph showing phage against S. marcescens reduced fly mortality. Flies that were pricked with S. marcescens were all dead within a day, whereas a sizeable portion of the flies that were pricked with both S. marcescens and the phage survived for five days after the treatment. Almost all of the control flies which were not treated in anyway survived till the end of the experiment. Log-rank test was used to compare the curves for statistical significance, asterisk denotes p<0.0001.
DETAILED DESCRIPTION
[0077] Provided herein are methods and compositions for agricultural use, e.g., for altering a level, activity, or metabolism of one or more microorganisms resident in a host nematode or arthropod (e.g., honeybee or silkworm), the alteration resulting in an increase in the fitness of the host. The invention features a composition that includes a modulating agent (e.g., phage, peptide, small molecule, antibiotic, or combinations thereof) that can alter the host's microbiota in a manner that is beneficial to the host. By promoting favorable microbial levels, microbial activity, microbial metabolism, and/or microbial diversity, the modulating agent described herein may be used to increase the fitness of a variety of beneficial nematodes or arthropods, such as bees and silkworms, utilized in agriculture and commerce.
[0078] The methods and compositions described herein are based in part on the examples which illustrate how different agents, for example imidacloprid-degrading microorganisms, fenitrothion-degrading microorganisms, and different phages can be used in insect hosts such as honeybees or Drosophila to indirectly improve the health of these hosts by altering the level, activity or metabolism of microorganisms within these hosts. Imidacloprid-degrading microorganisms are a representative example of neonicotinoid-degrading microorganisms and more generally are representative of insecticide- or pesticide-degrading microorganisms. Similarly, fenitrothion-degrading microorganisms are a representative example of organophosphorus insecticide-degrading microorganisms and more generally are representative of insecticide- or pesticide-degrading microorganisms. On this basis the present disclosure describes a variety of different approaches to the use of agents that alter a level, activity, or metabolism of one or more microorganisms resident in a host, the alteration resulting in a modulation in the host's fitness.
I. Hosts
[0079] i. Hosts
[0080] The host of any of the compositions or methods described herein may be any organism belonging to the phyla Nematoda (e.g., nematodes, e.g., beneficial nematodes) or Arthropoda (e.g., insects, e.g., beneficial insects), including any arthropods described herein. In some instances, the host is a beneficial insect or nematode (e.g., a pollinator, a natural competitor of a pest, or a producer of useful substances for humans). The term "beneficial insect" or "beneficial nematode," as used herein, refers to an insect or nematode that confers a benefit (e.g., economical and/or ecological) to humans, animals, an ecosystem, and/or the environment. For example, the host may be an insect that is involved in the production of a commercial product, including, but not limited to, insects cultivated to produce food (e.g., honey from honey bees, e.g., Apis mellifera), materials (such as silk from Bombyx mon), and/or substances (e.g., lac from Laccifer lacca or pigments from Dactylopius coccus and Cynipidae). Additionally, the host may include insects or nematodes that are used in agricultural applications, including insects that aid in the pollination of crops, spreading seeds, or pest control. Further, in some instances, the host may be an insect that is useful for waste disposal and/or organic recycling (e.g., earthworms, termites, or Diptera larvae).
[0081] In some instances, the host produces a useable product (e.g., honey, silk, beeswax, or shellac). In some instances, the host is a bee. Exemplary bee genera include, but are not limited to Apis, Bombus, Trigona, and Osmia. In some instances, the bee is a honeybee (e.g., an insect belonging to the genus Apis). In some instances, the honeybee is the species Apis mellifera (the European or Western honey bee), Apis cerana (the Asiatic, Eastern, or Himalayan honey bee), Apis dorsata (the "giant" honey bee), Apis florea (the "red dwarf" honey bee), Apis andreniformis (the "black dwarf" honey bee), or Apis nigrocincta. In some instances, the host is a silkworm. The silkworm may be a species in the family Bombycidae or Saturniidae. In some instances, the silkworm is Bombyx mori. In some instances, the host is a lac bug. The lac bug may be a species in the family Kerriidae. In some instances, the lac bug is Kerria lacca.
[0082] In some instances, the host aids in pollination of a plant (e.g., bees, beetles, wasps, flies, butterflies, or moths). In some examples, the host aiding in pollination of a plant is beetle. In some instances, the beetle is a species in the family Buprestidae, Cantharidae, Cerambycidae, Chrysomelidae, Cleridae, Coccinellidae, Elateridae, Melandryidae, Meloidae, Melyridae, Mordellidae, Nitidulidae, Oedemeridae, Scarabaeidae, or Staphyllinidae. In some instances, the host aiding in pollination of a plant is a butterfly or moth (e.g., Lepidoptera). In some instances, the butterfly or moth is a species in the family Geometridae, Hesperiidae, Lycaenidae, Noctuidae, Nymphalidae, Papilionidae, Pieridae, or Sphingidae. In some instances, the host aiding in pollination of a plant is a fly (e.g., Diptera). In some instances, the fly is in the family Anthomyiidae, Bibionidae, Bombyliidae, Calliphoridae, Cecidomiidae, Certopogonidae, Chrionomidae, Conopidae, Culicidae, Dolichopodidae, Empididae, Ephydridae, Lonchopteridae, Muscidae, Mycetophilidae, Phoridae, Simuliidae, Stratiomyidae, or Syrphidae. In some instances, the host aiding in pollination is an ant (e.g., Formicidae), sawfly (e.g., Tenthredinidae), or wasp (e.g., Sphecidae or Vespidae). In some instances, the host aiding in pollination of a plant is a bee. In some instances, the bee is in the family Andrenidae, Apidae, Colletidae, Halictidae, or Megachilidae.
[0083] In some instances, the host aids in pest control. In some instances, the host aiding in pest control is a predatory nematode. In particular examples, the nematode is a species of Heterorhabditis or Steinernema. In some instances, the host aiding in pest control is an insect. For example, the host aiding in pest control may be a species belonging to the family Braconidae (e.g., parasitoid wasps), Carabidae (e.g., ground beetles), Chrysopidae (e.g., green lacewings), Coccinellidae (e.g., ladybugs), Hemerobiidae (e.g., brown lacewings), Ichneumonidae (e.g., ichneumon wasps), Lampyridae (e.g., fireflies), Mantidae (e.g., praying mantises), Myrmeleontidae (e.g., antilions), Odonata (e.g., dragonflies and damselflies), or Syrphidae (e.g., hoverfly). In other instances, the host aiding in pest control is an insect that competes with an insect that is considered a pest (e.g., an agricultural pest). For example, the Mediterranean fruit fly, Ceratitis capitata is a common pest of fruits and vegetables worldwide. One way to control C. captitata is to release the sterilized male insect into the environment to compete with wild males to mate the females. In these instances, the host may be a sterilized male belonging to a species that is typically considered a pest.
[0084] In some instances, the host aids in degradation of waste or organic material. In some examples, the host aiding in degradation of waste or organic material belongs to Coleoptera or Diptera. In some instances, the host belonging to Diptera is in the family Calliphoridae, Curtonotidae, Drosophilidae, Fanniidae, Heleomyzidae, Milichiidae, Muscidae, Phoridae, Psychodidae, Scatopsidae, Sepsidae, Sphaeroceridae, Stratiomyidae, Syrphidae, Tephritidae, or Ulidiidae. In some instances, the host belonging to Coleoptera is in the family Carabidae, Hydrophilidae, Phalacaridae, Ptiliidae, or Staphylinidae.
[0085] In particular instances, the modulating agents disclosed herein may be used to increase the fitness of honeybee or silkworm hosts.
[0086] ii. Host Fitness
[0087] The methods and compositions provided herein may be used to increase the fitness of any of the hosts described herein. The increase in fitness may arise from any alterations in microorganisms resident in the host, wherein the alterations are a consequence of administration of a modulating agent and have beneficial or advantageous effects on the host.
[0088] In some instances, the increase in host fitness may manifest as an improvement in the physiology of the host (e.g., improved health or survival) as a consequence of administration of a modulating agent. In some instances, the fitness of an organism may be measured by one or more parameters, including, but not limited to, reproductive rate, lifespan, mobility, fecundity, body weight, metabolic rate or activity, or survival in comparison to a host organism to which the modulating agent has not been administered. For example, the methods or compositions provided herein may be effective to improve the overall health of the host or to improve the overall survival of the host in comparison to a host organism to which the modulating agent has not been administered. In some instances, the improved survival of the host is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% greater relative to a reference level (e.g., a level found in a host that does not receive a modulating agent). In some instances, the methods and compositions are effective to increase host reproduction (e.g., reproductive rate) in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods and compositions are effective to increase other physiological parameters, such as mobility, body weight, life span, fecundity, or metabolic rate, by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
[0089] In some instances, the increase in host fitness may manifest as an increased production of a product generated by said host in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the production of a product generated by the host, as described herein (e.g., honey, beeswax, beebread, propolis, silk, or lac), by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
[0090] In some instances, the increase in host fitness may manifest as an increase in the frequency or efficacy of a desired activity carried out by the host (e.g., pollination, predation on pests, seed spreading, or breakdown of waste or organic material) in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the frequency or efficacy of a desired activity carried out by the host (e.g., pollination, predation on pests, seed spreading, or breakdown of waste or organic material) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
[0091] In some instances, the increase in host fitness may manifest as an increase in the production of one or more nutrients in the host (e.g., vitamins, carbohydrates, amino acids, or polypeptides) in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the production of nutrients in the host (e.g., vitamins, carbohydrates, amino acids, or polypeptides) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent). In some instances, the methods or compositions provided herein may increase nutrients in the host by increasing the production of nutrients by one or more microorganisms (e.g., endosymbiont) in the host.
[0092] In some instances, the increase in host fitness may manifest as a decrease in the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12) and/or an increase in the host's resistance to a pesticidal agent (e.g., a pesticide listed in Table 12) in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods or compositions provided herein may be effective to decrease the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent). In some instances, the host's sensitivity to the pesticidal agent is altered by administering a modulating agent that degrades a pesticidal agent (e.g., a pesticidal-degrading bacteria, e.g., a neonicotinoid-degrading bacteria or an organophosphorus insecticide-degrading bacteria). The pesticidal agent may be any pesticidal agent known in the art, including insecticidal agents. In some instances, the pesticidal agent is a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion). In some instances, the methods or compositions provided herein may decrease the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12) by increasing the host's ability to metabolize or degrade the pesticidal agent into usable substrates.
[0093] In some instances, the host's sensitivity to the pesticidal agent is altered by administering a modulating agent that detoxifies a xenobiotic.
[0094] In some instances, the increase in host fitness may manifest as a decrease in the host's sensitivity to an allelochemical agent and/or an increase in the host's resistance to an allelochemical agent in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the host's resistance to an allelochemical agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent). In some instances, the allelochemical agent is caffeine, soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds. In some instances, the methods or compositions provided herein may decrease the host's sensitivity to an allelochemical agent by increasing the host's ability to metabolize or degrade the allelochemical agent into usable substrates.
[0095] In some instances, the methods or compositions provided herein may be effective to increase the host's resistance to parasites or pathogens (e.g., fungal, bacterial, or viral pathogens; or parasitic mites (e.g., Varroa destructor mite in honeybees)) in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the host's resistance to a pathogen or parasite (e.g., fungal, bacterial, or viral pathogens; or parasitic mites (e.g., Varroa destructor mite in honeybees)) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
[0096] In some instances, the increase in host fitness may manifest as other fitness advantages, such as improved tolerance to certain environmental factors (e.g., a high or low temperature tolerance), improved ability to survive in certain habitats, or an improved ability to sustain a certain diet (e.g., an improved ability to metabolize soy vs corn) in comparison to a host organism to which the modulating agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase host fitness in any plurality of ways described herein. Further, the modulating agent may increase host fitness in any number of host classes, orders, families, genera, or species (e.g., 1 host species, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 200, 250, 500, or more host species). In some instances, the modulating agent acts on a single host class, order, family, genus, or species.
[0097] Host fitness may be evaluated using any standard methods in the art. In some instances, host fitness may be evaluated by assessing an individual host. Alternatively, host fitness may be evaluated by assessing a host population. For example, an increase in host fitness may manifest as an increase in successful competition against other insects, thereby leading to an increase in the size of the host population.
[0098] iii. Host Invertebrates in Agriculture
[0099] By increasing the fitness of beneficial nematodes or insects, the modulating agents provided herein may be effective to promote the growth of plants that benefit from said hosts. The modulating agent may be delivered using any formulations and delivery methods described herein, in an amount and for a duration effective to increase the fitness of the hosts of interest and thereby benefit the plant (e.g., increase crop growth, increase crop yield, decrease pest infestation, and/or decrease damage to plants). This may or may not involve direct application of the modulating agent to the plant. For example, in instances where the primary host habitat is different than the region of plant growth, the modulating agent may be applied to either the primary host habitat, the plants of interest, or a combination of both.
[0100] In some instances, the plant may be an agricultural crop, such as a cereal, grain, legume, fruit, or vegetable crop. The compositions described herein may be delivered to the crop any time prior to or after harvesting the cereal, grain, legume, fruit, or vegetable. Crop yield is a measurement often used for, e.g., a cereal, grain, or legume and is normally measured in metric tons per hectare (or kilograms per hectare). Crop yield can also refer to the actual seed generation from the plant. In some instances, the modulating agent may be effective to increase crop yield (e.g., increase metric tons of cereal, grain, legume, fruit, or vegetable per hectare and/or increase seed generation) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a reference level (e.g., a crop to which the modulating agent has not been administered.
[0101] In some instances, the plant (e.g., crop) may be at risk of developing a pest infestation (e.g., by an insect) or may have already developed a pest infestation. The methods and compositions described herein may be used to reduce or prevent pest infestation in such crops by promoting the fitness of beneficial insects that prey on agricultural pests. In some instances, the modulating agent may be effective to reduce crop infestation (e.g., reduce the number of plants infested, reduce the pest population size, or reduce damage to plants) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a reference level (e.g., a crop to which the modulating agent has not been administered). In other instances, the modulating agent may be effective to prevent or reduce the likelihood of crop infestation by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a reference level (e.g., a crop to which the modulating agent has not been administered).
[0102] Any suitable plant tissues may benefit from the compositions and methods described herein, including, but not limited to, somatic embryos, pollen, leaves, stems, calli, stolons, microtubers, and shoots. The methods described herein may include treatment of angiosperm and gymnosperm plants such as acacia, alfalfa, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fava beans, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, sallow, soybean, spinach, spruce, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, a vine, walnut, watercress, watermelon, wheat, yams, yew, and zucchini.
II. Target Microorganisms
[0103] The microorganisms targeted by the modulating agent described herein may include any microorganism resident in or on the host, including, but not limited to, any bacteria and/or fungi described herein. Microorganisms resident in the host may include, for example, symbiotic (e.g., endosymbiotic microorganisms that provide beneficial nutrients or enzymes to the host), commensal, pathogenic, or parasitic microorganisms. A symbiotic microorganism (e.g., bacteria or fungi) may be an obligate symbiont of the host or a facultative symbiont of the host. Microorganisms resident in the host may be acquired by any mode of transmission, including vertical, horizontal, or multiple origins of transmission.
[0104] i. Bacteria
[0105] Exemplary bacteria that may be targeted in accordance with the methods and compositions provided herein, include, but are not limited to, Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Buchnera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacter spp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, and Escherichia spp. Non-limiting examples of bacteria that may be targeted by the methods and compositions provided herein are shown in Table 1.
TABLE-US-00001 TABLE 1 Examples of Target Bacteria and Host Insects Endosymbiont Host Insect Location 16S rRNA Snodgrassella alvi honeybee Ileum GAGAGTTTGATCCTGGCTCAGATTGAACGC (Apis TGGCGGCATGCCTTACACATGCAAGTCGAA mellifera) and CGGCAGCACGGAGAGCTTGCTCTCTGGTG Bombus spp. GCGAGTGGCGAACGGGTGAGTAATGCATC GGAACGTACCGAGTAATGGGGGATAACTG TCCGAAAGGATGGCTAATACCGCATACGCC CTGAGGGGGAAAGCGGGGGATCGAAAGAC CTCGCGTTATTTGAGCGGCCGATGTTGGAT TAGCTAGTTGGTGGGGTAAAGGCCTACCAA GGCGACGATCCATAGCGGGTCTGAGAGGA TGATCCGCCACATTGGGACTGAGACACGG CCCAAACTCCTACGGGAGGCAGCAGTGGG GAATTTTGGACAATGGGGGGAACCCTGATC CAGCCATGCCGCGTGTCTGAAGAAGGCCT TCGGGTTGTAAAGGACTTTTGTTAGGGAAG AAAAGCCGGGTGTTAATACCATCTGGTGCT GACGGTACCTAAAGAATAAGCACCGGCTAA CTACGTGCCAGCAGCCGCGGTAATACGTA GGGTGCGAGCGTTAATCGGAATTACTGGG CGTAAAGCGAGCGCAGACGGTTAATTAAGT CAGATGTGAAATCCCCGAGCTCAACTTGGG ACGTGCATTTGAAACTGGTTAACTAGAGTG TGTCAGAGGGAGGTAGAATTCCACGTGTAG CAGTGAAATGCGTAGAGATGTGGAGGAATA CCGATGGCGAAGGCAGCCTCCTGGGATAA CACTGACGTTCATGCTCGAAAGCGTGGGTA GCAAACAGGATTAGATACCCTGGTAGTCCA CGCCCTAAACGATGACAATTAGCTGTTGGG ACACTAGATGTCTTAGTAGCGAAGCTAACG CGTGAAATTGTCCGCCTGGGGAGTACGGT CGCAAGATTAAAACTCAAAGGAATTGACGG GGACCCGCACAAGCGGTGGATGATGTGGA TTAATTCGATGCAACGCGAAGAACCTTACC TGGTCTTGACATGTACGGAATCTCTTAGAG ATAGGAGAGTGCCTTCGGGAACCGTAACA CAGGTGCTGCATGGCTGTCGTCAGCTCGT GTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTCATTAGTTGCCATCA TTAAGTTGGGCACTCTAATGAGACTGCCGG TGACAAACCGGAGGAAGGTGGGGATGACG TCAAGTCCTCATGGCCCTTATGACCAGGGC TTCACACGTCATACAATGGTCGGTACAGAG GGTAGCGAAGCCGCGAGGTGAAGCCAATC TCAGAAAGCCGATCGTAGTCCGGATTGCAC TCTGCAACTCGAGTGCATGAAGTCGGAATC GCTAGTAATCGCAGGTCAGCATACTGCGGT GAATACGTTCCCGGGTCTTGTACACACCGC CCGTCACACCATGGGAGTGGGGGATACCA GAATTGGGTAGACTAACCGCAAGGAGGTC GCTTAACACGGTATGCTTCATGACTGGGGT GAAGTCGTAACAAGGTAGCCGTAG (SEQ ID NO: 1) Gilliamella apicola honeybee Ileum TTAAATTGAAGAGTTTGATCATGGCTCAGAT (Apis TGAACGCTGGCGGCAGGCTTAACACATGC mellifera) and AAGTCGAACGGTAACATGAGTGCTTGCACT Bombus spp. TGATGACGAGTGGCGGACGGGTGAGTAAA GTATGGGGATCTGCCGAATGGAGGGGGAC AACAGTTGGAAACGACTGCTAATACCGCAT AAAGTTGAGAGACCAAAGCATGGGACCTTC GGGCCATGCGCCATTTGATGAACCCATATG GGATTAGCTAGTTGGTAGGGTAATGGCTTA CCAAGGCGACGATCTCTAGCTGGTCTGAG AGGATGACCAGCCACACTGGAACTGAGAC ACGGTCCAGACTCCTACGGGAGGCAGCAG TGGGGAATATTGCACAATGGGGGAAACCCT GATGCAGCCATGCCGCGTGTATGAAGAAG GCCTTCGGGTTGTAAAGTACTTTCGGTGAT GAGGAAGGTGGTGTATCTAATAGGTGCATC AATTGACGTTAATTACAGAAGAAGCACCGG CTAACTCCGTGCCAGCAGCCGCGGTAATA CGGAGGGTGCGAGCGTTAATCGGAATGAC TGGGCGTAAAGGGCATGTAGGCGGATAAT TAAGTTAGGTGTGAAAGCCCTGGGCTCAAC CTAGGAATTGCACTTAAAACTGGTTAACTA GAGTATTGTAGAGGAAGGTAGAATTCCACG TGTAGCGGTGAAATGCGTAGAGATGTGGA GGAATACCGGTGGCGAAGGCGGCCTTCTG GACAGATACTGACGCTGAGATGCGAAAGC GTGGGGAGCAAACAGGATTAGATACCCTG GTAGTCCACGCTGTAAACGATGTCGATTTG GAGTTTGTTGCCTAGAGTGATGGGCTCCGA AGCTAACGCGATAAATCGACCGCCTGGGG AGTACGGCCGCAAGGTTAAAACTCAAATGA ATTGACGGGGGCCCGCACAAGCGGTGGAG CATGTGGTTTAATTCGATGCAACGCGAAGA ACCTTACCTGGTCTTGACATCCACAGAATC TTGCAGAGATGCGGGAGTGCCTTCGGGAA CTGTGAGACAGGTGCTGCATGGCTGTCGT CAGCTCGTGTTGTGAAATGTTGGGTTAAGT CCCGCAACGAGCGCAACCCTTATCCTTTGT TGCCATCGGTTAGGCCGGGAACTCAAAGG AGACTGCCGTTGATAAAGCGGAGGAAGGT GGGGACGACGTCAAGTCATCATGGCCCTT ACGACCAGGGCTACACACGTGCTACAATG GCGTATACAAAGGGAGGCGACCTCGCGAG AGCAAGCGGACCTCATAAAGTACGTCTAAG TCCGGATTGGAGTCTGCAACTCGACTCCAT GAAGTCGGAATCGCTAGTAATCGTGAATCA GAATGTCACGGTGAATACGTTCCCGGGCCT TGTACACACCGCCCGTCACACCATGGGAG TGGGTTGCACCAGAAGTAGATAGCTTAACC TTCGGGAGGGCGTTTACCACGGTGTGGTC CATGACTGGGGTGAAGTCGTAACAAGGTAA CCGTAGGGGAACCTGCGGTTGGATCACCT CCTTAC (SEQ ID NO: 2) Bartonella apis honeybee Gut AAGCCAAAATCAAATTTTCAACTTGAGAGTT (Apis TGATCCTGGCTCAGAACGAACGCTGGCGG mellifera) CAGGCTTAACACATGCAAGTCGAACGCACT TTTCGGAGTGAGTGGCAGACGGGTGAGTA ACGCGTGGGAATCTACCTATTTCTACGGAA TAACGCAGAGAAATTTGTGCTAATACCGTA TACGTCCTTCGGGAGAAAGATTTATCGGAG ATAGATGAGCCCGCGTTGGATTAGCTAGTT GGTGAGGTAATGGCCCACCAAGGCGACGA TCCATAGCTGGTCTGAGAGGATGACCAGC CACATTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGTGGGGAATATTG GACAATGGGCGCAAGCCTGATCCAGCCAT GCCGCGTGAGTGATGAAGGCCCTAGGGTT GTAAAGCTCTTTCACCGGTGAAGATAATGA CGGTAACCGGAGAAGAAGCCCCGGCTAAC TTCGTGCCAGCAGCCGCGGTAATACGAAG GGGGCTAGCGTTGTTCGGATTTACTGGGC GTAAAGCGCACGTAGGCGGATATTTAAGTC AGGGGTGAAATCCCGGGGCTCAACCCCGG AACTGCCTTTGATACTGGATATCTTGAGTAT GGAAGAGGTAAGTGGAATTCCGAGTGTAG AGGTGAAATTCGTAGATATTCGGAGGAACA CCAGTGGCGAAGGCGGCTTACTGGTCCAT TACTGACGCTGAGGTGCGAAAGCGTGGGG AGCAAACAGGATTAGATACCCTGGTAGTCC ACGCTGTAAACGATGAATGTTAGCCGTTGG ACAGTTTACTGTTCGGTGGCGCAGCTAACG CATTAAACATTCCGCCTGGGGAGTACGGTC GCAAGATTAAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGTGGAGCATGTGGTT TAATTCGAAGCAACGCGCAGAACCTTACCA GCCCTTGACATCCCGATCGCGGATGGTGG AGACACCGTCTTTCAGTTCGGCTGGATCGG TGACAGGTGCTGCATGGCTGTCGTCAGCT CGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTCGCCCTTAGTTGCCA TCATTTAGTTGGGCACTCTAAGGGGACTGC CGGTGATAAGCCGAGAGGAAGGTGGGGAT GACGTCAAGTCCTCATGGCCCTTACGGGCT GGGCTACACACGTGCTACAATGGTGGTGA CAGTGGGCAGCGAGACCGCGAGGTCGAG CTAATCTCCAAAAGCCATCTCAGTTCGGAT TGCACTCTGCAACTCGAGTGCATGAAGTTG GAATCGCTAGTAATCGTGGATCAGCATGCC ACGGTGAATACGTTCCCGGGCCTTGTACAC ACCGCCCGTCACACCATGGGAGTTGGTTTT ACCCGAAGGTGCTGTGCTAACCGCAAGGA GGCAGGCAACCACGGTAGGGTCAGCGACT GGGGTGAAGTCGTAACAAGGTAGCCGTAG GGGAACCTGCGGCTGGATCACCTCCTTTCT AAGGAAGATGAAGAATTGGAA (SEQ ID NO: 3) Parasaccharibacter honeybee Gut CTACCATGCAAGTCGCACGAAACCTTTCGG apium (Apis GGTTAGTGGCGGACGGGTGAGTAACGCGT mellifera) TAGGAACCTATCTGGAGGTGGGGGATAAC ATCGGGAAACTGGTGCTAATACCGCATGAT GCCTGAGGGCCAAAGGAGAGATCCGCCAT TGGAGGGGCCTGCGTTCGATTAGCTAGTT GGTTGGGTAAAGGCTGACCAAGGCGATGA TCGATAGCTGGTTTGAGAGGATGATCAGCC ACACTGGGACTGAGACACGGCCCAGACTC CTACGGGAGGCAGCAGTGGGGAATATTGG ACAATGGGGGCAACCCTGATCCAGCAATG CCGCGTGTGTGAAGAAGGTCTTCGGATTGT AAAGCACTTTCACTAGGGAAGATGATGACG GTACCTAGAGAAGAAGCCCCGGCTAACTTC GTGCCAGCAGCCGCGGTAATACGAAGGGG GCTAGCGTTGCTCGGAATGACTGGGCGTA AAGGGCGCGTAGGCTGTTTGTACAGTCAG ATGTGAAATCCCCGGGCTTAACCTGGGAAC TGCATTTGATACGTGCAGACTAGAGTCCGA GAGAGGGTTGTGGAATTCCCAGTGTAGAG GTGAAATTCGTAGATATTGGGAAGAACACC GGTTGCGAAGGCGGCAACCTGGCTNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNGAGCTAA CGCGTTAAGCACACCGCCTGGGGAGTACG GCCGCAAGGTTGAAACTCAAAGGAATTGAC GGGGGCCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGAAGCAACGCGCAGAACCTT ACCAGGGCTTGCATGGGGAGGCTGTATTC AGAGATGGATATTTCTTCGGACCTCCCGCA CAGGTGCTGCATGGCTGTCGTCAGCTCGT GTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTCTTTAGTTGCCATCA CGTCTGGGTGGGCACTCTAGAGAGACTGC CGGTGACAAGCCGGAGGAAGGTGGGGATG ACGTCAAGTCCTCATGGCCCTTATGTCCTG GGCTACACACGTGCTACAATGGCGGTGAC AGAGGGATGCTACATGGTGACATGGTGCT GATCTCAAAAAACCGTCTCAGTTCGGATTG TACTCTGCAACTCGAGTGCATGAAGGTGGA ATCGCTAGTAATCGCGGATCAGCATGCCGC GGTGAATACGTTCCCGGGCCTTGTACACAC CGCCCGTCACACCATGGGAGTTGGTTTGA CCTTAAGCCGGTGAGCGAACCGCAAGGAA CGCAGCCGACCACCGGTTCGGGTTCAGCG ACTGGGGGA (SEQ ID NO: 4) Lactobacillus honeybee Gut TTCCTTAGAAAGGAGGTGATCCAGCCGCAG kunkeei (Apis GTTCTCCTACGGCTACCTTGTTACGACTTC mellifera) ACCCTAATCATCTGTCCCACCTTAGACGAC TAGCTCCTAAAAGGTTACCCCATCGTCTTT GGGTGTTACAAACTCTCATGGTGTGACGGG CGGTGTGTACAAGGCCCGGGAACGTATTC ACCGTGGCATGCTGATCCACGATTACTAGT GATTCCAACTTCATGCAGGCGAGTTGCAGC CTGCAATCCGAACTGAGAATGGCTTTAAGA GATTAGCTTGACCTCGCGGTTTCGCGACTC GTTGTACCATCCATTGTAGCACGTGTGTAG CCCAGCTCATAAGGGGCATGATGATTTGAC GTCGTCCCCACCTTCCTCCGGTTTATCACC GGCAGTCTCACTAGAGTGCCCAACTAAATG CTGGCAACTAATAATAAGGGTTGCGCTCGT TGCGGGACTTAACCCAACATCTCACGACAC GAGCTGACGACAACCATGCACCACCTGTCA TTCTGTCCCCGAAGGGAACGCCCAATCTCT TGGGTTGGCAGAAGATGTCAAGAGCTGGT AAGGTTCTTCGCGTAGCATCGAATTAAACC ACATGCTCCACCACTTGTGCGGGCCCCCG TCAATTCCTTTGAGTTTCAACCTTGCGGTC GTACTCCCCAGGCGGAATACTTAATGCGTT AGCTGCGGCACTGAAGGGCGGAAACCCTC CAACACCTAGTATTCATCGTTTACGGCATG GACTACCAGGGTATCTAATCCTGTTCGCTA CCCATGCTTTCGAGCCTCAGCGTCAGTAAC AGACCAGAAAGCCGCCTTCGCCACTGGTG TTCTTCCATATATCTACGCATTTCACCGCTA CACATGGAGTTCCACTTTCCTCTTCTGTACT CAAGTTTTGTAGTTTCCACTGCACTTCCTCA GTTGAGCTGAGGGCTTTCACAGCAGACTTA CAAAACCGCCTGCGCTCGCTTTACGCCCAA TAAATCCGGACAACGCTTGCCACCTACGTA TTACCGCGGCTGCTGGCACGTAGTTAGCC
GTGGCTTTCTGGTTAAATACCGTCAAAGTG TTAACAGTTACTCTAACACTTGTTCTTCTTT AACAACAGAGTTTTACGATCCGAAAACCTT CATCACTCACGCGGCGTTGCTCCATCAGAC TTTCGTCCATTGTGGAAGATTCCCTACTGC TGCCTCCCGTAGGAGTCTGGGCCGTGTCT CAGTCCCAATGTGGCCGATTACCCTCTCAG GTCGGCTACGTATCATCGTCTTGGTGGGCT TTTATCTCACCAACTAACTAATACGGCGCG GGTCCATCCCAAAGTGATAGCAAAGCCATC TTTCAAGTTGGAACCATGCGGTTCCAACTA ATTATGCGGTATTAGCACTTGTTTCCAAATG TTATCCCCCGCTTCGGGGCAGGTTACCCAC GTGTTACTCACCAGTTCGCCACTCGCTCCG AATCCAAAAATCATTTATGCAAGCATAAAAT CAATTTGGGAGAACTCGTTCGACTTGCATG TATTAGGCACGCCGCCAGCGTTCGTCCTGA GCCAGGATCAAACTCTCATCTTAA (SEQ ID NO: 190) Lactobacillus Firm- honeybee Gut ACGAACGCTGGCGGCGTGCCTAATACATG 4 (Apis CAAGTCGAGCGCGGGAAGTCAGGGAAGCC mellifera) TTCGGGTGGAACTGGTGGAACGAGCGGCG GATGGGTGAGTAACACGTAGGTAACCTGC CCTAAAGCGGGGGATACCATCTGGAAACA GGTGCTAATACCGCATAAACCCAGCAGTCA CATGAGTGCTGGTTGAAAGACGGCTTCGG CTGTCACTTTAGGATGGACCTGCGGCGTAT TAGCTAGTTGGTGGAGTAACGGTTCACCAA GGCAATGATACGTAGCCGACCTGAGAGGG TAATCGGCCACATTGGGACTGAGACACGG CCCAAACTCCTACGGGAGGCAGCAGTAGG GAATCTTCCACAATGGACGCAAGTCTGATG GAGCAACGCCGCGTGGATGAAGAAGGTCT TCGGATCGTAAAATCCTGTTGTTGAAGAAG AACGGTTGTGAGAGTAACTGCTCATAACGT GACGGTAATCAACCAGAAAGTCACGGCTAA CTACGTGCCAGCAGCCGCGGTAATACGTA GGTGGCAAGCGTTGTCCGGATTTATTGGG CGTAAAGGGAGCGCAGGCGGTCTTTTAAG TCTGAATGTGAAAGCCCTCAGCTTAACTGA GGAAGAGCATCGGAAACTGAGAGACTTGA GTGCAGAAGAGGAGAGTGGAACTCCATGT GTAGCGGTGAAATGCGTAGATATATGGAAG AACACCAGTGGCGAAGGCGGCTCTCTGGT CTGTTACTGACGCTGAGGCTCGAAAGCATG GGTAGCGAACAGGATTAGATACCCTGGTAG TCCATGCCGTAAACGATGAGTGCTAAGTGT TGGGAGGTTTCCGCCTCTCAGTGCTGCAG CTAACGCATTAAGCACTCCGCCTGGGGAGT ACGACCGCAAGGTTGAAACTCAAAGGAATT GACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCAACGCGAAGAAC CTTACCAGGTCTTGACATCTCCTGCAAGCC TAAGAGATTAGGGGTTCCCTTCGGGGACA GGAAGACAGGTGGTGCATGGTTGTCGTCA GCTCGTGTCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCTTGTTACTAGTTG CCAGCATTAAGTTGGGCACTCTAGTGAGAC TGCCGGTGACAAACCGGAGGAAGGTGGGG ACGACGTCAAATCATCATGCCCCTTATGAC CTGGGCTACACACGTGCTACAATGGATGGT ACAATGAGAAGCGAACTCGCGAGGGGAAG CTGATCTCTGAAAACCATTCTCAGTTCGGA TTGCAGGCTGCAACTCGCCTGCATGAAGCT GGAATCGCTAGTAATCGCGGATCAGCATGC CGCGGTGAATACGTTCCCGGGCCTTGTAC ACACCGCCC (SEQ ID NO: 191) Enterococcus Bombyx mori Gut AGGTGATCCAGCCGCACCTTCCGATACGG CTACCTTGTTACGACTTCACCCCAATCATCT ATCCCACCTTAGGCGGCTGGCTCCAAAAAG GTTACCTCACCGACTTCGGGTGTTACAAAC TCTCGTGGTGTGACGGGCGGTGTGTACAA GGCCCGGGAACGTATTCACCGCGGCGTGC TGATCCGCGATTACTAGCGATTCCGGCTTC ATGCAGGCGAGTTGCAGCCTGCAATCCGA ACTGAGAGAAGCTTTAAGAGATTTGCATGA CCTCGCGGTCTAGCGACTCGTTGTACTTCC CATTGTAGCACGTGTGTAGCCCAGGTCATA AGGGGCATGATGATTTGACGTCATCCCCAC CTTCCTCCGGTTTGTCACCGGCAGTCTCGC TAGAGTGCCCAACTAAATGATGGCAACTAA CAATAAGGGTTGCGCTCGTTGCGGGACTTA ACCCAACATCTCACGACACGAGCTGACGAC AACCATGCACCACCTGTCACTTTGTCCCCG AAGGGAAAGCTCTATCTCTAGAGTGGTCAA AGGATGTCAAGACCTGGTAAGGTTCTTCGC GTTGCTTCGAATTAAACCACATGCTCCACC GCTTGTGCGGGCCCCCGTCAATTCCTTTGA GTTTCAACCTTGCGGTCGTACTCCCCAGGC GGAGTGCTTAATGCGTTTGCTGCAGCACTG AAGGGCGGAAACCCTCCAACACTTAGCACT CATCGTTTACGGCGTGGACTACCAGGGTAT CTAATCCTGTTTGCTCCCCACGCTTTCGAG CCTCAGCGTCAGTTACAGACCAGAGAGCC GCCTTCGCCACTGGTGTTCCTCCATATATC TACGCATTTCACCGCTACACATGGAATTCC ACTCTCCTCTTCTGCACTCAAGTCTCCCAG TTTCCAATGACCCTCCCCGGTTGAGCCGG GGGCTTTCACATCAGACTTAAGAAACCGCC TGCGCTCGCTTTACGCCCAATAAATCCGGA CAACGCTTGCCACCTACGTATTACCGCGGC TGCTGGCACGTAGTTAGCCGTGGCTTTCTG GTTAGATACCGTCAGGGGACGTTCAGTTAC TAACGTCCTTGTTCTTCTCTAACAACAGAGT TTTACGATCCGAAAACCTTCTTCACTCACG CGGCGTTGCTCGGTCAGACTTTCGTCCATT GCCGAAGATTCCCTACTGCTGCCTCCCGTA GGAGTCTGGGCCGTGTCTCAGTCCCAGTG TGGCCGATCACCCTCTCAGGTCGGCTATG CATCGTGGCCTTGGTGAGCCGTTACCTCAC CAACTAGCTAATGCACCGCGGGTCCATCCA TCAGCGACACCCGAAAGCGCCTTTCACTCT TATGCCATGCGGCATAAACTGTTATGCGGT ATTAGCACCTGTTTCCAAGTGTTATCCCCCT CTGATGGGTAGGTTACCCACGTGTTACTCA CCCGTCCGCCACTCCTCTTTCCAATTGAGT GCAAGCACTCGGGAGGAAAGAAGCGTTCG ACTTGCATGTATTAGGCACGCCGCCAGCGT TCGTCCTGAGCCAGGATCAAACTCT (SEQ ID NO: 5) Delftia Bombyx mori Gut CAGAAAGGAGGTGATCCAGCCGCACCTTC CGATACGGCTACCTTGTTACGACTTCACCC CAGTCACGAACCCCGCCGTGGTAAGCGCC CTCCTTGCGGTTAGGCTACCTACTTCTGGC GAGACCCGCTCCCATGGTGTGACGGGCGG TGTGTACAAGACCCGGGAACGTATTCACCG CGGCATGCTGATCCGCGATTACTAGCGATT CCGACTTCACGCAGTCGAGTTGCAGACTG CGATCCGGACTACGACTGGTTTTATGGGAT TAGCTCCCCCTCGCGGGTTGGCAACCCTC TGTACCAGCCATTGTATGACGTGTGTAGCC CCACCTATAAGGGCCATGAGGACTTGACGT CATCCCCACCTTCCTCCGGTTTGTCACCGG CAGTCTCATTAGAGTGCTCAACTGAATGTA GCAACTAATGACAAGGGTTGCGCTCGTTGC GGGACTTAACCCAACATCTCACGACACGAG CTGACGACAGCCATGCAGCACCTGTGTGC AGGTTCTCTTTCGAGCACGAATCCATCTCT GGAAACTTCCTGCCATGTCAAAGGTGGGTA AGGTTTTTCGCGTTGCATCGAATTAAACCA CATCATCCACCGCTTGTGCGGGTCCCCGT CAATTCCTTTGAGTTTCAACCTTGCGGCCG TACTCCCCAGGCGGTCAACTTCACGCGTTA GCTTCGTTACTGAGAAAACTAATTCCCAAC AACCAGTTGACATCGTTTAGGGCGTGGACT ACCAGGGTATCTAATCCTGTTTGCTCCCCA CGCTTTCGTGCATGAGCGTCAGTACAGGTC CAGGGGATTGCCTTCGCCATCGGTGTTCCT CCGCATATCTACGCATTTCACTGCTACACG CGGAATTCCATCCCCCTCTACCGTACTCTA GCCATGCAGTCACAAATGCAGTTCCCAGGT TGAGCCCGGGGATTTCACATCTGTCTTACA TAACCGCCTGCGCACGCTTTACGCCCAGTA ATTCCGATTAACGCTCGCACCCTACGTATT ACCGCGGCTGCTGGCACGTAGTTAGCCGG TGCTTATTCTTACGGTACCGTCATGGGCCC CCTGTATTAGAAGGAGCTTTTTCGTTCCGT ACAAAAGCAGTTTACAACCCGAAGGCCTTC ATCCTGCACGCGGCATTGCTGGATCAGGC TTTCGCCCATTGTCCAAAATTCCCCACTGC TGCCTCCCGTAGGAGTCTGGGCCGTGTCT CAGTCCCAGTGTGGCTGGTCGTCCTCTCA GACCAGCTACAGATCGTCGGCTTGGTAAG CTTTTATCCCACCAACTACCTAATCTGCCAT CGGCCGCTCCAATCGCGCGAGGCCCGAAG GGCCCCCGCTTTCATCCTCAGATCGTATGC GGTATTAGCTACTCTTTCGAGTAGTTATCCC CCACGACTGGGCACGTTCCGATGTATTACT CACCCGTTCGCCACTCGTCAGCGTCCGAA GACCTGTTACCGTTCGACTTGCATGTGTAA GGCATGCCGCCAGCGTTCAATCTGAGCCA GGATCAAACTCTACAGTTCGATCT (SEQ ID NO: 6) Pelomonas Bombyx mori Gut ATCCTGGCTCAGATTGAACGCTGGCGGCAT GCCTTACACATGCAAGTCGAACGGTAACAG GTTAAGCTGACGAGTGGCGAACGGGTGAG TAATATATCGGAACGTGCCCAGTCGTGGGG GATAACTGCTCGAAAGAGCAGCTAATACCG CATACGACCTGAGGGTGAAAGCGGGGGAT CGCAAGACCTCGCNNGATTGGAGCGGCCG ATATCAGATTAGGTAGTTGGTGGGGTAAAG GCCCACCAAGCCAACGATCTGTAGCTGGT CTGAGAGGACGACCAGCCACACTGGGACT GAGACACGGCCCAGACTCCTACGGGAGGC AGCAGTGGGGAATTTTGGACAATGGGCGC AAGCCTGATCCAGCCATGCCGCGTGCGGG AAGAAGGCCTTCGGGTTGTAAACCGCTTTT GTCAGGGAAGAAAAGGTTCTGGTTAATACC TGGGACTCATGACGGTACCTGAAGAATAAG CACCGGCTAACTACGTGCCAGCAGCCGCG GTAATACGTAGGGTGCAAGCGTTAATCGGA ATTACTGGGCGTAAAGCGTGCGCAGGCGG TTATGCAAGACAGAGGTGAAATCCCCGGG CTCAACCTGGGAACTGCCTTTGTGACTGCA TAGCTAGAGTACGGTAGAGGGGGATGGAA TTCCGCGTGTAGCAGTGAAATGCGTAGATA TGCGGAGGAACACCGATGGCGAAGGCAAT CCCCTGGACCTGTACTGACGCTCATGCAC GAAAGCGTGGGGAGCAAACAGGATTAGAT ACCCTGGTAGTCCACGCCCTAAACGATGTC AACTGGTTGTTGGGAGGGTTTCTTCTCAGT AACGTANNTAACGCGTGAAGTTGACCGCCT GGGGAGTACGGCCGCAAGGTTGAAACTCA AAGGAATTGACGGGGACCCGCACAAGCGG TGGATGATGTGGTTTAATTCGATGCAACGC GAAAAACCTTACCTACCCTTGACATGCCAG GAATCCTGAAGAGATTTGGGAGTGCTCGAA AGAGAACCTGGACACAGGTGCTGCATGGC CGTCGTCAGCTCGTGTCGTGAGATGTTGG GTTAAGTCCCGCAACGAGCGCAACCCTTGT CATTAGTTGCTACGAAAGGGCACTCTAATG AGACTGCCGGTGACAAACCGGAGGAAGGT GGGGATGACGTCAGGTCATCATGGCCCTT ATGGGTAGGGCTACACACGTCATACAATGG CCGGGACAGAGGGCTGCCAACCCGCGAG GGGGAGCTAATCCCAGAAACCCGGTCGTA GTCCGGATCGTAGTCTGCAACTCGACTGC GTGAAGTCGGAATCGCTAGTAATCGCGGAT CAGCTTGCCGCGGTGAATACGTTCCCGGG TCTTGTACACACCGCCCGTCACACCATGGG AGCGGGTTCTGCCAGAAGTAGTTAGCCTAA CCGCAAGGAGGGCGATTACCACGGCAGGG TTCGTGACTGGGGTGAAGTCGTAACAAGGT AGCCGTATCGGAAGGTGCGGCTGGATCAC (SEQ ID NO: 7)
[0106] Any number of bacterial species may be targeted by the compositions or methods described herein. For example, in some instances, the modulating agent may target a single bacterial species. In some instances, the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or more distinct bacterial species. In some instances, the modulating agent may target any one of about 1 to about 5, about 5 to about 10, about 10 to about 20, about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 10 to about 50, about 5 to about 20, or about 10 to about 100 distinct bacterial species. In some instances, the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more phyla, classes, orders, families, or genera of bacteria.
[0107] In some instances, the modulating agent may increase a population of one or more bacteria (e.g., symbiotic bacteria) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered. In some instances, the modulating agent may reduce the population of one or more bacteria (e.g., pathogenic or parasitic bacteria) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in the host in comparison to a host organism to which the modulating agent has not been administered. In some instances, the modulating agent may eradicate the population of a bacterium (e.g., a pathogenic or parasitic bacteria) in the host. In some instances, the modulating agent may increase the level of one or more symbiotic bacteria by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host and/or decreases the level of one or more pathogenic bacteria by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in the host in comparison to a host organism to which the modulating agent has not been administered.
[0108] In some instances, the modulating agent may alter the bacterial diversity and/or bacterial composition of the host. In some instances, the modulating agent may increase the bacterial diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered. In some instances, the modulating agent may decrease the bacterial diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered.
[0109] In some instances, the modulating agent may alter the function, activity, growth, and/or division of one or more bacterial cells. For example, the modulating agent may alter the expression of one or genes in the bacteria. In some instances, the modulating agent may alter the function of one or more proteins in the bacteria. In some instances, the modulating agent may alter the function of one or more cellular structures (e.g., the cell wall, the outer or inner membrane) in the bacteria. In some instances, the modulating agent may kill (e.g., lyse) the bacteria.
[0110] The target bacterium may reside in one or more parts of the insect. Further, the target bacteria may be intracellular or extracellular. In some instances, the bacteria reside in or on one or more parts of the host gut, including, e.g., the foregut, midgut, and/or hindgut. For example, in honey bees (e.g., Apis mellifera), bacterial symbionts confined to the hindguts of adults are acquired in the first few days following emergence of adults from the pupal stage, through social interactions with other adult worker bees in the colony. Honey bee gut inhabitants belong to a small number of distinctive lineages found only in honey bees and also in other Apis species and in Bombus species (bumble bees). In some instances, the target bacteria are resident in a honeybee. In some instances, one or more bacteria targeted in the honeybee is a Snodgrassella spp. (e.g., Snodgrasella alvi), a Gilliamella spp. (e.g., Gilliamella apicola), a Bartonella spp. (e.g., Bartonella apis), a Parasaccharibacter spp. (e.g., Parasaccharibacter apium), or a Lactobacillus spp. (e.g., Lactobacillus kunkeei, Lactobacillus Firm-4).
[0111] In some instances, the bacteria reside as an intracellular bacteria within a cell of the host insect. In some instances, the bacteria reside in a bacteriocyte of the host insect.
[0112] Changes to the populations of bacteria in the host may be determined by any methods known in the art, such as microarray, polymerase chain reaction (PCR), real-time PCR, flow cytometry, fluorescence microscopy, transmission electron microscopy, fluorescence in situ hybridization (e.g., FISH), spectrophotometry, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), and DNA sequencing. In some instances, a sample of the host treated with a modulating agent is sequenced (e.g., by metagenomics sequencing of 16S rRNA or rDNA) to determine the microbiome of the host after delivery or administration of the modulating agent. In some instances, a sample of a host that did not receive the modulating agent is also sequenced to provide a reference.
[0113] ii. Fungi
[0114] Exemplary fungi that may be targeted in accordance with the methods and compositions provided herein, include, but are not limited to Amylostereum areolatum, Epichloe spp, Pichia pinus, Hansenula capsulate, Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamyces bromatificus. Non-limiting examples of yeast and yeast-like symbionts found in insects include Candida, Metschnikowia, Debaromyces, Scheffersomyces shehatae and Scheffersomyces stipites, Starmerella, Pichia, Trichosporon, Cryptococcus, Pseudozyma, and yeast-like symbionts from the subphylum Pezizomycotina (e.g., Symbiotaphrina buneri and Symbiotaphrina kochii). Non-limiting examples of yeast that may be targeted by the methods and compositions herein are listed in Table 2.
TABLE-US-00002 TABLE 2 Examples of Yeast in Insects Insect Species Order: Family Yeast Location (Species) Stegobium paniceum Coleoptera: Anobiidae Mycetomes (=Sitodrepa panicea) (Saccharomyces) Cecae (Torulopsis buchnerii) Mycetome between foregut and midgut Mycetomes (Symbiotaphrina buchnerii) Mycetomes and digestive tube (Torulopsis buchnerii) Gut cecae (Symbiotaphrina buchnerii) Lasioderma serricorne Coleoptera: Anobiidae Mycetome between foregut and midgut (Symbiotaphrina kochii) Ernobius abietis Coleoptera: Anobiidae Mycetomes (Torulopsis karawaiewii) (Candida karawaiewii) Ernobius mollis Coleoptera: Anobiidae Mycetomes (Torulopsis ernobii) (Candida ernobii) Hemicoelus gibbicollis Coleoptera: Anobiidae Larval mycetomes Xestobium plumbeum Coleoptera: Anobiidae Mycetomes (Torulopsis xestobii) (Candida xestobii) Criocephalus rusticus Coleoptera: Cerambycidae Mycetomes Phoracantha Coleoptera: Cerambycidae Alimentary canal (Candida semipunctata guilliermondii, C. tenuis) Cecae around midgut (Candida guilliermondii) Harpium inquisitor Coleoptera: Cerambycidae Mycetomes (Candida rhagii) Harpium mordax Coleoptera: Cerambycidae Cecae around midgut (Candida tenuis) H. sycophanta Gaurotes virginea Coleoptera: Cerambycidae Cecae around midgut (Candida rhagii) Leptura rubra Coleoptera: Cerambycidae Cecae around midgut (Candida tenuis) Cecae around midgut (Candida parapsilosis) Leptura maculicornis Coleoptera: Cerambycidae Cecae around midgut (Candida parapsilosis) L. cerambyciformis Leptura sanguinolenta Coleoptera: Cerambycidae Cecae around midgut (Candida sp.) Rhagium bifasciatum Coleoptera: Cerambycidae Cecae around midgut (Candida tenuis) Rhagium inquisitor Coleoptera: Cerambycidae Cecae around midgut (Candida guilliermondii) Rhagium mordax Coleoptera: Cerambycidae Cecae around midgut (Candida) Carpophilus Coleoptera: Nitidulidae Intestinal tract (10 yeast species) hemipterus Odontotaenius Coleoptera: Passalidae Hindgut (Enteroramus dimorphus) disjunctus Odontotaenius Coleoptera: Passalidae Gut (Pichia stipitis, P. segobiensis, disjunctus Candida shehatae) Verres sternbergianus (C. ergatensis) Scarabaeus Coleoptera: Scarabaeidae Digestive tract (10 yeast species) semipunctatus Chironitis furcifer Unknown species Coleoptera: Scarabaeidae Guts (Trichosporon cutaneum) Dendroctonus and Ips Coleoptera: Scolytidae Alimentary canal (13 yeast species) spp. Dendroctonus frontalis Coleoptera: Scolytidae Midgut (Candida sp.) Ips sexdentatus Coleoptera: Scolytidae Digestive tract (Pichia bovis, P. rhodanensis) Hansenula holstii (Candida rhagii) Digestive tract (Candida pulcherina) Ips typographus Coleoptera: Scolytidae Alimentary canal Alimentary tracts (Hansenula capsulata, Candida parapsilosis) Guts and beetle homogenates (Hansenula holstii, H. capsulata, Candida diddensii, C. mohschtana, C. nitratophila, Cryptococcus albidus, C. laurentii) Trypodendron Coleoptera: Scolytidae Not specified lineatum Xyloterinus politus Coleoptera: Scolytidae Head, thorax, abdomen (Candida, Pichia, Saccharomycopsis) Periplaneta americana Dictyoptera: Blattidae Hemocoel (Candida sp. nov.) Blatta orientalis Dictyoptera: Blattidae Intestinal tract (Kluyveromyces blattae) Blatella germanica Dictyoptera: Blattellidae Hemocoel Cryptocercus Dictyoptera: Cryptocercidae Hindgut (1 yeast species) punctulatus Philophylla heraclei Diptera: Tephritidae Hemocoel Aedes (4 species) Diptera: Culicidae Internal microflora (9 yeast genera) Drosophila Diptera: Drosophilidae Alimentary canal (24 yeast species) pseudoobscura Drosophila (5 spp.) Diptera: Drosophilidae Crop (42 yeast species) Drosophila Diptera: Drosophilidae Crop (8 yeast species) melanogaster Drosophila (4 spp.) Diptera: Drosophilidae Crop (7 yeast species) Drosophila (6 spp.) Diptera: Drosophilidae Larval gut (17 yeast species) Drosophila (20 spp.) Diptera: Drosophilidae Crop (20 yeast species) Drosophila (8 species Diptera: Drosophilidae Crop (Kloeckera, Candida, groups) Kluyveromyces) Drosophila serido Diptera: Drosophilidae Crop (18 yeast species) Drosophila (6 spp.) Diptera: Drosophilidae Intestinal epithelium (Coccidiascus legeri) Protaxymia Diptera Unknown (Candida, Cryptococcus, melanoptera Sporoblomyces) Astegopteryx styraci Homoptera: Aphididae Hemocoel and fat body Tuberaphis sp. Homoptera: Aphididae Tissue sections Hamiltonaphis styraci Glyphinaphis bambusae Cerataphis sp. Hamiltonaphis styraci Homoptera: Aphididae Abdominal hemocoel Cofana unimaculata Homoptera: Cicadellidae Fat body Leofa unicolor Homoptera: Cicadellidae Fat body Lecaniines, etc. Homoptera: Coccoidea d Hemolymph, fatty tissue, etc. Lecanium sp. Homoptera: Coccidae Hemolymph, adipose tissue Ceroplastes (4 sp.) Homoptera: Coccidae Blood smears Laodelphax striatellus Homoptera: Delphacidae Fat body Eggs Eggs (Candida) Nilaparvata lugens Homoptera: Delphacidae Fat body Eggs (2 unidentified yeast species) Eggs, nymphs (Candida) Eggs (7 unidentified yeast species) Eggs (Candida) Nisia nervosa Homoptera: Delphacidae Fat body Nisia grandiceps Perkinsiella spp. Sardia rostrata Sogatella furcifera Sogatodes orizicola Homoptera: Delphacidae Fat body Amrasca devastans Homoptera: Jassidae Eggs, mycetomes, hemolymph Tachardina lobata Homoptera: Kerriidae Blood smears (Torulopsis) Laccifer (=Lakshadia) Homoptera: Kerriidae Blood smears (Torula variabilis) sp. Comperia merceti Hymenoptera: Encyrtidae Hemolymph, gut, poison gland Solenopsis invicta Hymenoptera: Formicidae Hemolymph (Myrmecomyces annellisae) S. quinquecuspis Solenopsis invicta Hymenoptera: Formicidae Fourth instar larvae (Candida parapsilosis, Yarrowia lipolytica) Gut and hemolymph (Candida parapsilosis, C. lipolytica, C. guillermondii, C. rugosa, Debaryomyces hansenii) Apis mellifera Hymenoptera: Apidae Digestive tracts (Torulopsis sp.) Intestinal tract (Torulopsis apicola) Digestive tracts (8 yeast species) Intestinal contents (12 yeast species) Intestinal contents (7 yeast species) Intestines (14 yeast species) Intestinal tract (Pichia melissophila) Intestinal tracts (7 yeast species) Alimentary canal (Hansenula silvicola) Crop and gut (13 yeast species) Apis mellifera Hymenoptera: Apidae Midguts (9 yeast genera) Anthophora Hymenoptera: Anthophoridae occidentalis Nomia melanderi Hymenoptera: Halictidae Halictus rubicundus Hymenoptera: Halictidae Megachile rotundata Hymenoptera: Megachilidae
[0115] Any number of fungal species may be targeted by the compositions or methods described herein. For example, in some instances, the modulating agent may target a single fungal species. In some instances, the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or more distinct fungal species. In some instances, the modulating agent may target any one of about 1 to about 5, about 5 to about 10, about 10 to about 20, about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 10 to about 50, about 5 to about 20, or about 10 to about 100 distinct fungal species. In some instances, the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more phyla, classes, orders, families, or genera of fungi.
[0116] In some instances, the modulating agent may increase a population of one or more fungi (e.g., symbiotic fungi) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered. In some instances, the modulating agent may reduce the population of one or more fungi (e.g., pathogenic or parasitic fungi) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered. In some instances, the modulating agent may eradicate the population of a fungi (e.g., a pathogenic or parasitic fungi) in the host. In some instances, the modulating agent may increase the level of one or more symbiotic fungi by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host and/or may decrease the level of one or more pathogenic fungi by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered.
[0117] In some instances, the modulating agent may alter the fungal diversity and/or fungal composition of the host. In some instances, the modulating agent may increase the fungal diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered. In some instances, the modulating agent may decrease the fungal diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered.
[0118] In some instances, the modulating agent may alter the function, activity, growth, and/or division of one or more fungi. For example, the modulating agent may alter the expression of one or more genes in the fungus. In some instances, the modulating agent may alter the function of one or more proteins in the fungus. In some instances, the modulating agent may alter the function of one or more cellular components in the fungus. In some instances, the modulating agent may kill the fungus.
[0119] Further, the target fungus may reside in one or more parts of the insect. In some instances, the fungus resides in or on one or more parts of the insect gut, including, e.g., the foregut, midgut, and/or hindgut. In some instances, the fungus lives extracellularly in the hemolymph, fat bodies or in specialized structures in the host.
[0120] Changes to the population of fungi in the host may be determined by any methods known in the art, such as microarray, polymerase chain reaction (PCR), real-time PCR, flow cytometry, fluorescence microscopy, transmission electron microscopy, fluorescence in situ hybridization (e.g., FISH), spectrophotometry, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), and DNA sequencing. In some instances, a sample of the host treated with modulating agent is sequenced (e.g., by metagenomics sequencing) to determine the microbiome of the host after delivery or administration of the modulating agent. In some instances, a sample of a host that did not receive the modulating agent is also sequenced to provide a reference.
III. Modulating Agents
[0121] The modulating agent of the methods and compositions provided herein may include a phage, a polypeptide, a small molecule, an antibiotic, a secondary metabolite, a bacterium, or any combination thereof.
[0122] i. Phage
[0123] The modulating agent described herein may include a phage (e.g., a lytic phage or a non-lytic phage). In some instances, an effective concentration of any phage described herein may altering a level, activity, or metabolism of one or more microorganisms (as described herein) resident in a host described herein, the alteration resulting in an increase in the host's fitness. In some instances, the modulating agent includes at least one phage selected from the order Tectiviridae, Myoviridae, Siphoviridae, Podoviridae, Caudovirales, Lipothrixviridae, Rudiviridae, or Ligamenvirales. In some instances, the composition includes at least one phage selected from the family Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and Tectiviridae. Further non-limiting examples of phages useful in the methods and compositions are listed in Table 3.
TABLE-US-00003 TABLE 3 Examples of Phage and Targeted Bacteria Phage and Accession # Target bacteria Target host SA1(NC_027991), phiP68 Staphylococcus Apidae family (NC_004679) sp. WO (AB036666.1) Wolbachia sp. Aedes aegypt; Drosophila melanogaster; Plasmodium sp; Teleogryllus taiwanemma; Bombyx mori KL1 (NC_018278), BcepNazgul Burkholderia sp. Riptortus sp.; Pyrrhocoris (NC_005091) PhiE125 (NC_003309) apterus. Fern (NC_028851), Xenia Paenibacillus bumble bees: Bombus (NC_028837), HB10c2 (NC_028758) larvae sp.; honey bees: A. mellifera CP2 (NC_020205), XP10 (NC_004902), Xanthomonas Plebeina denoiti; Apidae XP15 (NC_007024), phiL7 sp. family; Apis mellifera; (NC_012742) Drosphilidae family; and Chloropidae family PP1 (NC_019542), PM1 (NC_023865) Pectobacterium Apidae family carotovorum subsp. carotovorum .PHI.RSA1 (NC_009382), Ralstonia Bombyx mori .PHI.RSB1 (NC_011201), .PHI.RSL1 solanacearum (NC_010811), RSM1 (NC_008574) SF1 (NC_028807) Streptomyces Philantus sp.; Trachypus scabies sp ECML-4 (NC_025446), ECML-117 Escherichia coli Apidae family; (NC_025441), ECML-134 (NC_025449) Varroa destructor SSP5(JX274646.1), SSP6 Salmonella sp. Drosphilidae family (NC_004831), SFP10 (NC_016073), F18SE (NC_028698) .gamma. (NC_001416), Bcp1 (NC_024137) Bacillus sp. Gypsy moth; Lymantria dispar, Varroa destructor Phi1 (NC_009821) Enterococcus Schistocerca gragaria sp. .PHI.KMV (NC_005045), Pseudomonas Lymantria dispar; Apidae .PHI.EL(AJ697969.1), .PHI.KZ (NC_004629) sp. family A2 (NC_004112), phig1e (NC_004305) Lactobacilli sp. Apidae family; Drosophila family; Varroa destructor KLPN1 (NC_028760) Klebsiella sp C. capitata vB_AbaM_Acibel004 (NC_025462), Acinetobacter Schistocerca gragaria vB_AbaP_Acibel007 (NC_025457) sp.
[0124] In some instances, a modulating agent includes a lytic phage. Thus, after delivery of the lytic phage to a bacterial cell resident in the host, the phage causes lysis in the target bacterial cell. In some instances, the lytic phage targets and kills a bacterium resident in a host insect to increase the fitness of the host. Alternatively or additionally, the phage of the modulating agent may be a non-lytic phage (also referred to as lysogenic or temperate phage). Thus, after delivery of the non-lytic phage to a bacterial cell resident in the host, the bacterial cell may remain viable and able to stably maintain expression of genes encoded in the phage genome. In some instances, a non-lytic phage is used to alter gene expression in a bacterium resident in a host insect to increase the fitness of the host. In some instances, the modulating agent includes a mixture of lytic and non-lytic phage.
[0125] The modulating agent described herein may include phage with either a narrow or broad bacterial target range. In some instances, the phage has a narrow bacterial target range. In some instances, the phage is a promiscuous phage with a large bacterial target range. For example, the promiscuous phage may target at least about any of 5, 10, 20, 30, 40, 50, or more bacterium resident in the host. A phage with a narrow bacterial target range may target a specific bacterial strain in the host without affecting another, e.g., non-targeted, bacterium in the host. For example, the phage may target no more than about any of 50, 40, 30, 20, 10, 8, 6, 4, 2, or 1 bacterium resident in the host. For example, the compositions described herein may target the bacterial pathogen Paenibacillus larvae in honeybees but may not target other bacteria (e.g., symbiotic bacteria) including Lactobacillus Firm4, Lactobacillus Firm5, Bifidobacterium sp, Snodgrassella alvi, Gilliamella apicola, Bartonella apis, Parasaccharibacter apium, or Lactobacillus kunkeei. In some instances, the phage infects one or more specific bacteria by binding to a cell surface protein (e.g., OmpF, LamB, BtuB, ToIC, etc) or to a different recognition molecule (e.g., glycolipids, LPS, lipoteichoic acid, etc.).
[0126] The compositions described herein may include any number of phage, such as at least about any one of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more phage. In some instances, the composition includes phage from one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phage) families, one or more orders (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phage), or one or more species (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more phage). Compositions including one or more phage are also referred herein as "phage cocktails." Phage cocktails are useful because they allow for targeting of a wider host range of bacteria. Furthermore, they allow for each bacterial strain (i.e. subspecies) to be targeted by multiple orthogonal phages, thereby preventing or significantly delaying the onset of resistance. In some instances, a cocktail includes multiple phages targeting one bacterial species. In some instances, a cocktail includes multiple phages targeting multiple bacterial species. In some instances, a one-phage "cocktail" includes a single promiscuous phage (i.e. a phage with a large host range) targeting many strains within a species.
[0127] Suitable concentrations of the phage in the modulating agent described herein depends on factors such as efficacy, survival rate, transmissibility of the phage, number of distinct phage, and/or lysin types in the compositions, formulation, and methods of application of the composition. In some instances, the phage is in a liquid or a solid formulation. In some instances, the concentration of each phage in any of the compositions described herein is at least about any of 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10 or more pfu/ml. In some instances, the concentration of each phage in any of the compositions described herein is no more than about any of 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 pfu/ml. In some instances, the concentration of each phage in the composition is any of about 10.sup.2 to about 10.sup.3, about 10.sup.3 to about 10.sup.4, about 10.sup.4 to about 10.sup.5, about 10.sup.5 to about 10.sup.6, about 10.sup.7 to about 10.sup.8, about 10.sup.8 to about 10.sup.9, about 10.sup.2 to about 10.sup.4, about 10.sup.4 to about 10.sup.6, about 10.sup.6 to about 10.sup.9, or about 10.sup.3 to about 10.sup.8 pfu/ml. In some instances, wherein the composition includes at least two types of phages, the concentration of each type of the phages may be the same or different. For example, in some instances, the concentration of one phage in the cocktail is about 10.sup.8 pfu/ml and the concentration of a second phage in the cocktail is about 10.sup.6 pfu/ml.
[0128] A modulating agent including a phage as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of phage concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of phage concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of phage concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0129] As illustrated by Example 7 and 11, phages can be used as a modulating agents by eliminating bacterial pathogens such as Serratia marcescens, Erwinia catotovora, and Pseudomonas enzomophila from insect hosts, such as honeybees or silkworms.
[0130] ii. Polypeptides
[0131] Numerous polypeptides (e.g., a bacteriocin, R-type bacteriocin, nodule C-rich peptide, antimicrobial peptide, lysin, or bacteriocyte regulatory peptide) may be used in the compositions and methods described herein. In some instances, an effective concentration of any peptide or polypeptide described herein may alter a level, activity, or metabolism of one or more microorganisms (as described herein) resident in a host, the alteration resulting in an increase in the host's fitness. Polypeptides included herein may include naturally occurring polypeptides or recombinantly produced variants. For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a polypeptide described herein or a naturally occurring polypeptide.
[0132] A modulating agent comprising a polypeptide as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0133] The polypeptide modulating agents discussed hereinafter, namely bacteriocins, lysins, antimicrobial peptides, nodule C-rich peptides, and bacteriocyte regulatory peptides, can be used to alter the level, activity, or metabolism of target microorganisms as indicated in the section for increasing the fitness of insects, such as honeybees and silkworms.
[0134] (a) Bacteriocins
[0135] The modulating agent described herein may include a bacteriocin. In some instances, the bacteriocin is naturally produced by Gram-positive bacteria, such as Pseudomonas, Streptomyces, Bacillus, Staphylococcus, or lactic acid bacteria (LAB, such as Lactococcus lactis). In some instances, the bacteriocin is naturally produced by Gram-negative bacteria, such as Hafnia alvei, Citrobacter freundii, Klebsiella oxytoca, Klebsiella pneumonia, Enterobacter cloacae, Serratia plymithicum, Xanthomonas campestris, Erwinia carotovora, Ralstonia solanacearum, or Escherichia coli. Exemplary bacteriocins include, but are not limited to, Class I-IV LAB antibiotics (such as lantibiotics), colicins, microcins, and pyocins. Non-limiting examples of bacteriocins are listed in Table 4.
TABLE-US-00004 TABLE 4 Examples of Bacteriocins Class Name Producer Targets Sequence Class I Nisin Lactococcus Active on Gram-positive ITSISLCTPGCKT lactis bacteria: GALMGCNMKTA Enterococcus, TCHCSIHVSK Lactobacillus, (SEQ ID NO: 8) Lactococcus, Leuconostoc, Listeria, Clostridium Epidermin Staphylococcus Gram-positive bacteria IASKFICTPGCA epidermis KTGSFNSYCC (SEQ ID NO: 9) Class II Class II a Pediocin Pediococcus Pediococci, KYYGNGVTCG PA-1 acidilactici Lactobacilli, KHSCSVDWGK Leuconostoc, ATTCIINNGAMA Brochothrix thermosphacta, WATGGHQGNH Propionibacteria, KC Bacilli, (SEQ ID NO: 10) Enterococci, Staphylococci, Listeria clostridia, Listeria monocytogenes, Listeria innocua Class II b Enterocin Enterococcus Lactobacillus sakei, ATRSYGNGVYC P faecium Enterococcus faecium NNSKCWVNWG EAKENIAGIVISG WASGLAGMGH (SEQ ID NO: 11) Class II c Lactococc Streptococcus Gram-positive bacteria GTWDDIGQGIG in G lactis RVAYWVGKAM GNMSDVNQAS RINRKKKH (SEQ ID NO: 12) Class II d Lactacin-F Lactobacillus Lactobacilli, NRWGDTVLSAA johnsonii Enterococcus faecalis SGAGTGIKACK SFGPWGMAICG VGGAAIGGYFG YTHN (SEQ ID NO: 13) Class III Class III a Enterocin Enterococcus Broad spectrum: Gram MAKEFGIPAAVA AS-48 faecalis positive and Gram negative GTVLNVVEAGG bacteria. WVTTIVSILTAV GSGGLSLLAAA GRESIKAYLKKE IKKKGKRAVIAW (SEQ ID NO: 14) Class III b Aureocin Staphylococcus Broad spectrum: Gram MSWLNFLKYIAK A70 aureus positive and Gram negative YGKKAVSAAWK bacteria. YKGKVLEWLNV GPTLEWVWQKL KKIAGL (SEQ ID NO: 15) Class IV Garvicin A Lactococcus Broad spectrum: Gram IGGALGNALNGL garvieae positive and Gram negative GTWANMMNGG bacteria. GFVNQWQVYA NKGKINQYRPY (SEQ ID NO: 16) Unclassified Colicin V Escherichia Active against Escherichia MRTLTLNELDS coli coil (also closely related VSGGASGRDIA bacteria), MAIGTLSGQFV Enterobacteriaceae AGGIGAAAGGV AGGAIYDYAST HKPNPAMSPSG LGGTIKQKPEGI PSEAWNYAAGR LCNWSPNNLSD VCL (SEQ ID NO: 17)
[0136] In some instances, the bacteriocin is a colicin, a pyocin, or a microcin produced by Gram-negative bacteria. In some instances, the bacteriocin is a colicin. The colicin may be a group A colicin (e.g., uses the Tol system to penetrate the outer membrane of a target bacterium) or a group B colicin (e.g., uses the Ton system to penetrate the outer membrane of a target bacterium). In some instances, the bacteriocin is a microcin. The microcin may be a class I microcin (e.g., <5 kDa, has post-translational modifications) or a class II microcin (e.g., 5-10 kDa, with or without post-translational modifications). In some instances, the class II microcin is a class Ia microcin (e.g., requires more than one genes to synthesize and assemble functional peptides) or a class IIb microcin (e.g., linear peptides with or without post-translational modifications at C-terminus). In some instances, the bacteriocin is a pyocin. In some instances, the pyocin is an R-pyocin, F-pyocin, or S-pyocin.
[0137] In some instances, the bacteriocin is a class I, class II, class III, or class IV bacteriocin produced by Gram-positive bacteria. In some instances, the modulating agent includes a Class I bacteriocin (e.g., lanthionine-containing antibiotics (lantibiotics) produced by a Gram-positive bacteria). The class I bacteriocins or lantibiotic may be a low molecular weight peptide (e.g., less than about 5 kDa) and may possess post-translationally modified amino acid residues (e.g., lanthionine, p-methyllanthionine, or dehydrated amino acids).
[0138] In some instances, the bacteriocin is a Class II bacteriocin (e.g., non-lantibiotics produced by Gram-positive bacteria). Many are positively charged, non-lanthionine-containing peptides, which unlike lantibiotics, do not undergo extensive post-translational modification. The Class II bacteriocin may belong to one of the following subclasses: "pediocin-like" bacteriocins (e.g., pediocin PA-1 and carnobacteriocin X (Class Ia)); two-peptide bacteriocins (e.g., lactacin F and ABP-118 (Class IIb)); circular bacteriocins (e.g., carnocyclin A and enterocin AS-48 (Class IIc)); or unmodified, linear, non-pediocin-like bacteriocins (e.g., epidermicin NI01 and lactococcin A (Class IId)).
[0139] In some instances, the bacteriocin is a Class III bacteriocin (e.g., produced by Gram-positive bacteria). Class III bacteriocins may have a molecular weight greater than 10 kDa and may be heat unstable proteins. The Class III bacteriocins can be further subdivided into Group IIIA and Group IIIB bacteriocins. The Group IIIA bacteriocins include bacteriolytic enzymes which kill sensitive strains by lysis of the cell well, such as Enterolisin A. Group IIIB bacteriocins include non-lytic proteins, such as Caseicin 80, Helveticin J, and lactacin B.
[0140] In some instances, the bacteriocin is a Class IV bacteriocin (e.g., produced by Gram-positive bacteria). Class IV bacteriocins are a group of complex proteins, associated with other lipid or carbohydrate moieties, which appear to be required for activity. They are relatively hydrophobic and heat stable. Examples of Class IV bacteriocins leuconocin S, lactocin 27, and lactocin S.
[0141] In some instances, the bacteriocin is an R-type bacteriocin. R-type bacteriocins are contractile bacteriocidal protein complexes. Some R-type bacteriocins have a contractile phage-tail-like structure. The C-terminal region of the phage tail fiber protein determines target-binding specificity. They may attach to target cells through a receptor-binding protein, e.g., a tail fiber. Attachment is followed by sheath contraction and insertion of the core through the envelope of the target bacterium. The core penetration results in a rapid depolarization of the cell membrane potential and prompt cell death. Contact with a single R-type bacteriocin particle can result in cell death. An R-type bacteriocin, for example, may be thermolabile, mild acid resistant, trypsin resistant, sedimentable by centrifugation, resolvable by electron microscopy, or a combination thereof. Other R-type bacteriocins may be complex molecules including multiple proteins, polypeptides, or subunits, and may resemble a tail structure of bacteriophages of the myoviridae family. In naturally occurring R-type bacteriocins, the subunit structures may be encoded by a bacterial genome, such as that of C. difficile or P. aeruginosa and form R-type bacteriocins to serve as natural defenses against other bacteria. In some instances, the R-type bacteriocin is a pyocin. In some instances, the pyocin is an R-pyocin, F-pyocin, or S-pyocin.
[0142] In some instances, the bacteriocin is a functionally active variant of the bacteriocins described herein. In some instances, the variant of the bacteriocin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a bacteriocin described herein or a naturally occurring bacteriocin.
[0143] In some instances, the bacteriocin may be bioengineered, according to standard methods, to modulate their bioactivity, e.g., increase or decrease or regulate, or to specify their target microorganisms. In other instances, the bacteriocin is produced by the translational machinery (e.g. a ribosome, etc.) of a microbial cell. In some instances, the bacteriocin is chemically synthesized. Some bacteriocins can be derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (e.g., processing by a protease) to yield the polypeptide of the bacteriocin itself. As such, in some instances, the bacteriocin is produced from a precursor polypeptide. In some other instances, the bacteriocin includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
[0144] The bacteriocins described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type (e.g., classes) of bacteriocins, such as at least about any one of 1 bacteriocin, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more bacteriocins. Suitable concentrations of each bacteriocin in the compositions described herein depends on factors such as efficacy, stability of the bacteriocin, number of distinct bacteriocin types in the compositions, formulation, and methods of application of the composition. In some instances, each bacteriocin in a liquid composition is from about 0.01 ng/ml to about 100 mg/mL. In some instances, each bacteriocin in a solid composition is from about 0.01 ng/g to about 100 mg/g. In some instances, wherein the composition includes at least two types of bacteriocins, the concentration of each type of the bacteriocins may be the same or different. In some instances, the bacteriocin is provided in a composition including a bacterial cell that secretes the bacteriocin. In some instances, the bacteriocin is provided in a composition including a polypeptide (e.g., a polypeptide isolated from a bacterial cell).
[0145] Bacteriocins may neutralize (e.g., kill) at least one microorganism other than the individual bacterial cell in which the polypeptide is made, including cells clonally related to the bacterial cell and other microbial cells. As such, a bacterial cell may exert cytotoxic or growth-inhibiting effects on a plurality of microbial organisms by secreting bacteriocins. In some instances, the bacteriocin targets and kills one or more species of bacteria resident in the host via cytoplasmic membrane pore formation, cell wall interference (e.g., peptidoglycanase activity), or nuclease activity (e.g., DNase activity, 16S rRNase activity, or tRNase activity).
[0146] In some instances, the bacteriocin has a neutralizing activity. Neutralizing activity of bacteriocins may include, but is not limited to, arrest of microbial reproduction, or cytotoxicity. Some bacteriocins have cytotoxic activity, and thus can kill microbial organisms, for example bacteria, yeast, algae, and the like. Some bacteriocins can inhibit the reproduction of microbial organisms, for example bacteria, yeast, algae, and the like, for example by arresting the cell cycle.
[0147] In some instances, the bacteriocin has killing activity. The killing mechanism of bacteriocins is specific to each group of bacteriocins. In some instances, the bacteriocin has narrow-spectrum bioactivity. Bacteriocins are known for their very high potency against their target strains. Some bacteriocin activity is limited to strains that are closely related to the bacteriocin producer strain (narrow-spectrum bioactivity). In some instances, the bacteriocin has broad-spectrum bioactivity against a wide range of genera.
[0148] In some instances, bacteriocins interact with a receptor molecule or a docking molecule on the target bacterial cell membrane. For example, nisin is extremely potent against its target bacterial strains, showing antimicrobial activity even at a single-digit nanomolar concentration. The nisin molecule has been shown to bind to lipid II, which is the main transporter of peptidoglycan subunits from the cytoplasm to the cell wall
[0149] In some instances, the bacteriocin has anti-fungal activity. A number of bacteriocins with anti-yeast or anti-fungal activity have been identified. For example, bacteriocins from Bacillus have been shown to have neutralizing activity against some yeast strains (see, for example, Adetunji and Olaoye, Malaysian Journal of Microbiology 9:130-13, 2013). In another example, an Enterococcus faecalis peptide has been shown to have neutralizing activity against Candida species (see, for example, Shekh and Roy, BMC Microbiology 12:132, 2012). In another example, bacteriocins from Pseudomonas have been shown to have neutralizing activity against fungi, such as Curvularia lunata, Fusarium species, Helminthosporium species, and Biopolaris species (see, for example, Shalani and Srivastava, The Internet Journal of Microbiology Volume 5 Number 2, 2008). In another example, botrycidin AJ1316 and alirin B1 from B. subtilis have been shown to have antifungal activities.
[0150] A modulating agent including a bacteriocin as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of bacteriocin concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of bacteriocin concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of bacteriocin concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endoymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0151] (b) Lysins
[0152] The modulating agent described herein may include a lysin (e.g., also known as a murein hydrolase or peptidoglycan autolysin). Any lysin suitable for inhibiting a bacterium resident in the host may be used. In some instances, the lysin is one that can be naturally produced by a bacterial cell. In some instances, the lysin is one that can be naturally produced by a bacteriophage. In some instances, the lysin is obtained from a phage that inhibits a bacterium resident in the host. In some instances, the lysin is engineered based on a naturally occurring lysin. In some instances, the lysin is engineered to be secreted by a host bacterium, for example, by introducing a signal peptide to the lysin. In some instances, the lysin is used in combination with a phage holin. In some instances, a lysin is expressed by a recombinant bacterium host that is not sensitive to the lysin. In some instances, the lysin is used to inhibit a Gram-positive or Gram-negative bacterium resident in the host.
[0153] The lysin may be any class of lysin and may have one or more substrate specificities. For example, the lysin may be a glycosidase, an endopeptidase, a carboxypeptidase, or a combination thereof. In some instances, the lysin cleaves the .beta.-1-4 glycosidic bond in the sugar moiety of the cell wall, the amide bond connecting the sugar and peptide moieties of the bacterial cell wall, and/or the peptide bonds between the peptide moieties of the cell wall. The lysin may belong to one or more specific lysin groups, depending on the cleavage site within the peptidoglycan. In some instances, the lysin is a N-acetyl-.beta.-D-muramidase (e.g., lysozyme), lytic transglycosylase, N-acetyl-.beta.-D-glucosaminidase, N-acetylmuramyl-L-alanine amidase, L,D-endopeptidase, D,D-endopeptidase, D,D-carboxypeptidase, L,D-carboxypeptidase, or L,D-transpeptidase. Non-limiting examples of lysins and their activities are listed in Table 5.
TABLE-US-00005 TABLE 5 Examples of Lysins Target Bacteria Producer Lysins Activity Sequence S. pneumoniae Cp1 Cpl-1 Muramidase MVKKNDLFVDVSSHNGY DITGILEQMGTTNTIIKISES TTYLNPCLSAQVEQSNPI GFYHFARFGGDVAEAERE AQFFLDNVPMQVKYLVLD YEDDPSGDAQANTNACL RFMQMIADAGYKPIYYSY KPFTHDNVDYQQILAQFP NSLWIAGYGLNDGTANFE YFPSMDGIRWWQYSSNP FDKNIVLLDDEEDDKPKTA GTWKQDSKGWWFRRNN GSFPYNKWEKIGGVWYY FDSKGYCLTSEWLKDNEK WYYLKDNGAMATGWVLV GSEWYYMDDSGAMVTG WVKYKNNWYYMTNERGN MVSNEFIKSGKGWYFMNT NGELADNPSFTKEPDGLIT VA (SEQ ID NO: 18) S. pneumoniae Dp-1 Pal Amidase MGVDIEKGVAWMQARKG RVSYSMDFRDGPDSYDC SSSMYYALRSAGASSAG WAVNTEYMHAWLIENGY ELISENAPWDAKRGDIFIW GRKGASAGAGGHTGMFI DSDNIIHCNYAYDGISVND HDERWYYAGQPYYYVYR LTNANAQPAEKKLGWQK DATGFWYARANGTYPKD EFEYIEENKSWFYFDDQG YMLAEKWLKHTDGNWYW FDRDGYMATSWKRIGES WYYFNRDGSMVTGWIKY YDNWYYCDATNGDMKSN AFIRYNDGWYLLLPDGRL ADKPQFTVEPDGLITAKV (SEQ ID NO: 19) S. pyogenes C1 Cl Amidase N/A B. anthracis .gamma. PlyG Amidase MEIQKKLVDPSKYGTKCP YTMKPKYITVHNTYNDAP AENEVSYMISNNNEVSFHI AVDDKKAIQGIPLERNAW ACGDGNGSGNRQSISVEI CYSKSGGDRYYKAEDNA VDVVRQLMSMYNIPIENV RTHQSWSGKYCPHRMLA EGRWGAFIQKVKNGNVAT TSPTKQNIIQSGAFSPYET PDVMGALTSLKMTADFIL QSDGLTYFISKPTSDAQLK AMKEYLDRKGWWYEVK (SEQ ID NO: 20) B. anthracis Ames PlyPH Amidase N/A prophage E. faecalis and Phi1 PlyV12 Amidase N/A E. faecium S. aureus MR11 MV-L Endopeptidase MQAKLTKKEFIEWLKTSE and amidase GKQFNVDLWYGFQCFDY ANAGWKVLFGLLLKGLGA KDIPFANNFDGLATVYQN TPDFLAQPGDMVVFGSNY GAGYGHVAWVIEATLDYII VYEQNWLGGGWTDRIEQ PGWGWEKVTRRQHAYDF PMWFIRPNFKSETAPRSI QSPTQASKKETAKPQPKA VELKIIKDVVKGYDLPKRG GNPKGIVIHNDAGSKGAT AEAYRNGLVNAPLSRLEA GIAHSYVSGNTVWQALDE SQVGWHTANQLGNKYYY GIEVCQSMGADNATFLKN EQATFQECARLLKKWGLP ANRNTIRLHNEFTSTSCPH RSSVLHTGFDPVTRGLLP EDKQLQLKDYFIKQIRVYM DGKIPVATVSNESSASSN TVKPVASAWKRNKYGTYY MEENARFTNGNQPITVRKI GPFLSCPVAYQFQPGGY CDYTEVMLQDGHVWVGY TWEGQRYYLPIRTWNGS APPNQILGDLWGEIS (SEQ ID NO: 21) S. pyogenes C1 PlyC Amidase N/A S. agalactiae B30 GBS lysin Muramidase MVINIEQAIAWMASRKGK and VTYSMDYRNGPSSYDCS endopeptidase SSVYFALRSAGASDNGW AVNTEYEHDWLIKNGYVLI AENTNWNAQRGDIFIWGK RGASAGAFGHTGMFVDP DNIIHCNYGYNSITVNNHD EIWGYNGQPYVYAYRYS GKQSNAKVDNKSVVSKFE KELDVNTPLSNSNMPYYE ATISEDYYVESKPDVNSTD KELLVAGTRVRVYEKVKG WARIGAPQSNQWVEDAY LIDATDM (SEQ ID NO: 22) S. aureus P68 Lys16 Endopeptidase N/A S. aureus K LysK Amidase and MAKTQAEINKRLDAYAKG endopeptidase TVDSPYRVKKATSYDPSF GVMEAGAIDADGYYHAQ CQDLITDYVLWLTDNKVR TWGNAKDQIKQSYGTGFK IHENKPSTVPKKGWIAVFT SGSYEQWGHIGIVYDGGN TSTFTILEQNWNGYANKK PTKRVDNYYGLTHFIEIPV KAGTTVKKETAKKSASKT PAPKKKATLKVSKNHINYT MDKRGKKPEGMVIHNDA GRSSGQQYENSLANAGY ARYANGIAHYYGSEGYVW EAIDAKNQIAWHTGDGTG ANSGNFRFAGIEVCQSMS ASDAQFLKNEQAVFQFTA EKFKEWGLTPNRKTVRLH MEFVPTACPHRSMVLHTG FNPVTQGRPSQAIMNKLK DYFIKQIKNYMDKGTSSST VVKDGKTSSASTPATRPV TGSWKKNQYGTWYKPEN ATFVNGNQPIVTRIGSPFL NAPVGGNLPAGATIVYDE VCIQAGHIWIGYNAYNGN RVYCPVRTCQGVPPNQIP GVAWGVFK (SEQ ID NO: 23) L. A118 Ply118 Amidase MTSYYYSRSLANVNKLAD monocytogenes NTKAAARKLLDWSESNGI EVLIYETIRTKEQQAANVN SGASQTMRSYHLVGQAL DFVMAKGKTVDWGAYRS DKGKKFVAKAKSLGFEW GGDWSGFVDNPHLQFNY KGYGTDTFGKGASTSNSS KPSADTNTNSLGLVDYMN LNKLDSSFANRKKLATSY GIKNYSGTATQNTTLLAKL KAGKPHTPASKNTYYTEN PRKVKTLVQCDLYKSVDF TTKNQTGGTFPPGTVFTIS GMGKTKGGTPRLKTKSG YYLTANTKFVKKI (SEQ ID NO: 24) L. A511 Ply511 Amidase MVKYTVENKIIAGLPKGKL monocytogenes KGANFVIAHETANSKSTID NEVSYMTRNWKNAFVTH FVGGGGRVVQVANVNYV SWGAGQYANSYSYAQVE LCRTSNATTFKKDYEVYC QLLVDLAKKAGIPITLDSG SKTSDKGIKSHKWVADKL GGTTHQDPYAYLSSWGIS KAQFASDLAKVSGGGNT GTAPAKPSTPAPKPSTPS TNLDKLGLVDYMNAKKMD SSYSNRDKLAKQYGIANY SGTASQNTTLLSKIKGGAP KPSTPAPKPSTSTAKKIYF PPNKGNWSVYPTNKAPV KANAIGAINPTKFGGLTYTI QKDRGNGVYEIQTDQFG RVQVYGAPSTGAVIKK (SEQ ID NO: 25) L. A500 Ply500 Endopeptidase MALTEAWLIEKANRKLNA monocytogenes GGMYKITSDKTRNVIKKM AKEGIYLCVAQGYRSTAE QNALYAQGRTKPGAIVTN AKGGQSNHNYGVAVDLC LYTNDGKDVIWESTTSRW KKVVAAMKAEGFKWGGD WKSFKDYPHFELCDAVSG EKIPAATQNTNTNSNRYE GKVIDSAPLLPKMDFKSSP FRMYKVGTEFLVYDHNQY WYKTYIDDKLYYMYKSFC DVVAKKDAKGRIKVRIKSA KDLRIPVWNNIKLNSGKIK WYAPNVKLAWYNYRRGY LELWYPNDGWYYTAEYFL K (SEQ ID NO: 26) S. pneumoniae .PHI.Dp-1 Pal, S Endopeptidase N/A and amidase S. agalactiae LambdaSa1 LambdaSa1 Glycosidase MVINIEQAIAWMASRKGK prophage VTYSMDYRNGPSSYDCS SSVYFALRSAGASDNGW AVNTEYEHDWLIKNGYVLI AENTNWNAQRGDIFIWGK RGASAGAFGHTGMFVDP DNIIHCNYGYNSITVNNHD EIWGYNGQPYVYAYRYAR KQSNAKVDNQSVVSKFEK ELDVNTPLSNSNMPYYEA TISEDYYVESKPDVNSTDK ELLVAGTRVRVYEKVKGW ARIGAPQSNQWVEDAYLI DATDM (SEQ ID NO: 27) S. agalactiae LambdaSa2 LambdaSa2 Glycosidase MEINTEIAIAWMSARQGKV prophage and SYSMDYRDGPNSYDCSS endopeptidase SVYYALRSAGASSAGWA VNTEYMHDWLIKNGYELIA ENVDWNAVRGDIAIWGM RGHSSGAGGHVVMFIDPE NIIHCNWANNGITVNNYN QTAAASGWMYCYVYRLK SGASTQGKSLDTLVKETL AGNYGNGEARKAVLGNQ YEAVMSVINGKTTTNQKT VDQLVQEVIAGKHGNGEA RKKSLGSQYDAVQKRVTE LLKKQPSEPFKAQEVNKP TETKTSQTELTGQATATK EEGDLSFNGTILKKAVLDK ILGNCKKHDILPSYALTILH YEGLWGTSAVGKADNNW GGMTWTGQGNRPSGVTV TQGSARPSNEGGHYMHY ASVDDFLTDWFYLLRAGG SYKVSGAKTFSEAIKGMF KVGGAVYDYAASGFDSYI VGASSRLKAIEAENGSLD KFDKATDIGDGSKDKIDITI
EGIEVTINGITYELTKKPV (SEQ ID NO: 28) S. uberis (ATCC700407) Ply700 Amidase MTDSIQEMRKLQSIPVRY prophage DMGDRYGNDADRDGRIE MDCSSAVSKALGISMTNN TETLQQALPAIGYGKIHDA VDGTFDMQAYDVIIWAPR DGSSSLGAFGHVLIATSPT TAIHCNYGSDGITENDYNY IWEINGRPREIVFRKGVTQ TQATVTSQFERELDVNAR LTVSDKPYYEATLSEDYY VEAGPRIDSQDKELIKAGT RVRVYEKLNGWSRINHPE SAQWVEDSYLVDATEM (SEQ ID NO: 29) S. suis SMP LySMP Glycosidase N/A and endopeptidase B. anthracis Bcp1 PlyB Muramidase N/A S. aureus Phi11 and Phi11 lysin Amidase and MQAKLTKNEFIEWLKTSE Phi12 endopeptidase GKQFNVDLWYGFQCFDY ANAGWKVLFGLLLKGLGA KDIPFANNFDGLATVYQN TPDFLAQPGDMVVFGSNY GAGYGHVAWVIEATLDYII VYEQNWLGGGVVTDGIEQ PGWGWEKVTRRQHAYDF PMWFIRPNFKSETAPRSV QSPTQAPKKETAKPQPKA VELKIIKDVVKGYDLPKRG SNPKGIVIHNDAGSKGATA EAYRNGLVNAPLSRLEAGI AHSYVSGNTVWQALDES QVGWHTANQIGNKYYYGI EVCQSMGADNATFLKNE QATFQECARLLKKWGLPA NRNTIRLHNEFTSTSCPH RSSVLHTGFDPVTRGLLP EDKRLQLKDYFIKQIRAYM DGKIPVATVSNESSASSN TVKPVASAWKRNKYGTYY MEESARFTNGNQPITVRK VGPFLSCPVGYQFQPGG YCDYTEVMLQDGHVWVG YTWEGQRYYLPIRTWNG SAPPNQILGDLWGEIS (SEQ ID NO: 30) S. aureus .PHI.H5 LysH5 Amidase and MQAKLTKKEFIEWLKTSE endopeptidase GKQYNADGWYGFQCFDY ANAGWKALFGLLLKGVGA KDIPFANNFDGLATVYQN TPDFLAQPGDMVVFGSNY GAGYGHVAWVIEATLDYII VYEQNWLGGGVVTDGVQ QPGSGWEKVTRRQHAYD FPMWFIRPNFKSETAPRS VQSPTQASKKETAKPQPK AVELKIIKDVVKGYDLPKR GSNPNFIVIHNDAGSKGAT AEAYRNGLVNAPLSRLEA GIAHSYVSGNTVWQALDE SQVGWHTANQIGNKYGY GIEVCQSMGADNATFLKN EQATFQECARLLKKWGLP ANRNTIRLHNEFTSTSCPH RSSVLHTGFDPVTRGLLP EDKRLQLKDYFIKQIRAYM DGKIPVATVSNDSSASSN TVKPVASAWKRNKYGTYY MEESARFTNGNQPITVRK VGPFLSCPVGYQFQPGG YCDYTEVMLQDGHVWVG YTWEGQRYYLPIRTWNG SAPPNQILGDLWGEIS (SEQ ID NO: 31) S. warneri .PHI.WMY LysWMY Amidase and MKTKAQAKSWINSKIGKGI endopeptidase DWDGMYGYQCMDEAVD YIHHVTDGKVTMWGNAID APKNNFQGLCTVYTNTPE FRPAYGDVIVWSYGTFAT YGHIAIVVNPDPYGDLQYI TVLEQNWNGNGIYKTEFA TIRTHDYTGVSHFIRPKFA DEVKETAKTVNKLSVQKKI VTPKNSVERIKNYVKTSG YINGEHYELYNRGHKPKG VVIHNTAGTASATQEGQR LTNMTFQQLANGVAHVYI DKNTIYETLPEDRIAWHVA QQYGNTEFYGIEVCGSRN TDKEQFLANEQVAFQEAA RRLKSWGMRANRNTVRL HHTFSSTECPDMSMLLHT GYSMKNGKPTQDITNKCA DYFMKQINAYIDGKQPTST VVGSSSSNKLKAKNKDKS TGWNTNEYGTLWKKEHA TFTCGVRQGIVTRTTGPF TSCPQAGVLYYGQSVNY DTVCKQDGYVWISVVTTS DGYDVWMPIRTWDRSTD KVSEIWGTIS (SEQ ID NO: 32) Streptococci .PHI.NCTC PlyGBS Muramidase MATYQEYKSRSNGNAYDI (GBS) 11261 and DGSFGAQCWDGYADYCK endopeptidase YLGLPYANCTNTGYARDI WEQRHENGILNYFDEVEV MQAGDVAIFMVVDGVTPY SHVAIFDSDAGGGYGWFL GQNQGGANGAYNIVKIPY SATYPTAFRPKVFKNAVT VTGNIGLNKGDYFIDVSAY QQADLTTTCQQAGTTKTII KVSESIAWLSDRHQQQAN TSDPIGYYHFGRFGGDSA LAQREADLFLSNLPSKKV SYLVIDYEDSASADKQAN TNAVIAFMDKIASAGYKPI YYSYKPFTLNNIDYQKIIAK YPNSIWIAGYPDYEVRTEP LWEFFPSMDGVRWWQFT SVGVAGGLDKNIVLLADD SSKMDIPKVDKPQELTFY QKLATNTKLDNSNVPYYE ATLSTDYYVESKPNASSA DKEFIKAGTRVRVYEKVN GWSRINHPESAQWVEDS YLVNATDM (SEQ ID NO: 33) C. perfringens .PHI.3626 Ply3626 Amidase N/A C. difficile .PHI.CD27 CD27 lysin Amidase N/A E. faecalis .PHI.1 PlyV12 Amidase N/A A. naeslundii .PHI.Av-1- Av-1 lysin Putative N/A amidase/ muramidase L. gasseri .PHI.gaY LysgaY Muramidase N/A S. aureus .PHI.SA4 LysSA4 Amidase and N/A endopeptidase S. haemolyticus .PHI.SH2 SH2 Amidase and N/A endopeptidase B. thuringiensis .PHI.BtCS33 PlyBt33 Amidase N/A L. .PHI.P40 PlyP40 Amidase N/A monocytogenes L. .PHI.FWLLm3 LysZ5 Amidase MVKYTVENKIIAGLPKGKL monocytogenes KGANFVIAHETANSKSTID NEVSYMTRNWQNAFVTH FVGGGGRVVQVANVNYV SWGAGQYANSYSYAQVE LCRTSNATTFKKDYEVYC QLLVDLAKKAGIPITLDSG SKTSDKGIKSHKWVADKL GGTTHQDPYAYLSSWGIS KAQFASDLAKVSGGGNT GTAPAKPSTPSTNLDKLG LVDYMNAKKMDSSYSNR AKLAKQYGIANYSGTASQ NTTLLSKIKGGAPKPSTPA PKPSTSTAKKIYFPPNKGN WSVYPTNKAPVKANAIGAI NPTKFGGLTYTIQKDRGN GVYEIQTDQFGRVQVYGA PSTGAVIKK (SEQ ID NO: 34) B. cereus .PHI.BPS13 LysBPS13 Amidase MAKREKYIFDVEAEVGKA AKSIKSLEAELSKLQKLNK EIDATGGDRTEKEMLATL KAAKEVNAEYQKMQRILK DLSKYSGKVSRKEFNDSK VINNAKTSVQGGKVTDSF GQMLKNMERQINSVNKQ FDNHRKAMVDRGQQYTP HLKTNRKDSQGNSNPSM MGRNKSTTQDMEKAVDK FLNGQNEATTGLNQALYQ LKEISKLNRRSESLSRRAS ASGYMSFQQYSNFTGDR RTVQQTYGGLKTANRERV LELSGQATGISKELDRLNS KKGLTAREGEERKKLMRQ LEGIDAELTARKKLNSSLD ETTSNMEKFNQSLVDAQV SVKPERGTMRGMMYERA PAIALAIGGAITATIGKLYS EGGNHSKAMRPDEMYVG QQTGAVGANWRPNRTAT MRSGLGNHLGFTGQEMM EFQSNYLSANGYHGAED MKAATTGQATFARATGLG SDEVKDFFNTAYRSGGID GNQTKQFQNAFLGAMKQ SGAVGREKDQLKALNGIL SSMSQNRTVSNQDMMRT VGLQSAISSSGVASLQGT KGGALMEQLDNGIREGFN DPQMRVLFGQGTKYQGM GGRAALRKQMEKGISDPD NLNTLIDASKASAGQDPA EQAEVLATLASKMGVNMS SDQARGLIDLQPSGKLTK ENIDKVMKEGLKEGSIESA KRDKAYSESKASIDNSSE AATAKQATELNDMGSKLR QANAALGGLPAPLYTAIAA VVAFTAAVAGSALMFKGA SWLKGGMASKYGGKGGK GGKGGGTGGGGGAGGA AATGAGAAAGAGGVGAA AAGEVGAGVAAGGAAAG AAAGGSKLAGVGKGFMK GAGKLMLPLGILMGASEIM QAPEEAKGSAIGSAVGGI GGGIAGGAATGAIAGSFL GPIGTAVGGIAGGIAGGFA GSSLGETIGGWFDSGPKE DASAADKAKADASAAALA AAAGTSGAVGSSALQSQ MAQGITGAPNMSQVGSM ASALGISSGAMASALGISS GQENQIQTMTDKENTNTK KANEAKKGDNLSYERENI SMYERVLTRAEQILAQAR AQNGIMGVGGGGTAGAG GGINGFTGGGKLQFLAAG QKWSSSNLQQHDLGFTD QNLTAEDLDKWIDSKAPQ GSMMRGMGATFLKAGQE YGLDPRYLIAHAAEESGW GTSKIARDKGNFFGIGAFD DSPYSSAYEFKDGTGSAA ERGIMGGAKWISEKYYGK GNTTLDKMKAAGYATNAS WAPNIASIMAGAPTGSGS
GNVTATINVNVKGDEKVS DKLKNSSDMKKAGKDIGS LLGFYSREMTIA (SEQ ID NO: 35) S. aureus .PHI.GH15 LysGH15 Amidase and MAKTQAEINKRLDAYAKG endopeptidase TVDSPYRIKKATSYDPSFG VMEAGAIDADGYYHAQC QDLITDYVLWLTDNKVRT WGNAKDQIKQSYGTGFKI HENKPSTVPKKGWIAVFT SGSYQQWGHIGIVYDGG NTSTFTILEQNWNGYANK KPTKRVDNYYGLTHFIEIP VKAGTTVKKETAKKSASK TPAPKKKATLKVSKNHINY TMDKRGKKPEGMVIHNDA GRSSGQQYENSLANAGY ARYANGIAHYYGSEGYVW EAIDAKNQIAWHTGDGTG ANSGNFRFAGIEVCQSMS ASDAQFLKNEQAVFQFTA EKFKEWGLTPNRKTVRLH MEFVPTACPHRSMVLHTG FNPVTQGRPSQAIMNKLK DYFIKQIKNYMDKGTSSST VVKDGKTSSASTPATRPV TGSWKKNQYGTWYKPEN ATFVNGNQPIVTRIGSPFL NAPVGGNLPAGATIVYDE VCIQAGHIWIGYNAYNGD RVYCPVRTCQGVPPNHIP GVAWGVFK (SEQ ID NO: 36) S. aureus .PHI.vB SauS- HydH5 Endopeptidase N/A PLA88 and glycosidase E. faecalis .PHI.F168/08 Lys168 Endopeptidase N/A E. faecalis .PHI.F170/08 Lys170 Amidase N/A S. aureus .PHI.P-27/HP P-27/HP Nonspecified N/A C. perfringens .PHI.SM101 Psm Muramidase N/A C. sporogenes .PHI.8074-B1 C574L Amidase MKIGIDMGHTLSGADYGV VGLRPESVLTREVGTKVIY KLQKLGHVVVNCTVDKAS SVSESLYTRYYRANQANV DLFISIHFNATPGGTGTEV YTYAGRQLGEATRIRQEF KSLGLRDRGTKDGSGLAV IRNTKAKAMLVECCFCDN PNDMKLYNSESFSNAIVK GITGKLPNGESGNNNQG GNKVKAVVIYNEGADRRG AEYLADYLNCPTISNSRTF DYSCVEHVYAVGGKKEQ YTKYLKTLLSGANRYDTM QQILNFINGGK (SEQ ID NO: 37) S. typhimurium .PHI.SPN1S SPN1S Glycosidase MDINQFRRASGINEQLAA RWFPHITTAMNEFGITKPD DQAMFIAQVGHESGGFTR LQENFNYSVNGLSGFIRA GRITPDQANALGRKTYEK SLPLERQRAIANLVYSKR MGNNGPGDGWNYRGRG LIQITGLNNYRDCGNGLKV DLVAQPELLAQDEYAARS AAWFFSSKGCMKYTGDL VRVTQIINGGQNGIDDRRT RYAAARKVLAL (SEQ ID NO: 38) C. michiganensis .PHI.CMP1 CMP1 Peptidase N/A C. michiganensis .PHI.CN77 CN77 Peptidase MGYWGYPNGQIPNDKMA LYRGCLLRADAAAQAYAL QDAYTRATGKPLVILEGY RDLTRQKYLRNLYLSGRG NIAAVPGLSNHGWGLACD FAAPLNSSGSEEHRWMR QNAPLFGFDWARGKADN EPWHWEYGNVPVSRWA SLDVTPIDRNDMADITEGQ MQRIAVILLDTEIQTPLGPR LVKHALGDALLLGQANAN SIAEVPDKTWDVLVDHPL AKNEDGTPLKVRLGDVAK YEPLEHQNTRDAIAKLGTL QFTDKQLATIGAGVKPIDE ASLVKKIVDGVRALFGRAA A (SEQ ID NO: 39) A. baumannii .PHI.AB2 LysAB2 Glycosidase MILTKDGFSIIRNELFGGKL DQTQVDAINFIVAKATESG LTYPEAAYLLATIYHETGL PSGYRTMQPIKEAGSDSY LRSKKYYPYIGYGYVQLT WKENYERIGKLIGVDLIKN PEKALEPLIAIQIAIKGMLN GWFTGVGFRRKRPVSKY NKQQYVAARNIINGKDKA ELIAKYAIIFERALRSL (SEQ ID NO: 40) B. cereus .PHI.B4 LysB4 Endopeptidase MAMALQTLIDKANRKLNV SGMRKDVADRTRAVITQM HAQGIYICVAQGFRSFAE QNALYAQGRTKPGSIVTN ARGGQSNHNYGVAVDLC LYTQDGSDVIVVTVEGNFR KVIAAMKAQGFKWGGDW VSFKDYPHFELYDVVGGQ KPPADNGGAVDNGGGSG STGGSGGGSTGGGSTGG GYDSSWFTKETGTFVTNT SIKLRTAPFTSADVIATLPA GSPVNYNGFGIEYDGYV WIRQPRSNGYGYLATGES KGGKRQNYWGTFK (SEQ ID NO: 41) P. aeruginosa .PHI.KMV KMV45 Nonspecified N/A C. tyrobutyricum .PHI.CTP1 Ctp1I Glycosidase MKKIADISNLNGNVDVKLL FNLGYIGIIAKASEGGTFV DKYYKQNYTNTKAQGKIT GAYHFANFSTIAKAQQEA NFFLNCIAGTTPDFVVLDL EQQCTGDITDACLAFLNIV AKKFKCVVYCNSSFIKEHL NSKICAYPLWIANYGVATP AFTLVVTKYAMWQFTEKG QVSGISGYIDFSYITDEFIK YIKGEDEVENLVVYNDGA DQRAAEYLADRLACPTIN NARKFDYSNVKNVYAVG GNKEQYTSYLTTLIAGSTR YTTMQAVLDYIKNLK (SEQ ID NO: 42) P. aeruginosase .PHI.EL EL188 Transglycosyla N/A P. aeruginosase .PHI.KZ KZ144 Transglycosyla N/A S. aureus Staph- Ply187 Cell Wall MALPKTGKPTAKQVVDW ylococcus Hydrolase AINLIGSGVDVDGYYGRQ virus 187 CWDLPNYIFNRYWNFKTP GNARDMAWYRYPEGFKV FRNTSDFVPKPGDIAVWT GGNYNWNTWGHTGIVVG PSTKSYFYSVDQNWNNS NSYVGSPAAKIKHSYFGV THFVRPAYKAEPKPTPPA QNNPAPKDPEPSKKPESN KPIYKVVTKILFTTAHIEHV KANRFVHYITKSDNHNNK PNKIVIKNTNTALSTIDVYR YRDELDKDEIPHFFVDRLN VWACRPIEDSINGYHDSV VLSITETRTALSDNFKMNE IECLSLAESILKANNKKMS ASNIIVDNKAWRTFKLHTG KDSLKSSSFTSKDYQKAV NELIKLFNDKDKLLNNKPK DVVERIRIRTIVKENTKFVP SELKPRNNIRDKQDSKIDR VINNYTLKQALNIQYKLNP KPQTSNGVSWYNASVNQI KSAMDTTKIFNNNVQVYQ FLKLNQYQGIPVDKLNKLL VGKGTLANQGHAFADGC KKYNINEIYLIAHRFLESAN GTSFFASGKTGVYNYFGI GAFDNNPNNAMAFARSH GVVTSPTKAIIGGAEFVGK GYFNVGQNTLYRMRWNP QKPGTHQYATDISWAKVQ AQMISAMYKEIGLTGDYFI YDQYKK (SEQ ID NO: 43) P. uorescens .PHI.OBP OBPgp279 Glycosidase N/A L. .PHI.P35 PlyP35 Amidase MARKFTKAELVAKAEKKV monocytogenes GGLKPDVKKAVLSAVKEA YDRYGIGIIVSQGYRSIAE QNGLYAQGRTKPGNIVTN AKGGQSNHNFGVAVDFAI DLIDDGKIDSWQPSATIVN MMKRRGFKWGGDWKSF TDLPHFEACDWYRGERK YKVDTSEWKKKENINIVIK DVGYFQDKPQFLNSKSVR QWKHGTKVKLTKHNSHW YTGVVKDGNKSVRGYIYH SMAKVTSKNSDGSVNATI NAHAFCWDNKKLNGGDFI NLKRGFKGITHPASDGFY PLYFASRKKTFYIPRYMFD IKK (SEQ ID NO: 44) L. fermentum .PHI.OPYB5 Lyb5 Muramidase N/A S. pneumoniae .PHI.CP-7 Cp1-7 Muramidase MVKKNDLFVDVASHQGY DISGILEEAGTTNTIIKVSE STSYLNPCLSAQVSQSNPI GFYHFAWFGGNEEEAEA EARYFLDNVPTQVKYLVL DYEDHASASVQRNTTACL RFMQIIAEAGYTPIYYSYK PFTLDNVDYQQILAQFPN SLWIAGYGLNDGTANFEY FPSMDGIRWWQYSSNPF DKNIVLLDDEKEDNINNEN TLKSLTTVANEVIQGLWG NGQERYDSLANAGYDPQ AVQDKVNEILNAREIADLT TVANEVIQGLWGNGQER YDSLANAGYDPQAVQDK VNEILNAREIADLTTVANE VIQGLWGNGQERYDSLA NAGYDPQAVQDKVNELLS (SEQ ID NO: 45) P. .PHI.2-1 201y92- Glycosidase N/A chlororaphis201 1gp229 S. enterica .PHI.PVP-SE1) PVP- Glycosidase N/A SE1gp146 Corynebacterium .PHI.BFK20 BKF20 Amidase N/A E. faecalis .PHI.EFAP-1 EFAL-1 Amidase MKLKGILLSVVTTFGLLFG ATNVQAYEVNNEFNLQP WEGSQQLAYPNKIILHETA NPRATGRNEATYMKNNW FNAHTTAIVGDGGIVYKVA PEGNVSWGAGNANPYAP VQIELQHTNDPELFKANYK AYVDYTRDMGKKFGIPMT LDQGGSLWEKGVVSHQW VTDFVWGDHTDPYGYLA KMGISKAQLAHDLANGVS
GNTATPTPKPDKPKPTQP SKPSNKKRFNYRVDGLEY VNGMWQIYNEHLGKIDFN WTENGIPVEVVDKVNPAT GQPTKDQVLKVGDYFNF QENSTGVVQEQTPYMGY TLSHVQLPNEFIWLFTDSK QALMYQ (SEQ ID NO: 46) Lactobacilli lambdaSA2 LysA, Nonspecified N/A LysA2, and Lysga Y S. aureus SAL-1 Nonspecified N/A
[0154] In some instances, the lysin is a functionally active variant of the lysins described herein. In some instances, the variant of the lysin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a lysin described herein or a naturally occurring lysin.
[0155] In some instances, the lysin may be bioengineered to modulate its bioactivity, e.g., increase or decrease or regulate, or to specify a target microorganism. In some instances, the lysin is produced by the translational machinery (e.g. a ribosome, etc.) of a microbial cell. In some instances, the lysin is chemically synthesized. In some instances, the lysin is derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the polypeptide of the lysin itself. As such, in some instances, the lysin is produced from a precursor polypeptide. In some instances, the lysin includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
[0156] The lysins described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type (e.g., classes) of lysins, such as at least about any one of 1 lysin, 2, 3, 4, 5, 10, 15, 20, or more lysins. A suitable concentration of each lysin in the composition depends on factors such as efficacy, stability of the lysin, number of distinct lysin, the formulation, and methods of application of the composition. In some instances, each lysin in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL. In some instances, each lysin in a solid composition is from about 0.1 ng/g to about 100 mg/g. In some instances, wherein the composition includes at least two types of lysins, the concentration of each type of lysin may be the same or different.
[0157] A modulating agent including a lysin as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of lysin concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of lysin concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of lysin concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0158] (c) Antimicrobial Peptides
[0159] The modulating agent described herein may include an antimicrobial peptide (AMP). Any AMP suitable for inhibiting a microorganism resident in the host may be used. AMPs are a diverse group of molecules, which are divided into subgroups on the basis of their amino acid composition and structure.
[0160] The AMP may be derived or produced from any organism that naturally produces AMPs, including AMPs derived from plants (e.g., copsin), insects (e.g., drosocin, scorpion peptide (e.g., Uy192, UyCT3, D3, D10, Uy17, Uy192), mastoparan, poneratoxin, cecropin, moricin, melittin), frogs (e.g., magainin, dermaseptin, aurein), and mammals (e.g., cathelicidins, defensins and protegrins). For example, the AMP may be a scorpion peptide, such as Uy192 (5'-FLSTIWNGIKGLL-3'; SEQ ID NO: 193), UyCT3 (5'-LSAIWSGIKSLF-3; SEQ ID NO: 194'), D3 (5'-LWGKLWEGVKSLI-3'; SEQ ID NO: 195), and D10 (5'-FPFLKLSLKIPKSAIKSAIKRL-3'; SEQ ID NO: 196), Uy17 (5'-ILSAIWSGIKGLL-3'; SEQ ID NO: 197). Other non-limiting examples of AMPs are listed in Table 6.
TABLE-US-00006 TABLE 6 Examples of Antimicrobial Peptides Example Type Characteristic AMP Sequence Anionic rich in glutamic and dermcidin SSLLEKGLDGAKKAVGGLGKL peptides aspartic acid GKDAVEDLESVGKGAVHDVKD VLDSVL (SEQ ID NO: 47) Linear cationic lack cysteine cecropin A KWKLFKKIEKVGQNIRDGIIKAG .alpha.-helical PAVAVVGQATQIAK peptides (SEQ ID NO: 48) andropin MKYFSVLVVLTLILAIVDQSDAFI NLLDKVEDALHTGAQAGFKLIR PVERGATPKKSEKPEK (SEQ ID NO: 49) moricin MNILKFFFVFIVAMSLVSCSTAA PAKIPIKAIKTVGKAVGKGLRAI NIASTANDVFNFLKPKKRKH (SEQ ID NO: 50) ceratotoxin MANLKAVFLICIVAFIALQCVVA EPAAEDSVVVKRSIGSALKKAL PVAKKIGKIALPIAKAALPVAAG LVG (SEQ ID NO: 51) Cationic rich in proline, arginine, abaecin MKVVIFIFALLATICAAFAYVPLP peptide phenylalanine, glycine, NVPQPGRRPFPTFPGQGPFNP enriched for tryptophan KIKWPQGY specific amino (SEQ ID NO: 52) acid apidaecins KNFALAILVVTFVVAVFGNTNLD PPTRPTRLRREAKPEAEPGNN RPVYIPQPRPPHPRLRREAEPE AEPGNNRPVYIPQPRPPHPRL RREAELEAEPGNNRPVYISQP RPPHPRLRREAEPEAEPGNNR PVYIPQPRPPHPRLRREAELEA EPGNNRPVYISQPRPPHPRLR REAEPEAEPGNNRPVYIPQPR PPHPRLRREAEPEAEPGNNRP VYIPQPRPPHPRLRREAEPEAE PGNNRPVYIPQPRPPHPRLRR EAKPEAKPGNNRPVYIPQPRP PHPRI (SEQ ID NO: 53) prophenin METQRASLCLGRWSLWLLLLA LVVPSASAQALSYREAVLRAVD RLNEQSSEANLYRLLELDQPPK ADEDPGTPKPVSFTVKETVCP RPTRRPPELCDFKENGRVKQC VGTVTLDQIKDPLDITCNEGVR RFPVWVWPFLRRPRLRRQAFP PPNVPGPRFPPPNVPGPRFPP PNFPGPRFPPPNFPGPRFPPP NFPGPPFPPPIFPGPWFPPPPP FRPPPFGPPRFPGRR (SEQ ID NO: 54) indolicidin MQTQRASLSLGRWSLWLLLLG LVVPSASAQALSYREAVLRAVD QLNELSSEANLYRLLELDPPPK DNEDLGTRKPVSFTVKETVCP RTIQQPAEQCDFKEKGRVKQC VGTVTLDPSNDQFDLNCNELQ SVILPWKWPWWPWRRG (SEQ ID NO: 55) Anionic and contain 1-3 disulfide bond protegrin METQRASLCLGRWSLWLLLLA cationic LVVPSASAQALSYREAVLRAVD peptides that RLNEQSSEANLYRLLELDQPPK contain ADEDPGTPKPVSFTVKETVCP cysteine and RPTRQPPELCDFKENGRVKQC form disulfide VGTVTLDQIKDPLDITCNEVQG bonds VRGGRLCYCRRRFCVCVGRG (SEQ ID NO: 56) tachyplesins KWCFRVCYRGICYRRCR (SEQ ID NO: 57) defensin MKCATIVCTIAVVLAATLLNGSV QAAPQEEAALSGGANLNTLLD ELPEETHHAALENYRAKRATC DLASGFGVGSSLCAAHCIARR YRGGYCNSKAVCVCRN (SEQ ID NO: 58) drosomycin MMQIKYLFALFAVLMLVVLGAN EADADCLSGRYKGPCAVWDN ETCRRVCKEEGRSSGHCSPSL KCWCEGC (SEQ ID NO: 59)
[0161] The AMP may be active against any number of target microorganisms. In some instances, the AMP may have antibacterial and/or antifungal activities. In some instances, the AMP may have a narrow-spectrum bioactivity or abroad-spectrum bioactivity. For example, some AMPs target and kill only a few species of bacteria or fungi, while others are active against both gram-negative and gram-positive bacteria as well as fungi.
[0162] Further, the AMP may function through a number of known mechanisms of action. For example, the cytoplasmic membrane is a frequent target of AMPs, but AMPs may also interfere with DNA and protein synthesis, protein folding, and cell wall synthesis. In some instances, AMPs with net cationic charge and amphipathic nature disrupt bacterial membranes leading to cell lysis. In some instances, AMPs may enter cells and interact with intracellular target to interfere with DNA, RNA, protein, or cell wall synthesis. In addition to killing microorganisms, AMPs have demonstrated a number of immunomodulatory functions that are involved in the clearance of infection, including the ability to alter host gene expression, act as chemokines and/or induce chemokine production, inhibit lipopolysaccharide induced pro-inflammatory cytokine production, promote wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response.
[0163] In some instances, the AMP is a functionally active variant of the AMPs described herein. In some instances, the variant of the AMP has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of an AMP described herein or a naturally derived AMP.
[0164] In some instances, the AMP may be bioengineered to modulate its bioactivity, e.g., increase or decrease or regulate, or to specify a target microorganism. In some instances, the AMP is produced by the translational machinery (e.g. a ribosome, etc.) of a cell. In some instances, the AMP is chemically synthesized. In some instances, the AMP is derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the polypeptide of the AMP itself. As such, in some instances, the AMP is produced from a precursor polypeptide. In some instances, the AMP includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
[0165] The AMPs described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type (e.g., classes) of AMPs, such as at least about any one of 1 AMP, 2, 3, 4, 5, 10, 15, 20, or more AMPs. A suitable concentration of each AMP in the composition depends on factors such as efficacy, stability of the AMP, number of distinct AMP in the composition, the formulation, and methods of application of the composition. In some instances, each AMP in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL. In some instances, each AMP in a solid composition is from about 0.1 ng/g to about 100 mg/g. In some instances, wherein the composition includes at least two types of AMPs, the concentration of each type of AMP may be the same or different.
[0166] A modulating agent including an AMP as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of AMP concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of AMP concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of AMP concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0167] (d) Nodule C-Rich Peptides
[0168] The modulating agent described herein may include a nodule C-rich peptide (NCR peptide). NCR peptides are produced in certain leguminous plants and play an important role in the mutualistic, nitrogen-fixing symbiosis of the plants with bacteria from the Rhizobiaceae family (rhizobia), resulting in the formation of root nodules where plant cells contain thousands of intracellular endosymbionts. NCR peptides possess anti-microbial properties that direct an irreversible, terminal differentiation process of bacteria, e.g., to permeabilize the bacterial membrane, disrupt cell division, or inhibit protein synthesis. For example, in Medicago truncatula nodule cells infected with Sinorhizobium meliloti, hundreds of NCR peptides are produced which direct irreversible differentiation of the bacteria into large polyploid nitrogen-fixing bacteroids). Non-limiting examples of NCR peptides are listed in Table 7.
TABLE-US-00007 TABLE 7 Examples of NCR Peptides NAME Peptide sequence Producer >gi|152218086|gb|ABS31477. MTKIVVFIYVVILLLTIFHVSAKKKRYI Medicago truncatula 1| NCR 340 ECETHEDCSQVFMPPFVMRCVIHE CKIFNGEHLRY (SEQ ID NO: 60) >gi|152218084|gb|ABS31476. MAKIMKFVYNMIPFLSIFIITLQVNVV Medicago truncatula 1| NCR 339 VCEIDADCPQICMPPYEVRCVNHRC GWVNTDDSLFLTQEFTRSKQYIIS (SEQ ID NO: 61) >gi|152218082|gb|ABS31475. MYKVVESIFIRYMHRKPNMTKFFKF Medicago truncatula 1| NCR 338 VYTMFILISLFLVVTNANAHNCTDISD CSSNHCSYEGVSLCMNGQCICIYE (SEQ ID NO: 62) >gi|152218080|gb|ABS31474. MVETLRLFYIMILFVSLCLVVVDGES Medicago truncatula 1| NCR 337 KLEQTCSEDFECYIKNPHVPFGHLR CFEGFCQQLNGPA (SEQ ID NO: 63) >gi|152218078|gb|ABS31473. MAKIVNFVYSMIVFLFLFLVATKAAR Medicago truncatula 1| NCR 336 GYLCVTDSHCPPHMCPPGMEPRCV RRMCKCLPIGWRKYFVP (SEQ ID NO: 64) >gi|152218076|gb|ABS31472. MQIGKNMVETPKLDYVIIFFFLYFFF Medicago truncatula 1| NCR 335 RQMIILRLNTTFRPLNFKMLRFWGQ NRNIMKHRGQKVHFSLILSDCKTNK DCPKLRRANVRCRKSYCVPI (SEQ ID NO: 65) >gi|152218074|gb|ABS31471. MLRLYLVSYFLLKRTLLVSYFSYFST Medicago truncatula 1| NCR 334 YIIECKTDNDCPISQLKIYAWKCVKN GCHLFDVIPMMYE (SEQ ID NO: 66) >gi|152218072|gb|ABS31470. MAEILKFVYIVILFVSLLLIVVASEREC Medicago truncatula 1| NCR 333 VTDDDCEKLYPTNEYRMMCDSGYC MNLLNGKIIYLLCLKKKKFLIIISVLL (SEQ ID NO: 67) >gi|152218070|gb|ABS31469. MAEIIKFVYIMILCVSLLLIEVAGEECV Medicago truncatula 1| NCR 332 TDADCDKLYPDIRKPLMCSIGECYSL YKGKFSLSIISKTSFSLMVYNVVTLVI CLRLAYISLLLKFL (SEQ ID NO: 68) >gi|152218068|gb|ABS31468. MAEILKDFYAMNLFIFLIILPAKIRGET Medicago truncatula 1| NCR 331 LSLTHPKCHHIMLPSLFITEVFQRVT DDGCPKPVNHLRVVKCIEHICEYGY NYRPDFASQIPESTKMPRKRE (SEQ ID NO: 69) >gi|152218066|gb|ABS31467. MVEILKNFYAMNLFIFLIILAVKIRGAH Medicago truncatula 1| NCR 330 FPCVTDDDCPKPVNKLRVIKCIDHIC QYARNLPDFASEISESTKMPCKGE (SEQ ID NO: 70) >gi|152218064|gb|ABS31466. MFHAQAENMAKVSNFVCIMILFLALF Medicago truncatula 1| NCR 329 FITMNDAARFECREDSHCVTRIKCV LPRKPECRNYACGCYDSNKYR (SEQ ID NO: 71) >gi|152218062|gb|ABS31465. MQMRQNMATILNFVFVIILFISLLLVV Medicago truncatula 1| NCR 328 TKGYREPFSSFTEGPTCKEDIDCPSI SCVNPQVPKCIMFECHCKYIPTTLK (SEQ ID NO: 72) >gi|152218060|gb|ABS31464. MATILMYVYITILFISILTVLTEGLYEPL Medicago truncatula 1| NCR 327 YNFRRDPDCRRNIDCPSYLCVAPKV PRCIMFECHCKDIPSDH (SEQ ID NO: 73) >gi|152218058|gb|ABS31463. MTTSLKFVYVAILFLSLLLVVMGGIR Medicago truncatula 1| NCR 326 RFECRQDSDCPSYFCEKLTVPKCF WSKCYCK (SEQ ID NO: 74) >gi|152218056|gb|ABS31462. MTTSLKFVYVAILFLSLLLVVMGGIR Medicago truncatula 1| NCR 325 KKECRQDSDCPSYFCEKLTIAKCIHS TCLCK (SEQ ID NO: 75) >gi|152218054|gb|ABS31461. MQIGKNMVETPKLVYFIILFLSIFLCIT Medicago truncatula 1| NCR 324 VSNSSFSQIFNSACKTDKDCPKFGR VNVRCRKGNCVPI (SEQ ID NO: 76) >gi|152218046|gb|ABS31457. MTAILKKFINAVFLFIVLFLATTNVED Medicago truncatula 1| NCR 320 FVGGSNDECVYPDVFQCINNICKCV SHHRT (SEQ ID NO: 77) >gi|152218044|gb|ABS31456. MQKRKNMAQIIFYVYALIILFSPFLAA Medicago truncatula 1| NCR 319 RLVFVNPEKPCVTDADCDRYRHES AIYSDMFCKDGYCFIDYHHDPYP (SEQ ID NO: 78) >gi|152218042|gb|ABS31455. MQMRKNMAQILFYVYALLILFTPFLV Medicago truncatula 1| NCR 318 ARIMVVNPNNPCVTDADCQRYRHK LATRMICNQGFCLMDFTHDPYAPSL P (SEQ ID NO: 79) >gi|152218040|gb|ABS31454. MNHISKFVYALIIFLSIYLVVLDGLPIS Medicago truncatula 1| NCR 317 CKDHFECRRKINILRCIYRQEKPMCI NSICTCVKLL (SEQ ID NO: 80) >gi|152218038|gb|ABS31453. MQREKNMAKIFEFVYAMIIFILLFLVE Medicago truncatula 1| NCR 316 KNVVAYLKFECKTDDDCQKSLLKTY VWKCVKNECYFFAKK (SEQ ID NO: 81) >gi|152218036|gb|ABS31452. MAGIIKFVHVLIIFLSLFHVVKNDDGS Medicago truncatula 1| NCR 315 FCFKDSDCPDEMCPSPLKEMCYFL QCKCGVDTIA (SEQ ID NO: 82) >gi|152218034|gb|ABS31451. MANTHKLVSMILFIFLFLASNNVEGY Medicago truncatula 1| NCR 314 VNCETDADCPPSTRVKRFKCVKGE CRWTRMSYA (SEQ ID NO: 83) >gi|152218032|gb|ABS31450. MQRRKKKAQVVMFVHDLIICIYLFIVI Medicago truncatula 1| NCR 313 TTRKTDIRCRFYYDCPRLEYHFCECI EDFCAYIRLN (SEQ ID NO: 84) >gi|152218030|gb|ABS31449. MAKVYMFVYALIIFVSPFLLATFRTRL Medicago truncatula 1| NCR 312 PCEKDDDCPEAFLPPVMKCVNRFC QYEILE (SEQ ID NO: 85) >gi|152218028|gb|ABS31448. MIKQFSVCYIQMRRNMTTILKFPYIM Medicago truncatula 1| NCR 310 VICLLLLHVAAYEDFEKEIFDCKKDG DCDHMCVTPGIPKCTGYVCFCFENL (SEQ ID NO: 86) >gi|152218026|gb|ABS31447. MQRSRNMTTIFKFAYIMIICVFLLNIA Medicago truncatula 1| NCR 309 AQEIENGIHPCKKNEDCNHMCVMP GLPWCHENNLCFCYENAYGNTR (SEQ ID NO: 87) >gi|152218024|gb|ABS31446. MTIIIKFVNVLIIFLSLFHVAKNDDNKL Medicago truncatula 1| NCR 304 LLSFIEEGFLCFKDSDCPYNMCPSP LKEMCYFIKCVCGVYGPIRERRLYQ SHNPMIQ (SEQ ID NO: 88) >gi|152218022|gb|ABS31445. MRKNMTKILMIGYALMIFIFLSIAVSIT Medicago truncatula 1| NCR 303 GNLARASRKKPVDVIPCIYDHDCPR KLYFLERCVGRVCKYL (SEQ ID NO: 89) >gi|152218020|gb|ABS31444. MAHKLVYAITLFIFLFLIANNIEDDIFCI Medicago truncatula 1| NCR 301 TDNDCPPNTLVQRYRCINGKCNLSF VSYG (SEQ ID NO: 90) >gil152218018|gb|ABS31443. MDETLKFVYILILFVSLCLVVADGVK Medicago truncatula 1| NCR 300 NINRECTQTSDCYKKYPFIPWGKVR CVKGRCRLDM (SEQ ID NO: 91) >gil152218016|gb|ABS31442. MAKIIKFVYVLAIFFSLFLVAKNVNG Medicago truncatula 1| NCR 290 WTCVEDSDCPANICQPPMQRMCFY GECACVRSKFCT (SEQ ID NO: 92) >gil152218014|gb|ABS31441. MVKIIKFVYFMTLFLSMLLVTTKEDG Medicago truncatula 1| NCR 289 SVECIANIDCPQIFMLPFVMRCINFR CQIVNSEDT (SEQ ID NO: 93) >gil152218012|gb|ABS31440. MDEILKFVYTLIIFFSLFFAANNVDANI Medicago truncatula 1| NCR 286 MNCQSTFDCPRDMCSHIRDVICIFK KCKCAGGRYMPQVP (SEQ ID NO: 94) >gi|152218008|gb|ABS31438. MQRRKNMANNHMLIYAMIICLFPYL Medicago truncatula 1| NCR 278 VVTFKTAITCDCNEDCLNFFTPLDNL KCIDNVCEVFM (SEQ ID NO: 95) >gi|152218006|gb|ABS31437. MVNILKFIYVIIFFILMFFVLIDVDGHV Medicago truncatula 1| NCR 266 LVECIENRDCEKGMCKFPFIVRCLM DQCKCVRIHNLI (SEQ ID NO: 96) >gi|152218004|gb|ABS31436. MIIQFSIYYMQRRKLNMVEILKFSHA Medicago truncatula 1| NCR 265 LIIFLFLSALVTNANIFFCSTDEDCTW NLCRQPWVQKCRLHMCSCEKN (SEQ ID NO: 97) >gi|152218002|gb|ABS31435. MDEVFKFVYVMIIFPFLILDVATNAEK Medicago truncatula 1| NCR 263 IRRCFNDAHCPPDMCTLGVIPKCSR FTICIC (SEQ ID NO: 98) >gi|152218000|gb|ABS31434. MHRKPNMTKFFKFVYTMFILISLFLV Medicago truncatula 1| NCR 244 VTNANANNCTDTSDCSSNHCSYEG VSLCMNGQCICIYE (SEQ ID NO: 99) >gi|152217998|gb|ABS31433. MQMKKMATILKFVYLIILLIYPLLVVTE Medicago truncatula 1| NCR 239 ESHYMKFSICKDDTDCPTLFCVLPN VPKCIGSKCHCKLMVN (SEQ ID NO: 100) >gi|152217996|gb|ABS31432. MVETLRLFYIMILFVSLYLVVVDGVS Medicago truncatula 1| NCR 237 KLAQSCSEDFECYIKNPHAPFGQLR CFEGYCQRLDKPT (SEQ ID NO: 101) >gi|152217994|gb|ABS31431. MTTFLKVAYIMIICVFVLHLAAQVDS Medicago truncatula 1| NCR 228 QKRLHGCKEDRDCDNICSVHAVTK CIGNMCRCLANVK (SEQ ID NO: 102) >gi|152217992|gb|ABS31430. MRINRTPAIFKFVYTIIIYLFLLRVVAK Medicago truncatula 1| NCR 224 DLPFNICEKDEDCLEFCAHDKVAKC MLNICFCF (SEQ ID NO: 103)
>gi|152217990|gb|ABS31429. MAEILKILYVFIIFLSLILAVISQHPFTP Medicago truncatula 1| NCR 221 CETNADCKCRNHKRPDCLWHKCYC Y (SEQ ID NO: 104) >gi|152217988|gb|ABS31428. MRKSMATILKFVYVIMLFIYSLFVIES Medicago truncatula 1| NCR 217 FGHRFLIYNNCKNDTECPNDCGPHE QAKCILYACYCVE (SEQ ID NO: 105) >gi|152217986|gb|ABS31427. MNTILKFIFVVFLFLSIFLSAGNSKSY Medicago truncatula 1| NCR 209 GPCTTLQDCETHNWFEVCSCIDFEC KCWSLL (SEQ ID NO: 106) >gi|152217984|gb|ABS31426. MAEIIKFVYIMILCVSLLLIAEASGKEC Medicago truncatula 1| NCR 206 VTDADCENLYPGNKKPMFCNNTGY CMSLYKEPSRYM (SEQ ID NO: 107) >gi|152217982|gb|ABS31425. MAKIIKFVYIMILCVSLLLIVEAGGKEC Medicago truncatula 1| NCR 201 VTDVDCEKIYPGNKKPLICSTGYCYS LYEEPPRYHK (SEQ ID NO: 108) >gi|152217980|gb|ABS31424. MAKVTKFGYIIIHFLSLFFLAMNVAG Medicago truncatula 1| NCR 200 GRECHANSHCVGKITCVLPQKPEC WNYACVCYDSNKYR (SEQ ID NO: 109) >gi|152217978|gb|ABS31423. MAKIFNYVYALIMFLSLFLMGTSGMK Medicago truncatula 1| NCR 192 NGCKHTGHCPRKMCGAKTTKCRN NKCQCV (SEQ ID NO: 110) >gi|152217976|gb|ABS31422. MTEILKFVCVMIIFISSFIVSKSLNGG Medicago truncatula 1| NCR 189 GKDKCFRDSDCPKHMCPSSLVAKCI NRLCRCRRPELQVQLNP (SEQ ID NO: 111) >gi|152217974|gb|ABS31421. MAHIIMFVYALIYALIIFSSLFVRDGIP Medicago truncatula 1| NCR 187 CLSDDECPEMSHYSFKCNNKICEYD LGEMSDDDYYLEMSRE (SEQ ID NO: 112) >gi|152217972|gb|ABS31420. MYREKNMAKTLKFVYVIVLFLSLFLA Medicago truncatula 1| NCR 181 AKNIDGRVSYNSFIALPVCQTAADC PEGTRGRTYKCINNKCRYPKLLKPI Q (SEQ ID NO: 113) >gi|152217970|gb|ABS31419. MAHIFNYVYALLVFLSLFLMVTNGIHI Medicago truncatula 1| NCR 176 GCDKDRDCPKQMCHLNQTPKCLKN ICKCV (SEQ ID NO: 114) >gi|152217968|gb|ABS31418. MAEILKCFYTMNLFIFLIILPAKIREHI Medicago truncatula 1| NCR 175 QCVIDDDCPKSLNKLLIIKCINHVCQY VGNLPDFASQIPKSTKMPYKGE (SEQ ID NO: 115) >gi|152217966|gb|ABS31417. MAYISRIFYVLIIFLSLFFVVINGVKSL Medicago truncatula 1| NCR 173 LLIKVRSFIPCQRSDDCPRNLCVDQII PTCVWAKCKCKNYND (SEQ ID NO: 116) >gi|152217964|gb|ABS31416. MANVTKFVYIAIYFLSLFFIAKNDATA Medicago truncatula 1| NCR 172 TFCHDDSHCVTKIKCVLPRTPQCRN EACGCYHSNKFR (SEQ ID NO: 117) >gi|152217962|gb|ABS31415. MGEIMKFVYVMIIYLFMFNVATGSEF Medicago truncatula 1| NCR 171 IFTKKLTSCDSSKDCRSFLCYSPKFP VCKRGICECI (SEQ ID NO: 118) >gi|152217960|gb|ABS31414. MGEMFKFYTFILFVHLFLVVIFEDIG Medicago truncatula 1| NCR 169 HIKYCGIVDDCYKSKKPLFKIWKCVE NVCVLWYK (SEQ ID NO: 119) >gi|152217958|gb|ABS31413. MARTLKFVYSMILFLSLFLVANGLKIF Medicago truncatula 1| NCR 165 CIDVADCPKDLYPLLYKCIYNKCIVFT RIPFPFDWI (SEQ ID NO: 120) >gi|152217956|gb|ABS31412. MANITKFVYIAILFLSLFFIGMNDAAIL Medicago truncatula 1| NCR 159 ECREDSHCVTKIKCVLPRKPECRNN ACTCYKGGFSFHH (SEQ ID NO: 121) >gi|152217954|gb|ABS31411. MQRVKKMSETLKFVYVLILFISIFHVV Medicago truncatula 1| NCR 147 IVCDSIYFPVSRPCITDKDCPNMKHY KAKCRKGFCISSRVR (SEQ ID NO: 122) >gi|152217952|gb|ABS31410. MQIRKIMSGVLKFVYAIILFLFLFLVA Medicago truncatula 1| NCR 146 REVGGLETIECETDGDCPRSMIKM WNKNYRHKCIDGKCEWIKKLP (SEQ ID NO: 123) >gi|152217950|gb|ABS31409. MFVYDLILFISLILVVTGINAEADTSC Medicago truncatula 1| NCR 145 HSFDDCPWVAHHYRECIEGLCAYRI LY (SEQ ID NO: 124) >gi|152217948|gb|ABS31408. MQRRKKSMAKMLKFFFAIILLLSLFL Medicago truncatula 1| NCR 144 VATEVGGAYIECEVDDDCPKPMKN SHPDTYYKCVKHRCQWAWK (SEQ ID NO: 125) >gi|152217946|gb|ABS31407. MFVYTLIIFLFPSHVITNKIAIYCVSDD Medicago truncatula 1| NCR 140 DCLKTFTPLDLKCVDNVCEFNLRCK GKCGERDEKFVFLKALKKMDQKLVL EEQGNAREVKIPKKLLFDRIQVPTPA TKDQVEEDDYDDDDEEEEEEEDDV DMWFHLPDVVCH (SEQ ID NO: 126) >gi|152217944|gb|ABS31406. MAKFSMFVYALINFLSLFLVETAITNI Medicago truncatula 1| NCR 138 RCVSDDDCPKVIKPLVMKCIGNYCY FFMIYEGP (SEQ ID NO: 127) >gi|152217942|gb|ABS31405. MAHKFVYAIILFIFLFLVAKNVKGYVV Medicago truncatula 1| NCR 136 CRTVDDCPPDTRDLRYRCLNGKCK SYRLSYG (SEQ ID NO: 128) >gi|152217940|gb|ABS31404. MQRKKNMGQILIFVFALINFLSPILVE Medicago truncatula 1| NCR 129 MTTTTIPCTFIDDCPKMPLVVKCIDN FCNYFEIK (SEQ ID NO: 129) >gi|152217938|gb|ABS31403. MAQTLMLVYALIIFTSLFLVVISRQTD Medicago truncatula 1| NCR 128 IPCKSDDACPRVSSHHIECVKGFCT YWKLD (SEQ ID NO: 130) >gi|152217936|gb|ABS31402. MLRRKNTVQILMFVSALLIYIFLFLVIT Medicago truncatula 1| NCR 127 SSANIPCNSDSDCPWKIYYTYRCND GFCVYKSIDPSTIPQYMTDLIFPR (SEQ ID NO: 131) >gi|152217934|gb|ABS31401. MAVILKFVYIMIIFLFLLYVVNGTRCN Medicago truncatula 1| NCR 122 RDEDCPFICTGPQIPKCVSHICFCLS SGKEAY (SEQ ID NO: 132) >gi|152217932|gb|ABS31400. MDAILKFIYAMFLFLFLFVTTRNVEAL Medicago truncatula 1| NCR 121 FECNRDFVCGNDDECVYPYAVQCI HRYCKCLKSRN (SEQ ID NO: 133) >gi|152217930|gb|ABS31399. MQIGRKKMGETPKLVYVIILFLSIFLC Medicago truncatula 1| NCR 119 TNSSFSQMINFRGCKRDKDCPQFR GVNIRCRSGFCTPIDS (SEQ ID NO: 134) >gi|152217928|gb|ABS31398. MQMRKNMAQILFYVYALLILFSPFLV Medicago truncatula 1| NCR 118 ARIMVVNPNNPCVTDADCQRYRHK LATRMVCNIGFCLMDFTHDPYAPSL P (SEQ ID NO: 135) >gi|152217926|gb|ABS31397. MYVYYIQMGKNMAQRFMFIYALIIFL Medicago truncatula 1| NCR 111 SQFFVVINTSDIPNNSNRNSPKEDVF CNSNDDCPTILYYVSKCVYNFCEYW (SEQ ID NO: 136) >gi|152217924|gb|ABS31396. MAKIVNFVYSMIIFVSLFLVATKGGS Medicago truncatula 1| NCR 103 KPFLTRPYPCNTGSDCPQNMCPPG YKPGCEDGYCNHCYKRW (SEQ ID NO: 137) >gi|152217922|gb|ABS31395. MVRTLKFVYVIILILSLFLVAKGGGKK Medicago truncatula 1| NCR 101 IYCENAASCPRLMYPLVYKCLDNKC VKFMMKSRFV (SEQ ID NO: 138) >gi|152217920|gb|ABS31394. MARTLKFVYAVILFLSLFLVAKGDDV Medicago truncatula 1| NCR 96 KIKCVVAANCPDLMYPLVYKCLNGIC VQFTLTFPFV (SEQ ID NO: 139) >gi|152217918|gb|ABS31393. MSNTLMFVITFIVLVTLFLGPKNVYA Medicago truncatula 1| NCR 94 FQPCVTTADCMKTLKTDENIWYECI NDFCIPFPIPKGRK (SEQ ID NO: 140) >gi|152217916|gb|ABS31392. MKRVVNMAKIVKYVYVIIIFLSLFLVA Medicago truncatula 1| NCR 93 TKIEGYYYKCFKDSDCVKLLCRIPLR PKCMYRHICKCKVVLTQNNYVLT (SEQ ID NO: 141) >gi|152217914|gb|ABS31391. MKRGKNMSKILKFIYATLVLYLFLVV Medicago truncatula 1| NCR 90 TKASDDECKIDGDCPISWQKFHTYK CINQKCKWVLRFHEY (SEQ ID NO: 142) >gi|152217912|gb|ABS31390. MAKTLNFMFALILFISLFLVSKNVAIDI Medicago truncatula 1| NCR 88 FVCQTDADCPKSELSMYTWKCIDN ECNLFKVMQQMV (SEQ ID NO: 143) >gi|152217910|gb|ABS31389. MANTHKLVSMILFIFLFLVANNVEGY Medicago truncatula 1| NCR 86 VNCETDADCPPSTRVKRFKCVKGE CRWTRMSYA (SEQ ID NO: 144) >gi|152217908|gb|ABS31388. MAHFLMFVYALITCLSLFLVEMGHLS Medicago truncatula 1| NCR 77 IHCVSVDDCPKVEKPITMKCINNYCK YFVDHKL (SEQ ID NO: 145) >gi|1522179061gb|ABS31387. MNQIPMFGYTLIIFFSLFPVITNGDRI Medicago truncatula 1| NCR 76 PCVTNGDCPVMRLPLYMRCITYSCE LFFDGPNLCAVERI (SEQ ID NO: 146) >gi|152217904|gb|ABS31386. MRKDMARISLFVYALIIFFSLFFVLTN Medicago truncatula 1| NCR 74 GELEIRCVSDADCPLFPLPLHNRCID DVCHLFTS (SEQ ID NO: 147) >gi|152217902|gb|ABS31385. MAQILMFVYFLIIFLSLFLVESIKIFTE Medicago truncatula 1| NCR 68 HRCRTDADCPARELPEYLKCQGGM CRLLIKKD (SEQ ID NO: 148) >gi|152217900|gb|ABS31384. MARVISLFYALIIFLFLFLVATNGDLS Medicago truncatula 1| NCR 65 PCLRSGDCSKDECPSHLVPKCIGLT CYCI (SEQ ID NO: 149)
>gi|152217898|gb|ABS31383. MQRRKNMAQILLFAYVFIISISLFLVV Medicago truncatula 1| NCR 62 TNGVKIPCVKDTDCPTLPCPLYSKC VDGFCKMLSI (SEQ ID NO: 150) >gi|152217896|gb|ABS31382. MNHISKFVYALIIFLSVYLVVLDGRPV Medicago truncatula 1| NCR 57 SCKDHYDCRRKVKIVGCIFPQEKPM CINSMCTCIREIVP (SEQ ID NO: 151) >gi|152217894|gb|ABS31381. MKSQNHAKFISFYKNDLFKIFQNND Medicago truncatula 1| NCR 56 SHFKVFFALIIFLYTYLHVTNGVFVSC NSHIHCRVNNHKIGCNIPEQYLLCVN LFCLWLDY (SEQ ID NO: 152) >gi|152217892|gb|ABS31380. MTYISKVVYALIIFLSIYVGVNDCMLV Medicago truncatula 1| NCR 54 TCEDHFDCRQNVQQVGCSFREIPQ CINSICKCMKG (SEQ ID NO: 153) >gi|152217890|gb|ABS31379. MTHISKFVFALIIFLSIYVGVNDCKRIP Medicago truncatula 1| NCR 53 CKDNNDCNNNWQLLACRFEREVPR CINSICKCMPM (SEQ ID NO: 154) >gi|152217888|gb|ABS31378. MVQTPKLVYVIVLLLSIFLGMTICNSS Medicago truncatula 1| NCR 43 FSHFFEGACKSDKDCPKLHRSNVR CRKGQCVQI (SEQ ID NO: 155) >gi|152217886|gb|ABS31377. MTKILMLFYAMIVFHSIFLVASYTDEC Medicago truncatula 1| NCR 28 STDADCEYILCLFPIIKRCIHNHCKCV PMGSIEPMSTIPNGVHKFHIINN (SEQ ID NO: 156) >gi|152217884|gb|ABS31376. MAKTLNFVCAMILFISLFLVSKNVAL Medicago truncatula 1| NCR 26 YIIECKTDADCPISKLNMYNWRCIKS SCHLYKVIQFMV (SEQ ID NO: 157) >gi|152217882|gb|ABS31375. MQKEKNMAKTFEFVYAMIIFILLFLVE Medicago truncatula 1| NCR 24 NNFAAYIIECQTDDDCPKSQLEMFA WKCVKNGCHLFGMYEDDDDP (SEQ ID NO: 158) >gi|152217880|gb|ABS31374. MAATRKFIYVLSHFLFLFLVTKITDAR Medicago truncatula 1| NCR 21 VCKSDKDCKDIIIYRYILKCRNGECV KIKI (SEQ ID NO: 159) >gi|152217878|gb|ABS31373. MQRLDNMAKNVKFIYVIILLLFIFLVII Medicago truncatula 1| NCR 20 VCDSAFVPNSGPCTTDKDCKQVKG YIARCRKGYCMQSVKRTWSSYSR (SEQ ID NO: 160) >gi|152217876|gb|ABS31372. MKFIYIMILFLSLFLVQFLTCKGLTVP Medicago truncatula 1| NCR 19 CENPTTCPEDFCTPPMITRCINFICL CDGPEYAEPEYDGPEPEYDHKGDF LSVKPKIINENMMMRERHMMKEIEV (SEQ ID NO: 161) >gi|152217874|gb|ABS31371. MAQFLMFIYVLIIFLYLFYVEAAMFEL Medicago truncatula 1| NCR 12 TKSTIRCVTDADCPNVVKPLKPKCV DGFCEYT (SEQ ID NO: 162) >gi|1522178721gb|ABS31370. MKMRIHMAQIIMFFYALIIFLSPFLVD Medicago truncatula 1| NCR 10 RRSFPSSFVSPKSYTSEIPCKATRD CPYELYYETKCVDSLCTY (SEQ ID NO: 163)
[0169] Any NCR peptide known in the art is suitable for use in the methods or compositions described herein. NCR peptide-producing plants include but are not limited to Pisum sativum (pea), Astragalus sinicus (IRLC legumes), Phaseolus vulgaris (bean), Vigna unguiculata (cowpea), Medicago truncatula (barrelclover), and Lotus japonicus. For example, over 600 potential NCR peptides are predicted from the M. truncatula genome sequence and almost 150 different NCR peptides have been detected in cells isolated from root nodules by mass spectrometry.
[0170] The NCR peptides described herein may be mature or immature NCR peptides. Immature NCR peptides have a C-terminal signal peptide that is required for translocation into the endoplasmic reticulum and cleaved after translocation. The N-terminus of a NCR peptide includes a signal peptide, which may be cleavable, for targeting to a secretory pathway. NCR peptides are generally small peptides with disulfide bridges that stabilize their structure. Mature NCR peptides have a length in the range of about 20 to about 60 amino acids, about 25 to about 55 amino acids, about 30 to about 50 amino acids, about 35 to about 45 amino acids, or any range therebetween. NCR peptides may include a conserved sequence of cysteine residues with the rest of the peptide sequence highly variable. NCR peptides generally have about four or eight cysteines.
[0171] NCR peptides may be anionic, neutral, or cationic. In some instances, synthetic cationic NCR peptides having a pI greater than about eight possess antimicrobial activities. For example, NCR247 (pI=10.15, RNGCIVDPRCPYQQCRRPLYCRRR; SEQ ID NO: 164) and NCR335 (pI=11.22) are both effective against gram-negative and gram-positive bacteria as well as fungi. In some instances, neutral and/or anionic NCR peptides, such as NCR001 (MAQFLLFVYSLIIFLSLFFGEAAFERTETRMLTIPCTSDDNCPKVISPCHTKCFDGFCGWYIEGS- YEGP; SEQ ID NO: 165), do not possess antimicrobial activities at a pI greater than about 8.
[0172] In some instances, the NCR peptide is effective to kill bacteria. In some instances, the NCR peptide is effective to kill S. meliloti, Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Buchnera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacter spp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, or Escherichia spp.
[0173] In some instances, the NCR peptide is a functionally active variant of a NCR peptide described herein. In some instances, the variant of the NCR peptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a NCR peptide described herein or naturally derived NCR peptide.
[0174] In some instances, the NCR peptide may be bioengineered to modulate its bioactivity, e.g., increase or decrease or regulate, or to specify a target microorganism. In some instances, the NCR peptide is produced by the translational machinery (e.g. a ribosome, etc.) of a cell. In some instances, the NCR peptide is chemically synthesized. In some instances, the NCR peptide is derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the NCR peptide itself. As such, in some instances, the NCR peptide is produced from a precursor polypeptide. In some instances, the NCR peptide includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
[0175] The NCR peptide described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type of NCR peptides, such as at least about any one of 1 NCR peptide, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more NCR peptides. A suitable concentration of each NCR peptide in the composition depends on factors such as efficacy, stability of the NCR peptide, number of distinct NCR peptide, the formulation, and methods of application of the composition. In some instances, each NCR peptide in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL. In some instances, each NCR peptide in a solid composition is from about 0.1 ng/g to about 100 mg/g. In some instances, wherein the composition includes at least two types of NCR peptides, the concentration of each type of NCR peptide may be the same or different.
[0176] A modulating agent including a NCR peptide as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of NCR peptide concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of NCR peptide concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of NCR peptide concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0177] (e) Bacteriocyte Regulatory Peptides
[0178] The modulating agent described herein may include a bacteriocyte regulatory peptide (BRP). BRPs are peptides expressed in the bacteriocytes of insects. These genes are expressed first at a developmental time point coincident with the incorporation of symbionts and their bacteriocyte-specific expression is maintained throughout the insect's life. In some instances, the BRP has a hydrophobic amino terminal domain, which is predicted to be a signal peptide. In addition, some BRPs have a cysteine-rich domain. In some instances, the bacteriocyte regulatory peptide is a bacteriocyte-specific cysteine rich (BCR) protein. Bacteriocyte regulatory peptides have a length between about 40 and 150 amino acids. In some instances, the bacteriocyte regulatory peptide has a length in the range of about 45 to about 145, about 50 to about 140, about 55 to about 135, about 60 to about 130, about 65 to about 125, about 70 to about 120, about 75 to about 115, about 80 to about 110, about 85 to about 105, or any range therebetween. Non-limiting examples of BRPs and their activities are listed in Table 8.
TABLE-US-00008 TABLE 8 Examples of Bacteriocyte Regulatory Peptides Name Peptide Sequence Bacteriocyte-specific cysteine rich MKLLHGFLIIMLTMHLSIQYAYGGPFLTKYLCDRVCHKLC proteins BCR family, peptide BCR1 GDEFVCSCIQYKSLKGLWFPHCPTGKASVVLHNFLTSP (SEQ ID NO: 166) Bacteriocyte-specific cysteine rich MKLLYGFLIIMLTIHLSVQYFESPFETKYNCDTHCNKLCGK proteins BCR family, peptide BCR2 IDHCSCIQYHSMEGLWFPHCRTGSAAQMLHDFLSNP (SEQ ID NO: 167) Bacteriocyte-specific cysteine rich MSVRKNVLPTMFVVLLIMSPVTPTSVFISAVCYSGCGSLA proteins BCR family, peptide BCR3 LVCFVSNGITNGLDYFKSSAPLSTSETSCGEAFDTCTDH CLANFKF (SEQ ID NO: 168) Bacteriocyte-specific cysteine rich MRLLYGFLIIMLTIYLSVQDFDPTEFKGPFPTIEICSKYCAV proteins BCR family, peptide BCR4 VCNYTSRPCYCVEAAKERDQWFPYCYD (SEQ ID NO: 169) Bacteriocyte-specific cysteine rich MRLLYGFLIIMLTIHLSVQDIDPNTLRGPYPTKEICSKYCEY proteins BCR family, peptide BCR5 NVVCGASLPCICVQDARQLDHWFACCYDGGPEMLM (SEQ ID NO: 170) Secreted proteins SP family, peptide MKLFVVVVLVAVGIMFVFASDTAAAPTDYEDTNDMISLSS SP1 LVGDNSPYVRVSSADSGGSSKTSSKNPILGLLKSVIKLLT KIFGTYSDAAPAMPPIPPALRKNRGMLA (SEQ ID NO: 171) Secreted proteins SP family, peptide MVACKVILAVAVVFVAAVQGRPGGEPEWAAPIFAELKSV SP2 SDNITNLVGLDNAGEYATAAKNNLNAFAESLKTEAAVFSK SFEGKASASDVFKESTKNFQAVVDTYIKNLPKDLTLKDFT EKSEQALKYMVEHGTEITKKAQGNTETEKEIKEFFKKQIE NLIGQGKALQAKIAEAKKA (SEQ ID NO: 172) Secreted proteins SP family, peptide MKTSSSKVFASCVAIVCLASVANALPVQKSVAATTENPIV SP3 EKHGCRAHKNLVRQNVVDLKTYDSMLITNEVVQKQSNE VQSSEQSNEGQNSEQSNEGQNSEQSNEVQSSEHSNEG QNSKQSNEGQNSEQSNEVQSSEHSNEGQNSEQSNEVQ SSEHSNEGQNSKQSNEGQNSKQSNEVQSSEHWNEGQ NSKQSNEDQNSEQSNEGQNSKQSNEGQNSKQSNEDQ NSEQSNEGQNSKQSNEVQSSEQSNEGQNSKQSNEGQS SEQSNEGQNSKQSNEVQSPEEHYDLPDPESSYESEETK GSHESGDDSEHR (SEQ ID NO: 173) Secreted proteins SP family, peptide MKTIILGLCLFGALFWSTQSMPVGEVAPAVPAVPSEAVP SP4 QKQVEAKPETNAASPVSDAKPESDSKPVDAEVKPTVSEV KAESEQKPSGEPKPESDAKPVVASESKPESDPKPAAVVE SKPENDAVAPETNNDAKPENAAAPVSENKPATDAKAETE LIAQAKPESKPASDLKAEPEAAKPNSEVPVALPLNPTETK ATQQSVETNQVEQAAPAAAQADPAAAPAADPAPAPAAA PVAAEEAKLSESAPSTENKAAEEPSKPAEQQSAKPVEDA VPAASEISETKVSPAVPAVPEVPASPSAPAVADPVSAPEA EKNAEPAKAANSAEPAVQSEAKPAEDIQKSGAVVSAENP KPVEEQKPAEVAKPAEQSKSEAPAEAPKPTEQSAAEEPK KPESANDEKKEQHSVNKRDATKEKKPTDSIMKKQKQKK AN (SEQ ID NO: 174) Secreted proteins SP family, peptide MNGKIVLCFAVVFIGQAMSAATGTTPEVEDIKKVAEQMS SP5a QTFMSVANHLVGITPNSADAQKSIEKIRTIMNKGFTDMET EANKMKDIVRKNADPKLVEKYDELEKELKKHLSTAKDMF EDKVVKPIGEKVELKKITENVIKTTKDMEATMNKAIDGFKK Q (SEQ ID NO: 175) Secreted proteins SP family, peptide MHLFLALGLFIVCGMVDATFYNPRSQTFNQLMERRQRSI SP6 PIPYSYGYHYNPIEPSINVLDSLSEGLDSRINTFKPIYQNV KMSTQDVNSVPRTQYQPKNSLYDSEYISAKDIPSLFPEE DSYDYKYLGSPLNKYLTRPSTQESGIAINLVAIKETSVFDY GFPTYKSPYSSDSVWNFGSKIPNTVFEDPQSVESDPNTF KVSSPTIKIVKLLPETPEQESIITTTKNYELNYKTTQETPTE AELYPITSEEFQTEDEWHPMVPKENTTKDESSFITTEEPL TEDKSNSITIEKTQTEDESNSIEFNSIRTEEKSNSITTEENQ KEDDESMSTTSQETTTAFNLNDTFDTNRYSSSHESLMLR IRELMKNIADQQNKSQFRTVDNIPAKSQSNLSSDESTNQ QFEPQLVNGADTYK (SEQ ID NO: 176) Colepotericin A, ColA peptide MTRTMLFLACVAALYVCISATAGKPEEFAKLSDEAPSND QAMYESIQRYRRFVDGNRYNGGQQQQQQPKQWEVRP DLSRDQRGNTKAQVEINKKGDNHDINAGWGKNINGPDS HKDTWHVGGSVRW (SEQ ID NO: 177) RlpA type I MKETTVVWAKLFLILIILAKPLGLKAVNECKRLGNNSCRSH GECCSGFCFIEPGWALGVCKRLGTPKKSDDSNNGKNIEK NNGVHERIDDVFERGVCSYYKGPSITANGDVFDENEMTA AHRTLPFNTMVKVEGMGTSVVVKINDRKTAADGKVMLLS RAAAESLNIDENTGPVQCQLKFVLDGSGCTPDYGDTCVL HHECCSQNCFREMFSDKGFCLPK (SEQ ID NO: 192)
[0179] In some instances, the BRP alters the growth and/or activity of one or more bacteria resident in the bacteriocyte of the host. In some instances, the BRP may be bioengineered to modulate its bioactivity (e.g., increase, decrease, or regulate) or to specify a target microorganism. In some instances, the BRP is produced by the translational machinery (e.g. a ribosome, etc.) of a cell. In some instances, the BRP is chemically synthesized. In some instances, the BRP is derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the polypeptide of the BRP itself. As such, in some instances, the BRP is produced from a precursor polypeptide. In some instances, the BRP includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
[0180] Functionally active variants of the BRPs as described herein are also useful in the compositions and methods described herein. In some instances, the variant of the BRP has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a BRP described herein or naturally derived BRP.
[0181] The BRP described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type (e.g., classes) of BRPs, such as at least about any one of 1 BRP, 2, 3, 4, 5, 10, 15, 20, or more BRPs. A suitable concentration of each BRP in the composition depends on factors such as efficacy, stability of the BRP, number of distinct BRP, the formulation, and methods of application of the composition. In some instances, each BRP in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL. In some instances, each BRP in a solid composition is from about 0.1 ng/g to about 100 mg/g. In some instances, wherein the composition includes at least two types of BRPs, the concentration of each type of BRP may be the same or different.
[0182] A modulating agent including a BRP as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of BRP concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of BRP concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of BRP concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0183] iii. Small Molecules
[0184] Numerous small molecules (e.g., an antibiotic or a metabolite) may be used in the compositions and methods described herein. In some instances, an effective concentration of any small molecule described herein may alter the level, activity, or metabolism of one or more microorganisms (as described herein) resident in a host, the alteration resulting in an increase in the host's fitness.
[0185] A modulating agent comprising a small molecule as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of a small molecule concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of small molecule concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of a small molecule concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0186] The small molecules discussed hereinafter, namely antibiotics and secondary metabolites, can be used to alter the level, activity, or metabolism of target microorganisms as indicated in the sections for increasing the fitness of insects, such as honeybees and silkworms.
[0187] (a) Antibiotics
[0188] The modulating agent described herein may include an antibiotic. Any antibiotic known in the art may be used. Antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity.
[0189] The antibiotic described herein may target any bacterial function or growth processes and may be either bacteriostatic (e.g., slow or prevent bacterial growth) or bactericidal (e.g., kill bacteria). In some instances, the antibiotic is a bactericidal antibiotic. In some instances, the bactericidal antibiotic is one that targets the bacterial cell wall (e.g., penicillins and cephalosporins); one that targets the cell membrane (e.g., polymyxins); or one that inhibits essential bacterial enzymes (e.g., rifamycins, lipiarmycins, quinolones, and sulfonamides). In some instances, the bactericidal antibiotic is an aminoglycoside. In some instances, the antibiotic is a bacteriostatic antibiotic. In some instances the bacteriostatic antibiotic targets protein synthesis (e.g., macrolides, lincosamides and tetracyclines). Additional classes of antibiotics that may be used herein include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), or lipiarmycins (such as fidaxomicin). Examples of antibiotics include rifampicin, ciprofloxacin, doxycycline, ampicillin, and polymyxin B. Non-limiting examples of antibiotics are found in Table 9.
TABLE-US-00009 TABLE 9 Examples of Antibiotics Antibiotics Action Penicillins, cephalosporins, vancomycin Cell wall synthesis Polymixin, gramicidin Membrane active agent, disrupt cell membrane Tetracyclines, macrolides, chloramphenicol, Inhibit protein synthesis clindamycin, spectinomycin Sulfonamides Inhibit folate-dependent pathways Ciprofloxacin Inhibit DNA-gyrase Isoniazid, rifampicin, pyrazinamide, Antimycobacterial agents ethambutol, (myambutol)l, streptomycin
[0190] The antibiotic described herein may have any level of target specificity (e.g., narrow- or broad-spectrum). In some instances, the antibiotic is a narrow-spectrum antibiotic, and thus targets specific types of bacteria, such as gram-negative or gram-positive bacteria. Alternatively, the antibiotic may be a broad-spectrum antibiotic that targets a wide range of bacteria.
[0191] The antibiotics described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type (e.g., classes) of antibiotics, such as at least about any one of 1 antibiotic, 2, 3, 4, 5, 10, 15, 20, or more antibiotics (e.g., a combination of rifampicin and doxycycline, or a combination of ampicillin and rifampicin). A suitable concentration of each antibiotic in the composition depends on factors such as efficacy, stability of the antibiotic, number of distinct antibiotics, the formulation, and methods of application of the composition. In some instances, wherein the composition includes at least two types of antibiotics, the concentration of each type of antibiotic may be the same or different.
[0192] A modulating agent including an antibiotic as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0193] (b) Secondary Metabolites
[0194] In some instances, the modulating agent of the compositions and methods described herein includes a secondary metabolite. Secondary metabolites are derived from organic molecules produced by an organism. Secondary metabolites may act (i) as competitive agents used against bacteria, fungi, amoebae, plants, insects, and large animals; (ii) as metal transporting agents; (iii) as agents of symbiosis between microbes and plants, nematodes, insects, and higher animals; (iv) as sexual hormones; and (v) as differentiation effectors. Non-limiting examples of secondary metabolites are found in Table 10.
TABLE-US-00010 TABLE 10 Examples of Secondary Metabolites Phenyl- propanoids Alkaloids Terpenoids Quinones Steroids Polyketides Anthocyanins Acridines Carotenes Anthra- Cardiac Erythromycin quinones Coumarins Betalaines Monoterpenes Bezo- Glycosides Lovastatin and quinones other statins Flavonoids Quinolo- Sesquiterpenes Naphtho- Pregnenolone Discodermolide zidines quinones Hydroxy- Furono- Diterpenes Derivatives Aflatoxin B1 cinnamoyl quinones Derivatives Harringtonines Triterpenes Avermectins Isoflavonoids Isoquinolines Nystatin Lignans Indoles Rifamycin Phenolenones Purines Proantho- Pyridines cyanidins Stilbenes Tropane Tanins Alkaloids
[0195] The secondary metabolite used herein may include a metabolite from any known group of secondary metabolites. For example, secondary metabolites can be categorized into the following groups: alkaloids, terpenoids, flavonoids, glycosides, natural phenols (e.g., gossypol acetic acid), enals (e.g., trans-cinnamaldehyde), phenazines, biphenols and dibenzofurans, polyketides, fatty acid synthase peptides, nonribosomal peptides, ribosomally synthesized and post-translationally modified peptides, polyphenols, polysaccharides (e.g., chitosan), and biopolymers. For an in-depth review of secondary metabolites see, for example, Vining, Annu. Rev. Microbiol. 44:395-427, 1990.
[0196] Secondary metabolites useful for compositions and methods described herein include those that alter a natural function of an endosymbiont (e.g., primary or secondary endosymbiont), bacteriocyte, or extracellular symbiont. In some instances, one or more secondary metabolites described herein is isolated from a high throughput screening (HTS) for antimicrobial compounds. For example, a HTS screen identified 49 antibacterial extracts that have specificity against gram positive and gram negative bacteria from over 39,000 crude extracts from organisms growing in diverse ecosystems of one specific region. In some instances, the secondary metabolite is transported inside a bacteriocyte.
[0197] In some instances, the small molecule is an inhibitor of vitamin synthesis. In some instances, the vitamin synthesis inhibitor is a vitamin precursor analog. In certain instances, the vitamin precursor analog is pantothenol.
[0198] In some instances, the small molecule is an amino acid analog. In certain instances, the amino acid analog is L-canvanine, D-arginine, D-valine, D-methionine, D-phenylalanine, D-histidine, D-tryptophan, D-threonine, D-leucine, L-NG-nitroarginine, or a combination thereof.
[0199] In some instances, the small molecule is a natural antimicrobial compound, such as propionic acid, levulinic acid, trans-cinnemaldehdye, nisin, or low molecular weight chitosan. The secondary metabolite described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type (e.g., classes) of secondary metabolites, such as at least about any one of 1 secondary metabolite, 2, 3, 4, 5, 10, 15, 20, or more secondary metabolites. A suitable concentration of each secondary metabolite in the composition depends on factors such as efficacy, stability of the secondary metabolite, number of distinct secondary metabolites, the formulation, and methods of application of the composition. In some instances, wherein the composition includes at least two types of secondary metabolites, the concentration of each type of secondary metabolite may be the same or different.
[0200] A modulating agent including a secondary metabolite as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
[0201] iv. Bacteria as Modulating Agents
[0202] In some instances, the modulating agent described herein includes one or more bacteria. Numerous bacteria are useful in the compositions and methods described herein. In some instances, the agent is a bacterial species endogenously found in the host. In some instances, the bacterial modulating agent is an endosymbiotic bacterial species. Non-limiting examples of bacteria that may be used as modulating agents include all bacterial species described herein in Section II of the detailed description and those listed in Table 1 starting at page 15. For example, the modulating agent may be a bacterial species from any bacterial phyla present in insect guts, including Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Firmicutes (e.g., Lactobacillus and Bacillus spp.), Clostridia, Actinomycetes, Spirochetes, Verrucomicrobia, and Actinobacteria.
[0203] In some instances, the modulating agent is a bacterium that promotes microbial diversity or otherwise alters the microbiota of the host in a favorable manner. In one instance, bacteria may be provided to promote microbiome development in honey bees. For example, the modulating agent may include, for example, Bartonella apis, Parasaccharibacter apium, Frischella perrara, Snodgrassella alvi, Gilliamela apicola, Bifidobacterium spp, or Lactobacillus spp.
[0204] The bacterial modulating agents discussed herein can be used to alter the level, activity, or metabolism of target microorganisms as indicated in the sections for increasing the fitness of insects, such as, honeybees and silkworms.
[0205] In some instances, such bacterial modulating agents are bacteria which are capable of degrading pesticides as laid out in Table 12 including insecticides. Such insecticides include neonicotinoids such as imidacloprid, or organophosphorus insecticides, such as fenitrothion. In some instances, the pesticide-metabolizing bacteria are at a concentration of at least 100,000 cells/ml (e.g., at least about 100,000 cells/ml, at least about 150,000 cells/ml, at least about 200,000 cells/ml, at least about 250,000 cells/ml, at least about 300,000 cells/ml, at least about 350,000 cells/ml, at least about 400,000 cells/ml, at least about 450,000 cells/ml, or at least about 500,000 cells/ml).
[0206] Examples 1 to 3, 5, and 6 describe how imidacloprid and fenitrothion degrading microorganisms can be identified which can then be used a modulating agents in insect hosts, such as honeybees, giving the treated insect hosts a competitive advantage. Administering such pesticide-degrading microorganisms, for example imidacloprid- or fenitrothion-degrading microorganisms to insect hosts such as honeybees is understood to be encompassed by the alteration of a level, activity, or metabolism of one or more microorganisms resident in the host.
[0207] In some instances, such bacterial modulating agents are bacteria which are capable of producing nutrients, including amino acids (e.g., methionine or glutamate). The nutrient-producing bacteria may be naturally occurring bacteria, e.g., naturally occurring bacteria exogenous to the insect host. Such bacteria may be isolated from a population of bacteria, such as that found in an environmental sample. Bacteria can be isolated that produce one or more amino acids in a manner that increases production of amino acids in the host relative to a host who has not been administered the amino-acid producing bacteria. Amino acids that can be produced by the bacteria in the host include methionine, alanine, arginine, asparagine, asparatic acid, cysteine, glutamine, glutamic acid, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In certain instances, the amino acid-producing bacteria is a methionine-producing bacteria.
[0208] In some instances, the nutrient-producing bacteria (e.g., amino acid-producing bacteria, e.g., methionine-producing bacteria) are at a concentration of at least 100,000 cells/ml (e.g., at least about 100,000 cells/ml, at least about 150,000 cells/ml, at least about 200,000 cells/ml, at least about 250,000 cells/ml, at least about 300,000 cells/ml, at least about 350,000 cells/ml, at least about 400,000 cells/ml, at least about 450,000 cells/ml, or at least about 500,000 cells/ml).
[0209] Examples 8, 9, and 10 describe how methionine-producing microorganisms can be identified which can then be used as modulating agents in insect hosts, such as honeybees, or in the model organism Drosophila, to increase the fitness of the hosts (e.g., increase amino acid content (e.g., methionine content or glutamate content).
[0210] v. Modifications to Modulating Agents
[0211] (a) Fusions
[0212] Any of the modulating agents described herein may be fused or linked to an additional moiety. In some instances, the modulating agent includes a fusion of one or more additional moieties (e.g., 1 additional moiety, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional moieties). In some instances, the additional moiety is any one of the modulating agents described herein (e.g., a peptide, polypeptide, small molecule, or antibiotic). Alternatively, the additional moiety may not act as modulating agent itself but may instead serve a secondary function. For example, the additional moiety may to help the modulating agent access, bind, or become activated at a target site in the host (e.g., at a host gut or a host bacteriocyte) or at a target microorganism resident in the host (e.g., honeybee or silkworm).
[0213] In some instances, the additional moiety may help the modulating agent penetrate a target host cell or target microorganism resident in the host. For example, the additional moiety may include a cell penetrating peptide. Cell penetrating peptides (CPPs) may be natural sequences derived from proteins; chimeric peptides that are formed by the fusion of two natural sequences; or synthetic CPPs, which are synthetically designed sequences based on structure-activity studies. In some instances, CPPs have the capacity to ubiquitously cross cellular membranes (e.g., prokaryotic and eukaryotic cellular membranes) with limited toxicity. Further, CPPs may have the capacity to cross cellular membranes via energy-dependent and/or independent mechanisms, without the necessity of a chiral recognition by specific receptors. CPPs can be bound to any of the modulating agents described herein. For example, a CPP can be bound to an antimicrobial peptide (AMP), e.g., a scorpion peptide, e.g., UY192 fused to a cell penetrating peptide (e.g., YGRKKRRQRRRFLSTIWNGIKGLLFAM; SEQ ID NO: 198). Non-limiting examples of CPPs are listed in Table 11.
TABLE-US-00011 TABLE 11 Examples of Cell Penetrating Peptides (CPPs) Peptide Origin Sequence Protein-derived rit Antennapedia RQIKIWFQNRRMKWKK (SEQ ID NO: 178) Tat peptide Tat GRKKRRQRRRPPQ (SEQ ID NO: 179) pVEC Cadherin LLIILRRRIRKQAHAHSK (SEQ ID NO: 180) Chimeric Transportan Galanine/Mastoparan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 181) MPG HIV-gp41/SV40 T-antigen GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 182) Pep-1 HIV-reverse KETWWETWWTEWSQPKKKRKV (SEQ transcriptase/SV40 T- ID NO: 183) antigen Synthetic Polyarginines Based on Tat peptide (R).sub.n; 6 < .sub.n < 12 MAP de novo KLALKLALKALKAALKLA (SEQ ID NO: 184) R.sub.6W.sub.3 Based on penetratin RRWWRRWRR (SEQ ID NO: 185)
[0214] In other instances, the additional moiety helps the modulating agent bind a target microorganism (e.g., a fungi or bacterium) resident in the host. The additional moiety may include one or more targeting domains. In some instances, the targeting domain may target the modulating agent to one or more microorganisms (e.g., bacterium or fungus) resident in the gut of the host. In some instances, the targeting domain may target the modulating agent to a specific region of the host (e.g., host gut or bacteriocyte) to access microorganisms that are generally present in said region of the host. For example, the targeting domain may target the modulating agent to the foregut, midgut, or hindgut of the host. In other instances, the targeting domain may target the modulating agent to a bacteriocyte in the host and/or one or more specific bacteria resident in a host bacteriocyte. For example, the targeting domain may be Galanthus nivalis lectin or agglutinin (GNA) bound to a modulating agent described herein, e.g., an AMP, e.g., a scorpion peptide, e.g., Uy192.
[0215] (b) Pre- or Pro-Domains
[0216] In some instances, the modulating agent may include a pre- or pro-amino acid sequence. For example, the modulating agent may be an inactive protein or peptide that can be activated by cleavage or post-translational modification of a pre- or pro-sequence. In some instances, the modulating agent is engineered with an inactivating pre- or pro-sequence. For example, the pre- or pro-sequence may obscure an activation site on the modulating agent, e.g., a receptor binding site, or may induce a conformational change in the modulating agent. Thus, upon cleavage of the pre- or pro-sequence, the modulating agent is activated.
[0217] Alternatively, the modulating agent may include a pre- or pro-small molecule, e.g., an antibiotic. The modulating agent may be an inactive small molecule described herein that can be activated in a target environment inside the host. For example, the small molecule may be activated upon reaching a certain pH in the host gut.
[0218] (c) Linkers
[0219] In instances where the modulating agent is connected to an additional moiety, the modulating agent may further include a linker. For example, the linker may be a chemical bond, e.g., one or more covalent bonds or non-covalent bonds. In some instances, the linker may be a peptide linker (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 20, 25, 30, 35, 40, or more amino acids longer). The linker may be include any flexible, rigid, or cleavable linkers described herein.
[0220] A flexible peptide linker may include any of those commonly used in the art, including linkers having sequences having primarily Gly and Ser residues ("GS" linker). Flexible linkers may be useful for joining domains that require a certain degree of movement or interaction and may include small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids.
[0221] Alternatively, a peptide linker may be a rigid linker. Rigid linkers are useful to keep a fixed distance between moieties and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of the domains is critical to preserve the stability or bioactivity of one or more components in the fusion. Rigid linkers may, for example, have an alpha helix-structure or Pro-rich sequence, (XP).sub.n, with X designating any amino acid, preferably Ala, Lys, or Glu.
[0222] In yet other instances, a peptide linker may be a cleavable linker. In some instances, linkers may be cleaved under specific conditions, such as the presence of reducing reagents or proteases. In vivo cleavable linkers may utilize the reversible nature of a disulfide bond. One example includes a thrombin-sensitive sequence (e.g., PRS) between two Cys residues. In vitro thrombin treatment of CPRSC results in the cleavage of the thrombin-sensitive sequence, while the reversible disulfide linkage remains intact. Such linkers are known and described, e.g., in Chen et al., Adv. Drug Deliv. Rev. 65(10):1357-1369, 2013. Cleavage of linkers in fusions may also be carried out by proteases that are expressed in vivo under conditions in specific cells or tissues of the host or microorganisms resident in the host. In some instances, cleavage of the linker may release a free functional, modulating agent upon reaching a target site or cell.
[0223] Fusions described herein may alternatively be linked by a linking molecule, including a hydrophobic linker, such as a negatively charged sulfonate group; lipids, such as a poly (--CH2-) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof, non-carbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecule capable of covalently linking two or more molecules, e.g., two modulating agents. Non-covalent linkers may be used, such as hydrophobic lipid globules to which the modulating agent is linked, for example, through a hydrophobic region of the modulating agent or a hydrophobic extension of the modulating agent, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine or other hydrophobic residue. The modulating agent may be linked using charge-based chemistry, such that a positively charged moiety of the modulating agent is linked to a negative charge of another modulating agent or an additional moiety.
IV. Formulations and Compositions
[0224] The compositions described herein may be formulated either in pure form (e.g., the composition contains only the modulating agent) or together with one or more additional agents (such as excipient, delivery vehicle, carrier, diluent, stabilizer, etc.) to facilitate application or delivery of the compositions. Examples of suitable excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, and mineral oil.
[0225] In some instances, the composition includes a delivery vehicle or carrier. In some instances, the delivery vehicle includes an excipient. Exemplary excipients include, but are not limited to, solid or liquid carrier materials, solvents, stabilizers, slow-release excipients, colorings, and surface-active substances (surfactants). In some instances, the delivery vehicle is a stabilizing vehicle. In some instances, the stabilizing vehicle includes a stabilizing excipient. Exemplary stabilizing excipients include, but are not limited to, epoxidized vegetable oils, antifoaming agents, e.g. silicone oil, preservatives, viscosity regulators, binding agents and tackifiers. In some instances, the stabilizing vehicle is a buffer suitable for the modulating agent. In some instances, the composition is microencapsulated in a polymer bead delivery vehicle. In some instances, the stabilizing vehicle protects the modulating agent against UV and/or acidic conditions. In some instances, the delivery vehicle contains a pH buffer. In some instances, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
[0226] Depending on the intended objectives and prevailing circumstances, the composition may be formulated into emulsifiable concentrates, suspension concentrates, directly sprayable or dilutable solutions, coatable pastes, diluted emulsions, spray powders, soluble powders, dispersible powders, wettable powders, dusts, granules, encapsulations in polymeric substances, microcapsules, foams, aerosols, carbon dioxide gas preparations, tablets, resin preparations, paper preparations, nonwoven fabric preparations, or knitted or woven fabric preparations. In some instances, the composition is a liquid. In some instances, the composition is a solid. In some instances, the composition is an aerosol, such as in a pressurized aerosol can. In some instances, the composition is present in the waste (such as feces) of the pest. In some instances, the composition is present in or on a live pest.
[0227] In some instances, the delivery vehicle is the food or water of the host. In other instances, the delivery vehicle is a food source for the host. In some instances, the delivery vehicle is a food bait for the host. In some instances, the composition is a comestible agent consumed by the host. In some instances, the composition is delivered by the host to a second host, and consumed by the second host. In some instances, the composition is consumed by the host or a second host, and the composition is released to the surrounding of the host or the second host via the waste (such as feces) of the host or the second host. In some instances, the modulating agent is included in food bait intended to be consumed by a host or carried back to its colony.
[0228] In some instances, the modulating agent may make up about 0.1% to about 100% of the composition, such as any one of about 0.01% to about 100%, about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, about 50% to about 99%, or about 0.1% to about 90% of active ingredients (such as phage, lysin or bacteriocin). In some instances, the composition includes at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more active ingredients (such as phage, lysin or bacteriocin). In some instances, the concentrated agents are preferred as commercial products, the final user normally uses diluted agents, which have a substantially lower concentration of active ingredient.
[0229] Any of the formulations described herein may be used in the form of a bait, a coil, an electric mat, a smoking preparation, a fumigant, or a sheet.
[0230] i. Liquid Formulations
[0231] The compositions provided herein may be in a liquid formulation. Liquid formulations are generally mixed with water, but in some instances may be used with crop oil, diesel fuel, kerosene or other light oil as a carrier. The amount of active ingredient often ranges from about 0.5 to about 80 percent by weight.
[0232] An emulsifiable concentrate formulation may contain a liquid active ingredient, one or more petroleum-based solvents, and an agent that allows the formulation to be mixed with water to form an emulsion. Such concentrates may be used in agricultural, ornamental and turf, forestry, structural, food processing, livestock, and public health pest formulations. These may be adaptable to application equipment from small portable sprayers to hydraulic sprayers, low-volume ground sprayers, mist blowers, and low-volume aircraft sprayers. Some active ingredients are readily dissolve in a liquid carrier. When mixed with a carrier, they form a solution that does not settle out or separate, e.g., a homogenous solution. Formulations of these types may include an active ingredient, a carrier, and one or more other ingredients. Solutions may be used in any type of sprayer, indoors and outdoors.
[0233] In some instances, the composition may be formulated as an invert emulsion. An invert emulsion is a water-soluble active ingredient dispersed in an oil carrier. Invert emulsions require an emulsifier that allows the active ingredient to be mixed with a large volume of petroleum-based carrier, usually fuel oil. Invert emulsions aid in reducing drift. With other formulations, some spray drift results when water droplets begin to evaporate before reaching target surfaces; as a result the droplets become very small and lightweight. Because oil evaporates more slowly than water, invert emulsion droplets shrink less and more active ingredient reaches the target. Oil further helps to reduce runoff and improve rain resistance. It further serves as a sticker-spreader by improving surface coverage and absorption. Because droplets are relatively large and heavy, it is difficult to get thorough coverage on the undersides of foliage. Invert emulsions are most commonly used along rights-of-way where drift to susceptible non-target areas can be a problem.
[0234] A flowable or liquid formulation combines many of the characteristics of emulsifiable concentrates and wettable powders. Manufacturers use these formulations when the active ingredient is a solid that does not dissolve in either water or oil. The active ingredient, impregnated on a substance such as clay, is ground to a very fine powder. The powder is then suspended in a small amount of liquid. The resulting liquid product is quite thick. Flowables and liquids share many of the features of emulsifiable concentrates, and they have similar disadvantages. They require moderate agitation to keep them in suspension and leave visible residues, similar to those of wettable powders.
[0235] Flowables/liquids are easy to handle and apply. Because they are liquids, they are subject to spilling and splashing. They contain solid particles, so they contribute to abrasive wear of nozzles and pumps. Flowable and liquid suspensions settle out in their containers. Because flowable and liquid formulations tend to settle, packaging in containers of five gallons or less makes remixing easier.
[0236] Aerosol formulations contain one or more active ingredients and a solvent. Most aerosols contain a low percentage of active ingredients. There are two types of aerosol formulations--the ready-to-use type commonly available in pressurized sealed containers and those products used in electrical or gasoline-powered aerosol generators that release the formulation as a smoke or fog.
[0237] Ready to use aerosol formulations are usually small, self-contained units that release the formulation when the nozzle valve is triggered. The formulation is driven through a fine opening by an inert gas under pressure, creating fine droplets. These products are used in greenhouses, in small areas inside buildings, or in localized outdoor areas. Commercial models, which hold five to 5 pounds of active ingredient, are usually refillable.
[0238] Smoke or fog aerosol formulations are not under pressure. They are used in machines that break the liquid formulation into a fine mist or fog (aerosol) using a rapidly whirling disk or heated surface.
[0239] ii. Dry or Solid Formulations
[0240] Dry formulations can be divided into two types: ready-to-use and concentrates that must be mixed with water to be applied as a spray. Most dust formulations are ready to use and contain a low percentage of active ingredients (less than about 10 percent by weight), plus a very fine, dry inert carrier made from talc, chalk, clay, nut hulls, or volcanic ash. The size of individual dust particles varies. A few dust formulations are concentrates and contain a high percentage of active ingredients. Mix these with dry inert carriers before applying. Dusts are always used dry and can easily drift to non-target sites.
[0241] iii. Granule or Pellet Formulations
[0242] In some instances, the composition is formulated as granules. Granular formulations are similar to dust formulations, except granular particles are larger and heavier. The coarse particles may be made from materials such as clay, corncobs, or walnut shells. The active ingredient either coats the outside of the granules or is absorbed into them. The amount of active ingredient may be relatively low, usually ranging from about 0.5 to about 15 percent by weight. Granular formulations are most often used to apply to the soil, insects or nematodes living in the soil, or absorption into plants through the roots. Granular formulations are sometimes applied by airplane or helicopter to minimize drift or to penetrate dense vegetation. Once applied, granules may release the active ingredient slowly. Some granules require soil moisture to release the active ingredient. Granular formulations also are used to control larval mosquitoes and other aquatic pests. Granules are used in agricultural, structural, ornamental, turf, aquatic, right-of-way, and public health (biting insect) pest-control operations.
[0243] In some instances, the composition is formulated as pellets. Most pellet formulations are very similar to granular formulations; the terms are used interchangeably. In a pellet formulation, however, all the particles are the same weight and shape. The uniformity of the particles allows use with precision application equipment.
[0244] iv. Powders
[0245] In some instances, the composition is formulated as a powder. In some instances, the composition is formulated as a wettable powder. Wettable powders are dry, finely ground formulations that look like dusts. They usually must be mixed with water for application as a spray. A few products, however, may be applied either as a dust or as a wettable powder--the choice is left to the applicator. Wettable powders have about 1 to about 95 percent active ingredient by weight; in some cases more than about 50 percent. The particles do not dissolve in water. They settle out quickly unless constantly agitated to keep them suspended. They can be used for most pest problems and in most types of spray equipment where agitation is possible. Wettable powders have excellent residual activity. Because of their physical properties, most of the formulation remains on the surface of treated porous materials such as concrete, plaster, and untreated wood. In such cases, only the water penetrates the material.
[0246] In some instances, the composition is formulated as a soluble powder. Soluble powder formulations look like wettable powders. However, when mixed with water, soluble powders dissolve readily and form a true solution. After they are mixed thoroughly, no additional agitation is necessary. The amount of active ingredient in soluble powders ranges from about 15 to about 95 percent by weight; in some cases more than about 50 percent. Soluble powders have all the advantages of wettable powders and none of the disadvantages, except the inhalation hazard during mixing.
[0247] In some instances, the composition is formulated as a water-dispersible granule. Water-dispersible granules, also known as dry flowables, are like wettable powders, except instead of being dust-like, they are formulated as small, easily measured granules. Water-dispersible granules must be mixed with water to be applied. Once in water, the granules break apart into fine particles similar to wettable powders. The formulation requires constant agitation to keep it suspended in water. The percentage of active ingredient is high, often as much as 90 percent by weight. Water-dispersible granules share many of the same advantages and disadvantages of wettable powders, except they are more easily measured and mixed. Because of low dust, they cause less inhalation hazard to the applicator during handling
[0248] v. Bait
[0249] In some instances, the composition includes a bait. The bait can be in any suitable form, such as a solid, paste, pellet or powdered form. The bait can also be carried away by the host back to a population of said host (e.g., a colony or hive). The bait can then act as a food source for other members of the colony, thus providing an effective modulating agent for a large number of hosts and potentially an entire host colony.
[0250] The baits can be provided in a suitable "housing" or "trap." Such housings and traps are commercially available and existing traps can be adapted to include the compositions described herein. The housing or trap can be box-shaped for example, and can be provided in pre-formed condition or can be formed of foldable cardboard for example. Suitable materials for a housing or trap include plastics and cardboard, particularly corrugated cardboard. The inside surfaces of the traps can be lined with a sticky substance in order to restrict movement of the host once inside the trap. The housing or trap can contain a suitable trough inside which can hold the bait in place. A trap is distinguished from a housing because the host cannot readily leave a trap following entry, whereas a housing acts as a "feeding station" which provides the host with a preferred environment in which they can feed and feel safe from predators.
[0251] vi. Attractants
[0252] In some instances, the composition includes an attractant (e.g., a chemoattractant). The attractant may attract an adult host or immature host (e.g., larva) to the vicinity of the composition. Attractants include pheromones, a chemical that is secreted by an animal, especially an insect, which influences the behavior or development of others of the same species. Other attractants include sugar and protein hydrolysate syrups, yeasts, and rotting meat. Attractants also can be combined with an active ingredient and sprayed onto foliage or other items in the treatment area.
[0253] Various attractants are known which influence host behavior as a host's search for food, oviposition or mating sites, or mates. Attractants useful in the methods and compositions described herein include, for example, eugenol, phenethyl propionate, ethyl dimethylisobutyl-cyclopropane carboxylate, propyl benszodioxancarboxylate, cis-7,8-epoxy-2-methyloctadecane, trans-8,trans-0-dodecadienol, cis-9-tetradecenal (with cis-11-hexadecenal), trans-11-tetradecenal, cis-11-hexadecenal, (Z)-11,12-hexadecadienal, cis-7-dodecenyl acetate, cis-8-dodecenyul acetate, cis-9-dodecenyl acetate, cis-9-tetradecenyl acetate, cis-11-tetradecenyl acetate, trans-11-tetradecenyl acetate (with cis-11), cis-9,trans-11-tetradecadienyl acetate (with cis-9,trans-12), cis-9,trans-1 2-tetradecadienyl acetate, cis-7,cis-11-hexadecadienyl acetate (with cis-7,trans-11), cis-3,cis-13-octadecadienyl acetate, trans-3,cis-13-octadecadienyl acetate, anethole and isoamyl salicylate.
[0254] Means other than chemoattractants may also be used to attract insects, including lights in various wavelengths or colors.
[0255] vii. Nanocapsules/Microencapsulation/Liposomes
[0256] In some instances, the composition is provided in a microencapsulated formulation. Microencapsulated formulations are mixed with water and sprayed in the same manner as other sprayable formulations. After spraying, the plastic coating breaks down and slowly releases the active ingredient.
[0257] viii. Carriers
[0258] Any of the compositions described herein may be formulated to include the modulating agent described herein and an inert carrier. Such carrier can be a solid carrier, a liquid carrier, a gel carrier, and/or a gaseous carrier. In certain instances, the carrier can be a seed coating. The seed coating is any non-naturally occurring formulation that adheres, in whole or part, to the surface of the seed. The formulation may further include an adjuvant or surfactant. The formulation can also include one or more modulating agents to enlarge the action spectrum.
[0259] A solid carrier used for formulation includes finely-divided powder or granules of clay (e.g. kaolin clay, diatomaceous earth, bentonite, Fubasami clay, acid clay, etc.), synthetic hydrated silicon oxide, talc, ceramics, other inorganic minerals (e.g., sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, etc.), a substance which can be sublimated and is in the solid form at room temperature (e.g., 2,4,6-triisopropyl-1,3,5-trioxane, naphthalene, p-dichlorobenzene, camphor, adamantan, etc.); wool; silk; cotton; hemp; pulp; synthetic resins (e.g., polyethylene resins such as low-density polyethylene, straight low-density polyethylene and high-density polyethylene; ethylene-vinyl ester copolymers such as ethylene-vinyl acetate copolymers; ethylene-methacrylic acid ester copolymers such as ethylene-methyl methacrylate copolymers and ethylene-ethyl methacrylate copolymers; ethylene-acrylic acid ester copolymers such as ethylene-methyl acrylate copolymers and ethylene-ethyl acrylate copolymers; ethylene-vinylcarboxylic acid copolymers such as ethylene-acrylic acid copolymers; ethylene-tetracyclododecene copolymers; polypropylene resins such as propylene homopolymers and propylene-ethylene copolymers; poly-4-methylpentene-1, polybutene-1, polybutadiene, polystyrene; acrylonitrile-styrene resins; styrene elastomers such as acrylonitrile-butadiene-styrene resins, styrene-conjugated diene block copolymers, and styrene-conjugated diene block copolymer hydrides; fluororesins; acrylic resins such as poly(methyl methacrylate); polyamide resins such as nylon 6 and nylon 66; polyester resins such as polyethylene terephthalate, polyethylene naphthalate, polybutylene terephthalate, and polycyclohexylenedimethylene terephthalate; polycarbonates, polyacetals, polyacrylsulfones, polyarylates, hydroxybenzoic acid polyesters, polyetherimides, polyester carbonates, polyphenylene ether resins, polyvinyl chloride, polyvinylidene chloride, polyurethane, and porous resins such as foamed polyurethane, foamed polypropylene, or foamed ethylene, etc.), glasses, metals, ceramics, fibers, cloths, knitted fabrics, sheets, papers, yarn, foam, porous substances, and multifilaments.
[0260] A liquid carrier may include, for example, aromatic or aliphatic hydrocarbons (e.g., xylene, toluene, alkylnaphthalene, phenylxylylethane, kerosine, gas oil, hexane, cyclohexane, etc.), halogenated hydrocarbons (e.g., chlorobenzene, dichloromethane, dichloroethane, trichloroethane, etc.), alcohols (e.g., methanol, ethanol, isopropyl alcohol, butanol, hexanol, benzyl alcohol, ethylene glycol, etc.), ethers (e.g., diethyl ether, ethylene glycol dimethyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol monomethyl ether, tetrahydrofuran, dioxane, etc.), esters (e.g., ethyl acetate, butyl acetate, etc.), ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, etc.), nitriles (e.g., acetonitrile, isobutyronitrile, etc.), sulfoxides (e.g., dimethyl sulfoxide, etc.), amides (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, cyclic imides (e.g. N-methylpyrrolidone) alkylidene carbonates (e.g., propylene carbonate, etc.), vegetable oil (e.g., soybean oil, cottonseed oil, etc.), vegetable essential oils (e.g., orange oil, hyssop oil, lemon oil, etc.), or water.
[0261] A gaseous carrier may include, for example, butane gas, flon gas, liquefied petroleum gas (LPG), dimethyl ether, and carbon dioxide gas.
[0262] ix. Adjuvants
[0263] In some instances, the composition provided herein may include an adjuvant. Adjuvants are chemicals that do not possess activity. Adjuvants are either pre-mixed in the formulation or added to the spray tank to improve mixing or application or to enhance performance. They are used extensively in products designed for foliar applications. Adjuvants can be used to customize the formulation to specific needs and compensate for local conditions. Adjuvants may be designed to perform specific functions, including wetting, spreading, sticking, reducing evaporation, reducing volatilization, buffering, emulsifying, dispersing, reducing spray drift, and reducing foaming. No single adjuvant can perform all these functions, but compatible adjuvants often can be combined to perform multiple functions simultaneously.
[0264] Among nonlimiting examples of adjuvants included in the formulation are binders, dispersants and stabilizers, specifically, for example, casein, gelatin, polysaccharides (e.g., starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonite, sugars, synthetic water-soluble polymers (e.g., polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, etc.), PAP (acidic isopropyl phosphate), BHT (2,6-di-t-butyl-4-methylphenol), BHA (a mixture of 2-t-butyl-4-methoxyphenol and 3-t-butyl-4-methoxyphenol), vegetable oils, mineral oils, fatty acids and fatty acid esters.
[0265] x. Surfactants
[0266] In some instances, the composition provided herein includes a surfactant. Surfactants, also called wetting agents and spreaders, physically alter the surface tension of a spray droplet. For a formulation to perform its function properly, a spray droplet must be able to wet the foliage and spread out evenly over a leaf. Surfactants enlarge the area of formulation coverage, thereby increasing the pest's exposure to the chemical. Surfactants are particularly important when applying a formulation to waxy or hairy leaves. Without proper wetting and spreading, spray droplets often run off or fail to cover leaf surfaces adequately. Too much surfactant, however, can cause excessive runoff and reduce efficacy.
[0267] Surfactants are classified by the way they ionize or split apart into electrically charged atoms or molecules called ions. A surfactant with a negative charge is anionic. One with a positive charge is cationic, and one with no electrical charge is nonionic. Formulation activity in the presence of a nonionic surfactant can be quite different from activity in the presence of a cationic or anionic surfactant. Selecting the wrong surfactant can reduce the efficacy of a pesticide product and injure the target plant. Anionic surfactants are most effective when used with contact pesticides (pesticides that control the pest by direct contact rather than being absorbed systemically). Cationic surfactants should never be used as stand-alone surfactants because they usually are phytotoxic.
[0268] Nonionic surfactants, often used with systemic pesticides, help pesticide sprays penetrate plant cuticles. Nonionic surfactants are compatible with most pesticides, and most EPA-registered pesticides that require a surfactant recommend a nonionic type. Adjuvants include, but are not limited to, stickers, extenders, plant penetrants, compatibility agents, buffers or pH modifiers, drift control additives, defoaming agents, and thickeners.
[0269] Among nonlimiting examples of surfactants included in the compositions described herein are alkyl sulfate ester salts, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and polyoxyethylenated products thereof, polyethylene glycol ethers, polyvalent alcohol esters and sugar alcohol derivatives.
[0270] xi. Combinations
[0271] In formulations and in the use forms prepared from these formulations, the modulating agent may be in a mixture with other active compounds, such as pesticidal agents (e.g., insecticides, sterilants, acaricides, nematicides, molluscicides, or fungicides; see, e.g., pesticides listed in Table 12), attractants, growth-regulating substances, or herbicides. As used herein, the term "pesticidal agent" refers to any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest. A pesticide can be a chemical substance or biological agent used against pests including insects, mollusks, pathogens, weeds, nematodes, and microbes that compete with humans for food, destroy property, spread disease, or are a nuisance. The term "pesticidal agent" may further encompass other bioactive molecules such as antibiotics, antivirals pesticides, antifungals, antihelminthics, nutrients, pollen, sucrose, and/or agents that stun or slow insect movement.
[0272] In instances where the modulating agent is applied to plants, a mixture with other known compounds, such as herbicides, fertilizers, growth regulators, safeners, semiochemicals, or else with agents for improving plant properties is also possible.
V. Delivery
[0273] A host described herein can be exposed to any of the compositions described herein in any suitable manner that permits delivering or administering the composition to the insect. The modulating agent may be delivered either alone or in combination with other active or inactive substances and may be applied by, for example, spraying, microinjection, through plants, pouring, dipping, in the form of concentrated liquids, gels, solutions, suspensions, sprays, powders, pellets, briquettes, bricks and the like, formulated to deliver an effective concentration of the modulating agent.
[0274] Amounts and locations for application of the compositions described herein are generally determined by the habits of the host, the lifecycle stage at which the microorganisms of the host can be targeted by the modulating agent, the site where the application is to be made, and the physical and functional characteristics of the modulating agent. The modulating agents described herein may be administered to the insect by oral ingestion, but may also be administered by means which permit penetration through the cuticle or penetration of the insect respiratory system.
[0275] In some instances, the insect can be simply "soaked" or "sprayed" with a solution including the modulating agent. Alternatively, the modulating agent can be linked to a food component (e.g., comestible) of the insect for ease of delivery and/or in order to increase uptake of the modulating agent by the insect. Methods for oral introduction include, for example, directly mixing a modulating agent with the insect's food, spraying the modulating agent in the insect's habitat or field, as well as engineered approaches in which a species that is used as food is engineered to express a modulating agent, then fed to the insect to be affected. In some instances, for example, the modulating agent composition can be incorporated into, or overlaid on the top of, the insect's diet. For example, the modulating agent composition can be sprayed onto a field of crops which an insect inhabits.
[0276] In some instances, the composition is sprayed directly onto a plant e.g., crops, by e.g., backpack spraying, aerial spraying, crop spraying/dusting etc. In instances where the modulating agent is delivered to a plant, the plant receiving the modulating agent may be at any stage of plant growth. For example, formulated modulating agents can be applied as a seed-coating or root treatment in early stages of plant growth or as a total plant treatment at later stages of the crop cycle. In some instances, the modulating agent may be applied as a topical agent to a plant, such that the host insect ingests or otherwise comes in contact with the plant upon interacting with the plant.
[0277] Further, the modulating agent may be applied (e.g., in the soil in which a plant grows, or in the water that is used to water the plant) as a systemic agent that is absorbed and distributed through the tissues (e.g., stems or leafs) of a plant or animal host, such that an insect feeding thereon will obtain an effective dose of the modulating agent. In some instances, plants or food organisms may be genetically transformed to express the modulating agent such that a host feeding upon the plant or food organism will ingest the modulating agent.
[0278] Delayed or continuous release can also be accomplished by coating the modulating agent or a composition with the modulating agent(s) with a dissolvable or bioerodible coating layer, such as gelatin, which coating dissolves or erodes in the environment of use, to then make the modulating agent available, or by dispersing the agent in a dissolvable or erodible matrix. Such continuous release and/or dispensing means devices may be advantageously employed to consistently maintain an effective concentration of one or more of the modulating agents described herein in a specific host habitat.
[0279] The modulating agent can also be incorporated into the medium in which the insect grows, lives, reproduces, feeds, or infests. For example, a modulating agent can be incorporated into a food container, feeding station, protective wrapping, or a hive. For some applications the modulating agent may be bound to a solid support for application in powder form or in a "trap" or "feeding station." As an example, for applications where the composition is to be used in a trap or as bait for a particular host insect, the compositions may also be bound to a solid support or encapsulated in a time-release material. For example, in instances where the host is a honeybee, the compositions described herein can be administered by delivering the composition to a honeybee hive or at least one habitat where a honeybee grows, lives, reproduces, or feeds.
VI. Screening
[0280] Included herein are screening assays for identifying a modulating agent, wherein the modulating agent is effective to alter the microbiota of a host and thereby increase host fitness (e.g., insect or nematode fitness). For example, the screening assay may be used to identify one or more modulating agents that target specific microorganisms and/or specific hosts. Further, the screening assays may be used to identify one or more microorganisms with enhanced functionalities. For example, the screening assay may be effective to isolate one or more microorganisms with an enhanced ability to metabolize (e.g., degrade) a pesticide (e.g., a pesticide listed in Table 12 or an insecticide known in the art, e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion)) or plant allelochemical (e.g., caffeine, soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds). Delivery and colonization of an isolated microorganism in the host may increase the host's resistance to the pesticide (e.g., a pesticide listed in Table 12) or plant allelochemical, thereby increasing host fitness. The methods may also be useful for the isolation of microorganisms with an enhanced ability to colonize any of the hosts described herein.
[0281] For example, to screen for microorganisms that increase a host's resistance to a pesticide (e.g., a pesticide listed in Table 12), a starting culture may be used that includes microorganisms (e.g., bacteria) and high concentrations of a pesticide (e.g., a pesticide listed in Table 12 or an insecticide known in the art, e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion)). In some instances, the pesticide may be provided in the form of an environmental sample enriched with the pesticide (e.g., a soil sample). Alternatively, the pesticide (e.g., a pesticide listed in Table 12) may be provided in pure form or in combination with other carriers. Further, the one or more microorganism isolates may be inoculated directly into the media (e.g., from a laboratory strain) or may be an environmental sample including one or more microorganism species. The growth media may be either liquid or solid. In some instances, the pesticide of interest is the sole carbon or nitrogen source for the microorganisms in the media. The culture may be sub-cultured (e.g., inoculated into fresh media with high levels of the pesticide) any number of times to enrich for and/or isolate microbial strains (e.g., bacterial strains) capable of metabolizing the pesticide. The original culture or the subcultures may be assessed using any methods known in the art to test for alterations (e.g., decrease) in the levels of the pesticide in the sample (e.g., using HPLC). Isolates that reduce the concentration of the pesticide (e.g., a pesticide listed in Table 12 or a pesticide known in the art, e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion)) may be isolated for use as a modulating agent in any of the methods or compositions described herein.
[0282] The methods may be used to further select for microorganisms described herein, including those isolated from a screening assay, with an enhanced ability to colonize and survive in a host (e.g., insect, e.g., bee). For example, a host may be inoculated with a bacterial isolate (e.g., one with the ability to degrade a pesticide). The host may then be tested at regular intervals for the presence of the bacterial isolate (e.g., via culturing or 16s RNA from guts isolated from the host (e.g., honeybee)). Bacterial isolates that survive in the host (e.g., the midgut of the honeybee) may be isolated for use as a modulating agent in any of the methods or compositions described herein.
TABLE-US-00012 TABLE 12 Pesticides Aclonifen Acetamiprid Alanycarb Amidosulfuron Aminocyclopyrachlor Amisulbrom Anthraquinone Asulam, sodium salt Benfuracarb Bensulide beta-HCH; beta-BCH Bioresmethrin Blasticidin-S Borax; disodium tetraborate Boric acid Bromoxynil heptanoate Bromoxynil octanoate Carbosulfan Chlorantraniliprole Chlordimeform Chlorfluazuron Chlorphropham Climbazole Clopyralid Copper (II) hydroxide Cyflufenamid Cyhalothrin Cyhalothrin, gamma Decahydrate Diafenthiuron Dimefuron Dimoxystrobin Dinotefuran Diquat dichloride Dithianon E-Phosphamidon EPTC Ethaboxam Ethirimol Fenchlorazole-ethyl Fenothiocarb Fenitrothion Fenpropidin Fluazolate Flufenoxuron Flumetralin Fluxapyroxad Fuberidazole Glufosinate-ammonium Glyphosate Group: Borax, borate salts (see Group: Paraffin oils, Mineral Halfenprox Imiprothrin Imidacloprid Ipconazole Isopyrazam Isopyrazam Lenacil Magnesium phosphide Metaflumizone Metazachlor Metazachlor Metobromuron Metoxuron Metsulfuron-methyl Milbemectin Naled Napropamide Nicosulfuron Nitenpyram Nitrobenzene o-phenylphenol Oils Oxadiargyl Oxycarboxin Paraffin oil Penconazole Pendimethalin Penflufen Penflufen Pentachlorbenzene Penthiopyrad Penthiopyrad Pirimiphos-methyl Prallethrin Profenofos Proquinazid Prothiofos Pyraclofos Pyrazachlor Pyrazophos Pyridaben Pyridalyl Pyridiphenthion Pyrifenox Quinmerac Rotenone Sedaxane Sedaxane Silafluofen Sintofen Spinetoram Sulfoxaflor Temephos Thiocloprid Thiamethoxam Tolfenpyrad Tralomethrin Tributyltin compounds Tridiphane Triflumizole Validamycin Zinc phosphide
EXAMPLES
[0283] The following is an example of the methods of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.
Example 1: Isolation of Microorganisms that Degrade Imidacloprid, a Nicotinoid
[0284] This example demonstrates the acquisition of a library of microorganisms able to degrade imidacloprid, a neonicotinoid.
[0285] Experimental Design:
[0286] Four soil samples with elevated concentrations of imidacloprid are collected as described in Bonmatin et al. (Environ. Sci. Pollut. Res. Int. 22:35-67, 2015) and diluted in Kaufman and Kearney's minimal salts media (MSM) to enrich for imidacloprid-degrading microorganisms. Three variations of cultures for each soil sample include: 50 mL carbon-limited MSM with 83 mg/L imidacloprid as the sole carbon source; 50 mL nitrogen-limited MSM with imidacloprid as the sole nitrogen source and sodium citrate and sucrose added as supplemental carbon sources; and 50 mL MSM broth, with all components plus sodium citrate, sucrose, and imidacloprid. Each culture is inoculated with 2 g of the collected soil.
[0287] Cultures are maintained on a shaker at 100 rpm and 27.degree. C. Subcultures (1 mL of the cultured solution diluted in 25 mL of new medium) are made monthly in order to obtain soil-free enrichments. Aliquots of the cultures are periodically removed from the shaker flasks for HPLC analysis as described in Tago et al. (Microbes Environ. 21:58-64, 2006). For the HPLC assay, .apprxeq.7.0.times.10.sup.7 microorganisms are incubated in 1.0 mL of 20 mM sodium-potassium phosphate buffer (pH 7.0) with 1.0 mM imidacloprid. After 20 min of incubation at 30.degree. C., 1.0 mL of methanol is added. Imidacloprid and products of its partial degradation are quantitatively analyzed on a HPLC system (a 600E pump and 2487 dual absorbance detector; Waters). Retention times and peak areas of the HPLC profiles are compared with those of known standards Bonmatin et al. (Environ. Sci. Pollut. Res. Int. 22:35-67, 2015).
[0288] Mixed enrichment cultures from the soil that show a decrease of imidacloprid via HPLC analysis during incubation are spread-plated using dilutions of 10.sup.-1 to 10.sup.-6 on nitrogen-limited agar plates with 36 mg/kg imidacloprid and streptomycin.
[0289] After a one-week incubation at 27.degree. C., the plates are screened for colonies that morphologically appear different from one another (size differences). Individual colonies are grown into new agar plates with imidacloprid as the sole nitrogen source. After three weeks, single colonies are put into 25 mL of tryptic soy broth (TSB) with 25 mg/L imidacloprid.
[0290] After three days' growth, each of the isolates is centrifuged in conical Falcon tubes for 10 min at 6700.times.g. The supernatant is removed, and the isolates are re-suspended in sterile phosphate buffer for a total volume of 10 mL. A 2-mL sample of each isolate is inoculated into a nitrogen-limited MSM and a carbon-limited MSM cultures; both cultures with 30 mg/L of imidacloprid in 25 mL total. Non-inoculated controls are also made by inoculating 2 mL of phosphate buffer into each of the media types. All samples are wrapped in aluminum foil and placed on a shaker operated at 27.degree. C. and 100 rpm. The samples are then analyzed for imidacloprid concentration by HPLC.
[0291] After seven days of incubation, 25 mL of methanol is added to the flasks. The samples are then sonicated for six minutes each at 50% duty cycle and centrifuged at 6700.times.g. The supernatant is then analyzed and the imidacloprid is quantitatively measured by HPLC as described herein.
[0292] Isolates that reduce the concentration of imidacloprid are stored at -80 C in a solution of 50% glycerol.
Example 2: Selection of Imidacloprid Degrading Isolates that Survive in Bees' Midgut
[0293] This example demonstrates the ability to select for an imidacloprid-degrading microorganism from isolates described in Example 1 that are able to survive in the bee midgut.
[0294] Therapeutic design: Ten isolates described in Example 1 are formulated with 0 (negative control), 10.sup.2, 10.sup.5, or 10.sup.8 cfu/ml in 10 mL of 50% sucrose syrup.
[0295] Experimental Design:
[0296] About fifty bees per bacterial isolate are collected and kept in a wooden cage. Bacterial strains isolated in the previous example are cultivated and prepared for feeding as followed. Cell from each isolate are washed and diluted to an OD600 at 0.1 in MSM supplemented with 50% (w/v) sucrose syrup and incubated at 30.degree. C. for cell growth. The four concentrations of fresh bacterial cultures are administered to bees as feed (between 0-10.sup.8 cfu/ml) as follows. The different concentrations of isolates are placed in a 15 ml tube with six small pinched holes in which the bees project their proboscis and consume the isolate-containing solutions. Fresh isolate-containing cultures are prepared daily for use as feed and mortality of bees is scored every day during the entire course (7 days) of the experiment by counting and removing dead bees. After 7 days, the isolate-containing solution is replaced with 15% (w/v) sucrose for 48 h.
[0297] Ten bees are removed from each replicate every 24 hours for bacterial analysis; the first sample is removed before experiment onset, followed by seven additional sampling points. After each sampling, the bees are anaesthetized on ice and the midgut is removed. Replicates for each time point are pooled and homogenized in 150 .mu.l 1.times.PBS, then serially diluted and plated on nitrogen-limited agar plates with 36 mg/kg imidacloprid to grow out bacteria under selective growth conditions at 30.degree. C. for 2 days.
[0298] The bacterial isolates that survive in the bee midgut are selected for administration to increase bees' resistance to neonicotinoids.
Example 3: Increase Bees' Resistance to Neonicotinoids Through the Administration of Imidacloprid-Degrading Bacteria
[0299] This example demonstrates the ability to increase the resistance of bees to insecticides, and more specifically to neonicotinoids. Bees play an important role in pollinating flowering plants, and are the major type of pollinator in many ecosystems that contain flowering plants. It is estimated that one third of the human food supply depends on pollination by insects, birds and bats, most of which is accomplished by bees, especially the domesticated European honey bee. The number of colonies kept by beekeepers has declined, through urbanization, systematic pesticide use, and tracheal and Varroa mites. Through direct consumption of contaminated nectar and pollen from insecticide treated plants, neonicotinoids in the insecticides affect foraging, learning, and memory in worker bees. By introducing imidacloprid degrading bacteria into their microbiome, bees are expected to increase their resistance to neonicotinoids through improved behavior, such as increased foraging, hygienic behavior, and survival.
[0300] Therapeutic design: The bacterial isolate selected in Example 2 and identified as surviving in bees' midgut is formulated at 10.sup.8 organisms in 10 mL of 50% sucrose syrup.
[0301] Experimental Design:
[0302] The isolate selected in Example 2 is prepared at a concentration of 10.sup.8 cells/mL (see Example 2) and placed in a 15 ml tube with six small pinched holes in which the bees project their proboscis and consume the isolate-containing solution. Fresh isolate-containing cultures are prepared daily for use as feed. The pretreatment lasts for seven days in total.
[0303] Approximately 1500, 3000, or 7000 worker hives estimated by weight of worker bees, Apis mellifera, (0.5, 1, and 2 lbs., respectively, or 0.23, 0.45, and 0.1 kg) are pretreated with the isolate selected in Example 2 prepared at a concentration of 10.sup.8 cells/mL and placed in a 15 ml tube with six small pinched holes in which the bees project their proboscis and consume the isolate-containing solution. Fresh isolate-containing cultures are prepared daily for use as feed. The pretreatment lasts for seven days in total.
[0304] After the 7 day pretreatment, each worker hive is fed 2 to 4 grams of pollen supplement and sugar syrup (1:1) for 1-2 days before imidacloprid treatment. Smaller colonies (1500- and 3000-bees) are placed in glass-walled observation hives. The larger colonies (7000-bees) are placed in Ulster observation hives (Brushy Mountain Bee Supply, NC) containing a bottom box holding 4 standard frames and a division board feeder where treatment syrup is provided. All observation hives are housed in sheds maintained at constant temperature and relative humidity (23-25.degree. C. and 70%). Additionally, all hives contain an entrance leading to the outside allowing bees to freely forage in surrounding agricultural fields and urban residential neighborhoods.
[0305] When egg laying is visually confirmed, each colony is randomly assigned an imidacloprid treatment (0, 10, 20, 50, and 100 ppm) provided in 50% sucrose syrup. Colonies are given through a dispenser system similar to the one described in Example 2 with proportional amounts of sucrose solution with imidacloprid: 80, 160 and 320 mL for 1500-, 3000-, and 7000-bee colonies, respectively. Syrup is replenished every other day for 3 weeks. Syrup quantities are designed to supplement, but not sustain, the colonies, so bees are required to freely forage on other resources. Stock solutions of imidacloprid (100 ppm) are prepared using 99.5.+-.0.5% technical grade imidacloprid purchased from Chem Service, Inc (PS-2086) dissolved with agitation in 50% sucrose overnight. Stock solutions are prepared every two weeks and treatment solutions are prepared every week. Samples of treatment solutions (3-6 per dose) are randomly selected and tested for residue concentrations to confirm accuracy of dosage.
[0306] Measurements of Behavioral Phenotypes:
[0307] Foraging activity is measured by recording the number of workers entering and exiting the entrance of the bee colony during one-minute observations twice a day. Hygienic behavior is used as a measure to assess worker activity inside the hive, and is defined as the ability of worker bees to detect and remove diseased and mite-infested brood thereby limiting within-colony transmission of pathogens and parasites. Hygienic behavior is measured using a freeze-killed brood assay, in which a 3-inch (7.6 cm) polyvinyl chloride tube is gently pushed into a section of comb containing a large area of sealed pupal cells (taken from non-experimental field colonies). The number of empty cells is counted and recorded before pouring 400 ml of liquid nitrogen to freeze-kill roughly 160 pupae. The frame is then temporarily put into the bee colony and the proportion of pupae completely or partially removed from the cells is quantified after 24 hours to assess hygienic behavior. The removal of freeze-killed brood is correlated with the removal of diseased and parasitized brood.
[0308] Bees pretreated with the bacterial isolate selected in Example 2 demonstrate higher activity than bees treated with the control.
Example 4: Treatment of Varroa Mites that Infect Bees with Oxytetracycline Solutions
[0309] This example demonstrates the ability to kill, decrease the fitness of Varroa mites by treating them with an antibiotic solution. This example demonstrates that the effect of oxytetracycline on Varroa mites is mediated through the alteration of bacterial populations endogenous, such as Bacillus, to the Varroa mites that are sensitive to oxytetracycline.
[0310] Varroa mites are thought to be a leading cause for the wide spread Colony Collapse Disorder (CCD) that decimates domesticated honey bee colonies of Apis mellifera, around the world. They attach to the bees' abdomen and suck on their blood, depriving them of nutrients, and eventually killing them. While Varroa mites can be killed with chemically synthesized miticides, these types of chemicals must be kept away from comestible honey.
[0311] Therapeutic design: Oxytetracycline solution is formulated with 0 (negative control), 1, 10, or 50 .mu.g/ml in 10 mL of sterile water with 0.5% sucrose and essential amino acids.
[0312] Experimental Design:
[0313] To determine whether adult Varroa mites at the reproductive stage have different susceptibility compared to phoretic mites or their offspring because their cuticle is not hardened, Varroa mites living on adult bees, Apis mellifera, and mites associated with larvae and pupae are collected. This assay tests antibiotic solutions on different types of mites and determines how their fitness is altered by targeting endogenous microbes, such as Bacillus.
[0314] The brood mites are collected from combs (or pieces of combs) of Varroa mite-infested bee colonies. The mites are collected from cells where bee larvae develop.
[0315] Varroa mites are grouped per age of their brood host and assayed separately. The age of the brood is determined based on the morphology and pigmentation of the larva or the pupa as follows: Varroa mites collected from spinning larvae that are small enough to move around their cell are grouped into Group 1; Varroa mites collected from stretched larvae, which are too big to lay in the cell and start to stretch upright with their mouth in the direction of the cell opening, are grouped into Group 2; and Varroa mites collected from pupae are grouped into Group 3. Mites are stored on their host larva or pupa in glass Petri dishes until 50 units are collected. This ensures their feeding routine and physiological status remains unchanged. To prevent mites from straying from their host larva or pupa or climbing onto one another, only hosts at the same development stage are kept in the same dish.
[0316] The equipment--a stainless steel ring (56 mm inner diameter, 2-3 mm height) and 2 glass circles (62 mm diameter)--is cleaned with acetone and hexane or pentane to form the testing arena. The oxytetracycline solutions and control solution are applied on the equipment by spraying the glass disks and ring of the arena homogeneously. For this, a reservoir is loaded with 1 ml of the solutions; the distance of the sprayed surface from the bottom end of the tube is set at 11 mm and a 0.0275 inch nozzle is used. The pressure is adjusted (usually in the range 350-500 hPa) until the amount of solution deposited is 1.+-.0.05 mg/cm.sup.2. The antibiotic solutions are poured in their respective dishes, covering the whole bottom of the dishes, and residual liquid is evaporated under a fume hood. The ring is placed between the glass circles to build a cage. The cages are used within 60 hr of preparation, for not more than three assays, in order to control the exposure of mites to antibiotic solutions. 10 to 15 Varroa mites are introduced in this cage and the equipment pieces are bound together with droplets of melted wax. Mites collected from spinning larvae, stretched larvae, white eyed pupae and dark eyed with white and pale body are used.
[0317] After 4 hours, mites are transferred into a clean glass Petri dish (60 mm diameter) with two or three white eye pupae (4-5 days after capping) to feed on. The mites are observed under a dissecting microscope at 4 hr, 24 hr, and 48 hr after being treated with the antibiotic or the control solutions, and classified according to the following categories:
[0318] Mobile: they walk around when on their legs, whether after being poked by a needle.
[0319] Paralyzed: they move one or more appendages, unstimulated or after stimulation, but they cannot move around.
[0320] Dead: immobile and do not react to 3 subsequent stimulations.
[0321] A sterile toothpick or needle is used to stimulate the mites by touching their legs. New tooth picks or sterile needles are used for stimulating each group to avoid contamination between mite groups.
[0322] The assays are carried out at 32.5.degree. C. and 60-70% relative humidity. If the mortality in the controls exceeds 30%, the replicate is excluded. Each experiment is replicated with four series of cages.
[0323] The status of Bacillus in Varroa mite groups is assessed by PCR. Total DNA is isolated from control (non-oxytetracycline treated) and oxytetracyline treated individuals (whole body) using a DNA Kit (OMEGA, Bio-tek) according to the manufacturer's protocol. The primers for Bacillus, forward primer 5'-GAGGTAGACGAAGCGACCTG-3' (SEQ ID NO: 186) and reverse primer 5'-TTCCCTCACGGTACTGGTTC-3' (SEQ ID NO: 187), are designed based on 23S-5S rRNA sequences obtained from the Bacillus genome (Accession Number: AP007209.1) (Takeno et al., J. Bacteriol. 194(17):4767-4768, 2012) using Primer 5.0 software (Primer-E Ltd., Plymouth, UK). The PCR amplification cycles included an initial denaturation step at 95.degree. C. for 5 min, 35 cycles of 95.degree. C. for 1 min, 59.degree. C. for 1 min, and 72.degree. C. for 2 min, and a final extension step of 5 min at 72.degree. C. Amplification products from oxytetracyline treated and control samples are analyzed on 1% agarose gels, stained with SYBR safe, and visualized using an imaging System.
[0324] The survival rates of Varroa mites treated with an oxytetracyline solution are compared to the Varroa mites treated with the negative control.
[0325] The survival rate and the mobility of Varroa mites treated with oxytetracyline solution are decreased compared to the control.
Example 5: Isolation of Microorganisms that Degrade Fenitrothion, an Organophosphorus Insecticide
[0326] This Example demonstrates the acquisition of a library of microorganisms able to degrade fenitrothion, an organophosphorus insecticide.
[0327] Experimental Design
[0328] Soil samples are obtained from various regions where fenitrothion was previously utilized for insect control. Fenitrothion degrading bacteria will be isolated from the soil samples as described in (Microbes Environ. Vol. 21, No. 1, 58-64, 2006). Briefly, 1 g of the soil sample is mixed thoroughly with 9 ml of sterile distilled water. The soil particles are then centrifuged at 1000 rcf for 5 min, and 100 .mu.l of the supernatant is then plated onto fenitrothion with mineral salts medium (0.4 g/l of yeast extract, 0.4 g/l fenitrothion, 15 g/l bacteriological agar). The plates are cloudy when prepared as the fenitrothion is an emulsion.
[0329] Colonies that develop clear zones around them and are likely to be degrading or metabolizing fenitrothion, and these colonies are isolated and regrown on LB agar, nutrient agar, yeast glucose agar, TSA agar, BHI agar, or MRS agar. Once unique bacterial colonies are identified, their genomes are extracted using a genomic DNA extraction kit, (Qiagen, DNeasy Blood and Tissue Kit) as per the manufacturer's protocol.
[0330] The 16S rRNA gene is amplified using universal bacterial primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3'; SEQ ID NO: 188) and 1492R (5'-TACCTTGTTACGACTT-3'; SEQ ID NO: 189), and using PCR conditions of 94.degree. C. for 2 min, 30 cycles of 94.degree. C. for 1 min, 56.degree. C. for 1 min, and 72.degree. C. for 2 min, and a final extension of 72.degree. C. for 5 min. Gel electrophoresis is used to confirm that the amplicons are of the correct size (.about.1500 bp), and the products are excised from the gel and purified using a gel extraction kit (Qiagen, QIAquick) as per the manufacturer's protocol. The correct size amplicons are Sanger sequenced and BLAST is used to match the sequence against various repositories of 16s rRNA gene sequences to identify the bacteria.
[0331] To determine whether the isolated bacteria is degrading fenitrothion, .about.10.sup.7 bacterial cells are incubated in 1 ml of 20 mM sodium-potassium phosphate buffer (pH 7) with 1 mM fenitrothion, as described in PNAS, Vol. 109, No. 22, 8618-8622, 2012. After 30 min of incubation at 30.degree. C., the reaction is stopped by adding an equal volume of methanol. Then fenitrothion and its metabolite, 3-methyl-4-nitrophenol, are analyzed by HPLC. The retention times and peak areas of the HPLC profiles are compared to known standards.
[0332] Unique bacterial isolates that have fenitrothion degrading capabilities are then stored as frozen glycerol at -80.degree. C.
Example 6: Increasing Drosophila melanogaster's Resistance to Fenitrothion Through the Administration of Fenitrothion-Degrading Bacteria
[0333] This Example demonstrates the ability to protect an insect model, Drosophila melanogaster, from one or more negative effects of insecticides in their diet, more specifically fenitrothion. The following approach is a surrogate for other insects, such as bees or other insects disclosed herein, e.g., insects that are important pollinators for many flowering plant crops and vegetables. Many insecticides including fenitrothion have been shown to be toxic to bees.
[0334] Experimental Design:
[0335] Therapeutic design: The bacterial isolates selected in Example 5 are formulated at 10.sup.9 organisms in 100 .mu.l of fly food medium with and without fenitrothion.
[0336] The media used to rear flies is cornmeal, molasses and yeast medium (11 g/l yeast, 54 g/l yellow cornmeal, 5 g/l agar, 66 ml/molasses, and 4.8 ml/propionic acid). For experimental procedures, fenitrothion at 0, 10, and 100 p.p.m. or phosphate-buffered saline as negative controls are infused into sterile fly food medium.
[0337] Development Rate Assay
[0338] On day one, 10.sup.9 fenitrothion-degrading bacteria described in Example 5 are suspended in 100 .mu.l phosphate-buffered saline or equal volumes of saline (negative controls) are added to sterile fly food medium with or without fenitrothion. All are left to dry for a day at 25.degree. C. as described in Appl. Environ. Microbiol. Vol. 82, No. 20, 6204-6213, 2016.
[0339] On day two, fertilized embryos collected from flies are treated with 2% hypochlorite solution for 5 min and then washed with sterile water to remove any extracellular microbes from the embryos. 10 .mu.l of the embryo suspension in water (1:3 embryo:water suspension) is added to the bacteria-seeded or negative control fly food with or without the fenitrothion. The fly food cohorts with the embryos are maintained at 25.degree. C. with 12 h light and 12 dark cycle for the rest of the experiment.
[0340] The time to puparium formation and the number of pupa formed is measured in each cohort. The time to adult emergence and the rate of emergence is measured in each sample. From the time the first adult emerges from the pupa, the number of emerging adult flies are counted every 12 hours and rate of emergence is computed.
[0341] Embryos in the fenitrothion infused fly food seeded with Fenitrothion-degrading bacteria are expected to develop faster than the embryos on fenitrothion infused food without the fenitrothion-degrading bacteria.
[0342] Survival Assay
[0343] About 12 days before day one, sterile embryos are generated as described previously and raised on sterile fly food. Adults start to emerge from their pupae 11 days from embryo collection when raised in sterile fly food without fenitrothion at 25.degree. C. with 12 h light and 12 h dark cycle.
On day one, 10.sup.9 of the fenitrothion-degrading bacteria in phosphate-buffered saline are added to sterile fly food medium as described in a previous Example.
[0344] On day two, 10 newly emerged sterile adult males and females are introduced to bacteria-seeded or negative control fly food with or without fenitrothion. The fly food with the flies is maintained at 25.degree. C. with 12 h light and 12 dark cycle for the rest of the experiment. The number of surviving male and female flies are counted every day until all the flies are dead. Survival analysis is performed to assess the fitness benefit of fenitrothion-degrading bacteria on the fly survival.
[0345] Flies raised on fenitrothion infused fly food seeded with fenitrothion-degrading bacteria are expected to survive longer than flies raised on fenitrothion infused food without the fenitrothion-degrading bacteria.
Example 7: Elimination of Entomopathogenic Bacteria from Drosophila melanogaster Using Naturally Occurring Phages
[0346] This Example demonstrates the ability to eliminate insect bacterial pathogens (such as Serratia marcescens, Erwinia carotovora, and Pseudomonas entomophila) from Drosophila melanogaster using naturally occurring phages. This procedure can be useful as a surrogate assay for eliminating bacteria in other insect species, such as in bees.
[0347] Experimental Design:
[0348] Therapeutic design: Phage library collections are used having the following phage families: Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, Tectiviridae.
[0349] Multiple environmental samples (soil, pond sediments, and sewage water) are collected in sterile 1 L flasks over a period of 2 weeks and are immediately processed after collection and stored thereafter at 4.degree. C. Solid samples are homogenized in sterile double-strength difco luria broth (LB) or tryptic soy broth (TSB) supplemented with 2 mM CaCl2 to a final volume of 100 mL. The pH and phosphate levels are measured using phosphate test strips. For purification, all samples are centrifuged at 3000-6000 g for 10-15 min at 4.degree. C., and filtered through 0.2-.mu.m low protein filters to remove all remaining bacterial cells. The supernatant that contains the phage library is then stored at 4.degree. C. in the presence of chloroform in a glass bottle.
[0350] 20-30 ml of the phage library is diluted to a volume of 30-40 ml with LB-broth. The target bacterial strain (e.g., Serratia marcescens, Erwinia carotovora, and Pseudomonas entomophila) is added (50-200 .mu.l overnight culture grown in LB-broth) to enrich phages that target this specific bacterial strain in the culture. This culture is incubated overnight at 37.degree. C., shaken at 230 rpm. Bacteria from this enrichment culture are removed by centrifugation (3000-6000 g 15-20 min, 4.degree. C.) and filtered (0.2 or 0.45 .mu.m filter). 2.5 ml of the bacteria free culture is added to 2.5 ml of LB-broth and 50-100 .mu.l of the target bacteria are added back to the culture to further enrich the phages. The enrichment culture is grown overnight as above. A sample from this enrichment culture is centrifuged at 13,000 g for 15 min at room temperature and 10 .mu.l of the supernatant is plated on an LB-agar petri dish along with 100-300 .mu.l of the target bacteria and 3 ml of melted 0.7% soft-agar. The plates are incubated overnight at 37.degree. C.
[0351] Each of the plaques observed on the bacterial lawn are picked and transferred into 500 .mu.l of LB-broth. A sample from this plaque-stock is further plated on the target bacteria. Plaque-purification is performed three times for all discovered phages in order to isolate a single homogenous phage from the heterogeneous phage mix.
[0352] Lysates from plates with high-titer phages (>1.times.10{circumflex over ( )}10 PFU/ml) are prepared by harvesting overlay plates of a host bacterium strain exhibiting confluent lysis. After being flooded with 5 ml of buffer, the soft agar overlay is macerated, clarified by centrifugation, and filter sterilized. The resulting lysates are stored at 4.degree. C. High-titer phage lysates are further purified by isopycnic CsCl centrifugation, as described in Summer et al., J. Bacteriol. 192:179-190, 2010.
[0353] DNA is isolated from CsCl-purified phage suspensions as described in Summer, Methods Mol. Biol. 502:27-46, 2009. An individual isolated phage is sequenced as part of two pools of phage genomes by using a 454 pyrosequencing method. Briefly, phage genomic DNA is mixed in equimolar amounts to a final concentration of about 100 ng/L. The pooled DNA is sheared, ligated with a multiplex identifier (MID) tag specific for each of the pools, and sequenced by pyrosequencing using a full-plate reaction on a sequencer (Roche) according to the manufacturer's protocols. The pooled phage DNA is present in two sequencing reactions. The output corresponding to each of the pools is assembled individually by using software (454 Life Sciences), by adjusting the settings to include only reads with a single MID per assembly. The identity of individual contigs is determined by PCR using primers generated against contig sequences and individual phage genomic DNA preparations as the template. Sequence software (Gene Codes Corporation) is used for sequence assembly and editing.
[0354] Phage chromosomal end structures are determined experimentally. Cohesive (cos) ends for phages are determined by sequencing off the ends of the phage genome and sequencing the PCR products derived by amplification through the ligated junction of circularized genomic DNA, as described in Summer, Methods Mol. Biol. 502:27-46, 2009. Protein-coding regions are initially predicted using gene prediction software (Lukashin et al. Nucleic Acids Res. 26:1107-1115, 1998), refined through manual analysis in Artemis (Rutherford et al., Bioinformatics 16:944-945, 2000), and analyzed through the use of BLAST (E value cutoff of 0.005) (Camacho et al., BMC Bioinformatics 10:421, 2009). Proteins of particular interest are additionally analyzed by sequence searching software (Hunter et al., Nucleic Acids Res. 40:D306-D312, 2012).
[0355] Electron microscopy of CsCl-purified phage (>1.times.10{circumflex over ( )}11 PFU/ml) that lysed the Drosophila's pathogenic bacterial species is performed by diluting phage stock with the tryptic soy broth buffer. Phages are applied onto thin 400-mesh carbon-coated grids, stained with 2% (wt/vol) uranyl acetate, and air dried. Specimens are observed on a transmission electron microscope operating at an acceleration voltage of 100 kV. Five virions of each phage are measured to calculate mean values and standard deviations for dimensions of capsid and tail, where appropriate.
[0356] Incorporating Phages into a Meal
[0357] The media used to rear flies is cornmeal, molasses and yeast medium (11 g/l yeast, 54 g/l yellow cornmeal, 5 g/l agar, 66 ml/l molasses, and 4.8 ml/propionic acid). Phage solutions are infused into the fly food to obtain final concentrations of phages between 0 and 10.sup.8 pfu/ml.
[0358] S. Marcescens, Erwinia carotovora, and Pseudomonas entomphila bacteria are grown at 30.degree. C. in nutrient broth or LB broth.
[0359] Sterile fly embryos are generated by treating fertilized embryos collected from flies with 2% hypochlorite solution for 5 min and then washed with sterile water to remove any extracellular microbes. Fly larvae with the target bacteria are generated by seeding 10.sup.9 CFUs of bacteria in sterile fly food and adding sterile fly embryos to this food. After 2 days, ten S. marcescens infected first instar fly larvae are added to the fly food with a range (0-10.sup.8 pfu/ml) of the phage concentrations. The larvae are left to grow in the food with the phages for 3 days until they become third instars. The larvae are then collected and individually homogenized in nutrient broth or LB broth, and plated on nutrient agar or LB agar plates, and incubated at 30.degree. C. The number of CFUs of S. marcescens obtained from larvae in fly food with varying phage concentrations are recorded. This shows the number of live bacteria that were present in the flies.
[0360] The number of live bacteria are expected to be reduced in the larvae grown on fly food with the phages against the bacteria.
Example 8: Generation of a Library of Natural Microbes
[0361] This Example demonstrates the isolation of bacteria from soil that naturally produce the amino acid, methionine.
[0362] The medium used for isolation of microorganisms is Starch-Casein-Nitrate agar (Starch, 10.0 g; Casein, 0.003 g; KNO.sub.3, 0.02 g; NaCl, 0.02 g; MgSO.sub.4, 0.5 mg; CaCO.sub.3, 0.2 mg; FeSO.sub.4, 0.1 mg; Agar, 12.0 g; H.sub.2O, 1 L; pH 7.0) (Kuster and Williams, 1964). Each environmental soil sample (1.0 g) is suspended in 9 ml of sterile water, and 1 ml of the suspension is serially diluted ten-fold in sterile distilled water. One milliliter of the 10.sup.-5 dilution is inoculated onto the agar medium and incubated for 7 days at 30.degree. C. At the end of this period, the plates are observed for growth. White discrete and leathery colonies are picked and grown on new Starch-Casein-Nitrate agar plates to create a library of isolates. After 7 days of growth at 30.degree. C., the plates are kept at 4.degree. C.
Example 9: Screen for Isolates that Produce Methionine
[0363] This Example demonstrates the screening assay of isolates from Example 8 that naturally produce the amino acid, methionine.
[0364] Screening for Methionine Production:
[0365] A modified basal medium (K2HPO.sub.4, 0.3 g; KH.sub.2PO.sub.4, 0.7 g; Na.sub.2CO.sub.3, 1.0 g; CaCl.sub.2, 5.0 mg; MgSO.sub.4, 0.3 g; FeSO.sub.4, 1.0 mg; H.sub.2O, 1 L) with sucrose (20.0 g) and NH.sub.4Cl (10.0 g) is used for fermentation (Chay, B. P., Galvez, F. C. F., and Padolina, W. G. P. U. L. B. P. (1992). Methionine production by batch fermentation from various carbohydrates. ASEAN Food Journal (Malaysia)). The pH of the medium is 7.2.
[0366] Culture conditions: Two loops of the 7 day isolate culture of Example 8 are inoculated into a 250 ml Erlenmeyer flask with 30 ml of the fermentation medium. Methionine production is assayed after incubation of the flask for 5 days on a rotary shaker (160 rpm) at 30.degree. C. Duplicate flasks are prepared and non-inoculated flasks served as control in all experiments.
[0367] The presence of methionine in the culture broths of the isolates is examined by paper chromatography following a modified method of Khanna and Nag (Khanna et al., "Production of amino acids in vitro tissue culture," Indian Journal of Experimental Biology (1973)). The broth culture is centrifuged at 5000.times.g for 20 min and 2 .mu.L of the supernatant is applied 1.5 cm above one edge of Whatman No. 1 filter paper, with dimensions of 18 cm.times.22 cm. 1 .mu.L of volume of a standard methionine solution (0.1 mg/mL) is applied alongside with the supernatant, and the chromatogram is developed in a solvent mixture of n-butanol, acetic acid and water (4:1:1) for 18 h. The chromatogram is air-dried at room temperature, sprayed with 0.15% ninhydrin solution in butanol and dried again before heating at 60.degree. C. for 5 min in an oven. The value of the ninhydrin-positive spot (bluish-violet) of the supernatant that corresponds with the value of the standard methionine solution indicates presence of methionine in the broth culture. The concentration of methionine produced in the broth culture of the isolate is estimated as follows. The ninhydrin-positive spot of the supernatant of the isolate on the chromatogram is eluted in 10% ethanol and the spectrophotometric reading of the eluate at 520 nm recorded. The methionine concentration in the supernatant is determined from a standard curve. A plot of the values of optical densities against varying concentrations (0.1 to 0.9 mg/ml) of a methionine solution serve as the standard methionine curve.
[0368] Isolates that produce methionine are kept on fresh agar plates and a stock solution is created by suspending two loopfuls of microorganism in an aliquot of 50% glycerol solution.
Example 10: Administration of Amino Acid Producing Strain of Bacteria to Drosophila melanogaster Through Diet to Increase their Development Rate
[0369] This example demonstrates the ability to treat Drosophila melanogaster with amino acid producing bacteria to increase their development rate when raised on nutrient deprived diets. This experimental design can be extended to aid in the development of other insects, such as honeybees, which may lack nutrients in their diets.
[0370] Therapeutic Design:
[0371] Isolated bacteria Corynebacterium glutamicum that are glutamate or methionine producing, are formulated with a solution of 10.sup.9 colony forming units (CFUs) mixed to the feeding substrate for Drosophila flies.
[0372] Experimental Design:
[0373] Corynebacterium glutamicum strains that produce glutamate or methionine were grown in nutrient broth at 30.degree. C.
[0374] The media used to rear flies is cornmeal, molasses and yeast medium (11 g/l yeast, 54 g/l yellow cornmeal, 5 g/l agar, 66 ml/l molasses, and 4.8 ml/l propionic acid). All the components of the diet except propionic acid are heated together to 80.degree. C. in deionized water with constant mixing for 30 minutes and let to cool to 60.degree. C. Propionic acid is then mixed in and 50 ml of the diet is aliquoted into individual bottles and allowed to cool down and solidify. The flies are raised at 26.degree. C., 16:8 hour light:dark cycle, at around 60% humidity.
[0375] For the experimental setup to measure the larval growth rate, defined diet was used (Piper et al., 2014, Nature Methods). Defined diet eliminates the effects of batch to batch variation in the ingredients used for the cornmeal based diet. In addition, the defined diet allows for the exclusion of individual components to test their effects on fly development.
[0376] Development Rate Assay
[0377] On day one, 100 ul of a Corynebacterium glutamicum suspension in nutrient broth consisting of 10.sup.9 colony forming units (CFUs) were added to five replicates of fly food. As controls, nutrient broth without the bacteria was added to five more bottles of fly food. Fertilized embryos collected from fruit flies were treated with 2% hypochlorite solution for five minutes and then washed with sterile water to remove any extracellular microbes from the embryos. 10 ul of the embryo suspension in water (one:three embryo:water suspension) was added to all the bacteria seeded and control fly food bottles. The fly food with the embryos was maintained at 26.degree. C., 16:8 hour light:dark cycle, at around 60% humidity for the rest of the experiment. The time to adult emergence and the rate of emergence was measured in every replicate. From the time the first adult emerges from the pupa, the number of adult flies emerging was counted every 12 hours and rate of emergence was be computed.
[0378] Larval Mass Assay
[0379] To test whether the presence of bacteria producing amino acids can increase the body mass of developing larvae when raised on defined diet, we produced larvae that are axenic, and mono-associated with a single strain of bacterium. For these assays, three different bacteria were used, Corynebacterium glutamicum--a strain that produces glutamate, Corynebacterium glutamicum--a strain that produces methionine, and E. coli.
[0380] First, axenic embryos were generated. Fertilized embryos were collected from fruit flies over a 6 hour period on grape juice agar plates with yeast. To eliminate any bacterial contamination, the embryos were treated with 2% hypochlorite solution for five minutes and then washed thrice with sterile water. One volume of embryos was then suspended in 3 volumes of water.
[0381] The defined diet was aliquoted into vials and nine replicates were used for every condition being tested. The conditions were:
[0382] 1. No bacteria added to the food
[0383] 2. Food containing C. glutamicum, strain that produces glutamate (C.glu-Glu)
[0384] 3. Food containing C. glutamicum, a strain that produces methionine (C.glu-Met)
[0385] 4. Food containing E. coli
[0386] To each vial of the food that were in conditions 2, 3, and 4, 100 ul of overnight stationary phase cultures was added.
[0387] To each of the nine replicates in every condition, 10 ul of the sterile embryo+water suspension was added. The vials were then incubated at 26.degree. C., 60% humidity, 16:8 light:dark cycle.
[0388] After 13 days, 10-15 randomly chosen larvae from each replicate were sampled, and their areas were measured, as a proxy to their biomass and weight. The larvae were scooped out from the food with a sterile spatula, rinsed in water to clean the food from their bodies, and an image of every larvae sampled was acquired individually for every replicate in each condition. An Image J script was used to identify, outline, and measure the area of the larva in every image.
[0389] Amino Acid Producing Bacteria Treatment Increases Insect Development Rate.
[0390] Embryos that developed on diet that was seeded with the amino acid producing strain of bacterium reached adulthood significantly faster than those that were raised on the sterile diet (FIG. 1). Further, this effect was slightly stronger in female flies than in male files (FIGS. 2A and 2B).
[0391] Amino Acid Producing Bacteria Treatment Increases Larval Body Mass.
[0392] Larvae from the diet supplemented with C. glu-Met had the largest body size on average, followed by those in diet with C.glu-Glu, E. coli, and no bacteria (FIG. 3). This shows that augmenting the diet of insects with bacteria that produce amino acids produced insect biomass faster than un-supplemented diet.
[0393] Together this data demonstrates that augmenting the diet of insects with bacteria that were capable of producing amino acids produced insect biomass faster than un-supplemented diet. Extending this to other insects such as bees, supplementing their diet with bacteria that are capable of producing methionine can increase their fitness.
Example 11: Insects Treated with a Solution of Purified Phage
[0394] This Example demonstrates the isolation and purification of phages from environmental samples that targeted specific insect bacteria. This Example also demonstrates the efficacy of isolated phages against the target bacteria in vitro by plaque assays, by measuring their oxygen consumption rate, and the extracellular acidification rate. Finally, this Example demonstrates the efficacy of the phages in vivo, by measuring the ability of the phage to the target bacteria from flies by treating them with a phage isolated against the bacteria. This Example demonstrates that a pathogenic bacterium that decreased the fitness of an insect can be cleared using a phage to target the bacteria. Specifically, Serratia marcescens which is a pathogenic bacterium in flies can be cleared with the use of a phage that was isolated from garden compost.
[0395] There are several beneficial and commercially useful insects that are affected by naturally occurring bacterial pathogens. One such example is the bacterium Paenibacillus larvae which is the cause of foul brood disease in honey bees. Using a phage to treat foul brood disease would be of great value in mitigating huge losses due to foul brood disease.
[0396] Experimental Design
[0397] Isolation of Specific Bacteriophages from Natural Samples:
[0398] Bacteriophages against target bacteria were isolated from environmental source material. Briefly, a saturated culture of Serratia marcescens was diluted into fresh double-strength tryptic soy broth (TSB) and grown for .about.120 minutes to early log-phase at 24-26.degree. C., or into double-strength Luria-Bertani (LB) broth and grown for .about.90 min at 37.degree. C. Garden compost was prepared by homogenization in PBS and sterilized by 0.2 .mu.m filtration. Raw sewage was sterilized by 0.2 .mu.m filtration. One volume of filtered source material was added to log-phase bacterial cultures and incubation was continued for 24 h. Enriched source material was prepared by pelleting cultures and filtering supernatant fluid through 0.45 .mu.m membranes.
[0399] Phages were isolated by plating samples onto double-agar bacterial lawns. Stationary bacterial cultures were combined with molten 0.6% agar LB or TSB and poured onto 1.5% agar LB or TSB plates. After solidification, 2.5 .mu.L of phage sample dilutions were spotted onto the double-agar plates and allowed to absorb. Plates were then wrapped and incubated overnight at 25.degree. C. (TSA) or 37.degree. C. (LB), then assessed for the formation of visible plaques. Newly isolated plaques were purified by serial passaging of individual plaques on the target strain by picking plaques into SM Buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgSO4, 100 mM NaCl) and incubating for 15 min at 55.degree. C., then repeating the double-agar spotting method from above using the plaque suspension.
[0400] Bacteriophages were successfully isolated from both sewage and compost, as detailed above. Plaque formation was clearly evident after spotting samples onto lawns of the S. marcescens bacteria used for the enrichments.
[0401] Passaging, Quantification, and Propagation of Bacteriophages:
[0402] Propagation and generation of phage lysates for use in subsequent experiments was performed using bacteriophages isolated and purified as above. Briefly, saturated bacterial cultures were diluted 100-fold into fresh medium and grown for 60-120 minutes to achieve an early-logarithmic growth state for effective phage infection. Phage suspensions or lysates were added to early log phase cultures and incubation was continued until broth clearing, indicative of phage propagation and bacterial lysis, was observed, or until up to 24 h post-infection. Lysates were harvested by pelleting cells at 7,197.times.g for 20 min, then filtering the supernatant fluid through 0.45 or 0.2 .mu.m membranes. Filtered lysates were stored at 4.degree. C.
[0403] Enumeration of infective phage particles was performed using the double-agar spotting method. Briefly, a 1:10 dilution series of samples was performed in PBS and dilutions were spotted onto solidified double-agar plates prepared with the host bacteria as above. Plaque-forming units (PFU) were counted after overnight incubation to determine the approximate titer of samples.
[0404] In Vitro Analysis of Isolated Phages Measuring Bacterial Respiration:
[0405] A Seahorse XFe96 Analyzer (Agilent) was used to measure the effects of phages on bacteria by monitoring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) during infection. XFe96 plates were coated the day prior to experiments by 15 .mu.L of a 1 mg/mL poly-L-lysine stock per well and dried overnight at 28.degree. C. and XFe96 probes were equilibrated by placing into wells containing 200 .mu.L of XF Calibrant and incubating in the dark at room temperature. The following day, poly-L-lysine coated plates were washed twice with ddH2O. Saturated overnight cultures of E. coli BL21 (LB, 37.degree. C.) or S. marcescens (TSB, 25.degree. C.) were subcultured at 1:100 into the same media and grown with aeration for .about.2.5 h at 30.degree. C. Cultures were then diluted to O.D.600.about.nm 0.02 using the same media. Treatments were prepared by diluting stocks into SM Buffer at 10.times. final concentration and loading 20 .mu.L of the 10.times. solutions into the appropriate injection ports of the probe plate. While the probes were equilibrating in the XFe96 Flux Analyzer, bacterial plates were prepared by adding 90 .mu.L of bacterial suspensions or media controls and spun at 3,000 rpm for 10 min. Following centrifugation, an additional 90 .mu.L of the appropriate media were added gently to the wells so as not to disturb bacterial adherence, bringing the total volume to 180 .mu.L per well.
[0406] The XFe96 Flux Analyzer was run at .about.30.degree. C., following a Mix, Wait, Read cycling of 1:00, 0:30, 3:00. Four cycles were completed to permit equilibration/normalization of bacteria, then the 20 .mu.L treatments were injected and cycling continued as above, for a total time of approximately 6 h. Data were analyzed using the Seahorse XFe96 Wave software package.
[0407] The effects of isolated bacteriophages were assayed by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of bacteria with a Seahorse XFe96 Analyzer. When E. coli was infected with phage T7 and S. marcescens infected with the newly isolated .PHI.SmVL-C1, dramatic decreases in OCR were observed following brief bursts in this rate (FIG. 4). For both phages with both host organisms, the Seahorse assay permitted the detection of successful phage infection without the need for plaque assays. Thus, this method is applicable for detecting phage infection of a host organism not amenable to traditional phage detection methods.
[0408] SYBR Gold Transduction Assay for Infection Identification:
[0409] Bacteriophage preparations were prepared for staining by pretreating with nucleases to remove extraviral nucleic acids that could interfere with fluorescent signal interpretation. Briefly, MgCl2 was added to 10 mL of phage lysate at 10 mM final concentration, and RNase A (Qiagen) and DNase I (Sigma) were both added to final concentrations of 10 .mu.g/mL. Samples were incubated for 1 h at room temperature. After nuclease treatment, 5 mL of lysates were combined with 1 .mu.L of SYBR Gold (Thermo, 10,000.times.) and incubated at room temperature for .about.1.5 h. Excess dye was subsequently removed from samples using Amicon ultrafiltration columns. Briefly, Amicon columns (15 mL, 10 k MWCO) were washed by adding 10 mL of SM Buffer and spinning at 5,000.times.g, 4.degree. C. for 5 min. Labeled phage samples were then spun through the columns at 5,000.times.g, 4.degree. C. until the volume had decreased by approximately 10-fold (15-30 min). To wash samples, 5 mL SM Buffer was added to each reservoir and the spin repeated, followed by two additional washes. After the third wash, the retained samples were pipetted out from the Amicon reservoirs and brought up to approximately 1 mL using SM Buffer. To remove larger contaminants, washed and labeled phage samples were spun at 10,000.times.g for 2 min, and the supernatants were subsequently filtered through 0.2 .mu.m membranes into black microtubes and stored at 4.degree. C.
[0410] Saturated bacterial cultures (E. coli MG1655 grown in LB at 37.degree. C., S. marcescens and S. symbiotica grown in TSB at 26.degree. C.) were prepared by spinning down 1 mL aliquots and washing once with 1 mL PBS before a final resuspension using 1 mL PBS. Positive control labeled bacteria were stained by combining 500 .mu.L of washed bacteria with 1 .mu.L of SYBR Gold and incubating for 1 h in the dark at room temperature. Bacteria were pelleted by spinning at 8,000.times.g for 5 min and washed twice with an equal volume of PBS, followed by resuspension in a final volume of 500 .mu.L PBS. A volume of 25 .mu.L of stained bacteria was combined with 25 .mu.L of SM Buffer in a black microtube, to which 50 .mu.L of 10% formalin (5% final volume, .about.2% formaldehyde) was added and mixed by flicking. Samples were fixed at room temperature for .about.3 h and then washed using Amicon ultrafiltration columns. Briefly, 500 .mu.L of picopure water was added to Amicon columns (0.5 mL, 100 k MWCO) and spun at 14,000.times.g for 5 min to wash membranes. Fixed samples were diluted by adding 400 .mu.L of PBS and then transferred to pre-washed spin columns and spun at 14,000.times.g for 10 min. Columns were transferred to fresh collection tubes, and 500 .mu.L of PBS was added to dilute out fixative remaining in the retentate. Subsequently, two additional PBS dilutions were performed, for a total of three washes. The final retentates were diluted to roughly 100 .mu.L, then columns were inverted into fresh collection tubes and spun at 1,000.times.g for 2 min to collect samples. Washed samples were transferred to black microtubes and stored at 4.degree. C.
[0411] For transduction experiments and controls, 25 .mu.L of bacteria (or PBS) and 25 .mu.L of SYBR Gold labeled phage (or SM Buffer) were combined in black microtubes and incubated static for 15-20 min at room temperature to permit phage adsorption and injection into recipient bacteria. Immediately after incubation, 50 .mu.L of 10% formalin was added to samples and fixation was performed at room temperature for .about.4 h. Samples were washed with PBS using Amicon columns, as above.
[0412] Injection of bacteriophage nucleic acid was required for a phage to successfully infect a host bacterial cell. Coliphage P1kc labeled with SYBR Gold and co-incubated with S. marcescens revealed the presence of fluorescent bacteria by microscopy, validating the use of this assay in a phage isolation pipeline. As with the Seahorse assay, this approach provided an alternative to traditional phage methods to permit expansion to organisms not amenable to plaque assay. Additionally, the SYBR Gold transduction assay did not require bacterial growth, so is applicable to analysis of phages targeting difficult or even non-culturable organisms, including endosymbionts such as Buchnera.
[0413] Testing In Vivo Efficacy of the Phages Against S. marcescens in Drosophila melanogaster Flies
[0414] S. marcescens cultures were grown in Tryptic Soy Broth (TSB) at 30.degree. C. with constant shaking at 200 rpm.
[0415] The media used to rear fly stocks was cornmeal, molasses and yeast medium (11 g/l yeast, 54 g/l yellow cornmeal, 5 g/l agar, 66 ml/l molasses, and 4.8 ml/l propionic acid). All the components of the diet except propionic acid were heated together to 80.degree. C. in deionized water with constant mixing for 30 minutes and let to cool to 60.degree. C. Propionic acid was then mixed in and 50 ml of the diet was aliquoted into individual bottles and allowed to cool down and solidify. The flies were raised at 26.degree. C., 16:8 hour light:dark cycle, at around 60% humidity.
[0416] To infect the flies with S. marcescens, a fine needle (About 10 um wide tip) was dipped in a dense overnight stationary phase culture and the thorax of the flies was punctured. For this experiment, four replicates of 10 males and 10 females each were infected with S. marcescens using the needle puncturing method as the positive control for fly mortality. For the treatment group, four replicates of 10 males and 10 females each were pricked with S. marcescens and a phage solution containing about 108 phage particles/ml. Finally, two replicates of 10 males and 10 females each that were not pricked or treated in anyway were used as a negative control for mortality.
[0417] Flies in all conditions were placed in food bottles and incubated at 26.degree. C., 16:8 light:dark cycle, at 60% humidity. The number of alive and dead flies were counted every day for four days after the pricking. All The flies pricked with S. marcescens alone were all dead within 24 hours of the treatment. In comparison, more than 60% of the flies in the phage treatment group, and all the flies in the untreated control group were alive at that time point (FIG. 5). Further, most of the flies in the phage treatment group and the negative control group went on to survive for four more days when the experiment was terminated.
[0418] To ascertain the reason of death of the flies, dead flies from both the S. marcescens and S. marcescens+phage pricked flies were homogenized and plated out. Four dead flies from each of the four replicates of both the S. marcescens and the S. marcescens+phage treatment were homogenized in 100 ul of TSB. A 1:100 dilution was also produced by diluting the homogenate in TSB. 10 ul of the concentrated homogenate as well as the 1:100 dilution was plated out onto TSA plates, and incubated overnight at 30.degree. C. Upon inspection of the plates for bacteria growth, all the plates from the dead S. marcescens pricked flies had a lawn of bacteria growing on them, whereas the plates from the dead S. marcescens+phage pricked flies had no bacteria on them. This shows that in the absence of the phage, S. marcescens likely induced septic shock in the flies leading to their fatality. However, in the presence of the phage, the mortality may have been due to injury caused by the pricking with the needle.
Other Embodiments
[0419] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
Sequence CWU
1
1
19811505DNASnodgrassella alvi 1gagagtttga tcctggctca gattgaacgc tggcggcatg
ccttacacat gcaagtcgaa 60cggcagcacg gagagcttgc tctctggtgg cgagtggcga
acgggtgagt aatgcatcgg 120aacgtaccga gtaatggggg ataactgtcc gaaaggatgg
ctaataccgc atacgccctg 180agggggaaag cgggggatcg aaagacctcg cgttatttga
gcggccgatg ttggattagc 240tagttggtgg ggtaaaggcc taccaaggcg acgatccata
gcgggtctga gaggatgatc 300cgccacattg ggactgagac acggcccaaa ctcctacggg
aggcagcagt ggggaatttt 360ggacaatggg gggaaccctg atccagccat gccgcgtgtc
tgaagaaggc cttcgggttg 420taaaggactt ttgttaggga agaaaagccg ggtgttaata
ccatctggtg ctgacggtac 480ctaaagaata agcaccggct aactacgtgc cagcagccgc
ggtaatacgt agggtgcgag 540cgttaatcgg aattactggg cgtaaagcga gcgcagacgg
ttaattaagt cagatgtgaa 600atccccgagc tcaacttggg acgtgcattt gaaactggtt
aactagagtg tgtcagaggg 660aggtagaatt ccacgtgtag cagtgaaatg cgtagagatg
tggaggaata ccgatggcga 720aggcagcctc ctgggataac actgacgttc atgctcgaaa
gcgtgggtag caaacaggat 780tagataccct ggtagtccac gccctaaacg atgacaatta
gctgttggga cactagatgt 840cttagtagcg aagctaacgc gtgaaattgt ccgcctgggg
agtacggtcg caagattaaa 900actcaaagga attgacgggg acccgcacaa gcggtggatg
atgtggatta attcgatgca 960acgcgaagaa ccttacctgg tcttgacatg tacggaatct
cttagagata ggagagtgcc 1020ttcgggaacc gtaacacagg tgctgcatgg ctgtcgtcag
ctcgtgtcgt gagatgttgg 1080gttaagtccc gcaacgagcg caacccttgt cattagttgc
catcattaag ttgggcactc 1140taatgagact gccggtgaca aaccggagga aggtggggat
gacgtcaagt cctcatggcc 1200cttatgacca gggcttcaca cgtcatacaa tggtcggtac
agagggtagc gaagccgcga 1260ggtgaagcca atctcagaaa gccgatcgta gtccggattg
cactctgcaa ctcgagtgca 1320tgaagtcgga atcgctagta atcgcaggtc agcatactgc
ggtgaatacg ttcccgggtc 1380ttgtacacac cgcccgtcac accatgggag tgggggatac
cagaattggg tagactaacc 1440gcaaggaggt cgcttaacac ggtatgcttc atgactgggg
tgaagtcgta acaaggtagc 1500cgtag
150521541DNAGilliamella apicola 2ttaaattgaa
gagtttgatc atggctcaga ttgaacgctg gcggcaggct taacacatgc 60aagtcgaacg
gtaacatgag tgcttgcact tgatgacgag tggcggacgg gtgagtaaag 120tatggggatc
tgccgaatgg agggggacaa cagttggaaa cgactgctaa taccgcataa 180agttgagaga
ccaaagcatg ggaccttcgg gccatgcgcc atttgatgaa cccatatggg 240attagctagt
tggtagggta atggcttacc aaggcgacga tctctagctg gtctgagagg 300atgaccagcc
acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg 360aatattgcac
aatgggggaa accctgatgc agccatgccg cgtgtatgaa gaaggccttc 420gggttgtaaa
gtactttcgg tgatgaggaa ggtggtgtat ctaataggtg catcaattga 480cgttaattac
agaagaagca ccggctaact ccgtgccagc agccgcggta atacggaggg 540tgcgagcgtt
aatcggaatg actgggcgta aagggcatgt aggcggataa ttaagttagg 600tgtgaaagcc
ctgggctcaa cctaggaatt gcacttaaaa ctggttaact agagtattgt 660agaggaaggt
agaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaataccgg 720tggcgaaggc
ggccttctgg acagatactg acgctgagat gcgaaagcgt ggggagcaaa 780caggattaga
taccctggta gtccacgctg taaacgatgt cgatttggag tttgttgcct 840agagtgatgg
gctccgaagc taacgcgata aatcgaccgc ctggggagta cggccgcaag 900gttaaaactc
aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960gatgcaacgc
gaagaacctt acctggtctt gacatccaca gaatcttgca gagatgcggg 1020agtgccttcg
ggaactgtga gacaggtgct gcatggctgt cgtcagctcg tgttgtgaaa 1080tgttgggtta
agtcccgcaa cgagcgcaac ccttatcctt tgttgccatc ggttaggccg 1140ggaactcaaa
ggagactgcc gttgataaag cggaggaagg tggggacgac gtcaagtcat 1200catggccctt
acgaccaggg ctacacacgt gctacaatgg cgtatacaaa gggaggcgac 1260ctcgcgagag
caagcggacc tcataaagta cgtctaagtc cggattggag tctgcaactc 1320gactccatga
agtcggaatc gctagtaatc gtgaatcaga atgtcacggt gaatacgttc 1380ccgggccttg
tacacaccgc ccgtcacacc atgggagtgg gttgcaccag aagtagatag 1440cttaaccttc
gggagggcgt ttaccacggt gtggtccatg actggggtga agtcgtaaca 1500aggtaaccgt
aggggaacct gcggttggat cacctcctta c
154131528DNABartonella apis 3aagccaaaat caaattttca acttgagagt ttgatcctgg
ctcagaacga acgctggcgg 60caggcttaac acatgcaagt cgaacgcact tttcggagtg
agtggcagac gggtgagtaa 120cgcgtgggaa tctacctatt tctacggaat aacgcagaga
aatttgtgct aataccgtat 180acgtccttcg ggagaaagat ttatcggaga tagatgagcc
cgcgttggat tagctagttg 240gtgaggtaat ggcccaccaa ggcgacgatc catagctggt
ctgagaggat gaccagccac 300attgggactg agacacggcc cagactccta cgggaggcag
cagtggggaa tattggacaa 360tgggcgcaag cctgatccag ccatgccgcg tgagtgatga
aggccctagg gttgtaaagc 420tctttcaccg gtgaagataa tgacggtaac cggagaagaa
gccccggcta acttcgtgcc 480agcagccgcg gtaatacgaa gggggctagc gttgttcgga
tttactgggc gtaaagcgca 540cgtaggcgga tatttaagtc aggggtgaaa tcccggggct
caaccccgga actgcctttg 600atactggata tcttgagtat ggaagaggta agtggaattc
cgagtgtaga ggtgaaattc 660gtagatattc ggaggaacac cagtggcgaa ggcggcttac
tggtccatta ctgacgctga 720ggtgcgaaag cgtggggagc aaacaggatt agataccctg
gtagtccacg ctgtaaacga 780tgaatgttag ccgttggaca gtttactgtt cggtggcgca
gctaacgcat taaacattcc 840gcctggggag tacggtcgca agattaaaac tcaaaggaat
tgacgggggc ccgcacaagc 900ggtggagcat gtggtttaat tcgaagcaac gcgcagaacc
ttaccagccc ttgacatccc 960gatcgcggat ggtggagaca ccgtctttca gttcggctgg
atcggtgaca ggtgctgcat 1020ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc
ccgcaacgag cgcaaccctc 1080gcccttagtt gccatcattt agttgggcac tctaagggga
ctgccggtga taagccgaga 1140ggaaggtggg gatgacgtca agtcctcatg gcccttacgg
gctgggctac acacgtgcta 1200caatggtggt gacagtgggc agcgagaccg cgaggtcgag
ctaatctcca aaagccatct 1260cagttcggat tgcactctgc aactcgagtg catgaagttg
gaatcgctag taatcgtgga 1320tcagcatgcc acggtgaata cgttcccggg ccttgtacac
accgcccgtc acaccatggg 1380agttggtttt acccgaaggt gctgtgctaa ccgcaaggag
gcaggcaacc acggtagggt 1440cagcgactgg ggtgaagtcg taacaaggta gccgtagggg
aacctgcggc tggatcacct 1500cctttctaag gaagatgaag aattggaa
152841390DNAParasaccharibacter
apiummisc_feature(643)..(756)n is a, g, c, or t 4ctaccatgca agtcgcacga
aacctttcgg ggttagtggc ggacgggtga gtaacgcgtt 60aggaacctat ctggaggtgg
gggataacat cgggaaactg gtgctaatac cgcatgatgc 120ctgagggcca aaggagagat
ccgccattgg aggggcctgc gttcgattag ctagttggtt 180gggtaaaggc tgaccaaggc
gatgatcgat agctggtttg agaggatgat cagccacact 240gggactgaga cacggcccag
actcctacgg gaggcagcag tggggaatat tggacaatgg 300gggcaaccct gatccagcaa
tgccgcgtgt gtgaagaagg tcttcggatt gtaaagcact 360ttcactaggg aagatgatga
cggtacctag agaagaagcc ccggctaact tcgtgccagc 420agccgcggta atacgaaggg
ggctagcgtt gctcggaatg actgggcgta aagggcgcgt 480aggctgtttg tacagtcaga
tgtgaaatcc ccgggcttaa cctgggaact gcatttgata 540cgtgcagact agagtccgag
agagggttgt ggaattccca gtgtagaggt gaaattcgta 600gatattggga agaacaccgg
ttgcgaaggc ggcaacctgg ctnnnnnnnn nnnnnnnnnn 660nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnngagc taacgcgtta agcacaccgc 780ctggggagta cggccgcaag
gttgaaactc aaaggaattg acgggggccc gcacaagcgg 840tggagcatgt ggtttaattc
gaagcaacgc gcagaacctt accagggctt gcatggggag 900gctgtattca gagatggata
tttcttcgga cctcccgcac aggtgctgca tggctgtcgt 960cagctcgtgt cgtgagatgt
tgggttaagt cccgcaacga gcgcaaccct tgtctttagt 1020tgccatcacg tctgggtggg
cactctagag agactgccgg tgacaagccg gaggaaggtg 1080gggatgacgt caagtcctca
tggcccttat gtcctgggct acacacgtgc tacaatggcg 1140gtgacagagg gatgctacat
ggtgacatgg tgctgatctc aaaaaaccgt ctcagttcgg 1200attgtactct gcaactcgag
tgcatgaagg tggaatcgct agtaatcgcg gatcagcatg 1260ccgcggtgaa tacgttcccg
ggccttgtac acaccgcccg tcacaccatg ggagttggtt 1320tgaccttaag ccggtgagcg
aaccgcaagg aacgcagccg accaccggtt cgggttcagc 1380gactggggga
139051549DNAEnterococcus spp.
5aggtgatcca gccgcacctt ccgatacggc taccttgtta cgacttcacc ccaatcatct
60atcccacctt aggcggctgg ctccaaaaag gttacctcac cgacttcggg tgttacaaac
120tctcgtggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcgtgctg
180atccgcgatt actagcgatt ccggcttcat gcaggcgagt tgcagcctgc aatccgaact
240gagagaagct ttaagagatt tgcatgacct cgcggtctag cgactcgttg tacttcccat
300tgtagcacgt gtgtagccca ggtcataagg ggcatgatga tttgacgtca tccccacctt
360cctccggttt gtcaccggca gtctcgctag agtgcccaac taaatgatgg caactaacaa
420taagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac
480catgcaccac ctgtcacttt gtccccgaag ggaaagctct atctctagag tggtcaaagg
540atgtcaagac ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacatg ctccaccgct
600tgtgcgggcc cccgtcaatt cctttgagtt tcaaccttgc ggtcgtactc cccaggcgga
660gtgcttaatg cgtttgctgc agcactgaag ggcggaaacc ctccaacact tagcactcat
720cgtttacggc gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgagcct
780cagcgtcagt tacagaccag agagccgcct tcgccactgg tgttcctcca tatatctacg
840catttcaccg ctacacatgg aattccactc tcctcttctg cactcaagtc tcccagtttc
900caatgaccct ccccggttga gccgggggct ttcacatcag acttaagaaa ccgcctgcgc
960tcgctttacg cccaataaat ccggacaacg cttgccacct acgtattacc gcggctgctg
1020gcacgtagtt agccgtggct ttctggttag ataccgtcag gggacgttca gttactaacg
1080tccttgttct tctctaacaa cagagtttta cgatccgaaa accttcttca ctcacgcggc
1140gttgctcggt cagactttcg tccattgccg aagattccct actgctgcct cccgtaggag
1200tctgggccgt gtctcagtcc cagtgtggcc gatcaccctc tcaggtcggc tatgcatcgt
1260ggccttggtg agccgttacc tcaccaacta gctaatgcac cgcgggtcca tccatcagcg
1320acacccgaaa gcgcctttca ctcttatgcc atgcggcata aactgttatg cggtattagc
1380acctgtttcc aagtgttatc cccctctgat gggtaggtta cccacgtgtt actcacccgt
1440ccgccactcc tctttccaat tgagtgcaag cactcgggag gaaagaagcg ttcgacttgc
1500atgtattagg cacgccgcca gcgttcgtcc tgagccagga tcaaactct
154961541DNADelftia spp. 6cagaaaggag gtgatccagc cgcaccttcc gatacggcta
ccttgttacg acttcacccc 60agtcacgaac cccgccgtgg taagcgccct ccttgcggtt
aggctaccta cttctggcga 120gacccgctcc catggtgtga cgggcggtgt gtacaagacc
cgggaacgta ttcaccgcgg 180catgctgatc cgcgattact agcgattccg acttcacgca
gtcgagttgc agactgcgat 240ccggactacg actggtttta tgggattagc tccccctcgc
gggttggcaa ccctctgtac 300cagccattgt atgacgtgtg tagccccacc tataagggcc
atgaggactt gacgtcatcc 360ccaccttcct ccggtttgtc accggcagtc tcattagagt
gctcaactga atgtagcaac 420taatgacaag ggttgcgctc gttgcgggac ttaacccaac
atctcacgac acgagctgac 480gacagccatg cagcacctgt gtgcaggttc tctttcgagc
acgaatccat ctctggaaac 540ttcctgccat gtcaaaggtg ggtaaggttt ttcgcgttgc
atcgaattaa accacatcat 600ccaccgcttg tgcgggtccc cgtcaattcc tttgagtttc
aaccttgcgg ccgtactccc 660caggcggtca acttcacgcg ttagcttcgt tactgagaaa
actaattccc aacaaccagt 720tgacatcgtt tagggcgtgg actaccaggg tatctaatcc
tgtttgctcc ccacgctttc 780gtgcatgagc gtcagtacag gtccagggga ttgccttcgc
catcggtgtt cctccgcata 840tctacgcatt tcactgctac acgcggaatt ccatccccct
ctaccgtact ctagccatgc 900agtcacaaat gcagttccca ggttgagccc ggggatttca
catctgtctt acataaccgc 960ctgcgcacgc tttacgccca gtaattccga ttaacgctcg
caccctacgt attaccgcgg 1020ctgctggcac gtagttagcc ggtgcttatt cttacggtac
cgtcatgggc cccctgtatt 1080agaaggagct ttttcgttcc gtacaaaagc agtttacaac
ccgaaggcct tcatcctgca 1140cgcggcattg ctggatcagg ctttcgccca ttgtccaaaa
ttccccactg ctgcctcccg 1200taggagtctg ggccgtgtct cagtcccagt gtggctggtc
gtcctctcag accagctaca 1260gatcgtcggc ttggtaagct tttatcccac caactaccta
atctgccatc ggccgctcca 1320atcgcgcgag gcccgaaggg cccccgcttt catcctcaga
tcgtatgcgg tattagctac 1380tctttcgagt agttatcccc cacgactggg cacgttccga
tgtattactc acccgttcgc 1440cactcgtcag cgtccgaaga cctgttaccg ttcgacttgc
atgtgtaagg catgccgcca 1500gcgttcaatc tgagccagga tcaaactcta cagttcgatc t
154171502DNAPelomonas spp.misc_feature(192)..(193)n
is a, c, g, or tmisc_feature(832)..(833)n is a, c, g, or t 7atcctggctc
agattgaacg ctggcggcat gccttacaca tgcaagtcga acggtaacag 60gttaagctga
cgagtggcga acgggtgagt aatatatcgg aacgtgccca gtcgtggggg 120ataactgctc
gaaagagcag ctaataccgc atacgacctg agggtgaaag cgggggatcg 180caagacctcg
cnngattgga gcggccgata tcagattagg tagttggtgg ggtaaaggcc 240caccaagcca
acgatctgta gctggtctga gaggacgacc agccacactg ggactgagac 300acggcccaga
ctcctacggg aggcagcagt ggggaatttt ggacaatggg cgcaagcctg 360atccagccat
gccgcgtgcg ggaagaaggc cttcgggttg taaaccgctt ttgtcaggga 420agaaaaggtt
ctggttaata cctgggactc atgacggtac ctgaagaata agcaccggct 480aactacgtgc
cagcagccgc ggtaatacgt agggtgcaag cgttaatcgg aattactggg 540cgtaaagcgt
gcgcaggcgg ttatgcaaga cagaggtgaa atccccgggc tcaacctggg 600aactgccttt
gtgactgcat agctagagta cggtagaggg ggatggaatt ccgcgtgtag 660cagtgaaatg
cgtagatatg cggaggaaca ccgatggcga aggcaatccc ctggacctgt 720actgacgctc
atgcacgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780gccctaaacg
atgtcaactg gttgttggga gggtttcttc tcagtaacgt anntaacgcg 840tgaagttgac
cgcctgggga gtacggccgc aaggttgaaa ctcaaaggaa ttgacgggga 900cccgcacaag
cggtggatga tgtggtttaa ttcgatgcaa cgcgaaaaac cttacctacc 960cttgacatgc
caggaatcct gaagagattt gggagtgctc gaaagagaac ctggacacag 1020gtgctgcatg
gccgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080gcaacccttg
tcattagttg ctacgaaagg gcactctaat gagactgccg gtgacaaacc 1140ggaggaaggt
ggggatgacg tcaggtcatc atggccctta tgggtagggc tacacacgtc 1200atacaatggc
cgggacagag ggctgccaac ccgcgagggg gagctaatcc cagaaacccg 1260gtcgtagtcc
ggatcgtagt ctgcaactcg actgcgtgaa gtcggaatcg ctagtaatcg 1320cggatcagct
tgccgcggtg aatacgttcc cgggtcttgt acacaccgcc cgtcacacca 1380tgggagcggg
ttctgccaga agtagttagc ctaaccgcaa ggagggcgat taccacggca 1440gggttcgtga
ctggggtgaa gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc 1500ac
1502834PRTLactococcus lactis 8Ile Thr Ser Ile Ser Leu Cys Thr Pro Gly Cys
Lys Thr Gly Ala Leu1 5 10
15Met Gly Cys Asn Met Lys Thr Ala Thr Cys His Cys Ser Ile His Val
20 25 30Ser Lys922PRTStaphylococcus
epidermidis 9Ile Ala Ser Lys Phe Ile Cys Thr Pro Gly Cys Ala Lys Thr Gly
Ser1 5 10 15Phe Asn Ser
Tyr Cys Cys 201044PRTPediococcus acidilactici 10Lys Tyr Tyr
Gly Asn Gly Val Thr Cys Gly Lys His Ser Cys Ser Val1 5
10 15Asp Trp Gly Lys Ala Thr Thr Cys Ile
Ile Asn Asn Gly Ala Met Ala 20 25
30Trp Ala Thr Gly Gly His Gln Gly Asn His Lys Cys 35
401144PRTEnterococcus faecium 11Ala Thr Arg Ser Tyr Gly Asn Gly
Val Tyr Cys Asn Asn Ser Lys Cys1 5 10
15Trp Val Asn Trp Gly Glu Ala Lys Glu Asn Ile Ala Gly Ile
Val Ile 20 25 30Ser Gly Trp
Ala Ser Gly Leu Ala Gly Met Gly His 35
401239PRTStreptococcus lactis 12Gly Thr Trp Asp Asp Ile Gly Gln Gly Ile
Gly Arg Val Ala Tyr Trp1 5 10
15Val Gly Lys Ala Met Gly Asn Met Ser Asp Val Asn Gln Ala Ser Arg
20 25 30Ile Asn Arg Lys Lys Lys
His 351348PRTLactobacillus johnsonii 13Asn Arg Trp Gly Asp Thr Val
Leu Ser Ala Ala Ser Gly Ala Gly Thr1 5 10
15Gly Ile Lys Ala Cys Lys Ser Phe Gly Pro Trp Gly Met
Ala Ile Cys 20 25 30Gly Val
Gly Gly Ala Ala Ile Gly Gly Tyr Phe Gly Tyr Thr His Asn 35
40 451470PRTEnterococcus faecalis 14Met Ala Lys
Glu Phe Gly Ile Pro Ala Ala Val Ala Gly Thr Val Leu1 5
10 15Asn Val Val Glu Ala Gly Gly Trp Val
Thr Thr Ile Val Ser Ile Leu 20 25
30Thr Ala Val Gly Ser Gly Gly Leu Ser Leu Leu Ala Ala Ala Gly Arg
35 40 45Glu Ser Ile Lys Ala Tyr Leu
Lys Lys Glu Ile Lys Lys Lys Gly Lys 50 55
60Arg Ala Val Ile Ala Trp65 701551PRTStaphylococcus
aureus 15Met Ser Trp Leu Asn Phe Leu Lys Tyr Ile Ala Lys Tyr Gly Lys Lys1
5 10 15Ala Val Ser Ala
Ala Trp Lys Tyr Lys Gly Lys Val Leu Glu Trp Leu 20
25 30Asn Val Gly Pro Thr Leu Glu Trp Val Trp Gln
Lys Leu Lys Lys Ile 35 40 45Ala
Gly Leu 501643PRTLactococcus garvieae 16Ile Gly Gly Ala Leu Gly Asn
Ala Leu Asn Gly Leu Gly Thr Trp Ala1 5 10
15Asn Met Met Asn Gly Gly Gly Phe Val Asn Gln Trp Gln
Val Tyr Ala 20 25 30Asn Lys
Gly Lys Ile Asn Gln Tyr Arg Pro Tyr 35
4017103PRTEscherichia coli 17Met Arg Thr Leu Thr Leu Asn Glu Leu Asp Ser
Val Ser Gly Gly Ala1 5 10
15Ser Gly Arg Asp Ile Ala Met Ala Ile Gly Thr Leu Ser Gly Gln Phe
20 25 30Val Ala Gly Gly Ile Gly Ala
Ala Ala Gly Gly Val Ala Gly Gly Ala 35 40
45Ile Tyr Asp Tyr Ala Ser Thr His Lys Pro Asn Pro Ala Met Ser
Pro 50 55 60Ser Gly Leu Gly Gly Thr
Ile Lys Gln Lys Pro Glu Gly Ile Pro Ser65 70
75 80Glu Ala Trp Asn Tyr Ala Ala Gly Arg Leu Cys
Asn Trp Ser Pro Asn 85 90
95Asn Leu Ser Asp Val Cys Leu 10018339PRTStreptococcus phage
Cp1 18Met Val Lys Lys Asn Asp Leu Phe Val Asp Val Ser Ser His Asn Gly1
5 10 15Tyr Asp Ile Thr Gly
Ile Leu Glu Gln Met Gly Thr Thr Asn Thr Ile 20
25 30Ile Lys Ile Ser Glu Ser Thr Thr Tyr Leu Asn Pro
Cys Leu Ser Ala 35 40 45Gln Val
Glu Gln Ser Asn Pro Ile Gly Phe Tyr His Phe Ala Arg Phe 50
55 60Gly Gly Asp Val Ala Glu Ala Glu Arg Glu Ala
Gln Phe Phe Leu Asp65 70 75
80Asn Val Pro Met Gln Val Lys Tyr Leu Val Leu Asp Tyr Glu Asp Asp
85 90 95Pro Ser Gly Asp Ala
Gln Ala Asn Thr Asn Ala Cys Leu Arg Phe Met 100
105 110Gln Met Ile Ala Asp Ala Gly Tyr Lys Pro Ile Tyr
Tyr Ser Tyr Lys 115 120 125Pro Phe
Thr His Asp Asn Val Asp Tyr Gln Gln Ile Leu Ala Gln Phe 130
135 140Pro Asn Ser Leu Trp Ile Ala Gly Tyr Gly Leu
Asn Asp Gly Thr Ala145 150 155
160Asn Phe Glu Tyr Phe Pro Ser Met Asp Gly Ile Arg Trp Trp Gln Tyr
165 170 175Ser Ser Asn Pro
Phe Asp Lys Asn Ile Val Leu Leu Asp Asp Glu Glu 180
185 190Asp Asp Lys Pro Lys Thr Ala Gly Thr Trp Lys
Gln Asp Ser Lys Gly 195 200 205Trp
Trp Phe Arg Arg Asn Asn Gly Ser Phe Pro Tyr Asn Lys Trp Glu 210
215 220Lys Ile Gly Gly Val Trp Tyr Tyr Phe Asp
Ser Lys Gly Tyr Cys Leu225 230 235
240Thr Ser Glu Trp Leu Lys Asp Asn Glu Lys Trp Tyr Tyr Leu Lys
Asp 245 250 255Asn Gly Ala
Met Ala Thr Gly Trp Val Leu Val Gly Ser Glu Trp Tyr 260
265 270Tyr Met Asp Asp Ser Gly Ala Met Val Thr
Gly Trp Val Lys Tyr Lys 275 280
285Asn Asn Trp Tyr Tyr Met Thr Asn Glu Arg Gly Asn Met Val Ser Asn 290
295 300Glu Phe Ile Lys Ser Gly Lys Gly
Trp Tyr Phe Met Asn Thr Asn Gly305 310
315 320Glu Leu Ala Asp Asn Pro Ser Phe Thr Lys Glu Pro
Asp Gly Leu Ile 325 330
335Thr Val Ala19296PRTStreptococcus phage Dp-1 19Met Gly Val Asp Ile Glu
Lys Gly Val Ala Trp Met Gln Ala Arg Lys1 5
10 15Gly Arg Val Ser Tyr Ser Met Asp Phe Arg Asp Gly
Pro Asp Ser Tyr 20 25 30Asp
Cys Ser Ser Ser Met Tyr Tyr Ala Leu Arg Ser Ala Gly Ala Ser 35
40 45Ser Ala Gly Trp Ala Val Asn Thr Glu
Tyr Met His Ala Trp Leu Ile 50 55
60Glu Asn Gly Tyr Glu Leu Ile Ser Glu Asn Ala Pro Trp Asp Ala Lys65
70 75 80Arg Gly Asp Ile Phe
Ile Trp Gly Arg Lys Gly Ala Ser Ala Gly Ala 85
90 95Gly Gly His Thr Gly Met Phe Ile Asp Ser Asp
Asn Ile Ile His Cys 100 105
110Asn Tyr Ala Tyr Asp Gly Ile Ser Val Asn Asp His Asp Glu Arg Trp
115 120 125Tyr Tyr Ala Gly Gln Pro Tyr
Tyr Tyr Val Tyr Arg Leu Thr Asn Ala 130 135
140Asn Ala Gln Pro Ala Glu Lys Lys Leu Gly Trp Gln Lys Asp Ala
Thr145 150 155 160Gly Phe
Trp Tyr Ala Arg Ala Asn Gly Thr Tyr Pro Lys Asp Glu Phe
165 170 175Glu Tyr Ile Glu Glu Asn Lys
Ser Trp Phe Tyr Phe Asp Asp Gln Gly 180 185
190Tyr Met Leu Ala Glu Lys Trp Leu Lys His Thr Asp Gly Asn
Trp Tyr 195 200 205Trp Phe Asp Arg
Asp Gly Tyr Met Ala Thr Ser Trp Lys Arg Ile Gly 210
215 220Glu Ser Trp Tyr Tyr Phe Asn Arg Asp Gly Ser Met
Val Thr Gly Trp225 230 235
240Ile Lys Tyr Tyr Asp Asn Trp Tyr Tyr Cys Asp Ala Thr Asn Gly Asp
245 250 255Met Lys Ser Asn Ala
Phe Ile Arg Tyr Asn Asp Gly Trp Tyr Leu Leu 260
265 270Leu Pro Asp Gly Arg Leu Ala Asp Lys Pro Gln Phe
Thr Val Glu Pro 275 280 285Asp Gly
Leu Ile Thr Ala Lys Val 290 29520233PRTBacillus phage
gamma 20Met Glu Ile Gln Lys Lys Leu Val Asp Pro Ser Lys Tyr Gly Thr Lys1
5 10 15Cys Pro Tyr Thr
Met Lys Pro Lys Tyr Ile Thr Val His Asn Thr Tyr 20
25 30Asn Asp Ala Pro Ala Glu Asn Glu Val Ser Tyr
Met Ile Ser Asn Asn 35 40 45Asn
Glu Val Ser Phe His Ile Ala Val Asp Asp Lys Lys Ala Ile Gln 50
55 60Gly Ile Pro Leu Glu Arg Asn Ala Trp Ala
Cys Gly Asp Gly Asn Gly65 70 75
80Ser Gly Asn Arg Gln Ser Ile Ser Val Glu Ile Cys Tyr Ser Lys
Ser 85 90 95Gly Gly Asp
Arg Tyr Tyr Lys Ala Glu Asp Asn Ala Val Asp Val Val 100
105 110Arg Gln Leu Met Ser Met Tyr Asn Ile Pro
Ile Glu Asn Val Arg Thr 115 120
125His Gln Ser Trp Ser Gly Lys Tyr Cys Pro His Arg Met Leu Ala Glu 130
135 140Gly Arg Trp Gly Ala Phe Ile Gln
Lys Val Lys Asn Gly Asn Val Ala145 150
155 160Thr Thr Ser Pro Thr Lys Gln Asn Ile Ile Gln Ser
Gly Ala Phe Ser 165 170
175Pro Tyr Glu Thr Pro Asp Val Met Gly Ala Leu Thr Ser Leu Lys Met
180 185 190Thr Ala Asp Phe Ile Leu
Gln Ser Asp Gly Leu Thr Tyr Phe Ile Ser 195 200
205Lys Pro Thr Ser Asp Ala Gln Leu Lys Ala Met Lys Glu Tyr
Leu Asp 210 215 220Arg Lys Gly Trp Trp
Tyr Glu Val Lys225 23021481PRTStaphylococcus phage phi
MR11 21Met Gln Ala Lys Leu Thr Lys Lys Glu Phe Ile Glu Trp Leu Lys Thr1
5 10 15Ser Glu Gly Lys Gln
Phe Asn Val Asp Leu Trp Tyr Gly Phe Gln Cys 20
25 30Phe Asp Tyr Ala Asn Ala Gly Trp Lys Val Leu Phe
Gly Leu Leu Leu 35 40 45Lys Gly
Leu Gly Ala Lys Asp Ile Pro Phe Ala Asn Asn Phe Asp Gly 50
55 60Leu Ala Thr Val Tyr Gln Asn Thr Pro Asp Phe
Leu Ala Gln Pro Gly65 70 75
80Asp Met Val Val Phe Gly Ser Asn Tyr Gly Ala Gly Tyr Gly His Val
85 90 95Ala Trp Val Ile Glu
Ala Thr Leu Asp Tyr Ile Ile Val Tyr Glu Gln 100
105 110Asn Trp Leu Gly Gly Gly Trp Thr Asp Arg Ile Glu
Gln Pro Gly Trp 115 120 125Gly Trp
Glu Lys Val Thr Arg Arg Gln His Ala Tyr Asp Phe Pro Met 130
135 140Trp Phe Ile Arg Pro Asn Phe Lys Ser Glu Thr
Ala Pro Arg Ser Ile145 150 155
160Gln Ser Pro Thr Gln Ala Ser Lys Lys Glu Thr Ala Lys Pro Gln Pro
165 170 175Lys Ala Val Glu
Leu Lys Ile Ile Lys Asp Val Val Lys Gly Tyr Asp 180
185 190Leu Pro Lys Arg Gly Gly Asn Pro Lys Gly Ile
Val Ile His Asn Asp 195 200 205Ala
Gly Ser Lys Gly Ala Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val 210
215 220Asn Ala Pro Leu Ser Arg Leu Glu Ala Gly
Ile Ala His Ser Tyr Val225 230 235
240Ser Gly Asn Thr Val Trp Gln Ala Leu Asp Glu Ser Gln Val Gly
Trp 245 250 255His Thr Ala
Asn Gln Leu Gly Asn Lys Tyr Tyr Tyr Gly Ile Glu Val 260
265 270Cys Gln Ser Met Gly Ala Asp Asn Ala Thr
Phe Leu Lys Asn Glu Gln 275 280
285Ala Thr Phe Gln Glu Cys Ala Arg Leu Leu Lys Lys Trp Gly Leu Pro 290
295 300Ala Asn Arg Asn Thr Ile Arg Leu
His Asn Glu Phe Thr Ser Thr Ser305 310
315 320Cys Pro His Arg Ser Ser Val Leu His Thr Gly Phe
Asp Pro Val Thr 325 330
335Arg Gly Leu Leu Pro Glu Asp Lys Gln Leu Gln Leu Lys Asp Tyr Phe
340 345 350Ile Lys Gln Ile Arg Val
Tyr Met Asp Gly Lys Ile Pro Val Ala Thr 355 360
365Val Ser Asn Glu Ser Ser Ala Ser Ser Asn Thr Val Lys Pro
Val Ala 370 375 380Ser Ala Trp Lys Arg
Asn Lys Tyr Gly Thr Tyr Tyr Met Glu Glu Asn385 390
395 400Ala Arg Phe Thr Asn Gly Asn Gln Pro Ile
Thr Val Arg Lys Ile Gly 405 410
415Pro Phe Leu Ser Cys Pro Val Ala Tyr Gln Phe Gln Pro Gly Gly Tyr
420 425 430Cys Asp Tyr Thr Glu
Val Met Leu Gln Asp Gly His Val Trp Val Gly 435
440 445Tyr Thr Trp Glu Gly Gln Arg Tyr Tyr Leu Pro Ile
Arg Thr Trp Asn 450 455 460Gly Ser Ala
Pro Pro Asn Gln Ile Leu Gly Asp Leu Trp Gly Glu Ile465
470 475 480Ser22239PRTStreptococcus phage
B30 22Met Val Ile Asn Ile Glu Gln Ala Ile Ala Trp Met Ala Ser Arg Lys1
5 10 15Gly Lys Val Thr Tyr
Ser Met Asp Tyr Arg Asn Gly Pro Ser Ser Tyr 20
25 30Asp Cys Ser Ser Ser Val Tyr Phe Ala Leu Arg Ser
Ala Gly Ala Ser 35 40 45Asp Asn
Gly Trp Ala Val Asn Thr Glu Tyr Glu His Asp Trp Leu Ile 50
55 60Lys Asn Gly Tyr Val Leu Ile Ala Glu Asn Thr
Asn Trp Asn Ala Gln65 70 75
80Arg Gly Asp Ile Phe Ile Trp Gly Lys Arg Gly Ala Ser Ala Gly Ala
85 90 95Phe Gly His Thr Gly
Met Phe Val Asp Pro Asp Asn Ile Ile His Cys 100
105 110Asn Tyr Gly Tyr Asn Ser Ile Thr Val Asn Asn His
Asp Glu Ile Trp 115 120 125Gly Tyr
Asn Gly Gln Pro Tyr Val Tyr Ala Tyr Arg Tyr Ser Gly Lys 130
135 140Gln Ser Asn Ala Lys Val Asp Asn Lys Ser Val
Val Ser Lys Phe Glu145 150 155
160Lys Glu Leu Asp Val Asn Thr Pro Leu Ser Asn Ser Asn Met Pro Tyr
165 170 175Tyr Glu Ala Thr
Ile Ser Glu Asp Tyr Tyr Val Glu Ser Lys Pro Asp 180
185 190Val Asn Ser Thr Asp Lys Glu Leu Leu Val Ala
Gly Thr Arg Val Arg 195 200 205Val
Tyr Glu Lys Val Lys Gly Trp Ala Arg Ile Gly Ala Pro Gln Ser 210
215 220Asn Gln Trp Val Glu Asp Ala Tyr Leu Ile
Asp Ala Thr Asp Met225 230
23523495PRTStaphylococcus phage K 23Met Ala Lys Thr Gln Ala Glu Ile Asn
Lys Arg Leu Asp Ala Tyr Ala1 5 10
15Lys Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser
Tyr 20 25 30Asp Pro Ser Phe
Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly 35
40 45Tyr Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp
Tyr Val Leu Trp 50 55 60Leu Thr Asp
Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile65 70
75 80Lys Gln Ser Tyr Gly Thr Gly Phe
Lys Ile His Glu Asn Lys Pro Ser 85 90
95Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly
Ser Tyr 100 105 110Glu Gln Trp
Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser 115
120 125Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly
Tyr Ala Asn Lys Lys 130 135 140Pro Thr
Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu145
150 155 160Ile Pro Val Lys Ala Gly Thr
Thr Val Lys Lys Glu Thr Ala Lys Lys 165
170 175Ser Ala Ser Lys Thr Pro Ala Pro Lys Lys Lys Ala
Thr Leu Lys Val 180 185 190Ser
Lys Asn His Ile Asn Tyr Thr Met Asp Lys Arg Gly Lys Lys Pro 195
200 205Glu Gly Met Val Ile His Asn Asp Ala
Gly Arg Ser Ser Gly Gln Gln 210 215
220Tyr Glu Asn Ser Leu Ala Asn Ala Gly Tyr Ala Arg Tyr Ala Asn Gly225
230 235 240Ile Ala His Tyr
Tyr Gly Ser Glu Gly Tyr Val Trp Glu Ala Ile Asp 245
250 255Ala Lys Asn Gln Ile Ala Trp His Thr Gly
Asp Gly Thr Gly Ala Asn 260 265
270Ser Gly Asn Phe Arg Phe Ala Gly Ile Glu Val Cys Gln Ser Met Ser
275 280 285Ala Ser Asp Ala Gln Phe Leu
Lys Asn Glu Gln Ala Val Phe Gln Phe 290 295
300Thr Ala Glu Lys Phe Lys Glu Trp Gly Leu Thr Pro Asn Arg Lys
Thr305 310 315 320Val Arg
Leu His Met Glu Phe Val Pro Thr Ala Cys Pro His Arg Ser
325 330 335Met Val Leu His Thr Gly Phe
Asn Pro Val Thr Gln Gly Arg Pro Ser 340 345
350Gln Ala Ile Met Asn Lys Leu Lys Asp Tyr Phe Ile Lys Gln
Ile Lys 355 360 365Asn Tyr Met Asp
Lys Gly Thr Ser Ser Ser Thr Val Val Lys Asp Gly 370
375 380Lys Thr Ser Ser Ala Ser Thr Pro Ala Thr Arg Pro
Val Thr Gly Ser385 390 395
400Trp Lys Lys Asn Gln Tyr Gly Thr Trp Tyr Lys Pro Glu Asn Ala Thr
405 410 415Phe Val Asn Gly Asn
Gln Pro Ile Val Thr Arg Ile Gly Ser Pro Phe 420
425 430Leu Asn Ala Pro Val Gly Gly Asn Leu Pro Ala Gly
Ala Thr Ile Val 435 440 445Tyr Asp
Glu Val Cys Ile Gln Ala Gly His Ile Trp Ile Gly Tyr Asn 450
455 460Ala Tyr Asn Gly Asn Arg Val Tyr Cys Pro Val
Arg Thr Cys Gln Gly465 470 475
480Val Pro Pro Asn Gln Ile Pro Gly Val Ala Trp Gly Val Phe Lys
485 490 49524281PRTListeria
phage A118 24Met Thr Ser Tyr Tyr Tyr Ser Arg Ser Leu Ala Asn Val Asn Lys
Leu1 5 10 15Ala Asp Asn
Thr Lys Ala Ala Ala Arg Lys Leu Leu Asp Trp Ser Glu 20
25 30Ser Asn Gly Ile Glu Val Leu Ile Tyr Glu
Thr Ile Arg Thr Lys Glu 35 40
45Gln Gln Ala Ala Asn Val Asn Ser Gly Ala Ser Gln Thr Met Arg Ser 50
55 60Tyr His Leu Val Gly Gln Ala Leu Asp
Phe Val Met Ala Lys Gly Lys65 70 75
80Thr Val Asp Trp Gly Ala Tyr Arg Ser Asp Lys Gly Lys Lys
Phe Val 85 90 95Ala Lys
Ala Lys Ser Leu Gly Phe Glu Trp Gly Gly Asp Trp Ser Gly 100
105 110Phe Val Asp Asn Pro His Leu Gln Phe
Asn Tyr Lys Gly Tyr Gly Thr 115 120
125Asp Thr Phe Gly Lys Gly Ala Ser Thr Ser Asn Ser Ser Lys Pro Ser
130 135 140Ala Asp Thr Asn Thr Asn Ser
Leu Gly Leu Val Asp Tyr Met Asn Leu145 150
155 160Asn Lys Leu Asp Ser Ser Phe Ala Asn Arg Lys Lys
Leu Ala Thr Ser 165 170
175Tyr Gly Ile Lys Asn Tyr Ser Gly Thr Ala Thr Gln Asn Thr Thr Leu
180 185 190Leu Ala Lys Leu Lys Ala
Gly Lys Pro His Thr Pro Ala Ser Lys Asn 195 200
205Thr Tyr Tyr Thr Glu Asn Pro Arg Lys Val Lys Thr Leu Val
Gln Cys 210 215 220Asp Leu Tyr Lys Ser
Val Asp Phe Thr Thr Lys Asn Gln Thr Gly Gly225 230
235 240Thr Phe Pro Pro Gly Thr Val Phe Thr Ile
Ser Gly Met Gly Lys Thr 245 250
255Lys Gly Gly Thr Pro Arg Leu Lys Thr Lys Ser Gly Tyr Tyr Leu Thr
260 265 270Ala Asn Thr Lys Phe
Val Lys Lys Ile 275 28025341PRTListeria phage A511
25Met Val Lys Tyr Thr Val Glu Asn Lys Ile Ile Ala Gly Leu Pro Lys1
5 10 15Gly Lys Leu Lys Gly Ala
Asn Phe Val Ile Ala His Glu Thr Ala Asn 20 25
30Ser Lys Ser Thr Ile Asp Asn Glu Val Ser Tyr Met Thr
Arg Asn Trp 35 40 45Lys Asn Ala
Phe Val Thr His Phe Val Gly Gly Gly Gly Arg Val Val 50
55 60Gln Val Ala Asn Val Asn Tyr Val Ser Trp Gly Ala
Gly Gln Tyr Ala65 70 75
80Asn Ser Tyr Ser Tyr Ala Gln Val Glu Leu Cys Arg Thr Ser Asn Ala
85 90 95Thr Thr Phe Lys Lys Asp
Tyr Glu Val Tyr Cys Gln Leu Leu Val Asp 100
105 110Leu Ala Lys Lys Ala Gly Ile Pro Ile Thr Leu Asp
Ser Gly Ser Lys 115 120 125Thr Ser
Asp Lys Gly Ile Lys Ser His Lys Trp Val Ala Asp Lys Leu 130
135 140Gly Gly Thr Thr His Gln Asp Pro Tyr Ala Tyr
Leu Ser Ser Trp Gly145 150 155
160Ile Ser Lys Ala Gln Phe Ala Ser Asp Leu Ala Lys Val Ser Gly Gly
165 170 175Gly Asn Thr Gly
Thr Ala Pro Ala Lys Pro Ser Thr Pro Ala Pro Lys 180
185 190Pro Ser Thr Pro Ser Thr Asn Leu Asp Lys Leu
Gly Leu Val Asp Tyr 195 200 205Met
Asn Ala Lys Lys Met Asp Ser Ser Tyr Ser Asn Arg Asp Lys Leu 210
215 220Ala Lys Gln Tyr Gly Ile Ala Asn Tyr Ser
Gly Thr Ala Ser Gln Asn225 230 235
240Thr Thr Leu Leu Ser Lys Ile Lys Gly Gly Ala Pro Lys Pro Ser
Thr 245 250 255Pro Ala Pro
Lys Pro Ser Thr Ser Thr Ala Lys Lys Ile Tyr Phe Pro 260
265 270Pro Asn Lys Gly Asn Trp Ser Val Tyr Pro
Thr Asn Lys Ala Pro Val 275 280
285Lys Ala Asn Ala Ile Gly Ala Ile Asn Pro Thr Lys Phe Gly Gly Leu 290
295 300Thr Tyr Thr Ile Gln Lys Asp Arg
Gly Asn Gly Val Tyr Glu Ile Gln305 310
315 320Thr Asp Gln Phe Gly Arg Val Gln Val Tyr Gly Ala
Pro Ser Thr Gly 325 330
335Ala Val Ile Lys Lys 34026289PRTListeria phage A500 26Met
Ala Leu Thr Glu Ala Trp Leu Ile Glu Lys Ala Asn Arg Lys Leu1
5 10 15Asn Ala Gly Gly Met Tyr Lys
Ile Thr Ser Asp Lys Thr Arg Asn Val 20 25
30Ile Lys Lys Met Ala Lys Glu Gly Ile Tyr Leu Cys Val Ala
Gln Gly 35 40 45Tyr Arg Ser Thr
Ala Glu Gln Asn Ala Leu Tyr Ala Gln Gly Arg Thr 50 55
60Lys Pro Gly Ala Ile Val Thr Asn Ala Lys Gly Gly Gln
Ser Asn His65 70 75
80Asn Tyr Gly Val Ala Val Asp Leu Cys Leu Tyr Thr Asn Asp Gly Lys
85 90 95Asp Val Ile Trp Glu Ser
Thr Thr Ser Arg Trp Lys Lys Val Val Ala 100
105 110Ala Met Lys Ala Glu Gly Phe Lys Trp Gly Gly Asp
Trp Lys Ser Phe 115 120 125Lys Asp
Tyr Pro His Phe Glu Leu Cys Asp Ala Val Ser Gly Glu Lys 130
135 140Ile Pro Ala Ala Thr Gln Asn Thr Asn Thr Asn
Ser Asn Arg Tyr Glu145 150 155
160Gly Lys Val Ile Asp Ser Ala Pro Leu Leu Pro Lys Met Asp Phe Lys
165 170 175Ser Ser Pro Phe
Arg Met Tyr Lys Val Gly Thr Glu Phe Leu Val Tyr 180
185 190Asp His Asn Gln Tyr Trp Tyr Lys Thr Tyr Ile
Asp Asp Lys Leu Tyr 195 200 205Tyr
Met Tyr Lys Ser Phe Cys Asp Val Val Ala Lys Lys Asp Ala Lys 210
215 220Gly Arg Ile Lys Val Arg Ile Lys Ser Ala
Lys Asp Leu Arg Ile Pro225 230 235
240Val Trp Asn Asn Ile Lys Leu Asn Ser Gly Lys Ile Lys Trp Tyr
Ala 245 250 255Pro Asn Val
Lys Leu Ala Trp Tyr Asn Tyr Arg Arg Gly Tyr Leu Glu 260
265 270Leu Trp Tyr Pro Asn Asp Gly Trp Tyr Tyr
Thr Ala Glu Tyr Phe Leu 275 280
285Lys27239PRTStreptococcus prophage LambdaSa1 27Met Val Ile Asn Ile Glu
Gln Ala Ile Ala Trp Met Ala Ser Arg Lys1 5
10 15Gly Lys Val Thr Tyr Ser Met Asp Tyr Arg Asn Gly
Pro Ser Ser Tyr 20 25 30Asp
Cys Ser Ser Ser Val Tyr Phe Ala Leu Arg Ser Ala Gly Ala Ser 35
40 45Asp Asn Gly Trp Ala Val Asn Thr Glu
Tyr Glu His Asp Trp Leu Ile 50 55
60Lys Asn Gly Tyr Val Leu Ile Ala Glu Asn Thr Asn Trp Asn Ala Gln65
70 75 80Arg Gly Asp Ile Phe
Ile Trp Gly Lys Arg Gly Ala Ser Ala Gly Ala 85
90 95Phe Gly His Thr Gly Met Phe Val Asp Pro Asp
Asn Ile Ile His Cys 100 105
110Asn Tyr Gly Tyr Asn Ser Ile Thr Val Asn Asn His Asp Glu Ile Trp
115 120 125Gly Tyr Asn Gly Gln Pro Tyr
Val Tyr Ala Tyr Arg Tyr Ala Arg Lys 130 135
140Gln Ser Asn Ala Lys Val Asp Asn Gln Ser Val Val Ser Lys Phe
Glu145 150 155 160Lys Glu
Leu Asp Val Asn Thr Pro Leu Ser Asn Ser Asn Met Pro Tyr
165 170 175Tyr Glu Ala Thr Ile Ser Glu
Asp Tyr Tyr Val Glu Ser Lys Pro Asp 180 185
190Val Asn Ser Thr Asp Lys Glu Leu Leu Val Ala Gly Thr Arg
Val Arg 195 200 205Val Tyr Glu Lys
Val Lys Gly Trp Ala Arg Ile Gly Ala Pro Gln Ser 210
215 220Asn Gln Trp Val Glu Asp Ala Tyr Leu Ile Asp Ala
Thr Asp Met225 230
23528468PRTStreptococcus prophage LambdaSa2 28Met Glu Ile Asn Thr Glu Ile
Ala Ile Ala Trp Met Ser Ala Arg Gln1 5 10
15Gly Lys Val Ser Tyr Ser Met Asp Tyr Arg Asp Gly Pro
Asn Ser Tyr 20 25 30Asp Cys
Ser Ser Ser Val Tyr Tyr Ala Leu Arg Ser Ala Gly Ala Ser 35
40 45Ser Ala Gly Trp Ala Val Asn Thr Glu Tyr
Met His Asp Trp Leu Ile 50 55 60Lys
Asn Gly Tyr Glu Leu Ile Ala Glu Asn Val Asp Trp Asn Ala Val65
70 75 80Arg Gly Asp Ile Ala Ile
Trp Gly Met Arg Gly His Ser Ser Gly Ala 85
90 95Gly Gly His Val Val Met Phe Ile Asp Pro Glu Asn
Ile Ile His Cys 100 105 110Asn
Trp Ala Asn Asn Gly Ile Thr Val Asn Asn Tyr Asn Gln Thr Ala 115
120 125Ala Ala Ser Gly Trp Met Tyr Cys Tyr
Val Tyr Arg Leu Lys Ser Gly 130 135
140Ala Ser Thr Gln Gly Lys Ser Leu Asp Thr Leu Val Lys Glu Thr Leu145
150 155 160Ala Gly Asn Tyr
Gly Asn Gly Glu Ala Arg Lys Ala Val Leu Gly Asn 165
170 175Gln Tyr Glu Ala Val Met Ser Val Ile Asn
Gly Lys Thr Thr Thr Asn 180 185
190Gln Lys Thr Val Asp Gln Leu Val Gln Glu Val Ile Ala Gly Lys His
195 200 205Gly Asn Gly Glu Ala Arg Lys
Lys Ser Leu Gly Ser Gln Tyr Asp Ala 210 215
220Val Gln Lys Arg Val Thr Glu Leu Leu Lys Lys Gln Pro Ser Glu
Pro225 230 235 240Phe Lys
Ala Gln Glu Val Asn Lys Pro Thr Glu Thr Lys Thr Ser Gln
245 250 255Thr Glu Leu Thr Gly Gln Ala
Thr Ala Thr Lys Glu Glu Gly Asp Leu 260 265
270Ser Phe Asn Gly Thr Ile Leu Lys Lys Ala Val Leu Asp Lys
Ile Leu 275 280 285Gly Asn Cys Lys
Lys His Asp Ile Leu Pro Ser Tyr Ala Leu Thr Ile 290
295 300Leu His Tyr Glu Gly Leu Trp Gly Thr Ser Ala Val
Gly Lys Ala Asp305 310 315
320Asn Asn Trp Gly Gly Met Thr Trp Thr Gly Gln Gly Asn Arg Pro Ser
325 330 335Gly Val Thr Val Thr
Gln Gly Ser Ala Arg Pro Ser Asn Glu Gly Gly 340
345 350His Tyr Met His Tyr Ala Ser Val Asp Asp Phe Leu
Thr Asp Trp Phe 355 360 365Tyr Leu
Leu Arg Ala Gly Gly Ser Tyr Lys Val Ser Gly Ala Lys Thr 370
375 380Phe Ser Glu Ala Ile Lys Gly Met Phe Lys Val
Gly Gly Ala Val Tyr385 390 395
400Asp Tyr Ala Ala Ser Gly Phe Asp Ser Tyr Ile Val Gly Ala Ser Ser
405 410 415Arg Leu Lys Ala
Ile Glu Ala Glu Asn Gly Ser Leu Asp Lys Phe Asp 420
425 430Lys Ala Thr Asp Ile Gly Asp Gly Ser Lys Asp
Lys Ile Asp Ile Thr 435 440 445Ile
Glu Gly Ile Glu Val Thr Ile Asn Gly Ile Thr Tyr Glu Leu Thr 450
455 460Lys Lys Pro
Val46529236PRTUnknown(ATCC700407) prophage 29Met Thr Asp Ser Ile Gln Glu
Met Arg Lys Leu Gln Ser Ile Pro Val1 5 10
15Arg Tyr Asp Met Gly Asp Arg Tyr Gly Asn Asp Ala Asp
Arg Asp Gly 20 25 30Arg Ile
Glu Met Asp Cys Ser Ser Ala Val Ser Lys Ala Leu Gly Ile 35
40 45Ser Met Thr Asn Asn Thr Glu Thr Leu Gln
Gln Ala Leu Pro Ala Ile 50 55 60Gly
Tyr Gly Lys Ile His Asp Ala Val Asp Gly Thr Phe Asp Met Gln65
70 75 80Ala Tyr Asp Val Ile Ile
Trp Ala Pro Arg Asp Gly Ser Ser Ser Leu 85
90 95Gly Ala Phe Gly His Val Leu Ile Ala Thr Ser Pro
Thr Thr Ala Ile 100 105 110His
Cys Asn Tyr Gly Ser Asp Gly Ile Thr Glu Asn Asp Tyr Asn Tyr 115
120 125Ile Trp Glu Ile Asn Gly Arg Pro Arg
Glu Ile Val Phe Arg Lys Gly 130 135
140Val Thr Gln Thr Gln Ala Thr Val Thr Ser Gln Phe Glu Arg Glu Leu145
150 155 160Asp Val Asn Ala
Arg Leu Thr Val Ser Asp Lys Pro Tyr Tyr Glu Ala 165
170 175Thr Leu Ser Glu Asp Tyr Tyr Val Glu Ala
Gly Pro Arg Ile Asp Ser 180 185
190Gln Asp Lys Glu Leu Ile Lys Ala Gly Thr Arg Val Arg Val Tyr Glu
195 200 205Lys Leu Asn Gly Trp Ser Arg
Ile Asn His Pro Glu Ser Ala Gln Trp 210 215
220Val Glu Asp Ser Tyr Leu Val Asp Ala Thr Glu Met225
230 23530481PRTStaphylococcus phage Phi11 30Met Gln Ala
Lys Leu Thr Lys Asn Glu Phe Ile Glu Trp Leu Lys Thr1 5
10 15Ser Glu Gly Lys Gln Phe Asn Val Asp
Leu Trp Tyr Gly Phe Gln Cys 20 25
30Phe Asp Tyr Ala Asn Ala Gly Trp Lys Val Leu Phe Gly Leu Leu Leu
35 40 45Lys Gly Leu Gly Ala Lys Asp
Ile Pro Phe Ala Asn Asn Phe Asp Gly 50 55
60Leu Ala Thr Val Tyr Gln Asn Thr Pro Asp Phe Leu Ala Gln Pro Gly65
70 75 80Asp Met Val Val
Phe Gly Ser Asn Tyr Gly Ala Gly Tyr Gly His Val 85
90 95Ala Trp Val Ile Glu Ala Thr Leu Asp Tyr
Ile Ile Val Tyr Glu Gln 100 105
110Asn Trp Leu Gly Gly Gly Trp Thr Asp Gly Ile Glu Gln Pro Gly Trp
115 120 125Gly Trp Glu Lys Val Thr Arg
Arg Gln His Ala Tyr Asp Phe Pro Met 130 135
140Trp Phe Ile Arg Pro Asn Phe Lys Ser Glu Thr Ala Pro Arg Ser
Val145 150 155 160Gln Ser
Pro Thr Gln Ala Pro Lys Lys Glu Thr Ala Lys Pro Gln Pro
165 170 175Lys Ala Val Glu Leu Lys Ile
Ile Lys Asp Val Val Lys Gly Tyr Asp 180 185
190Leu Pro Lys Arg Gly Ser Asn Pro Lys Gly Ile Val Ile His
Asn Asp 195 200 205Ala Gly Ser Lys
Gly Ala Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val 210
215 220Asn Ala Pro Leu Ser Arg Leu Glu Ala Gly Ile Ala
His Ser Tyr Val225 230 235
240Ser Gly Asn Thr Val Trp Gln Ala Leu Asp Glu Ser Gln Val Gly Trp
245 250 255His Thr Ala Asn Gln
Ile Gly Asn Lys Tyr Tyr Tyr Gly Ile Glu Val 260
265 270Cys Gln Ser Met Gly Ala Asp Asn Ala Thr Phe Leu
Lys Asn Glu Gln 275 280 285Ala Thr
Phe Gln Glu Cys Ala Arg Leu Leu Lys Lys Trp Gly Leu Pro 290
295 300Ala Asn Arg Asn Thr Ile Arg Leu His Asn Glu
Phe Thr Ser Thr Ser305 310 315
320Cys Pro His Arg Ser Ser Val Leu His Thr Gly Phe Asp Pro Val Thr
325 330 335Arg Gly Leu Leu
Pro Glu Asp Lys Arg Leu Gln Leu Lys Asp Tyr Phe 340
345 350Ile Lys Gln Ile Arg Ala Tyr Met Asp Gly Lys
Ile Pro Val Ala Thr 355 360 365Val
Ser Asn Glu Ser Ser Ala Ser Ser Asn Thr Val Lys Pro Val Ala 370
375 380Ser Ala Trp Lys Arg Asn Lys Tyr Gly Thr
Tyr Tyr Met Glu Glu Ser385 390 395
400Ala Arg Phe Thr Asn Gly Asn Gln Pro Ile Thr Val Arg Lys Val
Gly 405 410 415Pro Phe Leu
Ser Cys Pro Val Gly Tyr Gln Phe Gln Pro Gly Gly Tyr 420
425 430Cys Asp Tyr Thr Glu Val Met Leu Gln Asp
Gly His Val Trp Val Gly 435 440
445Tyr Thr Trp Glu Gly Gln Arg Tyr Tyr Leu Pro Ile Arg Thr Trp Asn 450
455 460Gly Ser Ala Pro Pro Asn Gln Ile
Leu Gly Asp Leu Trp Gly Glu Ile465 470
475 480Ser31481PRTStaphylococcus phage PhiH5 31Met Gln
Ala Lys Leu Thr Lys Lys Glu Phe Ile Glu Trp Leu Lys Thr1 5
10 15Ser Glu Gly Lys Gln Tyr Asn Ala
Asp Gly Trp Tyr Gly Phe Gln Cys 20 25
30Phe Asp Tyr Ala Asn Ala Gly Trp Lys Ala Leu Phe Gly Leu Leu
Leu 35 40 45Lys Gly Val Gly Ala
Lys Asp Ile Pro Phe Ala Asn Asn Phe Asp Gly 50 55
60Leu Ala Thr Val Tyr Gln Asn Thr Pro Asp Phe Leu Ala Gln
Pro Gly65 70 75 80Asp
Met Val Val Phe Gly Ser Asn Tyr Gly Ala Gly Tyr Gly His Val
85 90 95Ala Trp Val Ile Glu Ala Thr
Leu Asp Tyr Ile Ile Val Tyr Glu Gln 100 105
110Asn Trp Leu Gly Gly Gly Trp Thr Asp Gly Val Gln Gln Pro
Gly Ser 115 120 125Gly Trp Glu Lys
Val Thr Arg Arg Gln His Ala Tyr Asp Phe Pro Met 130
135 140Trp Phe Ile Arg Pro Asn Phe Lys Ser Glu Thr Ala
Pro Arg Ser Val145 150 155
160Gln Ser Pro Thr Gln Ala Ser Lys Lys Glu Thr Ala Lys Pro Gln Pro
165 170 175Lys Ala Val Glu Leu
Lys Ile Ile Lys Asp Val Val Lys Gly Tyr Asp 180
185 190Leu Pro Lys Arg Gly Ser Asn Pro Asn Phe Ile Val
Ile His Asn Asp 195 200 205Ala Gly
Ser Lys Gly Ala Thr Ala Glu Ala Tyr Arg Asn Gly Leu Val 210
215 220Asn Ala Pro Leu Ser Arg Leu Glu Ala Gly Ile
Ala His Ser Tyr Val225 230 235
240Ser Gly Asn Thr Val Trp Gln Ala Leu Asp Glu Ser Gln Val Gly Trp
245 250 255His Thr Ala Asn
Gln Ile Gly Asn Lys Tyr Gly Tyr Gly Ile Glu Val 260
265 270Cys Gln Ser Met Gly Ala Asp Asn Ala Thr Phe
Leu Lys Asn Glu Gln 275 280 285Ala
Thr Phe Gln Glu Cys Ala Arg Leu Leu Lys Lys Trp Gly Leu Pro 290
295 300Ala Asn Arg Asn Thr Ile Arg Leu His Asn
Glu Phe Thr Ser Thr Ser305 310 315
320Cys Pro His Arg Ser Ser Val Leu His Thr Gly Phe Asp Pro Val
Thr 325 330 335Arg Gly Leu
Leu Pro Glu Asp Lys Arg Leu Gln Leu Lys Asp Tyr Phe 340
345 350Ile Lys Gln Ile Arg Ala Tyr Met Asp Gly
Lys Ile Pro Val Ala Thr 355 360
365Val Ser Asn Asp Ser Ser Ala Ser Ser Asn Thr Val Lys Pro Val Ala 370
375 380Ser Ala Trp Lys Arg Asn Lys Tyr
Gly Thr Tyr Tyr Met Glu Glu Ser385 390
395 400Ala Arg Phe Thr Asn Gly Asn Gln Pro Ile Thr Val
Arg Lys Val Gly 405 410
415Pro Phe Leu Ser Cys Pro Val Gly Tyr Gln Phe Gln Pro Gly Gly Tyr
420 425 430Cys Asp Tyr Thr Glu Val
Met Leu Gln Asp Gly His Val Trp Val Gly 435 440
445Tyr Thr Trp Glu Gly Gln Arg Tyr Tyr Leu Pro Ile Arg Thr
Trp Asn 450 455 460Gly Ser Ala Pro Pro
Asn Gln Ile Leu Gly Asp Leu Trp Gly Glu Ile465 470
475 480Ser32477PRTStaphylococcus phage phiWMY
32Met Lys Thr Lys Ala Gln Ala Lys Ser Trp Ile Asn Ser Lys Ile Gly1
5 10 15Lys Gly Ile Asp Trp Asp
Gly Met Tyr Gly Tyr Gln Cys Met Asp Glu 20 25
30Ala Val Asp Tyr Ile His His Val Thr Asp Gly Lys Val
Thr Met Trp 35 40 45Gly Asn Ala
Ile Asp Ala Pro Lys Asn Asn Phe Gln Gly Leu Cys Thr 50
55 60Val Tyr Thr Asn Thr Pro Glu Phe Arg Pro Ala Tyr
Gly Asp Val Ile65 70 75
80Val Trp Ser Tyr Gly Thr Phe Ala Thr Tyr Gly His Ile Ala Ile Val
85 90 95Val Asn Pro Asp Pro Tyr
Gly Asp Leu Gln Tyr Ile Thr Val Leu Glu 100
105 110Gln Asn Trp Asn Gly Asn Gly Ile Tyr Lys Thr Glu
Phe Ala Thr Ile 115 120 125Arg Thr
His Asp Tyr Thr Gly Val Ser His Phe Ile Arg Pro Lys Phe 130
135 140Ala Asp Glu Val Lys Glu Thr Ala Lys Thr Val
Asn Lys Leu Ser Val145 150 155
160Gln Lys Lys Ile Val Thr Pro Lys Asn Ser Val Glu Arg Ile Lys Asn
165 170 175Tyr Val Lys Thr
Ser Gly Tyr Ile Asn Gly Glu His Tyr Glu Leu Tyr 180
185 190Asn Arg Gly His Lys Pro Lys Gly Val Val Ile
His Asn Thr Ala Gly 195 200 205Thr
Ala Ser Ala Thr Gln Glu Gly Gln Arg Leu Thr Asn Met Thr Phe 210
215 220Gln Gln Leu Ala Asn Gly Val Ala His Val
Tyr Ile Asp Lys Asn Thr225 230 235
240Ile Tyr Glu Thr Leu Pro Glu Asp Arg Ile Ala Trp His Val Ala
Gln 245 250 255Gln Tyr Gly
Asn Thr Glu Phe Tyr Gly Ile Glu Val Cys Gly Ser Arg 260
265 270Asn Thr Asp Lys Glu Gln Phe Leu Ala Asn
Glu Gln Val Ala Phe Gln 275 280
285Glu Ala Ala Arg Arg Leu Lys Ser Trp Gly Met Arg Ala Asn Arg Asn 290
295 300Thr Val Arg Leu His His Thr Phe
Ser Ser Thr Glu Cys Pro Asp Met305 310
315 320Ser Met Leu Leu His Thr Gly Tyr Ser Met Lys Asn
Gly Lys Pro Thr 325 330
335Gln Asp Ile Thr Asn Lys Cys Ala Asp Tyr Phe Met Lys Gln Ile Asn
340 345 350Ala Tyr Ile Asp Gly Lys
Gln Pro Thr Ser Thr Val Val Gly Ser Ser 355 360
365Ser Ser Asn Lys Leu Lys Ala Lys Asn Lys Asp Lys Ser Thr
Gly Trp 370 375 380Asn Thr Asn Glu Tyr
Gly Thr Leu Trp Lys Lys Glu His Ala Thr Phe385 390
395 400Thr Cys Gly Val Arg Gln Gly Ile Val Thr
Arg Thr Thr Gly Pro Phe 405 410
415Thr Ser Cys Pro Gln Ala Gly Val Leu Tyr Tyr Gly Gln Ser Val Asn
420 425 430Tyr Asp Thr Val Cys
Lys Gln Asp Gly Tyr Val Trp Ile Ser Trp Thr 435
440 445Thr Ser Asp Gly Tyr Asp Val Trp Met Pro Ile Arg
Thr Trp Asp Arg 450 455 460Ser Thr Asp
Lys Val Ser Glu Ile Trp Gly Thr Ile Ser465 470
47533443PRTStreptococcus phage NCTC 11261 33Met Ala Thr Tyr Gln Glu
Tyr Lys Ser Arg Ser Asn Gly Asn Ala Tyr1 5
10 15Asp Ile Asp Gly Ser Phe Gly Ala Gln Cys Trp Asp
Gly Tyr Ala Asp 20 25 30Tyr
Cys Lys Tyr Leu Gly Leu Pro Tyr Ala Asn Cys Thr Asn Thr Gly 35
40 45Tyr Ala Arg Asp Ile Trp Glu Gln Arg
His Glu Asn Gly Ile Leu Asn 50 55
60Tyr Phe Asp Glu Val Glu Val Met Gln Ala Gly Asp Val Ala Ile Phe65
70 75 80Met Val Val Asp Gly
Val Thr Pro Tyr Ser His Val Ala Ile Phe Asp 85
90 95Ser Asp Ala Gly Gly Gly Tyr Gly Trp Phe Leu
Gly Gln Asn Gln Gly 100 105
110Gly Ala Asn Gly Ala Tyr Asn Ile Val Lys Ile Pro Tyr Ser Ala Thr
115 120 125Tyr Pro Thr Ala Phe Arg Pro
Lys Val Phe Lys Asn Ala Val Thr Val 130 135
140Thr Gly Asn Ile Gly Leu Asn Lys Gly Asp Tyr Phe Ile Asp Val
Ser145 150 155 160Ala Tyr
Gln Gln Ala Asp Leu Thr Thr Thr Cys Gln Gln Ala Gly Thr
165 170 175Thr Lys Thr Ile Ile Lys Val
Ser Glu Ser Ile Ala Trp Leu Ser Asp 180 185
190Arg His Gln Gln Gln Ala Asn Thr Ser Asp Pro Ile Gly Tyr
Tyr His 195 200 205Phe Gly Arg Phe
Gly Gly Asp Ser Ala Leu Ala Gln Arg Glu Ala Asp 210
215 220Leu Phe Leu Ser Asn Leu Pro Ser Lys Lys Val Ser
Tyr Leu Val Ile225 230 235
240Asp Tyr Glu Asp Ser Ala Ser Ala Asp Lys Gln Ala Asn Thr Asn Ala
245 250 255Val Ile Ala Phe Met
Asp Lys Ile Ala Ser Ala Gly Tyr Lys Pro Ile 260
265 270Tyr Tyr Ser Tyr Lys Pro Phe Thr Leu Asn Asn Ile
Asp Tyr Gln Lys 275 280 285Ile Ile
Ala Lys Tyr Pro Asn Ser Ile Trp Ile Ala Gly Tyr Pro Asp 290
295 300Tyr Glu Val Arg Thr Glu Pro Leu Trp Glu Phe
Phe Pro Ser Met Asp305 310 315
320Gly Val Arg Trp Trp Gln Phe Thr Ser Val Gly Val Ala Gly Gly Leu
325 330 335Asp Lys Asn Ile
Val Leu Leu Ala Asp Asp Ser Ser Lys Met Asp Ile 340
345 350Pro Lys Val Asp Lys Pro Gln Glu Leu Thr Phe
Tyr Gln Lys Leu Ala 355 360 365Thr
Asn Thr Lys Leu Asp Asn Ser Asn Val Pro Tyr Tyr Glu Ala Thr 370
375 380Leu Ser Thr Asp Tyr Tyr Val Glu Ser Lys
Pro Asn Ala Ser Ser Ala385 390 395
400Asp Lys Glu Phe Ile Lys Ala Gly Thr Arg Val Arg Val Tyr Glu
Lys 405 410 415Val Asn Gly
Trp Ser Arg Ile Asn His Pro Glu Ser Ala Gln Trp Val 420
425 430Glu Asp Ser Tyr Leu Val Asn Ala Thr Asp
Met 435 44034334PRTListeria phage FWLLm3 34Met Val
Lys Tyr Thr Val Glu Asn Lys Ile Ile Ala Gly Leu Pro Lys1 5
10 15Gly Lys Leu Lys Gly Ala Asn Phe
Val Ile Ala His Glu Thr Ala Asn 20 25
30Ser Lys Ser Thr Ile Asp Asn Glu Val Ser Tyr Met Thr Arg Asn
Trp 35 40 45Gln Asn Ala Phe Val
Thr His Phe Val Gly Gly Gly Gly Arg Val Val 50 55
60Gln Val Ala Asn Val Asn Tyr Val Ser Trp Gly Ala Gly Gln
Tyr Ala65 70 75 80Asn
Ser Tyr Ser Tyr Ala Gln Val Glu Leu Cys Arg Thr Ser Asn Ala
85 90 95Thr Thr Phe Lys Lys Asp Tyr
Glu Val Tyr Cys Gln Leu Leu Val Asp 100 105
110Leu Ala Lys Lys Ala Gly Ile Pro Ile Thr Leu Asp Ser Gly
Ser Lys 115 120 125Thr Ser Asp Lys
Gly Ile Lys Ser His Lys Trp Val Ala Asp Lys Leu 130
135 140Gly Gly Thr Thr His Gln Asp Pro Tyr Ala Tyr Leu
Ser Ser Trp Gly145 150 155
160Ile Ser Lys Ala Gln Phe Ala Ser Asp Leu Ala Lys Val Ser Gly Gly
165 170 175Gly Asn Thr Gly Thr
Ala Pro Ala Lys Pro Ser Thr Pro Ser Thr Asn 180
185 190Leu Asp Lys Leu Gly Leu Val Asp Tyr Met Asn Ala
Lys Lys Met Asp 195 200 205Ser Ser
Tyr Ser Asn Arg Ala Lys Leu Ala Lys Gln Tyr Gly Ile Ala 210
215 220Asn Tyr Ser Gly Thr Ala Ser Gln Asn Thr Thr
Leu Leu Ser Lys Ile225 230 235
240Lys Gly Gly Ala Pro Lys Pro Ser Thr Pro Ala Pro Lys Pro Ser Thr
245 250 255Ser Thr Ala Lys
Lys Ile Tyr Phe Pro Pro Asn Lys Gly Asn Trp Ser 260
265 270Val Tyr Pro Thr Asn Lys Ala Pro Val Lys Ala
Asn Ala Ile Gly Ala 275 280 285Ile
Asn Pro Thr Lys Phe Gly Gly Leu Thr Tyr Thr Ile Gln Lys Asp 290
295 300Arg Gly Asn Gly Val Tyr Glu Ile Gln Thr
Asp Gln Phe Gly Arg Val305 310 315
320Gln Val Tyr Gly Ala Pro Ser Thr Gly Ala Val Ile Lys Lys
325 330351278PRTBacillus phage BPS13 35Met Ala
Lys Arg Glu Lys Tyr Ile Phe Asp Val Glu Ala Glu Val Gly1 5
10 15Lys Ala Ala Lys Ser Ile Lys Ser
Leu Glu Ala Glu Leu Ser Lys Leu 20 25
30Gln Lys Leu Asn Lys Glu Ile Asp Ala Thr Gly Gly Asp Arg Thr
Glu 35 40 45Lys Glu Met Leu Ala
Thr Leu Lys Ala Ala Lys Glu Val Asn Ala Glu 50 55
60Tyr Gln Lys Met Gln Arg Ile Leu Lys Asp Leu Ser Lys Tyr
Ser Gly65 70 75 80Lys
Val Ser Arg Lys Glu Phe Asn Asp Ser Lys Val Ile Asn Asn Ala
85 90 95Lys Thr Ser Val Gln Gly Gly
Lys Val Thr Asp Ser Phe Gly Gln Met 100 105
110Leu Lys Asn Met Glu Arg Gln Ile Asn Ser Val Asn Lys Gln
Phe Asp 115 120 125Asn His Arg Lys
Ala Met Val Asp Arg Gly Gln Gln Tyr Thr Pro His 130
135 140Leu Lys Thr Asn Arg Lys Asp Ser Gln Gly Asn Ser
Asn Pro Ser Met145 150 155
160Met Gly Arg Asn Lys Ser Thr Thr Gln Asp Met Glu Lys Ala Val Asp
165 170 175Lys Phe Leu Asn Gly
Gln Asn Glu Ala Thr Thr Gly Leu Asn Gln Ala 180
185 190Leu Tyr Gln Leu Lys Glu Ile Ser Lys Leu Asn Arg
Arg Ser Glu Ser 195 200 205Leu Ser
Arg Arg Ala Ser Ala Ser Gly Tyr Met Ser Phe Gln Gln Tyr 210
215 220Ser Asn Phe Thr Gly Asp Arg Arg Thr Val Gln
Gln Thr Tyr Gly Gly225 230 235
240Leu Lys Thr Ala Asn Arg Glu Arg Val Leu Glu Leu Ser Gly Gln Ala
245 250 255Thr Gly Ile Ser
Lys Glu Leu Asp Arg Leu Asn Ser Lys Lys Gly Leu 260
265 270Thr Ala Arg Glu Gly Glu Glu Arg Lys Lys Leu
Met Arg Gln Leu Glu 275 280 285Gly
Ile Asp Ala Glu Leu Thr Ala Arg Lys Lys Leu Asn Ser Ser Leu 290
295 300Asp Glu Thr Thr Ser Asn Met Glu Lys Phe
Asn Gln Ser Leu Val Asp305 310 315
320Ala Gln Val Ser Val Lys Pro Glu Arg Gly Thr Met Arg Gly Met
Met 325 330 335Tyr Glu Arg
Ala Pro Ala Ile Ala Leu Ala Ile Gly Gly Ala Ile Thr 340
345 350Ala Thr Ile Gly Lys Leu Tyr Ser Glu Gly
Gly Asn His Ser Lys Ala 355 360
365Met Arg Pro Asp Glu Met Tyr Val Gly Gln Gln Thr Gly Ala Val Gly 370
375 380Ala Asn Trp Arg Pro Asn Arg Thr
Ala Thr Met Arg Ser Gly Leu Gly385 390
395 400Asn His Leu Gly Phe Thr Gly Gln Glu Met Met Glu
Phe Gln Ser Asn 405 410
415Tyr Leu Ser Ala Asn Gly Tyr His Gly Ala Glu Asp Met Lys Ala Ala
420 425 430Thr Thr Gly Gln Ala Thr
Phe Ala Arg Ala Thr Gly Leu Gly Ser Asp 435 440
445Glu Val Lys Asp Phe Phe Asn Thr Ala Tyr Arg Ser Gly Gly
Ile Asp 450 455 460Gly Asn Gln Thr Lys
Gln Phe Gln Asn Ala Phe Leu Gly Ala Met Lys465 470
475 480Gln Ser Gly Ala Val Gly Arg Glu Lys Asp
Gln Leu Lys Ala Leu Asn 485 490
495Gly Ile Leu Ser Ser Met Ser Gln Asn Arg Thr Val Ser Asn Gln Asp
500 505 510Met Met Arg Thr Val
Gly Leu Gln Ser Ala Ile Ser Ser Ser Gly Val 515
520 525Ala Ser Leu Gln Gly Thr Lys Gly Gly Ala Leu Met
Glu Gln Leu Asp 530 535 540Asn Gly Ile
Arg Glu Gly Phe Asn Asp Pro Gln Met Arg Val Leu Phe545
550 555 560Gly Gln Gly Thr Lys Tyr Gln
Gly Met Gly Gly Arg Ala Ala Leu Arg 565
570 575Lys Gln Met Glu Lys Gly Ile Ser Asp Pro Asp Asn
Leu Asn Thr Leu 580 585 590Ile
Asp Ala Ser Lys Ala Ser Ala Gly Gln Asp Pro Ala Glu Gln Ala 595
600 605Glu Val Leu Ala Thr Leu Ala Ser Lys
Met Gly Val Asn Met Ser Ser 610 615
620Asp Gln Ala Arg Gly Leu Ile Asp Leu Gln Pro Ser Gly Lys Leu Thr625
630 635 640Lys Glu Asn Ile
Asp Lys Val Met Lys Glu Gly Leu Lys Glu Gly Ser 645
650 655Ile Glu Ser Ala Lys Arg Asp Lys Ala Tyr
Ser Glu Ser Lys Ala Ser 660 665
670Ile Asp Asn Ser Ser Glu Ala Ala Thr Ala Lys Gln Ala Thr Glu Leu
675 680 685Asn Asp Met Gly Ser Lys Leu
Arg Gln Ala Asn Ala Ala Leu Gly Gly 690 695
700Leu Pro Ala Pro Leu Tyr Thr Ala Ile Ala Ala Val Val Ala Phe
Thr705 710 715 720Ala Ala
Val Ala Gly Ser Ala Leu Met Phe Lys Gly Ala Ser Trp Leu
725 730 735Lys Gly Gly Met Ala Ser Lys
Tyr Gly Gly Lys Gly Gly Lys Gly Gly 740 745
750Lys Gly Gly Gly Thr Gly Gly Gly Gly Gly Ala Gly Gly Ala
Ala Ala 755 760 765Thr Gly Ala Gly
Ala Ala Ala Gly Ala Gly Gly Val Gly Ala Ala Ala 770
775 780Ala Gly Glu Val Gly Ala Gly Val Ala Ala Gly Gly
Ala Ala Ala Gly785 790 795
800Ala Ala Ala Gly Gly Ser Lys Leu Ala Gly Val Gly Lys Gly Phe Met
805 810 815Lys Gly Ala Gly Lys
Leu Met Leu Pro Leu Gly Ile Leu Met Gly Ala 820
825 830Ser Glu Ile Met Gln Ala Pro Glu Glu Ala Lys Gly
Ser Ala Ile Gly 835 840 845Ser Ala
Val Gly Gly Ile Gly Gly Gly Ile Ala Gly Gly Ala Ala Thr 850
855 860Gly Ala Ile Ala Gly Ser Phe Leu Gly Pro Ile
Gly Thr Ala Val Gly865 870 875
880Gly Ile Ala Gly Gly Ile Ala Gly Gly Phe Ala Gly Ser Ser Leu Gly
885 890 895Glu Thr Ile Gly
Gly Trp Phe Asp Ser Gly Pro Lys Glu Asp Ala Ser 900
905 910Ala Ala Asp Lys Ala Lys Ala Asp Ala Ser Ala
Ala Ala Leu Ala Ala 915 920 925Ala
Ala Gly Thr Ser Gly Ala Val Gly Ser Ser Ala Leu Gln Ser Gln 930
935 940Met Ala Gln Gly Ile Thr Gly Ala Pro Asn
Met Ser Gln Val Gly Ser945 950 955
960Met Ala Ser Ala Leu Gly Ile Ser Ser Gly Ala Met Ala Ser Ala
Leu 965 970 975Gly Ile Ser
Ser Gly Gln Glu Asn Gln Ile Gln Thr Met Thr Asp Lys 980
985 990Glu Asn Thr Asn Thr Lys Lys Ala Asn Glu
Ala Lys Lys Gly Asp Asn 995 1000
1005Leu Ser Tyr Glu Arg Glu Asn Ile Ser Met Tyr Glu Arg Val Leu
1010 1015 1020Thr Arg Ala Glu Gln Ile
Leu Ala Gln Ala Arg Ala Gln Asn Gly 1025 1030
1035Ile Met Gly Val Gly Gly Gly Gly Thr Ala Gly Ala Gly Gly
Gly 1040 1045 1050Ile Asn Gly Phe Thr
Gly Gly Gly Lys Leu Gln Phe Leu Ala Ala 1055 1060
1065Gly Gln Lys Trp Ser Ser Ser Asn Leu Gln Gln His Asp
Leu Gly 1070 1075 1080Phe Thr Asp Gln
Asn Leu Thr Ala Glu Asp Leu Asp Lys Trp Ile 1085
1090 1095Asp Ser Lys Ala Pro Gln Gly Ser Met Met Arg
Gly Met Gly Ala 1100 1105 1110Thr Phe
Leu Lys Ala Gly Gln Glu Tyr Gly Leu Asp Pro Arg Tyr 1115
1120 1125Leu Ile Ala His Ala Ala Glu Glu Ser Gly
Trp Gly Thr Ser Lys 1130 1135 1140Ile
Ala Arg Asp Lys Gly Asn Phe Phe Gly Ile Gly Ala Phe Asp 1145
1150 1155Asp Ser Pro Tyr Ser Ser Ala Tyr Glu
Phe Lys Asp Gly Thr Gly 1160 1165
1170Ser Ala Ala Glu Arg Gly Ile Met Gly Gly Ala Lys Trp Ile Ser
1175 1180 1185Glu Lys Tyr Tyr Gly Lys
Gly Asn Thr Thr Leu Asp Lys Met Lys 1190 1195
1200Ala Ala Gly Tyr Ala Thr Asn Ala Ser Trp Ala Pro Asn Ile
Ala 1205 1210 1215Ser Ile Met Ala Gly
Ala Pro Thr Gly Ser Gly Ser Gly Asn Val 1220 1225
1230Thr Ala Thr Ile Asn Val Asn Val Lys Gly Asp Glu Lys
Val Ser 1235 1240 1245Asp Lys Leu Lys
Asn Ser Ser Asp Met Lys Lys Ala Gly Lys Asp 1250
1255 1260Ile Gly Ser Leu Leu Gly Phe Tyr Ser Arg Glu
Met Thr Ile Ala 1265 1270
127536495PRTStaphylococcus phage GH15 36Met Ala Lys Thr Gln Ala Glu Ile
Asn Lys Arg Leu Asp Ala Tyr Ala1 5 10
15Lys Gly Thr Val Asp Ser Pro Tyr Arg Ile Lys Lys Ala Thr
Ser Tyr 20 25 30Asp Pro Ser
Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly 35
40 45Tyr Tyr His Ala Gln Cys Gln Asp Leu Ile Thr
Asp Tyr Val Leu Trp 50 55 60Leu Thr
Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile65
70 75 80Lys Gln Ser Tyr Gly Thr Gly
Phe Lys Ile His Glu Asn Lys Pro Ser 85 90
95Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser
Gly Ser Tyr 100 105 110Gln Gln
Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser 115
120 125Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn
Gly Tyr Ala Asn Lys Lys 130 135 140Pro
Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu145
150 155 160Ile Pro Val Lys Ala Gly
Thr Thr Val Lys Lys Glu Thr Ala Lys Lys 165
170 175Ser Ala Ser Lys Thr Pro Ala Pro Lys Lys Lys Ala
Thr Leu Lys Val 180 185 190Ser
Lys Asn His Ile Asn Tyr Thr Met Asp Lys Arg Gly Lys Lys Pro 195
200 205Glu Gly Met Val Ile His Asn Asp Ala
Gly Arg Ser Ser Gly Gln Gln 210 215
220Tyr Glu Asn Ser Leu Ala Asn Ala Gly Tyr Ala Arg Tyr Ala Asn Gly225
230 235 240Ile Ala His Tyr
Tyr Gly Ser Glu Gly Tyr Val Trp Glu Ala Ile Asp 245
250 255Ala Lys Asn Gln Ile Ala Trp His Thr Gly
Asp Gly Thr Gly Ala Asn 260 265
270Ser Gly Asn Phe Arg Phe Ala Gly Ile Glu Val Cys Gln Ser Met Ser
275 280 285Ala Ser Asp Ala Gln Phe Leu
Lys Asn Glu Gln Ala Val Phe Gln Phe 290 295
300Thr Ala Glu Lys Phe Lys Glu Trp Gly Leu Thr Pro Asn Arg Lys
Thr305 310 315 320Val Arg
Leu His Met Glu Phe Val Pro Thr Ala Cys Pro His Arg Ser
325 330 335Met Val Leu His Thr Gly Phe
Asn Pro Val Thr Gln Gly Arg Pro Ser 340 345
350Gln Ala Ile Met Asn Lys Leu Lys Asp Tyr Phe Ile Lys Gln
Ile Lys 355 360 365Asn Tyr Met Asp
Lys Gly Thr Ser Ser Ser Thr Val Val Lys Asp Gly 370
375 380Lys Thr Ser Ser Ala Ser Thr Pro Ala Thr Arg Pro
Val Thr Gly Ser385 390 395
400Trp Lys Lys Asn Gln Tyr Gly Thr Trp Tyr Lys Pro Glu Asn Ala Thr
405 410 415Phe Val Asn Gly Asn
Gln Pro Ile Val Thr Arg Ile Gly Ser Pro Phe 420
425 430Leu Asn Ala Pro Val Gly Gly Asn Leu Pro Ala Gly
Ala Thr Ile Val 435 440 445Tyr Asp
Glu Val Cys Ile Gln Ala Gly His Ile Trp Ile Gly Tyr Asn 450
455 460Ala Tyr Asn Gly Asp Arg Val Tyr Cys Pro Val
Arg Thr Cys Gln Gly465 470 475
480Val Pro Pro Asn His Ile Pro Gly Val Ala Trp Gly Val Phe Lys
485 490 49537264PRTClostridium
phage phi8074-B1 37Met Lys Ile Gly Ile Asp Met Gly His Thr Leu Ser Gly
Ala Asp Tyr1 5 10 15Gly
Val Val Gly Leu Arg Pro Glu Ser Val Leu Thr Arg Glu Val Gly 20
25 30Thr Lys Val Ile Tyr Lys Leu Gln
Lys Leu Gly His Val Val Val Asn 35 40
45Cys Thr Val Asp Lys Ala Ser Ser Val Ser Glu Ser Leu Tyr Thr Arg
50 55 60Tyr Tyr Arg Ala Asn Gln Ala Asn
Val Asp Leu Phe Ile Ser Ile His65 70 75
80Phe Asn Ala Thr Pro Gly Gly Thr Gly Thr Glu Val Tyr
Thr Tyr Ala 85 90 95Gly
Arg Gln Leu Gly Glu Ala Thr Arg Ile Arg Gln Glu Phe Lys Ser
100 105 110Leu Gly Leu Arg Asp Arg Gly
Thr Lys Asp Gly Ser Gly Leu Ala Val 115 120
125Ile Arg Asn Thr Lys Ala Lys Ala Met Leu Val Glu Cys Cys Phe
Cys 130 135 140Asp Asn Pro Asn Asp Met
Lys Leu Tyr Asn Ser Glu Ser Phe Ser Asn145 150
155 160Ala Ile Val Lys Gly Ile Thr Gly Lys Leu Pro
Asn Gly Glu Ser Gly 165 170
175Asn Asn Asn Gln Gly Gly Asn Lys Val Lys Ala Val Val Ile Tyr Asn
180 185 190Glu Gly Ala Asp Arg Arg
Gly Ala Glu Tyr Leu Ala Asp Tyr Leu Asn 195 200
205Cys Pro Thr Ile Ser Asn Ser Arg Thr Phe Asp Tyr Ser Cys
Val Glu 210 215 220His Val Tyr Ala Val
Gly Gly Lys Lys Glu Gln Tyr Thr Lys Tyr Leu225 230
235 240Lys Thr Leu Leu Ser Gly Ala Asn Arg Tyr
Asp Thr Met Gln Gln Ile 245 250
255Leu Asn Phe Ile Asn Gly Gly Lys 26038209PRTSalmonella
phage SPN1S 38Met Asp Ile Asn Gln Phe Arg Arg Ala Ser Gly Ile Asn Glu Gln
Leu1 5 10 15Ala Ala Arg
Trp Phe Pro His Ile Thr Thr Ala Met Asn Glu Phe Gly 20
25 30Ile Thr Lys Pro Asp Asp Gln Ala Met Phe
Ile Ala Gln Val Gly His 35 40
45Glu Ser Gly Gly Phe Thr Arg Leu Gln Glu Asn Phe Asn Tyr Ser Val 50
55 60Asn Gly Leu Ser Gly Phe Ile Arg Ala
Gly Arg Ile Thr Pro Asp Gln65 70 75
80Ala Asn Ala Leu Gly Arg Lys Thr Tyr Glu Lys Ser Leu Pro
Leu Glu 85 90 95Arg Gln
Arg Ala Ile Ala Asn Leu Val Tyr Ser Lys Arg Met Gly Asn 100
105 110Asn Gly Pro Gly Asp Gly Trp Asn Tyr
Arg Gly Arg Gly Leu Ile Gln 115 120
125Ile Thr Gly Leu Asn Asn Tyr Arg Asp Cys Gly Asn Gly Leu Lys Val
130 135 140Asp Leu Val Ala Gln Pro Glu
Leu Leu Ala Gln Asp Glu Tyr Ala Ala145 150
155 160Arg Ser Ala Ala Trp Phe Phe Ser Ser Lys Gly Cys
Met Lys Tyr Thr 165 170
175Gly Asp Leu Val Arg Val Thr Gln Ile Ile Asn Gly Gly Gln Asn Gly
180 185 190Ile Asp Asp Arg Arg Thr
Arg Tyr Ala Ala Ala Arg Lys Val Leu Ala 195 200
205Leu39290PRTClavibacter phage CN77 39Met Gly Tyr Trp Gly
Tyr Pro Asn Gly Gln Ile Pro Asn Asp Lys Met1 5
10 15Ala Leu Tyr Arg Gly Cys Leu Leu Arg Ala Asp
Ala Ala Ala Gln Ala 20 25
30Tyr Ala Leu Gln Asp Ala Tyr Thr Arg Ala Thr Gly Lys Pro Leu Val
35 40 45Ile Leu Glu Gly Tyr Arg Asp Leu
Thr Arg Gln Lys Tyr Leu Arg Asn 50 55
60Leu Tyr Leu Ser Gly Arg Gly Asn Ile Ala Ala Val Pro Gly Leu Ser65
70 75 80Asn His Gly Trp Gly
Leu Ala Cys Asp Phe Ala Ala Pro Leu Asn Ser 85
90 95Ser Gly Ser Glu Glu His Arg Trp Met Arg Gln
Asn Ala Pro Leu Phe 100 105
110Gly Phe Asp Trp Ala Arg Gly Lys Ala Asp Asn Glu Pro Trp His Trp
115 120 125Glu Tyr Gly Asn Val Pro Val
Ser Arg Trp Ala Ser Leu Asp Val Thr 130 135
140Pro Ile Asp Arg Asn Asp Met Ala Asp Ile Thr Glu Gly Gln Met
Gln145 150 155 160Arg Ile
Ala Val Ile Leu Leu Asp Thr Glu Ile Gln Thr Pro Leu Gly
165 170 175Pro Arg Leu Val Lys His Ala
Leu Gly Asp Ala Leu Leu Leu Gly Gln 180 185
190Ala Asn Ala Asn Ser Ile Ala Glu Val Pro Asp Lys Thr Trp
Asp Val 195 200 205Leu Val Asp His
Pro Leu Ala Lys Asn Glu Asp Gly Thr Pro Leu Lys 210
215 220Val Arg Leu Gly Asp Val Ala Lys Tyr Glu Pro Leu
Glu His Gln Asn225 230 235
240Thr Arg Asp Ala Ile Ala Lys Leu Gly Thr Leu Gln Phe Thr Asp Lys
245 250 255Gln Leu Ala Thr Ile
Gly Ala Gly Val Lys Pro Ile Asp Glu Ala Ser 260
265 270Leu Val Lys Lys Ile Val Asp Gly Val Arg Ala Leu
Phe Gly Arg Ala 275 280 285Ala Ala
29040185PRTAcinetobacter phage phiAB2 40Met Ile Leu Thr Lys Asp Gly
Phe Ser Ile Ile Arg Asn Glu Leu Phe1 5 10
15Gly Gly Lys Leu Asp Gln Thr Gln Val Asp Ala Ile Asn
Phe Ile Val 20 25 30Ala Lys
Ala Thr Glu Ser Gly Leu Thr Tyr Pro Glu Ala Ala Tyr Leu 35
40 45Leu Ala Thr Ile Tyr His Glu Thr Gly Leu
Pro Ser Gly Tyr Arg Thr 50 55 60Met
Gln Pro Ile Lys Glu Ala Gly Ser Asp Ser Tyr Leu Arg Ser Lys65
70 75 80Lys Tyr Tyr Pro Tyr Ile
Gly Tyr Gly Tyr Val Gln Leu Thr Trp Lys 85
90 95Glu Asn Tyr Glu Arg Ile Gly Lys Leu Ile Gly Val
Asp Leu Ile Lys 100 105 110Asn
Pro Glu Lys Ala Leu Glu Pro Leu Ile Ala Ile Gln Ile Ala Ile 115
120 125Lys Gly Met Leu Asn Gly Trp Phe Thr
Gly Val Gly Phe Arg Arg Lys 130 135
140Arg Pro Val Ser Lys Tyr Asn Lys Gln Gln Tyr Val Ala Ala Arg Asn145
150 155 160Ile Ile Asn Gly
Lys Asp Lys Ala Glu Leu Ile Ala Lys Tyr Ala Ile 165
170 175Ile Phe Glu Arg Ala Leu Arg Ser Leu
180 18541262PRTBacillus phage B4 41Met Ala Met Ala
Leu Gln Thr Leu Ile Asp Lys Ala Asn Arg Lys Leu1 5
10 15Asn Val Ser Gly Met Arg Lys Asp Val Ala
Asp Arg Thr Arg Ala Val 20 25
30Ile Thr Gln Met His Ala Gln Gly Ile Tyr Ile Cys Val Ala Gln Gly
35 40 45Phe Arg Ser Phe Ala Glu Gln Asn
Ala Leu Tyr Ala Gln Gly Arg Thr 50 55
60Lys Pro Gly Ser Ile Val Thr Asn Ala Arg Gly Gly Gln Ser Asn His65
70 75 80Asn Tyr Gly Val Ala
Val Asp Leu Cys Leu Tyr Thr Gln Asp Gly Ser 85
90 95Asp Val Ile Trp Thr Val Glu Gly Asn Phe Arg
Lys Val Ile Ala Ala 100 105
110Met Lys Ala Gln Gly Phe Lys Trp Gly Gly Asp Trp Val Ser Phe Lys
115 120 125Asp Tyr Pro His Phe Glu Leu
Tyr Asp Val Val Gly Gly Gln Lys Pro 130 135
140Pro Ala Asp Asn Gly Gly Ala Val Asp Asn Gly Gly Gly Ser Gly
Ser145 150 155 160Thr Gly
Gly Ser Gly Gly Gly Ser Thr Gly Gly Gly Ser Thr Gly Gly
165 170 175Gly Tyr Asp Ser Ser Trp Phe
Thr Lys Glu Thr Gly Thr Phe Val Thr 180 185
190Asn Thr Ser Ile Lys Leu Arg Thr Ala Pro Phe Thr Ser Ala
Asp Val 195 200 205Ile Ala Thr Leu
Pro Ala Gly Ser Pro Val Asn Tyr Asn Gly Phe Gly 210
215 220Ile Glu Tyr Asp Gly Tyr Val Trp Ile Arg Gln Pro
Arg Ser Asn Gly225 230 235
240Tyr Gly Tyr Leu Ala Thr Gly Glu Ser Lys Gly Gly Lys Arg Gln Asn
245 250 255Tyr Trp Gly Thr Phe
Lys 26042274PRTClostridium phage phiCTP1 42Met Lys Lys Ile Ala
Asp Ile Ser Asn Leu Asn Gly Asn Val Asp Val1 5
10 15Lys Leu Leu Phe Asn Leu Gly Tyr Ile Gly Ile
Ile Ala Lys Ala Ser 20 25
30Glu Gly Gly Thr Phe Val Asp Lys Tyr Tyr Lys Gln Asn Tyr Thr Asn
35 40 45Thr Lys Ala Gln Gly Lys Ile Thr
Gly Ala Tyr His Phe Ala Asn Phe 50 55
60Ser Thr Ile Ala Lys Ala Gln Gln Glu Ala Asn Phe Phe Leu Asn Cys65
70 75 80Ile Ala Gly Thr Thr
Pro Asp Phe Val Val Leu Asp Leu Glu Gln Gln 85
90 95Cys Thr Gly Asp Ile Thr Asp Ala Cys Leu Ala
Phe Leu Asn Ile Val 100 105
110Ala Lys Lys Phe Lys Cys Val Val Tyr Cys Asn Ser Ser Phe Ile Lys
115 120 125Glu His Leu Asn Ser Lys Ile
Cys Ala Tyr Pro Leu Trp Ile Ala Asn 130 135
140Tyr Gly Val Ala Thr Pro Ala Phe Thr Leu Trp Thr Lys Tyr Ala
Met145 150 155 160Trp Gln
Phe Thr Glu Lys Gly Gln Val Ser Gly Ile Ser Gly Tyr Ile
165 170 175Asp Phe Ser Tyr Ile Thr Asp
Glu Phe Ile Lys Tyr Ile Lys Gly Glu 180 185
190Asp Glu Val Glu Asn Leu Val Val Tyr Asn Asp Gly Ala Asp
Gln Arg 195 200 205Ala Ala Glu Tyr
Leu Ala Asp Arg Leu Ala Cys Pro Thr Ile Asn Asn 210
215 220Ala Arg Lys Phe Asp Tyr Ser Asn Val Lys Asn Val
Tyr Ala Val Gly225 230 235
240Gly Asn Lys Glu Gln Tyr Thr Ser Tyr Leu Thr Thr Leu Ile Ala Gly
245 250 255Ser Thr Arg Tyr Thr
Thr Met Gln Ala Val Leu Asp Tyr Ile Lys Asn 260
265 270Leu Lys43628PRTStaphylococcus virus 187 43Met Ala
Leu Pro Lys Thr Gly Lys Pro Thr Ala Lys Gln Val Val Asp1 5
10 15Trp Ala Ile Asn Leu Ile Gly Ser
Gly Val Asp Val Asp Gly Tyr Tyr 20 25
30Gly Arg Gln Cys Trp Asp Leu Pro Asn Tyr Ile Phe Asn Arg Tyr
Trp 35 40 45Asn Phe Lys Thr Pro
Gly Asn Ala Arg Asp Met Ala Trp Tyr Arg Tyr 50 55
60Pro Glu Gly Phe Lys Val Phe Arg Asn Thr Ser Asp Phe Val
Pro Lys65 70 75 80Pro
Gly Asp Ile Ala Val Trp Thr Gly Gly Asn Tyr Asn Trp Asn Thr
85 90 95Trp Gly His Thr Gly Ile Val
Val Gly Pro Ser Thr Lys Ser Tyr Phe 100 105
110Tyr Ser Val Asp Gln Asn Trp Asn Asn Ser Asn Ser Tyr Val
Gly Ser 115 120 125Pro Ala Ala Lys
Ile Lys His Ser Tyr Phe Gly Val Thr His Phe Val 130
135 140Arg Pro Ala Tyr Lys Ala Glu Pro Lys Pro Thr Pro
Pro Ala Gln Asn145 150 155
160Asn Pro Ala Pro Lys Asp Pro Glu Pro Ser Lys Lys Pro Glu Ser Asn
165 170 175Lys Pro Ile Tyr Lys
Val Val Thr Lys Ile Leu Phe Thr Thr Ala His 180
185 190Ile Glu His Val Lys Ala Asn Arg Phe Val His Tyr
Ile Thr Lys Ser 195 200 205Asp Asn
His Asn Asn Lys Pro Asn Lys Ile Val Ile Lys Asn Thr Asn 210
215 220Thr Ala Leu Ser Thr Ile Asp Val Tyr Arg Tyr
Arg Asp Glu Leu Asp225 230 235
240Lys Asp Glu Ile Pro His Phe Phe Val Asp Arg Leu Asn Val Trp Ala
245 250 255Cys Arg Pro Ile
Glu Asp Ser Ile Asn Gly Tyr His Asp Ser Val Val 260
265 270Leu Ser Ile Thr Glu Thr Arg Thr Ala Leu Ser
Asp Asn Phe Lys Met 275 280 285Asn
Glu Ile Glu Cys Leu Ser Leu Ala Glu Ser Ile Leu Lys Ala Asn 290
295 300Asn Lys Lys Met Ser Ala Ser Asn Ile Ile
Val Asp Asn Lys Ala Trp305 310 315
320Arg Thr Phe Lys Leu His Thr Gly Lys Asp Ser Leu Lys Ser Ser
Ser 325 330 335Phe Thr Ser
Lys Asp Tyr Gln Lys Ala Val Asn Glu Leu Ile Lys Leu 340
345 350Phe Asn Asp Lys Asp Lys Leu Leu Asn Asn
Lys Pro Lys Asp Val Val 355 360
365Glu Arg Ile Arg Ile Arg Thr Ile Val Lys Glu Asn Thr Lys Phe Val 370
375 380Pro Ser Glu Leu Lys Pro Arg Asn
Asn Ile Arg Asp Lys Gln Asp Ser385 390
395 400Lys Ile Asp Arg Val Ile Asn Asn Tyr Thr Leu Lys
Gln Ala Leu Asn 405 410
415Ile Gln Tyr Lys Leu Asn Pro Lys Pro Gln Thr Ser Asn Gly Val Ser
420 425 430Trp Tyr Asn Ala Ser Val
Asn Gln Ile Lys Ser Ala Met Asp Thr Thr 435 440
445Lys Ile Phe Asn Asn Asn Val Gln Val Tyr Gln Phe Leu Lys
Leu Asn 450 455 460Gln Tyr Gln Gly Ile
Pro Val Asp Lys Leu Asn Lys Leu Leu Val Gly465 470
475 480Lys Gly Thr Leu Ala Asn Gln Gly His Ala
Phe Ala Asp Gly Cys Lys 485 490
495Lys Tyr Asn Ile Asn Glu Ile Tyr Leu Ile Ala His Arg Phe Leu Glu
500 505 510Ser Ala Asn Gly Thr
Ser Phe Phe Ala Ser Gly Lys Thr Gly Val Tyr 515
520 525Asn Tyr Phe Gly Ile Gly Ala Phe Asp Asn Asn Pro
Asn Asn Ala Met 530 535 540Ala Phe Ala
Arg Ser His Gly Trp Thr Ser Pro Thr Lys Ala Ile Ile545
550 555 560Gly Gly Ala Glu Phe Val Gly
Lys Gly Tyr Phe Asn Val Gly Gln Asn 565
570 575Thr Leu Tyr Arg Met Arg Trp Asn Pro Gln Lys Pro
Gly Thr His Gln 580 585 590Tyr
Ala Thr Asp Ile Ser Trp Ala Lys Val Gln Ala Gln Met Ile Ser 595
600 605Ala Met Tyr Lys Glu Ile Gly Leu Thr
Gly Asp Tyr Phe Ile Tyr Asp 610 615
620Gln Tyr Lys Lys62544291PRTListeria phage phiP35 44Met Ala Arg Lys Phe
Thr Lys Ala Glu Leu Val Ala Lys Ala Glu Lys1 5
10 15Lys Val Gly Gly Leu Lys Pro Asp Val Lys Lys
Ala Val Leu Ser Ala 20 25
30Val Lys Glu Ala Tyr Asp Arg Tyr Gly Ile Gly Ile Ile Val Ser Gln
35 40 45Gly Tyr Arg Ser Ile Ala Glu Gln
Asn Gly Leu Tyr Ala Gln Gly Arg 50 55
60Thr Lys Pro Gly Asn Ile Val Thr Asn Ala Lys Gly Gly Gln Ser Asn65
70 75 80His Asn Phe Gly Val
Ala Val Asp Phe Ala Ile Asp Leu Ile Asp Asp 85
90 95Gly Lys Ile Asp Ser Trp Gln Pro Ser Ala Thr
Ile Val Asn Met Met 100 105
110Lys Arg Arg Gly Phe Lys Trp Gly Gly Asp Trp Lys Ser Phe Thr Asp
115 120 125Leu Pro His Phe Glu Ala Cys
Asp Trp Tyr Arg Gly Glu Arg Lys Tyr 130 135
140Lys Val Asp Thr Ser Glu Trp Lys Lys Lys Glu Asn Ile Asn Ile
Val145 150 155 160Ile Lys
Asp Val Gly Tyr Phe Gln Asp Lys Pro Gln Phe Leu Asn Ser
165 170 175Lys Ser Val Arg Gln Trp Lys
His Gly Thr Lys Val Lys Leu Thr Lys 180 185
190His Asn Ser His Trp Tyr Thr Gly Val Val Lys Asp Gly Asn
Lys Ser 195 200 205Val Arg Gly Tyr
Ile Tyr His Ser Met Ala Lys Val Thr Ser Lys Asn 210
215 220Ser Asp Gly Ser Val Asn Ala Thr Ile Asn Ala His
Ala Phe Cys Trp225 230 235
240Asp Asn Lys Lys Leu Asn Gly Gly Asp Phe Ile Asn Leu Lys Arg Gly
245 250 255Phe Lys Gly Ile Thr
His Pro Ala Ser Asp Gly Phe Tyr Pro Leu Tyr 260
265 270Phe Ala Ser Arg Lys Lys Thr Phe Tyr Ile Pro Arg
Tyr Met Phe Asp 275 280 285Ile Lys
Lys 29045342PRTStreptococcus phage CP-7 45Met Val Lys Lys Asn Asp Leu
Phe Val Asp Val Ala Ser His Gln Gly1 5 10
15Tyr Asp Ile Ser Gly Ile Leu Glu Glu Ala Gly Thr Thr
Asn Thr Ile 20 25 30Ile Lys
Val Ser Glu Ser Thr Ser Tyr Leu Asn Pro Cys Leu Ser Ala 35
40 45Gln Val Ser Gln Ser Asn Pro Ile Gly Phe
Tyr His Phe Ala Trp Phe 50 55 60Gly
Gly Asn Glu Glu Glu Ala Glu Ala Glu Ala Arg Tyr Phe Leu Asp65
70 75 80Asn Val Pro Thr Gln Val
Lys Tyr Leu Val Leu Asp Tyr Glu Asp His 85
90 95Ala Ser Ala Ser Val Gln Arg Asn Thr Thr Ala Cys
Leu Arg Phe Met 100 105 110Gln
Ile Ile Ala Glu Ala Gly Tyr Thr Pro Ile Tyr Tyr Ser Tyr Lys 115
120 125Pro Phe Thr Leu Asp Asn Val Asp Tyr
Gln Gln Ile Leu Ala Gln Phe 130 135
140Pro Asn Ser Leu Trp Ile Ala Gly Tyr Gly Leu Asn Asp Gly Thr Ala145
150 155 160Asn Phe Glu Tyr
Phe Pro Ser Met Asp Gly Ile Arg Trp Trp Gln Tyr 165
170 175Ser Ser Asn Pro Phe Asp Lys Asn Ile Val
Leu Leu Asp Asp Glu Lys 180 185
190Glu Asp Asn Ile Asn Asn Glu Asn Thr Leu Lys Ser Leu Thr Thr Val
195 200 205Ala Asn Glu Val Ile Gln Gly
Leu Trp Gly Asn Gly Gln Glu Arg Tyr 210 215
220Asp Ser Leu Ala Asn Ala Gly Tyr Asp Pro Gln Ala Val Gln Asp
Lys225 230 235 240Val Asn
Glu Ile Leu Asn Ala Arg Glu Ile Ala Asp Leu Thr Thr Val
245 250 255Ala Asn Glu Val Ile Gln Gly
Leu Trp Gly Asn Gly Gln Glu Arg Tyr 260 265
270Asp Ser Leu Ala Asn Ala Gly Tyr Asp Pro Gln Ala Val Gln
Asp Lys 275 280 285Val Asn Glu Ile
Leu Asn Ala Arg Glu Ile Ala Asp Leu Thr Thr Val 290
295 300Ala Asn Glu Val Ile Gln Gly Leu Trp Gly Asn Gly
Gln Glu Arg Tyr305 310 315
320Asp Ser Leu Ala Asn Ala Gly Tyr Asp Pro Gln Ala Val Gln Asp Lys
325 330 335Val Asn Glu Leu Leu
Ser 34046328PRTEnterococcus phage EFAP-1 46Met Lys Leu Lys Gly
Ile Leu Leu Ser Val Val Thr Thr Phe Gly Leu1 5
10 15Leu Phe Gly Ala Thr Asn Val Gln Ala Tyr Glu
Val Asn Asn Glu Phe 20 25
30Asn Leu Gln Pro Trp Glu Gly Ser Gln Gln Leu Ala Tyr Pro Asn Lys
35 40 45Ile Ile Leu His Glu Thr Ala Asn
Pro Arg Ala Thr Gly Arg Asn Glu 50 55
60Ala Thr Tyr Met Lys Asn Asn Trp Phe Asn Ala His Thr Thr Ala Ile65
70 75 80Val Gly Asp Gly Gly
Ile Val Tyr Lys Val Ala Pro Glu Gly Asn Val 85
90 95Ser Trp Gly Ala Gly Asn Ala Asn Pro Tyr Ala
Pro Val Gln Ile Glu 100 105
110Leu Gln His Thr Asn Asp Pro Glu Leu Phe Lys Ala Asn Tyr Lys Ala
115 120 125Tyr Val Asp Tyr Thr Arg Asp
Met Gly Lys Lys Phe Gly Ile Pro Met 130 135
140Thr Leu Asp Gln Gly Gly Ser Leu Trp Glu Lys Gly Val Val Ser
His145 150 155 160Gln Trp
Val Thr Asp Phe Val Trp Gly Asp His Thr Asp Pro Tyr Gly
165 170 175Tyr Leu Ala Lys Met Gly Ile
Ser Lys Ala Gln Leu Ala His Asp Leu 180 185
190Ala Asn Gly Val Ser Gly Asn Thr Ala Thr Pro Thr Pro Lys
Pro Asp 195 200 205Lys Pro Lys Pro
Thr Gln Pro Ser Lys Pro Ser Asn Lys Lys Arg Phe 210
215 220Asn Tyr Arg Val Asp Gly Leu Glu Tyr Val Asn Gly
Met Trp Gln Ile225 230 235
240Tyr Asn Glu His Leu Gly Lys Ile Asp Phe Asn Trp Thr Glu Asn Gly
245 250 255Ile Pro Val Glu Val
Val Asp Lys Val Asn Pro Ala Thr Gly Gln Pro 260
265 270Thr Lys Asp Gln Val Leu Lys Val Gly Asp Tyr Phe
Asn Phe Gln Glu 275 280 285Asn Ser
Thr Gly Val Val Gln Glu Gln Thr Pro Tyr Met Gly Tyr Thr 290
295 300Leu Ser His Val Gln Leu Pro Asn Glu Phe Ile
Trp Leu Phe Thr Asp305 310 315
320Ser Lys Gln Ala Leu Met Tyr Gln 3254748PRTHomo
sapiens 47Ser Ser Leu Leu Glu Lys Gly Leu Asp Gly Ala Lys Lys Ala Val
Gly1 5 10 15Gly Leu Gly
Lys Leu Gly Lys Asp Ala Val Glu Asp Leu Glu Ser Val 20
25 30Gly Lys Gly Ala Val His Asp Val Lys Asp
Val Leu Asp Ser Val Leu 35 40
454837PRTHyalophora cecropia 48Lys Trp Lys Leu Phe Lys Lys Ile Glu Lys
Val Gly Gln Asn Ile Arg1 5 10
15Asp Gly Ile Ile Lys Ala Gly Pro Ala Val Ala Val Val Gly Gln Ala
20 25 30Thr Gln Ile Ala Lys
354962PRTDrosophila teissieri 49Met Lys Tyr Phe Ser Val Leu Val Val
Leu Thr Leu Ile Leu Ala Ile1 5 10
15Val Asp Gln Ser Asp Ala Phe Ile Asn Leu Leu Asp Lys Val Glu
Asp 20 25 30Ala Leu His Thr
Gly Ala Gln Ala Gly Phe Lys Leu Ile Arg Pro Val 35
40 45Glu Arg Gly Ala Thr Pro Lys Lys Ser Glu Lys Pro
Glu Lys 50 55 605066PRTBombyx mori
50Met Asn Ile Leu Lys Phe Phe Phe Val Phe Ile Val Ala Met Ser Leu1
5 10 15Val Ser Cys Ser Thr Ala
Ala Pro Ala Lys Ile Pro Ile Lys Ala Ile 20 25
30Lys Thr Val Gly Lys Ala Val Gly Lys Gly Leu Arg Ala
Ile Asn Ile 35 40 45Ala Ser Thr
Ala Asn Asp Val Phe Asn Phe Leu Lys Pro Lys Lys Arg 50
55 60Lys His655171PRTCeratitis capitata 51Met Ala Asn
Leu Lys Ala Val Phe Leu Ile Cys Ile Val Ala Phe Ile1 5
10 15Ala Leu Gln Cys Val Val Ala Glu Pro
Ala Ala Glu Asp Ser Val Val 20 25
30Val Lys Arg Ser Ile Gly Ser Ala Leu Lys Lys Ala Leu Pro Val Ala
35 40 45Lys Lys Ile Gly Lys Ile Ala
Leu Pro Ile Ala Lys Ala Ala Leu Pro 50 55
60Val Ala Ala Gly Leu Val Gly65 705253PRTApis
mellifera 52Met Lys Val Val Ile Phe Ile Phe Ala Leu Leu Ala Thr Ile Cys
Ala1 5 10 15Ala Phe Ala
Tyr Val Pro Leu Pro Asn Val Pro Gln Pro Gly Arg Arg 20
25 30Pro Phe Pro Thr Phe Pro Gly Gln Gly Pro
Phe Asn Pro Lys Ile Lys 35 40
45Trp Pro Gln Gly Tyr 5053283PRTApis mellifera 53Lys Asn Phe Ala Leu
Ala Ile Leu Val Val Thr Phe Val Val Ala Val1 5
10 15Phe Gly Asn Thr Asn Leu Asp Pro Pro Thr Arg
Pro Thr Arg Leu Arg 20 25
30Arg Glu Ala Lys Pro Glu Ala Glu Pro Gly Asn Asn Arg Pro Val Tyr
35 40 45Ile Pro Gln Pro Arg Pro Pro His
Pro Arg Leu Arg Arg Glu Ala Glu 50 55
60Pro Glu Ala Glu Pro Gly Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro65
70 75 80Arg Pro Pro His Pro
Arg Leu Arg Arg Glu Ala Glu Leu Glu Ala Glu 85
90 95Pro Gly Asn Asn Arg Pro Val Tyr Ile Ser Gln
Pro Arg Pro Pro His 100 105
110Pro Arg Leu Arg Arg Glu Ala Glu Pro Glu Ala Glu Pro Gly Asn Asn
115 120 125Arg Pro Val Tyr Ile Pro Gln
Pro Arg Pro Pro His Pro Arg Leu Arg 130 135
140Arg Glu Ala Glu Leu Glu Ala Glu Pro Gly Asn Asn Arg Pro Val
Tyr145 150 155 160Ile Ser
Gln Pro Arg Pro Pro His Pro Arg Leu Arg Arg Glu Ala Glu
165 170 175Pro Glu Ala Glu Pro Gly Asn
Asn Arg Pro Val Tyr Ile Pro Gln Pro 180 185
190Arg Pro Pro His Pro Arg Leu Arg Arg Glu Ala Glu Pro Glu
Ala Glu 195 200 205Pro Gly Asn Asn
Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His 210
215 220Pro Arg Leu Arg Arg Glu Ala Glu Pro Glu Ala Glu
Pro Gly Asn Asn225 230 235
240Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His Pro Arg Leu Arg
245 250 255Arg Glu Ala Lys Pro
Glu Ala Lys Pro Gly Asn Asn Arg Pro Val Tyr 260
265 270Ile Pro Gln Pro Arg Pro Pro His Pro Arg Ile
275 28054228PRTSus scrofa 54Met Glu Thr Gln Arg Ala Ser
Leu Cys Leu Gly Arg Trp Ser Leu Trp1 5 10
15Leu Leu Leu Leu Ala Leu Val Val Pro Ser Ala Ser Ala
Gln Ala Leu 20 25 30Ser Tyr
Arg Glu Ala Val Leu Arg Ala Val Asp Arg Leu Asn Glu Gln 35
40 45Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu
Glu Leu Asp Gln Pro Pro 50 55 60Lys
Ala Asp Glu Asp Pro Gly Thr Pro Lys Pro Val Ser Phe Thr Val65
70 75 80Lys Glu Thr Val Cys Pro
Arg Pro Thr Arg Arg Pro Pro Glu Leu Cys 85
90 95Asp Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val
Gly Thr Val Thr 100 105 110Leu
Asp Gln Ile Lys Asp Pro Leu Asp Ile Thr Cys Asn Glu Gly Val 115
120 125Arg Arg Phe Pro Trp Trp Trp Pro Phe
Leu Arg Arg Pro Arg Leu Arg 130 135
140Arg Gln Ala Phe Pro Pro Pro Asn Val Pro Gly Pro Arg Phe Pro Pro145
150 155 160Pro Asn Val Pro
Gly Pro Arg Phe Pro Pro Pro Asn Phe Pro Gly Pro 165
170 175Arg Phe Pro Pro Pro Asn Phe Pro Gly Pro
Arg Phe Pro Pro Pro Asn 180 185
190Phe Pro Gly Pro Pro Phe Pro Pro Pro Ile Phe Pro Gly Pro Trp Phe
195 200 205Pro Pro Pro Pro Pro Phe Arg
Pro Pro Pro Phe Gly Pro Pro Arg Phe 210 215
220Pro Gly Arg Arg22555144PRTBos taurus 55Met Gln Thr Gln Arg Ala
Ser Leu Ser Leu Gly Arg Trp Ser Leu Trp1 5
10 15Leu Leu Leu Leu Gly Leu Val Val Pro Ser Ala Ser
Ala Gln Ala Leu 20 25 30Ser
Tyr Arg Glu Ala Val Leu Arg Ala Val Asp Gln Leu Asn Glu Leu 35
40 45Ser Ser Glu Ala Asn Leu Tyr Arg Leu
Leu Glu Leu Asp Pro Pro Pro 50 55
60Lys Asp Asn Glu Asp Leu Gly Thr Arg Lys Pro Val Ser Phe Thr Val65
70 75 80Lys Glu Thr Val Cys
Pro Arg Thr Ile Gln Gln Pro Ala Glu Gln Cys 85
90 95Asp Phe Lys Glu Lys Gly Arg Val Lys Gln Cys
Val Gly Thr Val Thr 100 105
110Leu Asp Pro Ser Asn Asp Gln Phe Asp Leu Asn Cys Asn Glu Leu Gln
115 120 125Ser Val Ile Leu Pro Trp Lys
Trp Pro Trp Trp Pro Trp Arg Arg Gly 130 135
14056149PRTSus scrofa 56Met Glu Thr Gln Arg Ala Ser Leu Cys Leu Gly
Arg Trp Ser Leu Trp1 5 10
15Leu Leu Leu Leu Ala Leu Val Val Pro Ser Ala Ser Ala Gln Ala Leu
20 25 30Ser Tyr Arg Glu Ala Val Leu
Arg Ala Val Asp Arg Leu Asn Glu Gln 35 40
45Ser Ser Glu Ala Asn Leu Tyr Arg Leu Leu Glu Leu Asp Gln Pro
Pro 50 55 60Lys Ala Asp Glu Asp Pro
Gly Thr Pro Lys Pro Val Ser Phe Thr Val65 70
75 80Lys Glu Thr Val Cys Pro Arg Pro Thr Arg Gln
Pro Pro Glu Leu Cys 85 90
95Asp Phe Lys Glu Asn Gly Arg Val Lys Gln Cys Val Gly Thr Val Thr
100 105 110Leu Asp Gln Ile Lys Asp
Pro Leu Asp Ile Thr Cys Asn Glu Val Gln 115 120
125Gly Val Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe
Cys Val 130 135 140Cys Val Gly Arg
Gly1455717PRTTachypleus gigas 57Lys Trp Cys Phe Arg Val Cys Tyr Arg Gly
Ile Cys Tyr Arg Arg Cys1 5 10
15Arg58102PRTAnopheles gambiae 58Met Lys Cys Ala Thr Ile Val Cys Thr
Ile Ala Val Val Leu Ala Ala1 5 10
15Thr Leu Leu Asn Gly Ser Val Gln Ala Ala Pro Gln Glu Glu Ala
Ala 20 25 30Leu Ser Gly Gly
Ala Asn Leu Asn Thr Leu Leu Asp Glu Leu Pro Glu 35
40 45Glu Thr His His Ala Ala Leu Glu Asn Tyr Arg Ala
Lys Arg Ala Thr 50 55 60Cys Asp Leu
Ala Ser Gly Phe Gly Val Gly Ser Ser Leu Cys Ala Ala65 70
75 80His Cys Ile Ala Arg Arg Tyr Arg
Gly Gly Tyr Cys Asn Ser Lys Ala 85 90
95Val Cys Val Cys Arg Asn 1005970PRTDrosophila
melanogaster 59Met Met Gln Ile Lys Tyr Leu Phe Ala Leu Phe Ala Val Leu
Met Leu1 5 10 15Val Val
Leu Gly Ala Asn Glu Ala Asp Ala Asp Cys Leu Ser Gly Arg 20
25 30Tyr Lys Gly Pro Cys Ala Val Trp Asp
Asn Glu Thr Cys Arg Arg Val 35 40
45Cys Lys Glu Glu Gly Arg Ser Ser Gly His Cys Ser Pro Ser Leu Lys 50
55 60Cys Trp Cys Glu Gly Cys65
706063PRTMedicago truncatula 60Met Thr Lys Ile Val Val Phe Ile Tyr
Val Val Ile Leu Leu Leu Thr1 5 10
15Ile Phe His Val Ser Ala Lys Lys Lys Arg Tyr Ile Glu Cys Glu
Thr 20 25 30His Glu Asp Cys
Ser Gln Val Phe Met Pro Pro Phe Val Met Arg Cys 35
40 45Val Ile His Glu Cys Lys Ile Phe Asn Gly Glu His
Leu Arg Tyr 50 55 606176PRTMedicago
truncatula 61Met Ala Lys Ile Met Lys Phe Val Tyr Asn Met Ile Pro Phe Leu
Ser1 5 10 15Ile Phe Ile
Ile Thr Leu Gln Val Asn Val Val Val Cys Glu Ile Asp 20
25 30Ala Asp Cys Pro Gln Ile Cys Met Pro Pro
Tyr Glu Val Arg Cys Val 35 40
45Asn His Arg Cys Gly Trp Val Asn Thr Asp Asp Ser Leu Phe Leu Thr 50
55 60Gln Glu Phe Thr Arg Ser Lys Gln Tyr
Ile Ile Ser65 70 756276PRTMedicago
truncatula 62Met Tyr Lys Val Val Glu Ser Ile Phe Ile Arg Tyr Met His Arg
Lys1 5 10 15Pro Asn Met
Thr Lys Phe Phe Lys Phe Val Tyr Thr Met Phe Ile Leu 20
25 30Ile Ser Leu Phe Leu Val Val Thr Asn Ala
Asn Ala His Asn Cys Thr 35 40
45Asp Ile Ser Asp Cys Ser Ser Asn His Cys Ser Tyr Glu Gly Val Ser 50
55 60Leu Cys Met Asn Gly Gln Cys Ile Cys
Ile Tyr Glu65 70 756364PRTMedicago
truncatula 63Met Val Glu Thr Leu Arg Leu Phe Tyr Ile Met Ile Leu Phe Val
Ser1 5 10 15Leu Cys Leu
Val Val Val Asp Gly Glu Ser Lys Leu Glu Gln Thr Cys 20
25 30Ser Glu Asp Phe Glu Cys Tyr Ile Lys Asn
Pro His Val Pro Phe Gly 35 40
45His Leu Arg Cys Phe Glu Gly Phe Cys Gln Gln Leu Asn Gly Pro Ala 50
55 606467PRTMedicago truncatula 64Met Ala
Lys Ile Val Asn Phe Val Tyr Ser Met Ile Val Phe Leu Phe1 5
10 15Leu Phe Leu Val Ala Thr Lys Ala
Ala Arg Gly Tyr Leu Cys Val Thr 20 25
30Asp Ser His Cys Pro Pro His Met Cys Pro Pro Gly Met Glu Pro
Arg 35 40 45Cys Val Arg Arg Met
Cys Lys Cys Leu Pro Ile Gly Trp Arg Lys Tyr 50 55
60Phe Val Pro656596PRTMedicago truncatula 65Met Gln Ile Gly
Lys Asn Met Val Glu Thr Pro Lys Leu Asp Tyr Val1 5
10 15Ile Ile Phe Phe Phe Leu Tyr Phe Phe Phe
Arg Gln Met Ile Ile Leu 20 25
30Arg Leu Asn Thr Thr Phe Arg Pro Leu Asn Phe Lys Met Leu Arg Phe
35 40 45Trp Gly Gln Asn Arg Asn Ile Met
Lys His Arg Gly Gln Lys Val His 50 55
60Phe Ser Leu Ile Leu Ser Asp Cys Lys Thr Asn Lys Asp Cys Pro Lys65
70 75 80Leu Arg Arg Ala Asn
Val Arg Cys Arg Lys Ser Tyr Cys Val Pro Ile 85
90 956665PRTMedicago truncatula 66Met Leu Arg Leu
Tyr Leu Val Ser Tyr Phe Leu Leu Lys Arg Thr Leu1 5
10 15Leu Val Ser Tyr Phe Ser Tyr Phe Ser Thr
Tyr Ile Ile Glu Cys Lys 20 25
30Thr Asp Asn Asp Cys Pro Ile Ser Gln Leu Lys Ile Tyr Ala Trp Lys
35 40 45Cys Val Lys Asn Gly Cys His Leu
Phe Asp Val Ile Pro Met Met Tyr 50 55
60Glu656779PRTMedicago truncatula 67Met Ala Glu Ile Leu Lys Phe Val Tyr
Ile Val Ile Leu Phe Val Ser1 5 10
15Leu Leu Leu Ile Val Val Ala Ser Glu Arg Glu Cys Val Thr Asp
Asp 20 25 30Asp Cys Glu Lys
Leu Tyr Pro Thr Asn Glu Tyr Arg Met Met Cys Asp 35
40 45Ser Gly Tyr Cys Met Asn Leu Leu Asn Gly Lys Ile
Ile Tyr Leu Leu 50 55 60Cys Leu Lys
Lys Lys Lys Phe Leu Ile Ile Ile Ser Val Leu Leu65 70
756895PRTMedicago truncatula 68Met Ala Glu Ile Ile Lys Phe
Val Tyr Ile Met Ile Leu Cys Val Ser1 5 10
15Leu Leu Leu Ile Glu Val Ala Gly Glu Glu Cys Val Thr
Asp Ala Asp 20 25 30Cys Asp
Lys Leu Tyr Pro Asp Ile Arg Lys Pro Leu Met Cys Ser Ile 35
40 45Gly Glu Cys Tyr Ser Leu Tyr Lys Gly Lys
Phe Ser Leu Ser Ile Ile 50 55 60Ser
Lys Thr Ser Phe Ser Leu Met Val Tyr Asn Val Val Thr Leu Val65
70 75 80Ile Cys Leu Arg Leu Ala
Tyr Ile Ser Leu Leu Leu Lys Phe Leu 85 90
9569100PRTMedicago truncatula 69Met Ala Glu Ile Leu Lys
Asp Phe Tyr Ala Met Asn Leu Phe Ile Phe1 5
10 15Leu Ile Ile Leu Pro Ala Lys Ile Arg Gly Glu Thr
Leu Ser Leu Thr 20 25 30His
Pro Lys Cys His His Ile Met Leu Pro Ser Leu Phe Ile Thr Glu 35
40 45Val Phe Gln Arg Val Thr Asp Asp Gly
Cys Pro Lys Pro Val Asn His 50 55
60Leu Arg Val Val Lys Cys Ile Glu His Ile Cys Glu Tyr Gly Tyr Asn65
70 75 80Tyr Arg Pro Asp Phe
Ala Ser Gln Ile Pro Glu Ser Thr Lys Met Pro 85
90 95Arg Lys Arg Glu 1007078PRTMedicago
truncatula 70Met Val Glu Ile Leu Lys Asn Phe Tyr Ala Met Asn Leu Phe Ile
Phe1 5 10 15Leu Ile Ile
Leu Ala Val Lys Ile Arg Gly Ala His Phe Pro Cys Val 20
25 30Thr Asp Asp Asp Cys Pro Lys Pro Val Asn
Lys Leu Arg Val Ile Lys 35 40
45Cys Ile Asp His Ile Cys Gln Tyr Ala Arg Asn Leu Pro Asp Phe Ala 50
55 60Ser Glu Ile Ser Glu Ser Thr Lys Met
Pro Cys Lys Gly Glu65 70
757172PRTMedicago truncatula 71Met Phe His Ala Gln Ala Glu Asn Met Ala
Lys Val Ser Asn Phe Val1 5 10
15Cys Ile Met Ile Leu Phe Leu Ala Leu Phe Phe Ile Thr Met Asn Asp
20 25 30Ala Ala Arg Phe Glu Cys
Arg Glu Asp Ser His Cys Val Thr Arg Ile 35 40
45Lys Cys Val Leu Pro Arg Lys Pro Glu Cys Arg Asn Tyr Ala
Cys Gly 50 55 60Cys Tyr Asp Ser Asn
Lys Tyr Arg65 707278PRTMedicago truncatula 72Met Gln Met
Arg Gln Asn Met Ala Thr Ile Leu Asn Phe Val Phe Val1 5
10 15Ile Ile Leu Phe Ile Ser Leu Leu Leu
Val Val Thr Lys Gly Tyr Arg 20 25
30Glu Pro Phe Ser Ser Phe Thr Glu Gly Pro Thr Cys Lys Glu Asp Ile
35 40 45Asp Cys Pro Ser Ile Ser Cys
Val Asn Pro Gln Val Pro Lys Cys Ile 50 55
60Met Phe Glu Cys His Cys Lys Tyr Ile Pro Thr Thr Leu Lys65
70 757371PRTMedicago truncatula 73Met Ala Thr
Ile Leu Met Tyr Val Tyr Ile Thr Ile Leu Phe Ile Ser1 5
10 15Ile Leu Thr Val Leu Thr Glu Gly Leu
Tyr Glu Pro Leu Tyr Asn Phe 20 25
30Arg Arg Asp Pro Asp Cys Arg Arg Asn Ile Asp Cys Pro Ser Tyr Leu
35 40 45Cys Val Ala Pro Lys Val Pro
Arg Cys Ile Met Phe Glu Cys His Cys 50 55
60Lys Asp Ile Pro Ser Asp His65 707457PRTMedicago
truncatula 74Met Thr Thr Ser Leu Lys Phe Val Tyr Val Ala Ile Leu Phe Leu
Ser1 5 10 15Leu Leu Leu
Val Val Met Gly Gly Ile Arg Arg Phe Glu Cys Arg Gln 20
25 30Asp Ser Asp Cys Pro Ser Tyr Phe Cys Glu
Lys Leu Thr Val Pro Lys 35 40
45Cys Phe Trp Ser Lys Cys Tyr Cys Lys 50
557557PRTMedicago truncatula 75Met Thr Thr Ser Leu Lys Phe Val Tyr Val
Ala Ile Leu Phe Leu Ser1 5 10
15Leu Leu Leu Val Val Met Gly Gly Ile Arg Lys Lys Glu Cys Arg Gln
20 25 30Asp Ser Asp Cys Pro Ser
Tyr Phe Cys Glu Lys Leu Thr Ile Ala Lys 35 40
45Cys Ile His Ser Thr Cys Leu Cys Lys 50
557666PRTMedicago truncatula 76Met Gln Ile Gly Lys Asn Met Val Glu Thr
Pro Lys Leu Val Tyr Phe1 5 10
15Ile Ile Leu Phe Leu Ser Ile Phe Leu Cys Ile Thr Val Ser Asn Ser
20 25 30Ser Phe Ser Gln Ile Phe
Asn Ser Ala Cys Lys Thr Asp Lys Asp Cys 35 40
45Pro Lys Phe Gly Arg Val Asn Val Arg Cys Arg Lys Gly Asn
Cys Val 50 55 60Pro
Ile657757PRTMedicago truncatula 77Met Thr Ala Ile Leu Lys Lys Phe Ile Asn
Ala Val Phe Leu Phe Ile1 5 10
15Val Leu Phe Leu Ala Thr Thr Asn Val Glu Asp Phe Val Gly Gly Ser
20 25 30Asn Asp Glu Cys Val Tyr
Pro Asp Val Phe Gln Cys Ile Asn Asn Ile 35 40
45Cys Lys Cys Val Ser His His Arg Thr 50
557874PRTMedicago truncatula 78Met Gln Lys Arg Lys Asn Met Ala Gln Ile
Ile Phe Tyr Val Tyr Ala1 5 10
15Leu Ile Ile Leu Phe Ser Pro Phe Leu Ala Ala Arg Leu Val Phe Val
20 25 30Asn Pro Glu Lys Pro Cys
Val Thr Asp Ala Asp Cys Asp Arg Tyr Arg 35 40
45His Glu Ser Ala Ile Tyr Ser Asp Met Phe Cys Lys Asp Gly
Tyr Cys 50 55 60Phe Ile Asp Tyr His
His Asp Pro Tyr Pro65 707976PRTMedicago truncatula 79Met
Gln Met Arg Lys Asn Met Ala Gln Ile Leu Phe Tyr Val Tyr Ala1
5 10 15Leu Leu Ile Leu Phe Thr Pro
Phe Leu Val Ala Arg Ile Met Val Val 20 25
30Asn Pro Asn Asn Pro Cys Val Thr Asp Ala Asp Cys Gln Arg
Tyr Arg 35 40 45His Lys Leu Ala
Thr Arg Met Ile Cys Asn Gln Gly Phe Cys Leu Met 50 55
60Asp Phe Thr His Asp Pro Tyr Ala Pro Ser Leu Pro65
70 758064PRTMedicago truncatula 80Met Asn
His Ile Ser Lys Phe Val Tyr Ala Leu Ile Ile Phe Leu Ser1 5
10 15Ile Tyr Leu Val Val Leu Asp Gly
Leu Pro Ile Ser Cys Lys Asp His 20 25
30Phe Glu Cys Arg Arg Lys Ile Asn Ile Leu Arg Cys Ile Tyr Arg
Gln 35 40 45Glu Lys Pro Met Cys
Ile Asn Ser Ile Cys Thr Cys Val Lys Leu Leu 50 55
608167PRTMedicago truncatula 81Met Gln Arg Glu Lys Asn Met
Ala Lys Ile Phe Glu Phe Val Tyr Ala1 5 10
15Met Ile Ile Phe Ile Leu Leu Phe Leu Val Glu Lys Asn
Val Val Ala 20 25 30Tyr Leu
Lys Phe Glu Cys Lys Thr Asp Asp Asp Cys Gln Lys Ser Leu 35
40 45Leu Lys Thr Tyr Val Trp Lys Cys Val Lys
Asn Glu Cys Tyr Phe Phe 50 55 60Ala
Lys Lys658261PRTMedicago truncatula 82Met Ala Gly Ile Ile Lys Phe Val His
Val Leu Ile Ile Phe Leu Ser1 5 10
15Leu Phe His Val Val Lys Asn Asp Asp Gly Ser Phe Cys Phe Lys
Asp 20 25 30Ser Asp Cys Pro
Asp Glu Met Cys Pro Ser Pro Leu Lys Glu Met Cys 35
40 45Tyr Phe Leu Gln Cys Lys Cys Gly Val Asp Thr Ile
Ala 50 55 608359PRTMedicago
truncatula 83Met Ala Asn Thr His Lys Leu Val Ser Met Ile Leu Phe Ile Phe
Leu1 5 10 15Phe Leu Ala
Ser Asn Asn Val Glu Gly Tyr Val Asn Cys Glu Thr Asp 20
25 30Ala Asp Cys Pro Pro Ser Thr Arg Val Lys
Arg Phe Lys Cys Val Lys 35 40
45Gly Glu Cys Arg Trp Thr Arg Met Ser Tyr Ala 50
558463PRTMedicago truncatula 84Met Gln Arg Arg Lys Lys Lys Ala Gln Val
Val Met Phe Val His Asp1 5 10
15Leu Ile Ile Cys Ile Tyr Leu Phe Ile Val Ile Thr Thr Arg Lys Thr
20 25 30Asp Ile Arg Cys Arg Phe
Tyr Tyr Asp Cys Pro Arg Leu Glu Tyr His 35 40
45Phe Cys Glu Cys Ile Glu Asp Phe Cys Ala Tyr Ile Arg Leu
Asn 50 55 608557PRTMedicago
truncatula 85Met Ala Lys Val Tyr Met Phe Val Tyr Ala Leu Ile Ile Phe Val
Ser1 5 10 15Pro Phe Leu
Leu Ala Thr Phe Arg Thr Arg Leu Pro Cys Glu Lys Asp 20
25 30Asp Asp Cys Pro Glu Ala Phe Leu Pro Pro
Val Met Lys Cys Val Asn 35 40
45Arg Phe Cys Gln Tyr Glu Ile Leu Glu 50
558677PRTMedicago truncatula 86Met Ile Lys Gln Phe Ser Val Cys Tyr Ile
Gln Met Arg Arg Asn Met1 5 10
15Thr Thr Ile Leu Lys Phe Pro Tyr Ile Met Val Ile Cys Leu Leu Leu
20 25 30Leu His Val Ala Ala Tyr
Glu Asp Phe Glu Lys Glu Ile Phe Asp Cys 35 40
45Lys Lys Asp Gly Asp Cys Asp His Met Cys Val Thr Pro Gly
Ile Pro 50 55 60Lys Cys Thr Gly Tyr
Val Cys Phe Cys Phe Glu Asn Leu65 70
758773PRTMedicago truncatula 87Met Gln Arg Ser Arg Asn Met Thr Thr Ile
Phe Lys Phe Ala Tyr Ile1 5 10
15Met Ile Ile Cys Val Phe Leu Leu Asn Ile Ala Ala Gln Glu Ile Glu
20 25 30Asn Gly Ile His Pro Cys
Lys Lys Asn Glu Asp Cys Asn His Met Cys 35 40
45Val Met Pro Gly Leu Pro Trp Cys His Glu Asn Asn Leu Cys
Phe Cys 50 55 60Tyr Glu Asn Ala Tyr
Gly Asn Thr Arg65 708885PRTMedicago truncatula 88Met Thr
Ile Ile Ile Lys Phe Val Asn Val Leu Ile Ile Phe Leu Ser1 5
10 15Leu Phe His Val Ala Lys Asn Asp
Asp Asn Lys Leu Leu Leu Ser Phe 20 25
30Ile Glu Glu Gly Phe Leu Cys Phe Lys Asp Ser Asp Cys Pro Tyr
Asn 35 40 45Met Cys Pro Ser Pro
Leu Lys Glu Met Cys Tyr Phe Ile Lys Cys Val 50 55
60Cys Gly Val Tyr Gly Pro Ile Arg Glu Arg Arg Leu Tyr Gln
Ser His65 70 75 80Asn
Pro Met Ile Gln 858969PRTMedicago truncatula 89Met Arg Lys
Asn Met Thr Lys Ile Leu Met Ile Gly Tyr Ala Leu Met1 5
10 15Ile Phe Ile Phe Leu Ser Ile Ala Val
Ser Ile Thr Gly Asn Leu Ala 20 25
30Arg Ala Ser Arg Lys Lys Pro Val Asp Val Ile Pro Cys Ile Tyr Asp
35 40 45His Asp Cys Pro Arg Lys Leu
Tyr Phe Leu Glu Arg Cys Val Gly Arg 50 55
60Val Cys Lys Tyr Leu659058PRTMedicago truncatula 90Met Ala His Lys
Leu Val Tyr Ala Ile Thr Leu Phe Ile Phe Leu Phe1 5
10 15Leu Ile Ala Asn Asn Ile Glu Asp Asp Ile
Phe Cys Ile Thr Asp Asn 20 25
30Asp Cys Pro Pro Asn Thr Leu Val Gln Arg Tyr Arg Cys Ile Asn Gly
35 40 45Lys Cys Asn Leu Ser Phe Val Ser
Tyr Gly 50 559161PRTMedicago truncatula 91Met Asp Glu
Thr Leu Lys Phe Val Tyr Ile Leu Ile Leu Phe Val Ser1 5
10 15Leu Cys Leu Val Val Ala Asp Gly Val
Lys Asn Ile Asn Arg Glu Cys 20 25
30Thr Gln Thr Ser Asp Cys Tyr Lys Lys Tyr Pro Phe Ile Pro Trp Gly
35 40 45Lys Val Arg Cys Val Lys Gly
Arg Cys Arg Leu Asp Met 50 55
609262PRTMedicago truncatula 92Met Ala Lys Ile Ile Lys Phe Val Tyr Val
Leu Ala Ile Phe Phe Ser1 5 10
15Leu Phe Leu Val Ala Lys Asn Val Asn Gly Trp Thr Cys Val Glu Asp
20 25 30Ser Asp Cys Pro Ala Asn
Ile Cys Gln Pro Pro Met Gln Arg Met Cys 35 40
45Phe Tyr Gly Glu Cys Ala Cys Val Arg Ser Lys Phe Cys Thr
50 55 609361PRTMedicago truncatula
93Met Val Lys Ile Ile Lys Phe Val Tyr Phe Met Thr Leu Phe Leu Ser1
5 10 15Met Leu Leu Val Thr Thr
Lys Glu Asp Gly Ser Val Glu Cys Ile Ala 20 25
30Asn Ile Asp Cys Pro Gln Ile Phe Met Leu Pro Phe Val
Met Arg Cys 35 40 45Ile Asn Phe
Arg Cys Gln Ile Val Asn Ser Glu Asp Thr 50 55
609467PRTMedicago truncatula 94Met Asp Glu Ile Leu Lys Phe Val
Tyr Thr Leu Ile Ile Phe Phe Ser1 5 10
15Leu Phe Phe Ala Ala Asn Asn Val Asp Ala Asn Ile Met Asn
Cys Gln 20 25 30Ser Thr Phe
Asp Cys Pro Arg Asp Met Cys Ser His Ile Arg Asp Val 35
40 45Ile Cys Ile Phe Lys Lys Cys Lys Cys Ala Gly
Gly Arg Tyr Met Pro 50 55 60Gln Val
Pro659562PRTMedicago truncatula 95Met Gln Arg Arg Lys Asn Met Ala Asn Asn
His Met Leu Ile Tyr Ala1 5 10
15Met Ile Ile Cys Leu Phe Pro Tyr Leu Val Val Thr Phe Lys Thr Ala
20 25 30Ile Thr Cys Asp Cys Asn
Glu Asp Cys Leu Asn Phe Phe Thr Pro Leu 35 40
45Asp Asn Leu Lys Cys Ile Asp Asn Val Cys Glu Val Phe Met
50 55 609665PRTMedicago truncatula
96Met Val Asn Ile Leu Lys Phe Ile Tyr Val Ile Ile Phe Phe Ile Leu1
5 10 15Met Phe Phe Val Leu Ile
Asp Val Asp Gly His Val Leu Val Glu Cys 20 25
30Ile Glu Asn Arg Asp Cys Glu Lys Gly Met Cys Lys Phe
Pro Phe Ile 35 40 45Val Arg Cys
Leu Met Asp Gln Cys Lys Cys Val Arg Ile His Asn Leu 50
55 60Ile659774PRTMedicago truncatula 97Met Ile Ile Gln
Phe Ser Ile Tyr Tyr Met Gln Arg Arg Lys Leu Asn1 5
10 15Met Val Glu Ile Leu Lys Phe Ser His Ala
Leu Ile Ile Phe Leu Phe 20 25
30Leu Ser Ala Leu Val Thr Asn Ala Asn Ile Phe Phe Cys Ser Thr Asp
35 40 45Glu Asp Cys Thr Trp Asn Leu Cys
Arg Gln Pro Trp Val Gln Lys Cys 50 55
60Arg Leu His Met Cys Ser Cys Glu Lys Asn65
709858PRTMedicago truncatula 98Met Asp Glu Val Phe Lys Phe Val Tyr Val
Met Ile Ile Phe Pro Phe1 5 10
15Leu Ile Leu Asp Val Ala Thr Asn Ala Glu Lys Ile Arg Arg Cys Phe
20 25 30Asn Asp Ala His Cys Pro
Pro Asp Met Cys Thr Leu Gly Val Ile Pro 35 40
45Lys Cys Ser Arg Phe Thr Ile Cys Ile Cys 50
559964PRTMedicago truncatula 99Met His Arg Lys Pro Asn Met Thr Lys
Phe Phe Lys Phe Val Tyr Thr1 5 10
15Met Phe Ile Leu Ile Ser Leu Phe Leu Val Val Thr Asn Ala Asn
Ala 20 25 30Asn Asn Cys Thr
Asp Thr Ser Asp Cys Ser Ser Asn His Cys Ser Tyr 35
40 45Glu Gly Val Ser Leu Cys Met Asn Gly Gln Cys Ile
Cys Ile Tyr Glu 50 55
6010069PRTMedicago truncatula 100Met Gln Met Lys Lys Met Ala Thr Ile Leu
Lys Phe Val Tyr Leu Ile1 5 10
15Ile Leu Leu Ile Tyr Pro Leu Leu Val Val Thr Glu Glu Ser His Tyr
20 25 30Met Lys Phe Ser Ile Cys
Lys Asp Asp Thr Asp Cys Pro Thr Leu Phe 35 40
45Cys Val Leu Pro Asn Val Pro Lys Cys Ile Gly Ser Lys Cys
His Cys 50 55 60Lys Leu Met Val
Asn6510164PRTMedicago truncatula 101Met Val Glu Thr Leu Arg Leu Phe Tyr
Ile Met Ile Leu Phe Val Ser1 5 10
15Leu Tyr Leu Val Val Val Asp Gly Val Ser Lys Leu Ala Gln Ser
Cys 20 25 30Ser Glu Asp Phe
Glu Cys Tyr Ile Lys Asn Pro His Ala Pro Phe Gly 35
40 45Gln Leu Arg Cys Phe Glu Gly Tyr Cys Gln Arg Leu
Asp Lys Pro Thr 50 55
6010263PRTMedicago truncatula 102Met Thr Thr Phe Leu Lys Val Ala Tyr Ile
Met Ile Ile Cys Val Phe1 5 10
15Val Leu His Leu Ala Ala Gln Val Asp Ser Gln Lys Arg Leu His Gly
20 25 30Cys Lys Glu Asp Arg Asp
Cys Asp Asn Ile Cys Ser Val His Ala Val 35 40
45Thr Lys Cys Ile Gly Asn Met Cys Arg Cys Leu Ala Asn Val
Lys 50 55 6010361PRTMedicago
truncatula 103Met Arg Ile Asn Arg Thr Pro Ala Ile Phe Lys Phe Val Tyr Thr
Ile1 5 10 15Ile Ile Tyr
Leu Phe Leu Leu Arg Val Val Ala Lys Asp Leu Pro Phe 20
25 30Asn Ile Cys Glu Lys Asp Glu Asp Cys Leu
Glu Phe Cys Ala His Asp 35 40
45Lys Val Ala Lys Cys Met Leu Asn Ile Cys Phe Cys Phe 50
55 6010454PRTMedicago truncatula 104Met Ala Glu Ile
Leu Lys Ile Leu Tyr Val Phe Ile Ile Phe Leu Ser1 5
10 15Leu Ile Leu Ala Val Ile Ser Gln His Pro
Phe Thr Pro Cys Glu Thr 20 25
30Asn Ala Asp Cys Lys Cys Arg Asn His Lys Arg Pro Asp Cys Leu Trp
35 40 45His Lys Cys Tyr Cys Tyr
5010565PRTMedicago truncatula 105Met Arg Lys Ser Met Ala Thr Ile Leu Lys
Phe Val Tyr Val Ile Met1 5 10
15Leu Phe Ile Tyr Ser Leu Phe Val Ile Glu Ser Phe Gly His Arg Phe
20 25 30Leu Ile Tyr Asn Asn Cys
Lys Asn Asp Thr Glu Cys Pro Asn Asp Cys 35 40
45Gly Pro His Glu Gln Ala Lys Cys Ile Leu Tyr Ala Cys Tyr
Cys Val 50 55
60Glu6510658PRTMedicago truncatula 106Met Asn Thr Ile Leu Lys Phe Ile Phe
Val Val Phe Leu Phe Leu Ser1 5 10
15Ile Phe Leu Ser Ala Gly Asn Ser Lys Ser Tyr Gly Pro Cys Thr
Thr 20 25 30Leu Gln Asp Cys
Glu Thr His Asn Trp Phe Glu Val Cys Ser Cys Ile 35
40 45Asp Phe Glu Cys Lys Cys Trp Ser Leu Leu 50
5510764PRTMedicago truncatula 107Met Ala Glu Ile Ile Lys Phe
Val Tyr Ile Met Ile Leu Cys Val Ser1 5 10
15Leu Leu Leu Ile Ala Glu Ala Ser Gly Lys Glu Cys Val
Thr Asp Ala 20 25 30Asp Cys
Glu Asn Leu Tyr Pro Gly Asn Lys Lys Pro Met Phe Cys Asn 35
40 45Asn Thr Gly Tyr Cys Met Ser Leu Tyr Lys
Glu Pro Ser Arg Tyr Met 50 55
6010864PRTMedicago truncatula 108Met Ala Lys Ile Ile Lys Phe Val Tyr Ile
Met Ile Leu Cys Val Ser1 5 10
15Leu Leu Leu Ile Val Glu Ala Gly Gly Lys Glu Cys Val Thr Asp Val
20 25 30Asp Cys Glu Lys Ile Tyr
Pro Gly Asn Lys Lys Pro Leu Ile Cys Ser 35 40
45Thr Gly Tyr Cys Tyr Ser Leu Tyr Glu Glu Pro Pro Arg Tyr
His Lys 50 55 6010964PRTMedicago
truncatula 109Met Ala Lys Val Thr Lys Phe Gly Tyr Ile Ile Ile His Phe Leu
Ser1 5 10 15Leu Phe Phe
Leu Ala Met Asn Val Ala Gly Gly Arg Glu Cys His Ala 20
25 30Asn Ser His Cys Val Gly Lys Ile Thr Cys
Val Leu Pro Gln Lys Pro 35 40
45Glu Cys Trp Asn Tyr Ala Cys Val Cys Tyr Asp Ser Asn Lys Tyr Arg 50
55 6011055PRTMedicago truncatula 110Met
Ala Lys Ile Phe Asn Tyr Val Tyr Ala Leu Ile Met Phe Leu Ser1
5 10 15Leu Phe Leu Met Gly Thr Ser
Gly Met Lys Asn Gly Cys Lys His Thr 20 25
30Gly His Cys Pro Arg Lys Met Cys Gly Ala Lys Thr Thr Lys
Cys Arg 35 40 45Asn Asn Lys Cys
Gln Cys Val 50 5511169PRTMedicago truncatula 111Met
Thr Glu Ile Leu Lys Phe Val Cys Val Met Ile Ile Phe Ile Ser1
5 10 15Ser Phe Ile Val Ser Lys Ser
Leu Asn Gly Gly Gly Lys Asp Lys Cys 20 25
30Phe Arg Asp Ser Asp Cys Pro Lys His Met Cys Pro Ser Ser
Leu Val 35 40 45Ala Lys Cys Ile
Asn Arg Leu Cys Arg Cys Arg Arg Pro Glu Leu Gln 50 55
60Val Gln Leu Asn Pro6511269PRTMedicago truncatula
112Met Ala His Ile Ile Met Phe Val Tyr Ala Leu Ile Tyr Ala Leu Ile1
5 10 15Ile Phe Ser Ser Leu Phe
Val Arg Asp Gly Ile Pro Cys Leu Ser Asp 20 25
30Asp Glu Cys Pro Glu Met Ser His Tyr Ser Phe Lys Cys
Asn Asn Lys 35 40 45Ile Cys Glu
Tyr Asp Leu Gly Glu Met Ser Asp Asp Asp Tyr Tyr Leu 50
55 60Glu Met Ser Arg Glu6511377PRTMedicago truncatula
113Met Tyr Arg Glu Lys Asn Met Ala Lys Thr Leu Lys Phe Val Tyr Val1
5 10 15Ile Val Leu Phe Leu Ser
Leu Phe Leu Ala Ala Lys Asn Ile Asp Gly 20 25
30Arg Val Ser Tyr Asn Ser Phe Ile Ala Leu Pro Val Cys
Gln Thr Ala 35 40 45Ala Asp Cys
Pro Glu Gly Thr Arg Gly Arg Thr Tyr Lys Cys Ile Asn 50
55 60Asn Lys Cys Arg Tyr Pro Lys Leu Leu Lys Pro Ile
Gln65 70 7511456PRTMedicago truncatula
114Met Ala His Ile Phe Asn Tyr Val Tyr Ala Leu Leu Val Phe Leu Ser1
5 10 15Leu Phe Leu Met Val Thr
Asn Gly Ile His Ile Gly Cys Asp Lys Asp 20 25
30Arg Asp Cys Pro Lys Gln Met Cys His Leu Asn Gln Thr
Pro Lys Cys 35 40 45Leu Lys Asn
Ile Cys Lys Cys Val 50 5511577PRTMedicago truncatula
115Met Ala Glu Ile Leu Lys Cys Phe Tyr Thr Met Asn Leu Phe Ile Phe1
5 10 15Leu Ile Ile Leu Pro Ala
Lys Ile Arg Glu His Ile Gln Cys Val Ile 20 25
30Asp Asp Asp Cys Pro Lys Ser Leu Asn Lys Leu Leu Ile
Ile Lys Cys 35 40 45Ile Asn His
Val Cys Gln Tyr Val Gly Asn Leu Pro Asp Phe Ala Ser 50
55 60Gln Ile Pro Lys Ser Thr Lys Met Pro Tyr Lys Gly
Glu65 70 7511670PRTMedicago truncatula
116Met Ala Tyr Ile Ser Arg Ile Phe Tyr Val Leu Ile Ile Phe Leu Ser1
5 10 15Leu Phe Phe Val Val Ile
Asn Gly Val Lys Ser Leu Leu Leu Ile Lys 20 25
30Val Arg Ser Phe Ile Pro Cys Gln Arg Ser Asp Asp Cys
Pro Arg Asn 35 40 45Leu Cys Val
Asp Gln Ile Ile Pro Thr Cys Val Trp Ala Lys Cys Lys 50
55 60Cys Lys Asn Tyr Asn Asp65
7011764PRTMedicago truncatula 117Met Ala Asn Val Thr Lys Phe Val Tyr Ile
Ala Ile Tyr Phe Leu Ser1 5 10
15Leu Phe Phe Ile Ala Lys Asn Asp Ala Thr Ala Thr Phe Cys His Asp
20 25 30Asp Ser His Cys Val Thr
Lys Ile Lys Cys Val Leu Pro Arg Thr Pro 35 40
45Gln Cys Arg Asn Glu Ala Cys Gly Cys Tyr His Ser Asn Lys
Phe Arg 50 55 6011862PRTMedicago
truncatula 118Met Gly Glu Ile Met Lys Phe Val Tyr Val Met Ile Ile Tyr Leu
Phe1 5 10 15Met Phe Asn
Val Ala Thr Gly Ser Glu Phe Ile Phe Thr Lys Lys Leu 20
25 30Thr Ser Cys Asp Ser Ser Lys Asp Cys Arg
Ser Phe Leu Cys Tyr Ser 35 40
45Pro Lys Phe Pro Val Cys Lys Arg Gly Ile Cys Glu Cys Ile 50
55 6011961PRTMedicago truncatula 119Met Gly Glu
Met Phe Lys Phe Ile Tyr Thr Phe Ile Leu Phe Val His1 5
10 15Leu Phe Leu Val Val Ile Phe Glu Asp
Ile Gly His Ile Lys Tyr Cys 20 25
30Gly Ile Val Asp Asp Cys Tyr Lys Ser Lys Lys Pro Leu Phe Lys Ile
35 40 45Trp Lys Cys Val Glu Asn Val
Cys Val Leu Trp Tyr Lys 50 55
6012063PRTMedicago truncatula 120Met Ala Arg Thr Leu Lys Phe Val Tyr Ser
Met Ile Leu Phe Leu Ser1 5 10
15Leu Phe Leu Val Ala Asn Gly Leu Lys Ile Phe Cys Ile Asp Val Ala
20 25 30Asp Cys Pro Lys Asp Leu
Tyr Pro Leu Leu Tyr Lys Cys Ile Tyr Asn 35 40
45Lys Cys Ile Val Phe Thr Arg Ile Pro Phe Pro Phe Asp Trp
Ile 50 55 6012166PRTMedicago
truncatula 121Met Ala Asn Ile Thr Lys Phe Val Tyr Ile Ala Ile Leu Phe Leu
Ser1 5 10 15Leu Phe Phe
Ile Gly Met Asn Asp Ala Ala Ile Leu Glu Cys Arg Glu 20
25 30Asp Ser His Cys Val Thr Lys Ile Lys Cys
Val Leu Pro Arg Lys Pro 35 40
45Glu Cys Arg Asn Asn Ala Cys Thr Cys Tyr Lys Gly Gly Phe Ser Phe 50
55 60His His6512268PRTMedicago truncatula
122Met Gln Arg Val Lys Lys Met Ser Glu Thr Leu Lys Phe Val Tyr Val1
5 10 15Leu Ile Leu Phe Ile Ser
Ile Phe His Val Val Ile Val Cys Asp Ser 20 25
30Ile Tyr Phe Pro Val Ser Arg Pro Cys Ile Thr Asp Lys
Asp Cys Pro 35 40 45Asn Met Lys
His Tyr Lys Ala Lys Cys Arg Lys Gly Phe Cys Ile Ser 50
55 60Ser Arg Val Arg6512372PRTMedicago truncatula
123Met Gln Ile Arg Lys Ile Met Ser Gly Val Leu Lys Phe Val Tyr Ala1
5 10 15Ile Ile Leu Phe Leu Phe
Leu Phe Leu Val Ala Arg Glu Val Gly Gly 20 25
30Leu Glu Thr Ile Glu Cys Glu Thr Asp Gly Asp Cys Pro
Arg Ser Met 35 40 45Ile Lys Met
Trp Asn Lys Asn Tyr Arg His Lys Cys Ile Asp Gly Lys 50
55 60Cys Glu Trp Ile Lys Lys Leu Pro65
7012454PRTMedicago truncatula 124Met Phe Val Tyr Asp Leu Ile Leu Phe Ile
Ser Leu Ile Leu Val Val1 5 10
15Thr Gly Ile Asn Ala Glu Ala Asp Thr Ser Cys His Ser Phe Asp Asp
20 25 30Cys Pro Trp Val Ala His
His Tyr Arg Glu Cys Ile Glu Gly Leu Cys 35 40
45Ala Tyr Arg Ile Leu Tyr 5012569PRTMedicago truncatula
125Met Gln Arg Arg Lys Lys Ser Met Ala Lys Met Leu Lys Phe Phe Phe1
5 10 15Ala Ile Ile Leu Leu Leu
Ser Leu Phe Leu Val Ala Thr Glu Val Gly 20 25
30Gly Ala Tyr Ile Glu Cys Glu Val Asp Asp Asp Cys Pro
Lys Pro Met 35 40 45Lys Asn Ser
His Pro Asp Thr Tyr Tyr Lys Cys Val Lys His Arg Cys 50
55 60Gln Trp Ala Trp Lys65126140PRTMedicago truncatula
126Met Phe Val Tyr Thr Leu Ile Ile Phe Leu Phe Pro Ser His Val Ile1
5 10 15Thr Asn Lys Ile Ala Ile
Tyr Cys Val Ser Asp Asp Asp Cys Leu Lys 20 25
30Thr Phe Thr Pro Leu Asp Leu Lys Cys Val Asp Asn Val
Cys Glu Phe 35 40 45Asn Leu Arg
Cys Lys Gly Lys Cys Gly Glu Arg Asp Glu Lys Phe Val 50
55 60Phe Leu Lys Ala Leu Lys Lys Met Asp Gln Lys Leu
Val Leu Glu Glu65 70 75
80Gln Gly Asn Ala Arg Glu Val Lys Ile Pro Lys Lys Leu Leu Phe Asp
85 90 95Arg Ile Gln Val Pro Thr
Pro Ala Thr Lys Asp Gln Val Glu Glu Asp 100
105 110Asp Tyr Asp Asp Asp Asp Glu Glu Glu Glu Glu Glu
Glu Asp Asp Val 115 120 125Asp Met
Trp Phe His Leu Pro Asp Val Val Cys His 130 135
14012760PRTMedicago truncatula 127Met Ala Lys Phe Ser Met Phe
Val Tyr Ala Leu Ile Asn Phe Leu Ser1 5 10
15Leu Phe Leu Val Glu Thr Ala Ile Thr Asn Ile Arg Cys
Val Ser Asp 20 25 30Asp Asp
Cys Pro Lys Val Ile Lys Pro Leu Val Met Lys Cys Ile Gly 35
40 45Asn Tyr Cys Tyr Phe Phe Met Ile Tyr Glu
Gly Pro 50 55 6012858PRTMedicago
truncatula 128Met Ala His Lys Phe Val Tyr Ala Ile Ile Leu Phe Ile Phe Leu
Phe1 5 10 15Leu Val Ala
Lys Asn Val Lys Gly Tyr Val Val Cys Arg Thr Val Asp 20
25 30Asp Cys Pro Pro Asp Thr Arg Asp Leu Arg
Tyr Arg Cys Leu Asn Gly 35 40
45Lys Cys Lys Ser Tyr Arg Leu Ser Tyr Gly 50
5512961PRTMedicago truncatula 129Met Gln Arg Lys Lys Asn Met Gly Gln Ile
Leu Ile Phe Val Phe Ala1 5 10
15Leu Ile Asn Phe Leu Ser Pro Ile Leu Val Glu Met Thr Thr Thr Thr
20 25 30Ile Pro Cys Thr Phe Ile
Asp Asp Cys Pro Lys Met Pro Leu Val Val 35 40
45Lys Cys Ile Asp Asn Phe Cys Asn Tyr Phe Glu Ile Lys 50
55 6013057PRTMedicago truncatula 130Met
Ala Gln Thr Leu Met Leu Val Tyr Ala Leu Ile Ile Phe Thr Ser1
5 10 15Leu Phe Leu Val Val Ile Ser
Arg Gln Thr Asp Ile Pro Cys Lys Ser 20 25
30Asp Asp Ala Cys Pro Arg Val Ser Ser His His Ile Glu Cys
Val Lys 35 40 45Gly Phe Cys Thr
Tyr Trp Lys Leu Asp 50 5513177PRTMedicago truncatula
131Met Leu Arg Arg Lys Asn Thr Val Gln Ile Leu Met Phe Val Ser Ala1
5 10 15Leu Leu Ile Tyr Ile Phe
Leu Phe Leu Val Ile Thr Ser Ser Ala Asn 20 25
30Ile Pro Cys Asn Ser Asp Ser Asp Cys Pro Trp Lys Ile
Tyr Tyr Thr 35 40 45Tyr Arg Cys
Asn Asp Gly Phe Cys Val Tyr Lys Ser Ile Asp Pro Ser 50
55 60Thr Ile Pro Gln Tyr Met Thr Asp Leu Ile Phe Pro
Arg65 70 7513259PRTMedicago truncatula
132Met Ala Val Ile Leu Lys Phe Val Tyr Ile Met Ile Ile Phe Leu Phe1
5 10 15Leu Leu Tyr Val Val Asn
Gly Thr Arg Cys Asn Arg Asp Glu Asp Cys 20 25
30Pro Phe Ile Cys Thr Gly Pro Gln Ile Pro Lys Cys Val
Ser His Ile 35 40 45Cys Phe Cys
Leu Ser Ser Gly Lys Glu Ala Tyr 50 5513362PRTMedicago
truncatula 133Met Asp Ala Ile Leu Lys Phe Ile Tyr Ala Met Phe Leu Phe Leu
Phe1 5 10 15Leu Phe Val
Thr Thr Arg Asn Val Glu Ala Leu Phe Glu Cys Asn Arg 20
25 30Asp Phe Val Cys Gly Asn Asp Asp Glu Cys
Val Tyr Pro Tyr Ala Val 35 40
45Gln Cys Ile His Arg Tyr Cys Lys Cys Leu Lys Ser Arg Asn 50
55 6013467PRTMedicago truncatula 134Met Gln Ile
Gly Arg Lys Lys Met Gly Glu Thr Pro Lys Leu Val Tyr1 5
10 15Val Ile Ile Leu Phe Leu Ser Ile Phe
Leu Cys Thr Asn Ser Ser Phe 20 25
30Ser Gln Met Ile Asn Phe Arg Gly Cys Lys Arg Asp Lys Asp Cys Pro
35 40 45Gln Phe Arg Gly Val Asn Ile
Arg Cys Arg Ser Gly Phe Cys Thr Pro 50 55
60Ile Asp Ser6513576PRTMedicago truncatula 135Met Gln Met Arg Lys
Asn Met Ala Gln Ile Leu Phe Tyr Val Tyr Ala1 5
10 15Leu Leu Ile Leu Phe Ser Pro Phe Leu Val Ala
Arg Ile Met Val Val 20 25
30Asn Pro Asn Asn Pro Cys Val Thr Asp Ala Asp Cys Gln Arg Tyr Arg
35 40 45His Lys Leu Ala Thr Arg Met Val
Cys Asn Ile Gly Phe Cys Leu Met 50 55
60Asp Phe Thr His Asp Pro Tyr Ala Pro Ser Leu Pro65 70
7513677PRTMedicago truncatula 136Met Tyr Val Tyr Tyr Ile
Gln Met Gly Lys Asn Met Ala Gln Arg Phe1 5
10 15Met Phe Ile Tyr Ala Leu Ile Ile Phe Leu Ser Gln
Phe Phe Val Val 20 25 30Ile
Asn Thr Ser Asp Ile Pro Asn Asn Ser Asn Arg Asn Ser Pro Lys 35
40 45Glu Asp Val Phe Cys Asn Ser Asn Asp
Asp Cys Pro Thr Ile Leu Tyr 50 55
60Tyr Val Ser Lys Cys Val Tyr Asn Phe Cys Glu Tyr Trp65
70 7513767PRTMedicago truncatula 137Met Ala Lys Ile Val
Asn Phe Val Tyr Ser Met Ile Ile Phe Val Ser1 5
10 15Leu Phe Leu Val Ala Thr Lys Gly Gly Ser Lys
Pro Phe Leu Thr Arg 20 25
30Pro Tyr Pro Cys Asn Thr Gly Ser Asp Cys Pro Gln Asn Met Cys Pro
35 40 45Pro Gly Tyr Lys Pro Gly Cys Glu
Asp Gly Tyr Cys Asn His Cys Tyr 50 55
60Lys Arg Trp6513862PRTMedicago truncatula 138Met Val Arg Thr Leu Lys
Phe Val Tyr Val Ile Ile Leu Ile Leu Ser1 5
10 15Leu Phe Leu Val Ala Lys Gly Gly Gly Lys Lys Ile
Tyr Cys Glu Asn 20 25 30Ala
Ala Ser Cys Pro Arg Leu Met Tyr Pro Leu Val Tyr Lys Cys Leu 35
40 45Asp Asn Lys Cys Val Lys Phe Met Met
Lys Ser Arg Phe Val 50 55
6013962PRTMedicago truncatula 139Met Ala Arg Thr Leu Lys Phe Val Tyr Ala
Val Ile Leu Phe Leu Ser1 5 10
15Leu Phe Leu Val Ala Lys Gly Asp Asp Val Lys Ile Lys Cys Val Val
20 25 30Ala Ala Asn Cys Pro Asp
Leu Met Tyr Pro Leu Val Tyr Lys Cys Leu 35 40
45Asn Gly Ile Cys Val Gln Phe Thr Leu Thr Phe Pro Phe Val
50 55 6014065PRTMedicago truncatula
140Met Ser Asn Thr Leu Met Phe Val Ile Thr Phe Ile Val Leu Val Thr1
5 10 15Leu Phe Leu Gly Pro Lys
Asn Val Tyr Ala Phe Gln Pro Cys Val Thr 20 25
30Thr Ala Asp Cys Met Lys Thr Leu Lys Thr Asp Glu Asn
Ile Trp Tyr 35 40 45Glu Cys Ile
Asn Asp Phe Cys Ile Pro Phe Pro Ile Pro Lys Gly Arg 50
55 60Lys6514176PRTMedicago truncatula 141Met Lys Arg
Val Val Asn Met Ala Lys Ile Val Lys Tyr Val Tyr Val1 5
10 15Ile Ile Ile Phe Leu Ser Leu Phe Leu
Val Ala Thr Lys Ile Glu Gly 20 25
30Tyr Tyr Tyr Lys Cys Phe Lys Asp Ser Asp Cys Val Lys Leu Leu Cys
35 40 45Arg Ile Pro Leu Arg Pro Lys
Cys Met Tyr Arg His Ile Cys Lys Cys 50 55
60Lys Val Val Leu Thr Gln Asn Asn Tyr Val Leu Thr65
70 7514266PRTMedicago truncatula 142Met Lys Arg Gly Lys
Asn Met Ser Lys Ile Leu Lys Phe Ile Tyr Ala1 5
10 15Thr Leu Val Leu Tyr Leu Phe Leu Val Val Thr
Lys Ala Ser Asp Asp 20 25
30Glu Cys Lys Ile Asp Gly Asp Cys Pro Ile Ser Trp Gln Lys Phe His
35 40 45Thr Tyr Lys Cys Ile Asn Gln Lys
Cys Lys Trp Val Leu Arg Phe His 50 55
60Glu Tyr6514364PRTMedicago truncatula 143Met Ala Lys Thr Leu Asn Phe
Met Phe Ala Leu Ile Leu Phe Ile Ser1 5 10
15Leu Phe Leu Val Ser Lys Asn Val Ala Ile Asp Ile Phe
Val Cys Gln 20 25 30Thr Asp
Ala Asp Cys Pro Lys Ser Glu Leu Ser Met Tyr Thr Trp Lys 35
40 45Cys Ile Asp Asn Glu Cys Asn Leu Phe Lys
Val Met Gln Gln Met Val 50 55
6014459PRTMedicago truncatula 144Met Ala Asn Thr His Lys Leu Val Ser Met
Ile Leu Phe Ile Phe Leu1 5 10
15Phe Leu Val Ala Asn Asn Val Glu Gly Tyr Val Asn Cys Glu Thr Asp
20 25 30Ala Asp Cys Pro Pro Ser
Thr Arg Val Lys Arg Phe Lys Cys Val Lys 35 40
45Gly Glu Cys Arg Trp Thr Arg Met Ser Tyr Ala 50
5514559PRTMedicago truncatula 145Met Ala His Phe Leu Met Phe Val
Tyr Ala Leu Ile Thr Cys Leu Ser1 5 10
15Leu Phe Leu Val Glu Met Gly His Leu Ser Ile His Cys Val
Ser Val 20 25 30Asp Asp Cys
Pro Lys Val Glu Lys Pro Ile Thr Met Lys Cys Ile Asn 35
40 45Asn Tyr Cys Lys Tyr Phe Val Asp His Lys Leu
50 5514666PRTMedicago truncatula 146Met Asn Gln Ile Pro
Met Phe Gly Tyr Thr Leu Ile Ile Phe Phe Ser1 5
10 15Leu Phe Pro Val Ile Thr Asn Gly Asp Arg Ile
Pro Cys Val Thr Asn 20 25
30Gly Asp Cys Pro Val Met Arg Leu Pro Leu Tyr Met Arg Cys Ile Thr
35 40 45Tyr Ser Cys Glu Leu Phe Phe Asp
Gly Pro Asn Leu Cys Ala Val Glu 50 55
60Arg Ile6514761PRTMedicago truncatula 147Met Arg Lys Asp Met Ala Arg
Ile Ser Leu Phe Val Tyr Ala Leu Ile1 5 10
15Ile Phe Phe Ser Leu Phe Phe Val Leu Thr Asn Gly Glu
Leu Glu Ile 20 25 30Arg Cys
Val Ser Asp Ala Asp Cys Pro Leu Phe Pro Leu Pro Leu His 35
40 45Asn Arg Cys Ile Asp Asp Val Cys His Leu
Phe Thr Ser 50 55 6014860PRTMedicago
truncatula 148Met Ala Gln Ile Leu Met Phe Val Tyr Phe Leu Ile Ile Phe Leu
Ser1 5 10 15Leu Phe Leu
Val Glu Ser Ile Lys Ile Phe Thr Glu His Arg Cys Arg 20
25 30Thr Asp Ala Asp Cys Pro Ala Arg Glu Leu
Pro Glu Tyr Leu Lys Cys 35 40
45Gln Gly Gly Met Cys Arg Leu Leu Ile Lys Lys Asp 50
55 6014956PRTMedicago truncatula 149Met Ala Arg Val Ile
Ser Leu Phe Tyr Ala Leu Ile Ile Phe Leu Phe1 5
10 15Leu Phe Leu Val Ala Thr Asn Gly Asp Leu Ser
Pro Cys Leu Arg Ser 20 25
30Gly Asp Cys Ser Lys Asp Glu Cys Pro Ser His Leu Val Pro Lys Cys
35 40 45Ile Gly Leu Thr Cys Tyr Cys Ile
50 5515062PRTMedicago truncatula 150Met Gln Arg Arg
Lys Asn Met Ala Gln Ile Leu Leu Phe Ala Tyr Val1 5
10 15Phe Ile Ile Ser Ile Ser Leu Phe Leu Val
Val Thr Asn Gly Val Lys 20 25
30Ile Pro Cys Val Lys Asp Thr Asp Cys Pro Thr Leu Pro Cys Pro Leu
35 40 45Tyr Ser Lys Cys Val Asp Gly Phe
Cys Lys Met Leu Ser Ile 50 55
6015166PRTMedicago truncatula 151Met Asn His Ile Ser Lys Phe Val Tyr Ala
Leu Ile Ile Phe Leu Ser1 5 10
15Val Tyr Leu Val Val Leu Asp Gly Arg Pro Val Ser Cys Lys Asp His
20 25 30Tyr Asp Cys Arg Arg Lys
Val Lys Ile Val Gly Cys Ile Phe Pro Gln 35 40
45Glu Lys Pro Met Cys Ile Asn Ser Met Cys Thr Cys Ile Arg
Glu Ile 50 55 60Val
Pro6515286PRTMedicago truncatula 152Met Lys Ser Gln Asn His Ala Lys Phe
Ile Ser Phe Tyr Lys Asn Asp1 5 10
15Leu Phe Lys Ile Phe Gln Asn Asn Asp Ser His Phe Lys Val Phe
Phe 20 25 30Ala Leu Ile Ile
Phe Leu Tyr Thr Tyr Leu His Val Thr Asn Gly Val 35
40 45Phe Val Ser Cys Asn Ser His Ile His Cys Arg Val
Asn Asn His Lys 50 55 60Ile Gly Cys
Asn Ile Pro Glu Gln Tyr Leu Leu Cys Val Asn Leu Phe65 70
75 80Cys Leu Trp Leu Asp Tyr
8515362PRTMedicago truncatula 153Met Thr Tyr Ile Ser Lys Val Val Tyr
Ala Leu Ile Ile Phe Leu Ser1 5 10
15Ile Tyr Val Gly Val Asn Asp Cys Met Leu Val Thr Cys Glu Asp
His 20 25 30Phe Asp Cys Arg
Gln Asn Val Gln Gln Val Gly Cys Ser Phe Arg Glu 35
40 45Ile Pro Gln Cys Ile Asn Ser Ile Cys Lys Cys Met
Lys Gly 50 55 6015463PRTMedicago
truncatula 154Met Thr His Ile Ser Lys Phe Val Phe Ala Leu Ile Ile Phe Leu
Ser1 5 10 15Ile Tyr Val
Gly Val Asn Asp Cys Lys Arg Ile Pro Cys Lys Asp Asn 20
25 30Asn Asp Cys Asn Asn Asn Trp Gln Leu Leu
Ala Cys Arg Phe Glu Arg 35 40
45Glu Val Pro Arg Cys Ile Asn Ser Ile Cys Lys Cys Met Pro Met 50
55 6015560PRTMedicago truncatula 155Met Val
Gln Thr Pro Lys Leu Val Tyr Val Ile Val Leu Leu Leu Ser1 5
10 15Ile Phe Leu Gly Met Thr Ile Cys
Asn Ser Ser Phe Ser His Phe Phe 20 25
30Glu Gly Ala Cys Lys Ser Asp Lys Asp Cys Pro Lys Leu His Arg
Ser 35 40 45Asn Val Arg Cys Arg
Lys Gly Gln Cys Val Gln Ile 50 55
6015677PRTMedicago truncatula 156Met Thr Lys Ile Leu Met Leu Phe Tyr Ala
Met Ile Val Phe His Ser1 5 10
15Ile Phe Leu Val Ala Ser Tyr Thr Asp Glu Cys Ser Thr Asp Ala Asp
20 25 30Cys Glu Tyr Ile Leu Cys
Leu Phe Pro Ile Ile Lys Arg Cys Ile His 35 40
45Asn His Cys Lys Cys Val Pro Met Gly Ser Ile Glu Pro Met
Ser Thr 50 55 60Ile Pro Asn Gly Val
His Lys Phe His Ile Ile Asn Asn65 70
7515764PRTMedicago truncatula 157Met Ala Lys Thr Leu Asn Phe Val Cys Ala
Met Ile Leu Phe Ile Ser1 5 10
15Leu Phe Leu Val Ser Lys Asn Val Ala Leu Tyr Ile Ile Glu Cys Lys
20 25 30Thr Asp Ala Asp Cys Pro
Ile Ser Lys Leu Asn Met Tyr Asn Trp Arg 35 40
45Cys Ile Lys Ser Ser Cys His Leu Tyr Lys Val Ile Gln Phe
Met Val 50 55 6015872PRTMedicago
truncatula 158Met Gln Lys Glu Lys Asn Met Ala Lys Thr Phe Glu Phe Val Tyr
Ala1 5 10 15Met Ile Ile
Phe Ile Leu Leu Phe Leu Val Glu Asn Asn Phe Ala Ala 20
25 30Tyr Ile Ile Glu Cys Gln Thr Asp Asp Asp
Cys Pro Lys Ser Gln Leu 35 40
45Glu Met Phe Ala Trp Lys Cys Val Lys Asn Gly Cys His Leu Phe Gly 50
55 60Met Tyr Glu Asp Asp Asp Asp Pro65
7015957PRTMedicago truncatula 159Met Ala Ala Thr Arg Lys
Phe Ile Tyr Val Leu Ser His Phe Leu Phe1 5
10 15Leu Phe Leu Val Thr Lys Ile Thr Asp Ala Arg Val
Cys Lys Ser Asp 20 25 30Lys
Asp Cys Lys Asp Ile Ile Ile Tyr Arg Tyr Ile Leu Lys Cys Arg 35
40 45Asn Gly Glu Cys Val Lys Ile Lys Ile
50 5516075PRTMedicago truncatula 160Met Gln Arg Leu Asp
Asn Met Ala Lys Asn Val Lys Phe Ile Tyr Val1 5
10 15Ile Ile Leu Leu Leu Phe Ile Phe Leu Val Ile
Ile Val Cys Asp Ser 20 25
30Ala Phe Val Pro Asn Ser Gly Pro Cys Thr Thr Asp Lys Asp Cys Lys
35 40 45Gln Val Lys Gly Tyr Ile Ala Arg
Cys Arg Lys Gly Tyr Cys Met Gln 50 55
60Ser Val Lys Arg Thr Trp Ser Ser Tyr Ser Arg65 70
75161102PRTMedicago truncatula 161Met Lys Phe Ile Tyr Ile Met
Ile Leu Phe Leu Ser Leu Phe Leu Val1 5 10
15Gln Phe Leu Thr Cys Lys Gly Leu Thr Val Pro Cys Glu
Asn Pro Thr 20 25 30Thr Cys
Pro Glu Asp Phe Cys Thr Pro Pro Met Ile Thr Arg Cys Ile 35
40 45Asn Phe Ile Cys Leu Cys Asp Gly Pro Glu
Tyr Ala Glu Pro Glu Tyr 50 55 60Asp
Gly Pro Glu Pro Glu Tyr Asp His Lys Gly Asp Phe Leu Ser Val65
70 75 80Lys Pro Lys Ile Ile Asn
Glu Asn Met Met Met Arg Glu Arg His Met 85
90 95Met Lys Glu Ile Glu Val
10016259PRTMedicago truncatula 162Met Ala Gln Phe Leu Met Phe Ile Tyr Val
Leu Ile Ile Phe Leu Tyr1 5 10
15Leu Phe Tyr Val Glu Ala Ala Met Phe Glu Leu Thr Lys Ser Thr Ile
20 25 30Arg Cys Val Thr Asp Ala
Asp Cys Pro Asn Val Val Lys Pro Leu Lys 35 40
45Pro Lys Cys Val Asp Gly Phe Cys Glu Tyr Thr 50
5516370PRTMedicago truncatula 163Met Lys Met Arg Ile His Met Ala
Gln Ile Ile Met Phe Phe Tyr Ala1 5 10
15Leu Ile Ile Phe Leu Ser Pro Phe Leu Val Asp Arg Arg Ser
Phe Pro 20 25 30Ser Ser Phe
Val Ser Pro Lys Ser Tyr Thr Ser Glu Ile Pro Cys Lys 35
40 45Ala Thr Arg Asp Cys Pro Tyr Glu Leu Tyr Tyr
Glu Thr Lys Cys Val 50 55 60Asp Ser
Leu Cys Thr Tyr65 7016441PRTMedicago truncatula 164Thr
Arg Met Leu Thr Ile Pro Cys Thr Ser Asp Asp Asn Cys Pro Lys1
5 10 15Val Ile Ser Pro Cys His Thr
Lys Cys Phe Asp Gly Phe Cys Gly Trp 20 25
30Tyr Ile Glu Gly Ser Tyr Glu Gly Pro 35
4016569PRTMedicago truncatula 165Met Ala Gln Phe Leu Leu Phe Val Tyr
Ser Leu Ile Ile Phe Leu Ser1 5 10
15Leu Phe Phe Gly Glu Ala Ala Phe Glu Arg Thr Glu Thr Arg Met
Leu 20 25 30Thr Ile Pro Cys
Thr Ser Asp Asp Asn Cys Pro Lys Val Ile Ser Pro 35
40 45Cys His Thr Lys Cys Phe Asp Gly Phe Cys Gly Trp
Tyr Ile Glu Gly 50 55 60Ser Tyr Glu
Gly Pro6516678PRTBuchnera aphidicola 166Met Lys Leu Leu His Gly Phe Leu
Ile Ile Met Leu Thr Met His Leu1 5 10
15Ser Ile Gln Tyr Ala Tyr Gly Gly Pro Phe Leu Thr Lys Tyr
Leu Cys 20 25 30Asp Arg Val
Cys His Lys Leu Cys Gly Asp Glu Phe Val Cys Ser Cys 35
40 45Ile Gln Tyr Lys Ser Leu Lys Gly Leu Trp Phe
Pro His Cys Pro Thr 50 55 60Gly Lys
Ala Ser Val Val Leu His Asn Phe Leu Thr Ser Pro65 70
7516777PRTBuchnera aphidicola 167Met Lys Leu Leu Tyr Gly Phe
Leu Ile Ile Met Leu Thr Ile His Leu1 5 10
15Ser Val Gln Tyr Phe Glu Ser Pro Phe Glu Thr Lys Tyr
Asn Cys Asp 20 25 30Thr His
Cys Asn Lys Leu Cys Gly Lys Ile Asp His Cys Ser Cys Ile 35
40 45Gln Tyr His Ser Met Glu Gly Leu Trp Phe
Pro His Cys Arg Thr Gly 50 55 60Ser
Ala Ala Gln Met Leu His Asp Phe Leu Ser Asn Pro65 70
7516886PRTBuchnera aphidicola 168Met Ser Val Arg Lys Asn Val
Leu Pro Thr Met Phe Val Val Leu Leu1 5 10
15Ile Met Ser Pro Val Thr Pro Thr Ser Val Phe Ile Ser
Ala Val Cys 20 25 30Tyr Ser
Gly Cys Gly Ser Leu Ala Leu Val Cys Phe Val Ser Asn Gly 35
40 45Ile Thr Asn Gly Leu Asp Tyr Phe Lys Ser
Ser Ala Pro Leu Ser Thr 50 55 60Ser
Glu Thr Ser Cys Gly Glu Ala Phe Asp Thr Cys Thr Asp His Cys65
70 75 80Leu Ala Asn Phe Lys Phe
8516969PRTBuchnera aphidicola 169Met Arg Leu Leu Tyr Gly Phe
Leu Ile Ile Met Leu Thr Ile Tyr Leu1 5 10
15Ser Val Gln Asp Phe Asp Pro Thr Glu Phe Lys Gly Pro
Phe Pro Thr 20 25 30Ile Glu
Ile Cys Ser Lys Tyr Cys Ala Val Val Cys Asn Tyr Thr Ser 35
40 45Arg Pro Cys Tyr Cys Val Glu Ala Ala Lys
Glu Arg Asp Gln Trp Phe 50 55 60Pro
Tyr Cys Tyr Asp6517077PRTBuchnera aphidicola 170Met Arg Leu Leu Tyr Gly
Phe Leu Ile Ile Met Leu Thr Ile His Leu1 5
10 15Ser Val Gln Asp Ile Asp Pro Asn Thr Leu Arg Gly
Pro Tyr Pro Thr 20 25 30Lys
Glu Ile Cys Ser Lys Tyr Cys Glu Tyr Asn Val Val Cys Gly Ala 35
40 45Ser Leu Pro Cys Ile Cys Val Gln Asp
Ala Arg Gln Leu Asp His Trp 50 55
60Phe Ala Cys Cys Tyr Asp Gly Gly Pro Glu Met Leu Met65
70 75171108PRTBuchnera aphidicola 171Met Lys Leu Phe Val
Val Val Val Leu Val Ala Val Gly Ile Met Phe1 5
10 15Val Phe Ala Ser Asp Thr Ala Ala Ala Pro Thr
Asp Tyr Glu Asp Thr 20 25
30Asn Asp Met Ile Ser Leu Ser Ser Leu Val Gly Asp Asn Ser Pro Tyr
35 40 45Val Arg Val Ser Ser Ala Asp Ser
Gly Gly Ser Ser Lys Thr Ser Ser 50 55
60Lys Asn Pro Ile Leu Gly Leu Leu Lys Ser Val Ile Lys Leu Leu Thr65
70 75 80Lys Ile Phe Gly Thr
Tyr Ser Asp Ala Ala Pro Ala Met Pro Pro Ile 85
90 95Pro Pro Ala Leu Arg Lys Asn Arg Gly Met Leu
Ala 100 105172178PRTBuchnera aphidicola 172Met
Val Ala Cys Lys Val Ile Leu Ala Val Ala Val Val Phe Val Ala1
5 10 15Ala Val Gln Gly Arg Pro Gly
Gly Glu Pro Glu Trp Ala Ala Pro Ile 20 25
30Phe Ala Glu Leu Lys Ser Val Ser Asp Asn Ile Thr Asn Leu
Val Gly 35 40 45Leu Asp Asn Ala
Gly Glu Tyr Ala Thr Ala Ala Lys Asn Asn Leu Asn 50 55
60Ala Phe Ala Glu Ser Leu Lys Thr Glu Ala Ala Val Phe
Ser Lys Ser65 70 75
80Phe Glu Gly Lys Ala Ser Ala Ser Asp Val Phe Lys Glu Ser Thr Lys
85 90 95Asn Phe Gln Ala Val Val
Asp Thr Tyr Ile Lys Asn Leu Pro Lys Asp 100
105 110Leu Thr Leu Lys Asp Phe Thr Glu Lys Ser Glu Gln
Ala Leu Lys Tyr 115 120 125Met Val
Glu His Gly Thr Glu Ile Thr Lys Lys Ala Gln Gly Asn Thr 130
135 140Glu Thr Glu Lys Glu Ile Lys Glu Phe Phe Lys
Lys Gln Ile Glu Asn145 150 155
160Leu Ile Gly Gln Gly Lys Ala Leu Gln Ala Lys Ile Ala Glu Ala Lys
165 170 175Lys
Ala173311PRTBuchnera aphidicola 173Met Lys Thr Ser Ser Ser Lys Val Phe
Ala Ser Cys Val Ala Ile Val1 5 10
15Cys Leu Ala Ser Val Ala Asn Ala Leu Pro Val Gln Lys Ser Val
Ala 20 25 30Ala Thr Thr Glu
Asn Pro Ile Val Glu Lys His Gly Cys Arg Ala His 35
40 45Lys Asn Leu Val Arg Gln Asn Val Val Asp Leu Lys
Thr Tyr Asp Ser 50 55 60Met Leu Ile
Thr Asn Glu Val Val Gln Lys Gln Ser Asn Glu Val Gln65 70
75 80Ser Ser Glu Gln Ser Asn Glu Gly
Gln Asn Ser Glu Gln Ser Asn Glu 85 90
95Gly Gln Asn Ser Glu Gln Ser Asn Glu Val Gln Ser Ser Glu
His Ser 100 105 110Asn Glu Gly
Gln Asn Ser Lys Gln Ser Asn Glu Gly Gln Asn Ser Glu 115
120 125Gln Ser Asn Glu Val Gln Ser Ser Glu His Ser
Asn Glu Gly Gln Asn 130 135 140Ser Glu
Gln Ser Asn Glu Val Gln Ser Ser Glu His Ser Asn Glu Gly145
150 155 160Gln Asn Ser Lys Gln Ser Asn
Glu Gly Gln Asn Ser Lys Gln Ser Asn 165
170 175Glu Val Gln Ser Ser Glu His Trp Asn Glu Gly Gln
Asn Ser Lys Gln 180 185 190Ser
Asn Glu Asp Gln Asn Ser Glu Gln Ser Asn Glu Gly Gln Asn Ser 195
200 205Lys Gln Ser Asn Glu Gly Gln Asn Ser
Lys Gln Ser Asn Glu Asp Gln 210 215
220Asn Ser Glu Gln Ser Asn Glu Gly Gln Asn Ser Lys Gln Ser Asn Glu225
230 235 240Val Gln Ser Ser
Glu Gln Ser Asn Glu Gly Gln Asn Ser Lys Gln Ser 245
250 255Asn Glu Gly Gln Ser Ser Glu Gln Ser Asn
Glu Gly Gln Asn Ser Lys 260 265
270Gln Ser Asn Glu Val Gln Ser Pro Glu Glu His Tyr Asp Leu Pro Asp
275 280 285Pro Glu Ser Ser Tyr Glu Ser
Glu Glu Thr Lys Gly Ser His Glu Ser 290 295
300Gly Asp Asp Ser Glu His Arg305
310174431PRTBuchnera aphidicola 174Met Lys Thr Ile Ile Leu Gly Leu Cys
Leu Phe Gly Ala Leu Phe Trp1 5 10
15Ser Thr Gln Ser Met Pro Val Gly Glu Val Ala Pro Ala Val Pro
Ala 20 25 30Val Pro Ser Glu
Ala Val Pro Gln Lys Gln Val Glu Ala Lys Pro Glu 35
40 45Thr Asn Ala Ala Ser Pro Val Ser Asp Ala Lys Pro
Glu Ser Asp Ser 50 55 60Lys Pro Val
Asp Ala Glu Val Lys Pro Thr Val Ser Glu Val Lys Ala65 70
75 80Glu Ser Glu Gln Lys Pro Ser Gly
Glu Pro Lys Pro Glu Ser Asp Ala 85 90
95Lys Pro Val Val Ala Ser Glu Ser Lys Pro Glu Ser Asp Pro
Lys Pro 100 105 110Ala Ala Val
Val Glu Ser Lys Pro Glu Asn Asp Ala Val Ala Pro Glu 115
120 125Thr Asn Asn Asp Ala Lys Pro Glu Asn Ala Ala
Ala Pro Val Ser Glu 130 135 140Asn Lys
Pro Ala Thr Asp Ala Lys Ala Glu Thr Glu Leu Ile Ala Gln145
150 155 160Ala Lys Pro Glu Ser Lys Pro
Ala Ser Asp Leu Lys Ala Glu Pro Glu 165
170 175Ala Ala Lys Pro Asn Ser Glu Val Pro Val Ala Leu
Pro Leu Asn Pro 180 185 190Thr
Glu Thr Lys Ala Thr Gln Gln Ser Val Glu Thr Asn Gln Val Glu 195
200 205Gln Ala Ala Pro Ala Ala Ala Gln Ala
Asp Pro Ala Ala Ala Pro Ala 210 215
220Ala Asp Pro Ala Pro Ala Pro Ala Ala Ala Pro Val Ala Ala Glu Glu225
230 235 240Ala Lys Leu Ser
Glu Ser Ala Pro Ser Thr Glu Asn Lys Ala Ala Glu 245
250 255Glu Pro Ser Lys Pro Ala Glu Gln Gln Ser
Ala Lys Pro Val Glu Asp 260 265
270Ala Val Pro Ala Ala Ser Glu Ile Ser Glu Thr Lys Val Ser Pro Ala
275 280 285Val Pro Ala Val Pro Glu Val
Pro Ala Ser Pro Ser Ala Pro Ala Val 290 295
300Ala Asp Pro Val Ser Ala Pro Glu Ala Glu Lys Asn Ala Glu Pro
Ala305 310 315 320Lys Ala
Ala Asn Ser Ala Glu Pro Ala Val Gln Ser Glu Ala Lys Pro
325 330 335Ala Glu Asp Ile Gln Lys Ser
Gly Ala Val Val Ser Ala Glu Asn Pro 340 345
350Lys Pro Val Glu Glu Gln Lys Pro Ala Glu Val Ala Lys Pro
Ala Glu 355 360 365Gln Ser Lys Ser
Glu Ala Pro Ala Glu Ala Pro Lys Pro Thr Glu Gln 370
375 380Ser Ala Ala Glu Glu Pro Lys Lys Pro Glu Ser Ala
Asn Asp Glu Lys385 390 395
400Lys Glu Gln His Ser Val Asn Lys Arg Asp Ala Thr Lys Glu Lys Lys
405 410 415Pro Thr Asp Ser Ile
Met Lys Lys Gln Lys Gln Lys Lys Ala Asn 420
425 430175160PRTBuchnera aphidicola 175Met Asn Gly Lys
Ile Val Leu Cys Phe Ala Val Val Phe Ile Gly Gln1 5
10 15Ala Met Ser Ala Ala Thr Gly Thr Thr Pro
Glu Val Glu Asp Ile Lys 20 25
30Lys Val Ala Glu Gln Met Ser Gln Thr Phe Met Ser Val Ala Asn His
35 40 45Leu Val Gly Ile Thr Pro Asn Ser
Ala Asp Ala Gln Lys Ser Ile Glu 50 55
60Lys Ile Arg Thr Ile Met Asn Lys Gly Phe Thr Asp Met Glu Thr Glu65
70 75 80Ala Asn Lys Met Lys
Asp Ile Val Arg Lys Asn Ala Asp Pro Lys Leu 85
90 95Val Glu Lys Tyr Asp Glu Leu Glu Lys Glu Leu
Lys Lys His Leu Ser 100 105
110Thr Ala Lys Asp Met Phe Glu Asp Lys Val Val Lys Pro Ile Gly Glu
115 120 125Lys Val Glu Leu Lys Lys Ile
Thr Glu Asn Val Ile Lys Thr Thr Lys 130 135
140Asp Met Glu Ala Thr Met Asn Lys Ala Ile Asp Gly Phe Lys Lys
Gln145 150 155
160176415PRTBuchnera aphidicola 176Met His Leu Phe Leu Ala Leu Gly Leu
Phe Ile Val Cys Gly Met Val1 5 10
15Asp Ala Thr Phe Tyr Asn Pro Arg Ser Gln Thr Phe Asn Gln Leu
Met 20 25 30Glu Arg Arg Gln
Arg Ser Ile Pro Ile Pro Tyr Ser Tyr Gly Tyr His 35
40 45Tyr Asn Pro Ile Glu Pro Ser Ile Asn Val Leu Asp
Ser Leu Ser Glu 50 55 60Gly Leu Asp
Ser Arg Ile Asn Thr Phe Lys Pro Ile Tyr Gln Asn Val65 70
75 80Lys Met Ser Thr Gln Asp Val Asn
Ser Val Pro Arg Thr Gln Tyr Gln 85 90
95Pro Lys Asn Ser Leu Tyr Asp Ser Glu Tyr Ile Ser Ala Lys
Asp Ile 100 105 110Pro Ser Leu
Phe Pro Glu Glu Asp Ser Tyr Asp Tyr Lys Tyr Leu Gly 115
120 125Ser Pro Leu Asn Lys Tyr Leu Thr Arg Pro Ser
Thr Gln Glu Ser Gly 130 135 140Ile Ala
Ile Asn Leu Val Ala Ile Lys Glu Thr Ser Val Phe Asp Tyr145
150 155 160Gly Phe Pro Thr Tyr Lys Ser
Pro Tyr Ser Ser Asp Ser Val Trp Asn 165
170 175Phe Gly Ser Lys Ile Pro Asn Thr Val Phe Glu Asp
Pro Gln Ser Val 180 185 190Glu
Ser Asp Pro Asn Thr Phe Lys Val Ser Ser Pro Thr Ile Lys Ile 195
200 205Val Lys Leu Leu Pro Glu Thr Pro Glu
Gln Glu Ser Ile Ile Thr Thr 210 215
220Thr Lys Asn Tyr Glu Leu Asn Tyr Lys Thr Thr Gln Glu Thr Pro Thr225
230 235 240Glu Ala Glu Leu
Tyr Pro Ile Thr Ser Glu Glu Phe Gln Thr Glu Asp 245
250 255Glu Trp His Pro Met Val Pro Lys Glu Asn
Thr Thr Lys Asp Glu Ser 260 265
270Ser Phe Ile Thr Thr Glu Glu Pro Leu Thr Glu Asp Lys Ser Asn Ser
275 280 285Ile Thr Ile Glu Lys Thr Gln
Thr Glu Asp Glu Ser Asn Ser Ile Glu 290 295
300Phe Asn Ser Ile Arg Thr Glu Glu Lys Ser Asn Ser Ile Thr Thr
Glu305 310 315 320Glu Asn
Gln Lys Glu Asp Asp Glu Ser Met Ser Thr Thr Ser Gln Glu
325 330 335Thr Thr Thr Ala Phe Asn Leu
Asn Asp Thr Phe Asp Thr Asn Arg Tyr 340 345
350Ser Ser Ser His Glu Ser Leu Met Leu Arg Ile Arg Glu Leu
Met Lys 355 360 365Asn Ile Ala Asp
Gln Gln Asn Lys Ser Gln Phe Arg Thr Val Asp Asn 370
375 380Ile Pro Ala Lys Ser Gln Ser Asn Leu Ser Ser Asp
Glu Ser Thr Asn385 390 395
400Gln Gln Phe Glu Pro Gln Leu Val Asn Gly Ala Asp Thr Tyr Lys
405 410 415177126PRTSitophilus
zeamais 177Met Thr Arg Thr Met Leu Phe Leu Ala Cys Val Ala Ala Leu Tyr
Val1 5 10 15Cys Ile Ser
Ala Thr Ala Gly Lys Pro Glu Glu Phe Ala Lys Leu Ser 20
25 30Asp Glu Ala Pro Ser Asn Asp Gln Ala Met
Tyr Glu Ser Ile Gln Arg 35 40
45Tyr Arg Arg Phe Val Asp Gly Asn Arg Tyr Asn Gly Gly Gln Gln Gln 50
55 60Gln Gln Gln Pro Lys Gln Trp Glu Val
Arg Pro Asp Leu Ser Arg Asp65 70 75
80Gln Arg Gly Asn Thr Lys Ala Gln Val Glu Ile Asn Lys Lys
Gly Asp 85 90 95Asn His
Asp Ile Asn Ala Gly Trp Gly Lys Asn Ile Asn Gly Pro Asp 100
105 110Ser His Lys Asp Thr Trp His Val Gly
Gly Ser Val Arg Trp 115 120
12517816PRTAcyrthosiphon pisum 178Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
Arg Met Lys Trp Lys Lys1 5 10
1517913PRTArtificial SequenceTat peptide 179Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Pro Pro Gln1 5
1018018PRTHuman immunodeficiency virus 1 180Leu Leu Ile Ile Leu Arg Arg
Arg Ile Arg Lys Gln Ala His Ala His1 5 10
15Ser Lys18127PRTArtificial SequenceTransportan 181Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu1
5 10 15Lys Ala Leu Ala Ala Leu Ala
Lys Lys Ile Leu 20 2518227PRTArtificial
SequenceMPG 182Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr
Met Gly1 5 10 15Ala Trp
Ser Gln Pro Lys Lys Lys Arg Lys Val 20
2518321PRTArtificial SequencePep-1 183Lys Glu Thr Trp Trp Glu Thr Trp Trp
Thr Glu Trp Ser Gln Pro Lys1 5 10
15Lys Lys Arg Lys Val 2018418PRTArtificial
SequenceMAP 184Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala
Leu Lys1 5 10 15Leu
Ala1859PRTArtificial SequenceR6W3 185Arg Arg Trp Trp Arg Arg Trp Arg Arg1
518620DNAArtificial SequenceBacillus, forward primer
186gaggtagacg aagcgacctg
2018720DNAArtificial SequenceBacillus, reverse primer 187ttccctcacg
gtactggttc
2018820DNAArtificial SequenceUniversal bacterial primer 27F 188agagtttgat
cmtggctcag
2018916DNAArtificial SequenceUniversal bacterial primer 1492R
189taccttgtta cgactt
161901583DNALactobacillus kunkeei 190ttccttagaa aggaggtgat ccagccgcag
gttctcctac ggctaccttg ttacgacttc 60accctaatca tctgtcccac cttagacgac
tagctcctaa aaggttaccc catcgtcttt 120gggtgttaca aactctcatg gtgtgacggg
cggtgtgtac aaggcccggg aacgtattca 180ccgtggcatg ctgatccacg attactagtg
attccaactt catgcaggcg agttgcagcc 240tgcaatccga actgagaatg gctttaagag
attagcttga cctcgcggtt tcgcgactcg 300ttgtaccatc cattgtagca cgtgtgtagc
ccagctcata aggggcatga tgatttgacg 360tcgtccccac cttcctccgg tttatcaccg
gcagtctcac tagagtgccc aactaaatgc 420tggcaactaa taataagggt tgcgctcgtt
gcgggactta acccaacatc tcacgacacg 480agctgacgac aaccatgcac cacctgtcat
tctgtccccg aagggaacgc ccaatctctt 540gggttggcag aagatgtcaa gagctggtaa
ggttcttcgc gtagcatcga attaaaccac 600atgctccacc acttgtgcgg gcccccgtca
attcctttga gtttcaacct tgcggtcgta 660ctccccaggc ggaatactta atgcgttagc
tgcggcactg aagggcggaa accctccaac 720acctagtatt catcgtttac ggcatggact
accagggtat ctaatcctgt tcgctaccca 780tgctttcgag cctcagcgtc agtaacagac
cagaaagccg ccttcgccac tggtgttctt 840ccatatatct acgcatttca ccgctacaca
tggagttcca ctttcctctt ctgtactcaa 900gttttgtagt ttccactgca cttcctcagt
tgagctgagg gctttcacag cagacttaca 960aaaccgcctg cgctcgcttt acgcccaata
aatccggaca acgcttgcca cctacgtatt 1020accgcggctg ctggcacgta gttagccgtg
gctttctggt taaataccgt caaagtgtta 1080acagttactc taacacttgt tcttctttaa
caacagagtt ttacgatccg aaaaccttca 1140tcactcacgc ggcgttgctc catcagactt
tcgtccattg tggaagattc cctactgctg 1200cctcccgtag gagtctgggc cgtgtctcag
tcccaatgtg gccgattacc ctctcaggtc 1260ggctacgtat catcgtcttg gtgggctttt
atctcaccaa ctaactaata cggcgcgggt 1320ccatcccaaa gtgatagcaa agccatcttt
caagttggaa ccatgcggtt ccaactaatt 1380atgcggtatt agcacttgtt tccaaatgtt
atcccccgct tcggggcagg ttacccacgt 1440gttactcacc agttcgccac tcgctccgaa
tccaaaaatc atttatgcaa gcataaaatc 1500aatttgggag aactcgttcg acttgcatgt
attaggcacg ccgccagcgt tcgtcctgag 1560ccaggatcaa actctcatct taa
15831911395DNALactobacillus Firm-4
191acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg cgggaagtca gggaagcctt
60cgggtggaac tggtggaacg agcggcggat gggtgagtaa cacgtaggta acctgcccta
120aagcggggga taccatctgg aaacaggtgc taataccgca taaacccagc agtcacatga
180gtgctggttg aaagacggct tcggctgtca ctttaggatg gacctgcggc gtattagcta
240gttggtggag taacggttca ccaaggcaat gatacgtagc cgacctgaga gggtaatcgg
300ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc
360acaatggacg caagtctgat ggagcaacgc cgcgtggatg aagaaggtct tcggatcgta
420aaatcctgtt gttgaagaag aacggttgtg agagtaactg ctcataacgt gacggtaatc
480aaccagaaag tcacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg
540ttgtccggat ttattgggcg taaagggagc gcaggcggtc ttttaagtct gaatgtgaaa
600gccctcagct taactgagga agagcatcgg aaactgagag acttgagtgc agaagaggag
660agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa
720ggcggctctc tggtctgtta ctgacgctga ggctcgaaag catgggtagc gaacaggatt
780agataccctg gtagtccatg ccgtaaacga tgagtgctaa gtgttgggag gtttccgcct
840ctcagtgctg cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa
900actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca
960acgcgaagaa ccttaccagg tcttgacatc tcctgcaagc ctaagagatt aggggttccc
1020ttcggggaca ggaagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg
1080gttaagtccc gcaacgagcg caacccttgt tactagttgc cagcattaag ttgggcactc
1140tagtgagact gccggtgaca aaccggagga aggtggggac gacgtcaaat catcatgccc
1200cttatgacct gggctacaca cgtgctacaa tggatggtac aatgagaagc gaactcgcga
1260ggggaagctg atctctgaaa accattctca gttcggattg caggctgcaa ctcgcctgca
1320tgaagctgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc
1380ttgtacacac cgccc
1395192220PRTSitophilus zeamais 192Met Lys Glu Thr Thr Val Val Trp Ala
Lys Leu Phe Leu Ile Leu Ile1 5 10
15Ile Leu Ala Lys Pro Leu Gly Leu Lys Ala Val Asn Glu Cys Lys
Arg 20 25 30Leu Gly Asn Asn
Ser Cys Arg Ser His Gly Glu Cys Cys Ser Gly Phe 35
40 45Cys Phe Ile Glu Pro Gly Trp Ala Leu Gly Val Cys
Lys Arg Leu Gly 50 55 60Thr Pro Lys
Lys Ser Asp Asp Ser Asn Asn Gly Lys Asn Ile Glu Lys65 70
75 80Asn Asn Gly Val His Glu Arg Ile
Asp Asp Val Phe Glu Arg Gly Val 85 90
95Cys Ser Tyr Tyr Lys Gly Pro Ser Ile Thr Ala Asn Gly Asp
Val Phe 100 105 110Asp Glu Asn
Glu Met Thr Ala Ala His Arg Thr Leu Pro Phe Asn Thr 115
120 125Met Val Lys Val Glu Gly Met Gly Thr Ser Val
Val Val Lys Ile Asn 130 135 140Asp Arg
Lys Thr Ala Ala Asp Gly Lys Val Met Leu Leu Ser Arg Ala145
150 155 160Ala Ala Glu Ser Leu Asn Ile
Asp Glu Asn Thr Gly Pro Val Gln Cys 165
170 175Gln Leu Lys Phe Val Leu Asp Gly Ser Gly Cys Thr
Pro Asp Tyr Gly 180 185 190Asp
Thr Cys Val Leu His His Glu Cys Cys Ser Gln Asn Cys Phe Arg 195
200 205Glu Met Phe Ser Asp Lys Gly Phe Cys
Leu Pro Lys 210 215
22019313PRTPandinum imperator 193Phe Leu Ser Thr Ile Trp Asn Gly Ile Lys
Gly Leu Leu1 5 1019413PRTUrodacus
yaschenkoi 194Ile Leu Ser Ala Ile Trp Ser Gly Ile Lys Ser Leu Phe1
5 1019513PRTScorpiops tibetanus 195Leu Trp Gly
Lys Leu Trp Glu Gly Val Lys Ser Leu Ile1 5
1019622PRTApostichopus japonicus 196Phe Pro Phe Leu Lys Leu Ser Leu Lys
Ile Pro Lys Ser Ala Ile Lys1 5 10
15Ser Ala Ile Lys Arg Leu 2019713PRTUrodacus
yaschenkoi 197Ile Leu Ser Ala Ile Trp Ser Gly Ile Lys Gly Leu Leu1
5 1019827PRTArtificial SequenceUy192 + cell
penetrating peptide 198Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Phe
Leu Ser Thr Ile1 5 10
15Trp Asn Gly Ile Lys Gly Leu Leu Phe Ala Met 20
25
User Contributions:
Comment about this patent or add new information about this topic: