COMPOSITIONS, METHODS AND USES FOR A NOVEL FAMILY OF PEPTIDES

Abstract

ludes compositions and methods for the characterization and use of novel peptide from Brevibacillus sp., and peptides related thereto, including an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide that have two or more D-amino acids used as, e.g., an antimicrobial or even a feed additive.

Description

[0001]This application claims priority to U.S. Provisional Pat. Application Ser. No. 60/540,569, filed Jan. 30, 2004, and U.S. patent application Ser. No. 11/046,560, filed Jan. 28, 2005, now U.S. Pat. No. ______, issued ______, relevant portions incorporated herein by reference. Without limiting the scope of the invention, its background is described in connection with antibiotics and feed additives.

[0002]This application claims priority to U.S. Provisional Patent Application Ser. No. 60/540,569, filed Jan. 30, 2004, relevant portions incorporated herein by reference. Without limiting the scope of the invention, its background is described in connection with antibiotics and feed additives.

TECHNICAL FIELD OF THE INVENTION

[0003]The present invention relates in general to the field of novel isolated and purified peptides, and more particularly, to the identification, characterization and use of a novel group of peptides from the newly discovered organism Brevibacillus texasporus .

BACKGROUND OF THE INVENTION

[0004]Antibiotic overuse has led to widespread bacterial drug resistance. Novel antibiotics are needed to combat infections caused by bacterial resistant to conventional antibiotics. It is well known that microbes produce a huge variety of antibiotics to wage chemical warfare against competing microbes.

[0005]Many peptide antibiotics of microbial origin are synthesized by non-ribosomal peptide synthases (RPS) and they contain unusual amino acids. NRPS enzymes usually have a co-linear modular architecture (Mootz, et al., 2002). The N-terminal to C-terminal order and specificities of the individual modules correspond to the sequential order and identities of the amino acid residues in the peptide product. Each NRPS module recognizes a specific amino acid and catalyzes stepwise condensation to form a growing peptide chain. The identity of the amino acid recognized by a particular module can be predicted by comparisons to other modules of known specificities (Challis, et al., 2000). Such strict correlation made it possible to identify genes encoding the NRPS enzymes for a number of microbial non-ribosomal peptides with known structures, as demonstrated by the identification of the mycobactin biosynthesis operon in the genome of Mycobacterium tuberculosis (Quadri, et al., 1998). Nevertheless, the art recognizes the continuing need to isolate, identify and characterize novel antimicrobial agents.

[0006]Examples of feed additives are widely known in the art. For example, U.S. Pat. No. 6,682,762 issued to Register, discloses one such Poultry and livestock feed additive. Briefly, this patent teaches a poultry and livestock feed additive composition containing 36 wt. % electrolytes, roughage and mineral oil to increase their dietary electrolyte balance. Addition of the electrolyte additive composition improves breeder hen performance as to egg production, body weight, and reduced mortality from heat stress. Broiler chickens on this diet result in increased processing yield, feed conversion and body weight. A method of preparing this dietary electrolyte feed for poultry and livestock is also described.

[0007]Yet another example of a feed additive is a taught by Nagai, et al., in U.S. Pat. No. 6,503,544, which teaches an animal feed additive that includes at least two components selected from the group consisting of the following three components (a), (b) and (c): (a) at least one herb selected from Pine Needle, Hawthorn Fruit, Bighead Atractylodes Rhizome, Milkvetch Root, Skullcap Root, Tangerine Fruit and Mint Siftings; (b) a live bacteria mixture composed of a yeast cell wall and a live bacteria preparation containing Lactobacillus acidophilus and/or Enterococcus faecium; and (c) an organic acid.

[0008]Feed additives may also include the byproducts of fermentation and other precesses, such as those taught by U.S. Pat. No. 5,863,574 issued to Julien for a feed additive for ruminant animals containing fungal and/or bacterial fermentation byproducts. The feed additive for ruminants, includes dried fungal and/or bacterial fermentation by products which provide glutamic acid fermentation solubles, dried corn fermentation solubles, or a mixture of dried glutamic acid fermentation solubles and dried corn fermentation solubles, wherein the dried solubles have been dried to a total moisture content of less than 30% by weight at a temperature not less than about 80° F. and not more than about 900° F.

SUMMARY OF THE INVENTION

[0009]In one embodiment, the present invention is a feed additive that includes an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus sp. The carboxy-terminus --COOH group of the C-terminal Valine of the peptide may be reduced to --CH₂OH, and may confer protease resistance to the peptide. The peptide feed additive may be stable at a pH of 1.0, at a pH 13.0, resistant to proteases or combinations thereof. Examples of the peptide may be selected from one or more of SEQ ID NOS: 1 to 20 (collectively called the BT peptides). It has been found that the peptide kills, gram positive bacteria, gram negative bacteria, fungi, protozoa or combinations thereof. The peptide may be isolated from Brevibacillus texasporus (ATCC PTA-5854) and may be added to feed at between about 0.5 and about 100 ppm. In one use, the peptide was added at between about 6 and about 12 ppm and demonstrated statistically significant growth stimulation.

[0010]The additive peptide may be added to a feed adapted for use by one or more of poultry, livestock, farm-raised fish, crabs, shrimp and fresh-water turtles. For example, the peptide may be included in a cereal-based animal feed, e.g., at least one cereal selected from barley, soya, wheat, triticale, rye and maize; and an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus sp. The peptide-based feed additive may be include at between about 1 and 1000 ppm of an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide having two or more D-amino acids isolated from Brevibacillus sp. In fact, the present invention may be used with any of a large variety of feeds.

[0011]The present invention also includes an antimicrobial peptide that has two or more D-amino acids, carboxy-terminus reduced pH and heat stable isolated from Brevibacillus sp. For example, the present invention includes a biologically pure culture of microorganism Brevibacillus texasporus deposit No. ATCC PTA-5854) that produces an antimicrobial peptide that is carboxy-terminus reduced heat stable, amino terminus-methylated peptide and may include two or more D-amino acids. The feed additive may even be an isolated and purified microorganism of ATCC PTA-5854. The additive may be mixed with a feed for livestock selected from the group consisting of a milk replacer, a grower feed, a finisher feed, a pre-starter feed and a starter feed.

[0012]The present invention also includes a method for increasing body weight gain efficiency and feed efficiency in animals, by providing one or more of the BT peptides in an effective amount sufficient to increase growth in an animal feed. The animal feed is adapted for feeding livestock selected from the group consisting of, e.g., cattle, swine, chicken, horse, turkey, sheep, goat, farm-raised fish, crab, shrimp and turtle. Examples of feeds also include those for feeding birds selected from the group consisting of, e.g., chicken, turkey, duck, quail, Cornish hens, and pigeon. As such, the feed may be selected from the group consisting of, e.g., a cereal, soybean meal, isolated soybean protein, isolated soybean oil, isolated soybean fat, skimmed milk, fish meal, meat meal, bone meal, blood meal, blood plasma protein, whey, rice bran, wheat bran, a sweetener, a mineral, a vitamin, salt, and grass. Daily dose of the peptide ranges from about 0.01 to about 10 grams per kg body weight of the animal.

[0013]In yet another embodiment, the present invention is a broad spectrum antimicrobial compound for topical use comprising a peptide having two or more D-amino acids, carboxy-terminus reduced, pH and heat stable isolated from Brevibacillus sp. For example, the peptide may have the sequence Me₂Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH₂OH (SEQ ID NO.: 1), or any one of SEQ ID NOS.: 1-20.

[0014]Yet another embodiment is an isolated and purified nucleic acid having the sequence of BT operon (SEQ ID NO.: 21) that produces a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. The isolated and purified nucleic acid that encode one or more polypeptide sequences for BT operon proteins (SEQ ID NOS.: 22 to 28) that include one or more enzymes used to make a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. The invention also includes those isolated nucleic acids having at least 75% homology to SEQ ID NO.: 21. More specifically, the nucleic acid may encodes one or more polypeptide sequences for peptide synthesis operon proteins (SEQ ID NOS.: 22 to 28) that are enzymes used to make a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. One or more BT operon polypeptides are expressed from SEQ ID NO.:21 and comprise one or more enzymes used to make a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids.

[0015]An isolated bacterial sample for use with the present invention may include an isolated bacterial strain of Brevibacillus texasporus E58. Another embodiment is an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide having two or more D-amino acids isolated from Brevibacillus sp that inhibits the growth of at least one bacterium selected from the group consisting of: Staphylococcus, Enterococcus, Pneumococcus, Bacilli, Methanococcus, Haemophilus, Archaeoglobus, Borrelia, Synedrocyptis, Mycobacteria, Pseudomonas and E. coli. A bacteria may be transformed with an isolated and purified nucleic acid having the sequence of BT operon (SEQ ID NO.: 21) that produces a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. The protein expressed from the nucleic acid may include one or more BT operon proteins, or those related thereto. A vector may be modified or isolated that includes an isolated and purified nucleic acid having the sequence of BT operon (SEQ ID NO.: 21) that produces a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. One or more proteins may be expressed from the nucleic acid that encodes one or more BT operon proteins. The feed additive may also include an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide that has greater than 75% sequence homology to SEQ ID NOS.: 1-20.

[0016]The present invention also relates to peptides, and non-ribosomal peptide synthases that synthesize these peptides containing unusual amino acids and other types of modifications. The invention also includes methods of producing and using the peptides alone or synergistically with conventional antibiotics in the treatment and prevention of various microbial infections and protozoal infections and disorders related to such infections; tumor cell proliferation, growth and spread; or as an immune modulating agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0018]FIG. 1A is a gel that shows the purification of peptide BT. Tricine gel separation of BT and the associated antibiotic activity. Chloroform extracted peptides were separated on a precast 16.5% Tricine gel (purchase from BioRad). One gel was stained with coomassie blue (left) to show peptide bands. Another gel (right) was overlaid with agar embedded with Bacillus cereus. Clear zones in the bacterial lawn correspond to the species that collapses into a single band with a molecular weight of ˜1500. Molecular weight markers are as follows: triosephosphate isomerase 26.6 kD, myoglobin 17.0 kD, alpha-lactalbumin 14.4 kD, aprotinin 6.5 kD, insulin b chain, oxidized 3.5 kD, and bacitracin 1.4 kD.

[0019]FIG. 1B is a graph of mass spectrometry of chloroform-extracted BT Chloroform-extracted BT was ionized by addition of sodium chloride and then subjected to mass spectrometry analysis. Five ionized BT isomer groups (BT1555, BT1571, BT1583, BT1599 and BT1613) were detected and labeled.

[0020]FIG. 1C is a graph of mass spectrometry of purified BT1583. Fraction 33 of the C18 reverse phase HPLC was subjected to mass spectrometry analysis. Only protonated, sodium and potassium ionized BT1583 were detected;

[0021]FIG. 2A is a graph of BT1583 tandem mass spectrometry data. FIG. 2B is a partial BT1583 sequence structure deduced from amino acid composition and MS/MS experiments (Tables 1 and 2) (for complete motif and sequences see Tables 5 and 6, respectively);

[0022]FIGS. 3A to 3D are maps of the BT NRPS operon. FIG. 3A is a map of the construction of a supercontig from two contigs linked by a mate pair. Contig1 and contig 2 share a mate pair from a clone. The contigs are ordered and arranged to form a supercontig, which contains the sequences of contig 1 and contig 2, separated by an unsequenced gap region;

[0023]FIG. 3B is a map of the region sequenced in this work and the location of 9 ORFs found in the region. Six ORFs btA through btF encode the BT NRPS subunits (BtA, BtB, BtC, BtD, BtE and BtF);

[0024]FIG. 3C is a map of the domain organization of the BT NRPS subunits. The predicted amino acid substrate specificity of each module is marked in each A-domain;

[0025]FIG. 3D is a Phylogenetic tree of a multiple sequence alignment of all 13 binding pocket constituents as described in Table 3. The putative specificity was assigned using the partial BT1583 sequence. It is shown that those binding pockets of A-domains that supposedly activate the same or similar substrate cluster together;

[0026]FIG. 3E is the nucleic acid sequence of the BT operon (SEQ ID NO.:21);

[0027]FIG. 3F is the amino acid sequence of BtA (SEQ ID NO.:22);

[0028]FIG. 3G is the amino acid sequence of BtB (SEQ ID NO.:23);

[0029]FIG. 3H is the amino acid sequence of BtC (SEQ ID NO.:24);

[0030]FIG. 3I is the amino acid sequence of BtD (SEQ ID NO.:25);

[0031]FIG. 3J is the amino acid sequence of BtE (SEQ ID NO.:26);

[0032]FIG. 3K is the amino acid sequence of BtF (SEQ ID NO.:27);

[0033]FIG. 3L is the amino acid sequence of BtG (SEQ ID NO.:28);

[0034]FIGS. 4A to 4E are sequence alignment of conserved motifs and alignments of the adenylation, consensation, thilation, epimerization and reductase domains from the BT NRPS modules, respectively. Conserved motifs were identified according to (Marahiel, 1997). Consensus sequences were placed under each alignment. Residues agree with consensus were black shaded. All 12 C-domains were aligned together, with the * symbols indicate the start C domains that are known to be less conserved;

[0035]FIGS. 5A to 5E are ATP-PPi exchange assays for the relative substrate specificities of the purified A-domains of Modules 8, 5, 7, 4 and 2, respectively, obtained from the ATP-PPi exchange assays were listed A) to D), respectively. The highest activity was defined as 100%. All 20 proteinogenic amino acids and L-Orn were tested in each assay, and background was usually below 1%. Apparent Km of the A-domains toward specific amino acids were listed underneath; and

[0036]FIG. 6A is a summary of synthetic BT variants and FIG. 6B is a correlation between the BT variants and their properties for antibiotic activity and Pronase resistance as previously described.

DETAILED DESCRIPTION OF THE INVENTION

[0037]While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0038]To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0039]As used herein, the term "domestic animal" describes, e.g., swine, cattle, horse, goat, sheep, deer, dog, cat and any of a number of useful rodents. The term "poultry" as used herein includes, e.g., chicken, hen, quail, turkey, guinea fowl and so forth. The term "nursery fishes" used here includes, e.g., carp, catfish, rainbow trout, ayu (sweet fish), eel, tilapia, conger, salmon, trout, red seabream, yellow tail, flounder, globefish, and so forth. A number of other animals are contemplated to also be useful, e.g., shrimp and prawn.

[0040]As used herein, the terms "additive" and "feed additive" are used to describe compositions from bacteria that may be used in conjunction with animal feed as feed additive resulting in an improvement of the health of livestock, poultry and fish, and a reduction of economic loss due to reduced or low weight and/or increasing the rate of growth (e.g., weight) of existing health animals. For example, the feed additive of the present invention may be used from bacterial isolates, partially or wholly degraded bacteria, isolated, isolated and purified from bacteria and/or synthesized synthetically in whole or in part. The additive or feed additive for the domestic animals, poultry and fishes may be of powder, grain or liquid form and will be used in accordance with the feeding condition and installations of the farm and the target animal.

[0041]Suitable animal feedstuffs include, e.g., green feed, silages, dried green feed, roots, tubers, fleshy fruits, grains and seeds, brewer's grains, pomace, brewer's yeast, distiller's spent grains, milling byproducts, byproducts of the production of sugar, starch and oil recovery and various food wastes. The feed additive of the present invention may be used alone or in conjunction with other well-known feed additives such as antioxidants or mixtures of various substances (mineral mixtures, vitamin mixtures) that can be added to such feeds for enhancement. Specific feeds may also adapted for certain animal species depending on age and stages of development.

[0042]Base feeds suitable for use in conjunction with the peptides of the present invention may be prepared as is well-known to the artisan skilled in the art of preparing feeds, e.g., they may use those as described in Kirk-Othmer, Encyclopedia of Chemical Technology, 4th Ed., vol. 10, pp. 288-300, Wiley, N.Y., 1993, relevant portions incorporated herein by reference. For example, the base feed may include one or more of the following ingredients: corn, sorghum, barley, wheat, soybean, peanut, canola, fish meal, milk products, fats and oils, vitamins and minerals.

[0043]The present inventor recognized that an NRPS operon may be a source of information that allows one to learn certain structural details of the peptide product. The identification of the BT NRPS operon results in critical refinements of the BT1583 peptide structure. Soil microorganisms were screened for strains that produce novel antibiotics. A Bacillus spp. E58 (ATCC PTA-5854) was isolated for its ability to produce an antibiotic BT against Staphylococcal aureus that cause life-threatening hospital-acquired infections in immunity-compromised patients The strain was named Brevibacillus texasporus based on its relatedness to Brevibacillus laterosporus.

[0044]The modules of an NRPS are composed of smaller units or "domains" that each carries out a specific role in the recognition, activation, modification or joining of amino acid precursors to form the peptide product. One type of domain, the adenylation (A-) domain, is responsible for selectively recognizing and activating the amino acid that is to be incorporated by a particular module of the NRPS. Through analysis of the substrate-binding pocket of the A-domain of the PheA subunit of the Gramicidin S NRPS in combination with sequence comparison with other A-domains, it was possible to define ten residues that are the main determinants of the substrate specificity for an A-domain (Conti, et al., 1997; Stachelhaus, et al., 1999). The ten residues are considered an NRPS `codon`. The NRPS codon collection is still growing as new NRPS codons continue to be discovered. The present invention includes the isolated and purified nucleic acids and the proteins encoded thereby for a group of novel NRPS codons for Valine, Lysine, Ornithine and Tyrosine.

[0045]The amino acid activation step is ATP-dependent and involves the transient formation of an amino-acyl-adenylate. The activated amino acid is covalently attached to the peptide synthase through another type of domain, the thiolation (T-) domain that is generally located adjacent to the A-domain. The T-domain is post-translationally modified by the covalent attachment of a phosphopantetheinyl prosthetic arm to a conserved serine residue. The activated amino acid substrates are tethered onto the NRPS via a thioester bond to the phosphopantetheinyl prosthetic arm of the respective T-domains. Amino acids joined to successive units of the NRPS are subsequently covalently linked together by the formation of amide bonds catalyzed by another type of domain, the condensation (C-) domain. NRPS modules can also occasionally contain additional functional domains that carry out auxiliary reactions, the most common being epimerization of an amino acid substrate from the L- to the D-form. This reaction is catalyzed by a domain referred to as an epimerization (E-) domain that is generally located adjacent to the T-domain of a given NRPS module. Thus, a typical NRPS module has the following domain organization: C-A-T-(E).

[0046]Product assembly by NRPS involves three distinct phases, namely chain initiation, chain elongation, and chain termination (Keating & Walsh, 1999). Peptide chain initiation is carried out by specialized modules termed a "starter module" that comprises an A-domain and a T-domain. Elongation modules have, in addition, a C-domain that is located upstream of the A-domain. It has been experimentally demonstrated that such elongation domains cannot initiate peptide bond formation due to interference by the C-domain (Linne & Marahiel, 2000). All the growing peptide intermediates are covalently tethered to the NRPS during translocations as an elongating series of acyl-5-enzyme intermediates. To release the mature peptide product from the NRPS, the terminal acyl-5-enzyme bond must be broken. This process is the chain termination step and is usually catalyzed by a C-terminal thioesterase (TE) domain. Thioesterase-mediated release of the mature peptide from the NRPS enzyme involves the transient formation of an acyl-O-TE intermediate that is then hydrolyzed or hydrolyzed and concomitantly cyclized to release the mature peptide (Keating, et al., 2001). An alternative termination scheme involves reduction of the tethered C-terminal residue by a reductase (R-) domain that resides in the last NRPS module, resulting in release of a peptide with an alcoholic C-terminal residue (Gaitatzis, et al., 2001; Kessler, et al., 2004). Such reductase-mediated termination/C-terminal modification occurs in BT biosynthesis and contributes to super protease resistance of the BT peptides.

[0047]Identification and isolation of the NRPS operon was useful to the studies of a peptide antibiotic, however, identification of a specific NRPS operon remains a challenging task. Identification of an NRPS operon traditionally starts with identification of clones in a genomic BAC or cosmid library by hybridization with DNA probes from known NRPS genes or by gene fragments amplified by PCR of genomic DNA using degenerate primers. Because the amino acid sequences of NRPS domains are usually quite similar, such approaches can be successful, however, because probes or primers are often imperfect, some NRPS operons can be missed. Moreover, microbes often contain multiple NRPS operons, so that the probes or primers may reveal some NRPS operons but not the one sought. This often results in ill-fated efforts devoted towards an incorrect gene (Hopwood, 1997). A novel in silica approach was used as described herein to allow rapid and accurate identification of an NRPS operon.

[0048]Materials and Methods. Partial purification of BT. E58 B. texasporus cells were grown in one liter of LB in a 37° C. air shaker for three days. The culture was spun in a clinical centrifuge at 3000 rpm for 15 minutes. The supernatant was collected and 500 grams of ammonium sulfate was added and dissolved. The sample was spun in the clinical centrifuge at 3000 rpm for 15 minutes. The pellets were dissolved in 200 ml of distilled water. The solution was then boiled for 15 minutes and then cooled on ice. The sample was filtered with a 0.2 micron filter (Nalgen). The filtrate was mixed with 0.2 liter of chloroform at room temperature for 20 minutes with a stir bar. The mixture was separated into two phases through centrifugation in the clinical centrifuge at 3000 rpm for 15 minutes. The organic phase was collected and dried in a vacuum evaporator.

[0049]C18 reverse phase HPLC. The dried chloroform extract was dissolved in 2 ml of sterile distilled water. The solution was fractionated on a C18 reverse phase HPLC column in a gradient from 30% B to 55% Solution B (Solution B is 0.075% TFA in acetonitrile, Solution A is 0.1% TFA in water). Resultant fractions were dried and dissolved in sterile distilled water and analyzed for anti-S. aureus activity in a plate clear zone assay The peak fraction (Fraction 33) was subjected to amino acid composition, mass spectrometry, tandem mass spectrometry and chirality analyses.

[0050]Amino acid composition. Amino acid analysis was performed by the Protein Chemistry Laboratory at Texas A&M University in College Station, Tex. Samples were mixed with internal standards, dried in glass tubes in a vacuum concentrator and subjected to vapor phase hydrolysis by 6N HCl at 110° C. for 24 hours under argon atmosphere in the presence of phenol. The samples were subsequently reconstituted in borate buffer and transferred to a Hewlett Packard AminoQuant II system for automated derivatization and loading. The AminoQuant analyzes peptides and proteins by pre-column derivatization of hydrolyzed samples with o-phthalaldehyde (OPA) and 9-fluoromethyl-chloroformate (FMOC). The derivatized amino acids are separated by reverse phase HPLC and detected by UV absorbance with a diode array detector or by fluorescence using an in-line fluorescence detector.

[0051]Mass spectrometry and tandem mass spectrometry. Detection of D-form amino acid residues. The chiral analysis of amino acid residues in BT was performed by Commonwealth Biotechnologies, Inc. of Richmond, Va. BT was subjected to hydrolysis in 6N HCl in vacuum for 18 hours at 110° C. The amino acids were derivatized to FMOC amino acids and separated by HPLC chromatography. The elution profile of each amino acid was then determined on a chiral column. For both types of chromatography columns, peaks were identified by comparisons with appropriate standards.

[0052]Genomic DNA preparation. Log-phase E58 cells were harvested from an LB culture and lysed with Lysis Buffer [10 mM Tris (pH 8.0), 100 mM EDTA, 0.5% SDS]. RNase A was added to digest contaminating RNA. Genomic DNA was extracted with phenol/choloroform and then precipitated with ethanol. Dried DNA was resuspended in TE and an aliquot was run in 0.5% agarose gel for quality control.

[0053]Library construction and genome sequencing. The E58 genomic library construction, shot-gun sequencing and the assembly were performed by Agencourt Biosciences Corporation (Beverly, Mass.). Briefly, the whole genome library was constructed with an average insert length around 5 kb. 10,000 such clones were subject to automated DNA sequencing from both ends of the insert. 16,901 successfully sequenced reads were collected and assembled.

[0054]Nucleotide sequences and data analysis. All BLAST analyses against E58 genome were performed by use of WU BLAST software package (version 2.0) installed on a local computer (Gish, W. 1996-2003. http://blast.wustl.edu). Amino acid sequence homology searches were performed by use of the BLAST server at the National Center for Biotechnology Information (Bethesda, Md.) and nonredundant protein sequence database with default parameter values (Altschul, et al., 1990). Amino acid sequence alignments were performed by use of the CLUSTALW program (Thompson, et al., 1994) running at NPS (web server at Institute of Biology and Chemistry of Proteins (Lyon, France).

[0055]The BT NRPS operon. The BT NRPS operon (Supercontig 3) contained 11 contigs, spanning a region of at least 46 kb. There were unsequenced regions, regions that were just sequenced once, and regions with bad sequencing quality. Also, carboxyl region of the thirteenth module was not covered by Supercontig 3. Three rounds of primer extension sequencing and one round of genome walking were performed to achieve the finishing of the NRPS operon. All original sequencing reads in Superontig 3 were extracted and reassembled using the SeqMan (Lasergene, DNASTAR Inc.). The default parameters were used for the reassembly. A higher stringency adopted by SeqMan caused the reassembled Supercotig 3 to break into 17 contigs with 16 unsequenced gaps. All contigs were further examined manually for single coverage and bad quality regions. Primers were designed to sequence into a gap as well as to obtain additional reads in the single coverage and low quality regions. New sequencing reads were joined with the original reads to create a new supercotig. The new supercotig was checked again for gaps, single coverage and low quality regions. After three rounds of such primer extension and reassembly, the putative BT NRPS operon was assembled into a single contig of 48,997 bp in length. To verify the assembled sequence, an EcoRI plus HindIII double digestion was performed with 20 clones that collectively spanned the whole region. Resultant digestion patterns were in perfect agreement with the restriction map predicted by the contig (data not shown). To sequence the downstream region of the contig, genome walking was successfully performed with E58 genomic DNA using GenomeWalker kit from Clontech. The effort resulted in a DNA sequence of 50,674 bp covering the putative BT NRPS operon (Genbank accession # ______).

[0056]Cloning, overexpression, and purification of His10-tagged BT A-domain proteins. DNA fragments encoding the A-domains of the BT NRPS Modules 8, 5, 7, 4 and 2 (Bt8A, 8t5A, Bt7A, Bt4A and Bt2A) were PCR-amplified and the PCR products were inserted into the His10-tag recombinant protein expression vector pET16b (Novagen). The A-domain borders were determined as defined by (Konz, et al., 1999). The expression constructs were transformed into the E. coli BL21-AI strain (Invitrogen). Transformants were grown in L-broth at 37° C. to an A600 of 0.6 and then induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) plus 0.2% L-arabinose. The cells were allowed to grow for two additional hours at 30° C. before being harvested. Purification of the His10-tag recombinant proteins was achieved using the TALON metal affinity resins (BD Biosciences) under conditions recommended in the manual with modifications. Briefly, the E. coli cells were broken by sonication. Cell lysates were cleared by centrifugation at 25,000×g for one hour. His-tagged recombinant proteins were then incubated with the TALON resin, washed, and eluted with 500 mM imidazole. Eluted proteins were dialyzed against a buffer (50 mM HEPES, pH 8.0, 100 mM sodium chloride, 10 mM magnesium chloride, and 1 mM EDTA) and then analyzed with SDS PAGE plus Coomassie Blue staining. The recombinant proteins displayed apparent molecular weights compatible with calculated ones, and they appeared to be purified to homogeneity. Concentrations of the purified proteins were determined by using the calculated molar extinction coefficient for the A₂₈₀.

[0057]ATP-PPi exchange assay. ATP-PPi exchange assays were performed to determine the substrate specificity of an A-domain. ATP-PPi exchanges were assayed as previously described (Stachelhaus, et al., 1998) with minor modifications. The assay mixture contained 50 mM HEPES (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM amino acid, 0.05 mM PPi, 0.15 μCi tetrasodium [³²P]pyrophosphate. Exchange was initiated by addition of purified recombinant A-domain proteins to a total volume of 0.1 ml. The protein concentrations were 0.2 μM for Module 4 and Module 5 A-domains while 2 μM for Module 7 and Module 8 A-domains. After incubation at 37° C. for 15 min, the reaction was stopped by addition of 0.5 ml of Terminaton Mix (100 mM tetrasodium pyrophosphate, 3.5% HClO4, and 1.6% [w/v] activated charcoal). The charcoal was pelleted by centrifugation, washed first with 40 mM pyrophosphate plus 1.4% perchloric acid and then with water, and was re-suspended in 0.5 ml of water. The charcoal/water suspension was added to a scintillation vial containing 5.0 ml of scintillation fluid, and the bound radioactivity was determined by liquid scintillation counting. The apparent Km values were determined with substrate concentrations ranging from 0.1 to 10 mM.

[0058]MIC determination assays. Staphylococcal aureus was grown to mid-log phase in LB at 37° C., and diluted by 500-fold with fresh LB and dispensed into 96-well micro-titer plates. Different concentrations of peptides were added, and the micro-titer plates were incubated at 37° C. with shaking. A minimal inhibition concentration (MIC) was determined as the lowest peptide concentration that produced a clear well. All experiments were performed in triplicates, and highly consistent MICs were obtained.

[0059]Identification of the BT peptides. The bacterial strain E58 was isolated from soil in an effort to identify soil microorganisms that produce novel antibiotics against Staphylococcus aureus. E58 was found to be closely related to Brevibacillus laterosporus based on the 16S rDNA sequence homology (98.5% identity). E58 was named Brevibacillus texasporus and deposited to ATCC (catalog number PTA-5854). The antibiotic produced by E58 was named BT and its activity could be detected in the supernatant of a liquid E58 LB culture. Cell-free culture supernatant was, therefore, the starting material for BT purification. The antibiotic activity was precipitated by ammonium sulfate, which suggested that the antibiotic be a protein or peptide (data not shown). The activity was further extracted into chloroform, indicating that BT is made of small molecules. The antibiotic chloroform extract was evaporated in a vacuum evaporator, dissolved in water and then run on a SDS tricine gel. The two halves of a gel with identical lanes in each half were either stained for proteins/peptides or overlaid with agar embedded with BT-sensitive bacteria Bacillus cereus to test for antibiotic activity (FIG. 1A). Three species were visible after staining: the Bromophenol Blue dye originated from the gel loading buffer, an unknown peptide with a mass <1.4 kD and a third species with an antibiotic activity. This third species ran as a ˜1.5 kD band at low concentrations (clearly visible on the original gel) and were later shown made up of a group of related peptides (see below). Their apparent masses increased with concentration suggesting that the peptides aggregate at higher concentrations. An antibiotic activity was seen associated with the peptides at higher concentrations, and we therefore concluded that the peptides likely conferred the BT antibiotic activity. The peptides were referred as the BT peptides. The BT peptides apparently were not toxic to B. cereus at the lower concentrations in this assay. Since the smallest detectable BT band ran at ˜1.5 kD, therefore the BT peptides contained approximately 13 residues.

[0060]The chloroform-extracted BT was subject to a mass spectrometry assay. A group of peptides were detected in a range between 1550 and 1650 Daltons (FIG. 1B). The main species showed a molecular weight of 1583, and it was named BT1583. The other peptides were later shown to be isomers of BT1583 (Tables 5 and 6).

[0061]Partial BT Sequence Determination. The chloroform-extracted BT was purified further by C18 reverse phase HPLC (see Materials and Methods for details). BT1583 was purified to homogeneity in Fraction 33 of the C18 HPLC (FIG. 1C). An amino acid composition analysis of BT1583 (Fraction 33) showed BT1583 contained residues of Tyr, Lys, Leu, Ile, Val and Orn. BT1583 was refractory to N-terminal sequencing and resistant to degradation by aminopeptidase M, suggesting that a non-standard N-terminal residue. BT1583 was also resistant to cleavage by carboxypeptidase Y, suggesting a non-standard C-terminal amino acid. Carboxyl-terminal sequencing was, therefore, not attempted.

[0062]Tandem mass spectrometry (MS/MS) was then chosen to sequence the BT1583 peptide. MS/MS data were obtained for BT1583 and they are shown in FIG. 2A and Table 2. The MS/MS data indicated that BT1583 contained 13 amino acid residues that correlated well with the amino acid composition. As expected, the masses of Residues 1 and 13 did not correspond to any standard amino acids. The last residue showed a mass of 103 daltons, which appeared to be compatible with a Valine having its C-terminus reduced from a carboxylic acid to an alcohol. The presence of a C-terminal alcoholic Valine was further confirmed by the presence of a reductase domain in the 13th Valine-specific module of the BT NRPS (see below). The identity of the N-terminal residue was more difficult to determine. Nonetheless, an N-terminal residue with a mass of 198 seemed to be compatible with the N,N-methylated form of Bmt {4-methyl-4-[(E)-2-butenyl]-4,N-methyl-Threonine} (Offenzeller, et al., 1996; Offenzeller, et al., 1993)

[0063]The presence of Ornithine in BT1583 indicated that BT1583 could not be synthesized by ribosomes. The presence of D-amino acids would strengthen this idea. We chose to assess the chiral properties of two of the most abundant residues in BT1583, Val and Leu. Chiral analyses revealed uniform L-Val residues but both L- and D-Leu residues at a ratio of 2:1.

[0064]The above biochemical and structural analyses were able to provide us with a partial BT1583 peptide sequence (Table 2 and FIG. 2B). The structures of the N- and C-terminal residues were not fully determined. Isoleucine and Leucine could not be distinguished. The position of the D-form Leu was not specified. Chiral properties of other residues in the peptide were not determined.

[0065]Shot-gun sequencing of the E58 genome. To better understand the structure and biosynthesis of the BT1583 peptide, we decided to identify the gene or operon that is responsible for the BT biosynthesis. The presence of non-proteinogenic Ornithine and D-form amino acids in the peptide led us to believe that BT1583 was synthesized by the NRPS in vivo (Marahiel, 1997). Most of the NRPS genes are co-linear reflecting a strict correlation between NRPS modules and the amino acid residues in the peptide product. If the BT NRPS operon is co-linear, it should encode 13 modules corresponding to the 13 amino acid residues in the BT1583 peptide. Assuming that on average, each module is encoded by an average 3.5 kb DNA fragment, a DNA fragment of 46 kb long would be necessary to accommodate the BT NRPS operon. As mentioned before, the traditional method to identify an NRPS operon involves probing a cosmid library with a generic probe. Since an imperfect generic probe may miss the target gene and there are usually multiple NRPS operons in a bacterial genome, such method frequently causes researchers to chase the wrong NRPS operon. To avoid such pitfall, we developed a genomic approach that provides an unbiased in silica overview of all NRPS operons in a genome to allow direct comparisons of the NRPS operons and therefore rational candidate operon selection. This novel approach resulted in rapid and accurate identification of the BT NRPS operon.

[0066]The E58 genome was estimated to be 5 Mb. An E58 genomic library was constructed with an average insert size of 5 kb. The whole genome was sequenced for a two-fold coverage. After sequence assembly, the E58 genome was represented by 1919 contigs with sizes ranging from 700 bp to 22.6 kb and 932 singlets. Such coverage would allow 99.995% of the genome to be represented by clones. Also, the average length of the gap between two neighboring contigs would be as small as 250 bp so that supercontigs could be constructed (see below). Moreover, supercontigs at such resolution would contain sufficient information to allow accurate in silica NRPS operon identification.

[0067]In silica identification of the BT NRPS operon. A three-step procedure was used to select the candidate BT NRPS operon. First, all contigs and singlets were searched for sequences encoding NRPS modules. Since E58 is related to B. subtilis, the putative peptide synthetase PPS1 from B. subtilis was chosen as the query sequence for BLAST analysis against a database containing all assembled E58 contigs. 128 contigs showed translated amino acid sequence similarities to PPS1, with P-values arranging from 0 to 1.

[0068]Second, supercontigs were constructed from the 128 contigs. Two sequencing reads from the ends of the same insert form a mate pair. A supercontig is a collection of contigs joined mate pairs that reside in different contigs. Identification of mate pairs allowed neighboring contigs to be ordered and orientated to form a supercontig (FIG. 3A). 31 supercontigs were successfully constructed to represent the whole E58 NRPS operon portfolio.

[0069]The candidate BT NRPS operon was selected from the E58 NRPS operon portfolio. The 31 supercontigs were examined for the possibility of harboring the BT NRPS operon, and Supercontig 3 (whose genetic features based on finished sequence are shown in FIGS. 3B and 3C) was chosen as the candidate based on the following analyses.

[0070]Supercontig 3 potentially contained DNA sequence encoding 13 NRPS modules. Available information regarding the A-domain substrate specificities of Supercontig 3 showed compatibility with the partial BT1583 sequence. Complete sets of substrate specificity-conferring amino acid residues could be identified for eleven modules (except Modules 2 and 13 due to incomplete DNA sequence). Although not all specificity predictions could be made, good correlations were established between predicted NRPS amino acid substrates and the partial BT1583 sequence. Specifically, Module 4 was predicted to incorporate Ile, and Modules 9 and 12 were predicted to incorporate Leu (Table 3, see below for details). The partial BT1583 sequence had Leu or Ile at Positions 4, 9 and 12. Phylogenetic analysis of the substrate conferring amino acids of the eleven modules showed that modules expected to incorporate the same or highly similar amino acid did group together (FIG. 3D). For example, Modules 5, 6 and 8 that were all predicted to incorporate Val formed a cluster. Modules 7, 10 and 3 that were predicted to incorporate similar cationic amino acids (Lys and Orn respectively) formed another cluster.

[0071]The E-domain positions in the NRPS encoded by Supercontig 3 showed compatibility with the partial BT1583 peptide structure. Four E-domains were found in Modules 3, 7, 9 and 11 (FIG. 3C). Their positions were consistent with the aforementioned BT1583 chiral properties of all L-form Val residues and a 2:1 L- to D-form Leu residue ratio.

[0072]Supercontig 3 was therefore identified as the candidate locus for the BT NRPS operon. Primer extensions and genome walking were performed to obtain high quality sequence of the locus. The efforts resulted in a contig of 51,821 bp covering the putative BT NRPS operon (Genbank accession #), see FIG. 3F.

[0073]Putative BT NRPS subunits. Ten open reading frames (ORFs) were identified in the sequenced region through translation analysis and blast searches (Altschul, et al., 1997) (FIG. 3B). The middle six ORFs (named btA through btF) were predicted to encode six subunits of the BT NRPS (BtA through BtF), and their coordinates are listed in Table 4. Sequence analysis of the putative subunits confirmed the modular structure of a typical co-linear NRPS (FIG. 3C). The modules, each containing an A-domain and a T-domain, are linked by a C-- domain. The loading module BtA has an A-domain followed by a T-domain. There are two noticeable overall features for the putative BT NRPS subunits. First, four out of six subunits exhibit a two-module structure. Second, all auxiliary E-domains are present at the end rather than in the middle of the putative NRPS subunits. Sequence alignments of conserved domains are shown in FIG. 4.

[0074]A reductase domain in Module 13. A domain of about 500 amino acids was identified at the C-terminus of BtF or Module 13. BLAST analysis showed that it has high similarity with several NADPH-dependent reductases from other NRPSs and polyketide synthetases. Its alignment with the reductase domains from MxcG of S. aurantiaca and Lys2 of S. cereviciae is shown in FIG. 4E. A similar reductase domain has also been identified in the Gramicidin A NRPS (Kessler, et al., 2004). All three reductases have been experimentally demonstrated to reduce their substrates to corresponding aldehydes in an NADPH-dependent reaction (Gaitatzis, et al., 2001; Kessler, et al., 2004; Sagisaka & Shimura, 1959). For myxochelin A and gramicidin A, the aldehydes are further reduced to alcohols. The exact mechanism for the second reduction step has not been identified. Either those reductase themselves or another proteins carry out the second reduction step, or the second reduction step is spontaneous. The MS/MS experiment suggested that the C-terminal residue of BT1583 might be the alcoholic form of Valine (FIG. 2B). The A-domain specificity prediction of the last putative BT NRPS module and the presence of a reductase domain in the module confirmed this proposal.

[0075]btG encodes an ABC transporter. btG is an ORF that is immediately downstream of btF, and it is transcribed in the same direction as are other bt ORFs. The initiation codon ATG is located 61 bp downstream of the btF stop codon. Translated amino acid sequence showed high similarity to members of the ATP-binding cassette (ABC) transporter super-family (data not shown). ABC transporter ORFs are found in typical NRPS operons. Their roles have been proposed to provide host with resistance to the peptide antibiotic product by pumping the peptide out of the cells. The exact role of the putative BtG ABC transporter needs to be established.

[0076]BT1583 peptide sequence refinement. The substrate specificity-conferring residues (Stachelhaus, et al., 1999) were extracted from all 13 A-domains and were compared to the collection of the amino acid-binding pocket constituents in the public NRPS codon database (raynam.chmjhu.edu/˜nrps/index.html) (Challis, et al., 2000). Substrate specificity predictions were made based on the sequence alignments and they are listed in Table 3. The amino acid-binding pocket constituents of the first module showed a perfect match with an NRPS codon for Threonine/Dehydrothreonine, and it was predicted that Module 1 incorporates a Threonine derivative. N,N-methylated Bmt was proposed to be the N-terminal amino acid residue according to the MS/MS data (FIG. 2B and Table 2). Although the two proposals do not agree with each other 100%, both call for a Threonine derivative as the N-terminal amino acid residue.

[0077]As mentioned before three unambiguous specificity assignments could be made for Module 4 (Ile), Module 9 (Leu) and Module 12 (Leu) according to the NRPS codon database. These assignments were compatible with the partial BT1583 sequence and accordingly Positions 4, 9 and 12 of the BT1583 peptide were refined to Ile, Leu and Leu respectively. Since the only Ile of the BT1583 peptide had been assigned to Position 4, the remainder Leu was assigned to Position 2 of the BT1583 peptide. The A-domain specificity of Module 2 was therefore deduced to be Leu. These assignments in conjunction with the E-domain positional information allowed us to refine the BT1583 peptide sequence to (CH₃)₂-Bmt-Leu-_dOrn-Ile-Val-Val-_dLys-Val-_dLeu-Ly- s-_dTyr-Leu-Val-CH₂OH.

[0078]Novel NRPS codons in BT biosynthesis. The amino acid-binding pocket constituents of Modules 5, 6 and 8 are identical. They differ with those of Module 13 by only one residue. No good matches were found for these sets of amino acid-binding pocket constituents in the NRPS codon database. However, they showed similarities to certain Ile, Leu or Val NRPS codons in the database. Since the partial BT1583 peptide sequence had Val residues at Positions 5, 6, 8 and 13, Modules 5, 6, 8 and 13 were deduced to incorporate Val. The amino acid-binding pocket constituents of Modules 5, 6, 8 and 13 represent potential novel NRPS codons for Val.

[0079]The amino acid-binding pocket constituents of Modules 7 and 10 are identical and they differ with those of Module 3 by only one residue. No match was found for these sets of amino acid-binding pocket constituents in the NRPS codon database. Since the partial BT1583 peptide sequence had Lys residues at Positions 7 and 10, the specificities of these modules were deduced to be Lys. Likewise the partial BT1583 peptide sequence had an Orn residue (which is highly similar to Lys in structure) at Position 3, and the specificity of Module 3 was therefore deduced to be Orn. The amino acid-binding pocket constituents of Modules 7 and 10 represent potentially the first NRPS codon for Lys, while those of Module 3 represent a potential novel NRPS codon for Orn.

[0080]The specificity prediction for Module 11 was quite ambiguous according the NRPS codon database. No good match was found for this set of amino acid-binding pocket constituents in the NRPS codon database. However, it showed similarities to certain Phe, Trp or Tyr NRPS codons in the database (data not shown). Since the partial BT1583 peptide sequence had Tyr residues at Position 11, the A-domain specificity of Module 11 was therefore deduced to be Tyr. The amino acid-binding pocket constituents of Module 11 represent a potential novel NRPS codon for Tyr.

[0081]Identity verification of the BT NRPS operon. Since the BT biosynthesis involves novel NRPS codons, experimental establishment of the novel codons (especially the novel Valine and Lysine codons) is critical to verifying the identity of the BT NRPS operon. In addition, since the placement of Ile at position 4 in BT1583 affects the placement of three Leu residues, the Module 4 codon also needed to be tested.

[0082]Since a purified recombinant A-domain of an NRPS module can selectively and efficiently activate the cognate amino acid substrate of the NRPS module in an ATP-PPi exchange assay (Konz, et al., 1999; Mootz & Marahiel, 1997), ATP-PPi exchange assays have been used to experimentally establish NRPS module specificities and novel NRPS codons. Recombinant A-domains of Modules 8, 5, 7, 4 and 2 of the BT NRPS were produced and purified as described in Methods and Materials. Almost completely soluble recombinant A-domain proteins were obtained. A-domain specificities were determined in ATP-PPi exchange and aa Km assays (see Methods and Materials), and the results are shown in FIG. 5. All 20 proteinogenic amino acids and L-Orn were tested for each A-domain protein, and background noise in the experiments was usually below 1%.

[0083]The Module 8 A-domain protein was shown to activate L-Val (100%), with minor activation of L-Lys (10%) and L-Ile (4%). The apparent K_m was determined to be 2.75 mM for L-Val. These results confirmed the novel Valine NRPS codon. Similarly, the Module 5 A-domain protein was found to activate L-Val (100%), L-Ile (23%), and L-Leu (17%). The apparent K_m was determined to be 1.11 mM for L-Val and 2.78 mM for L-Ile, clearly showing that L-Val is the preferred substrate for Module 5.

[0084]L-Lys was the only amino acid that activated by the Module 7 A-domain protein. The apparent K_m value was determined to be 1.12 mM. These results established the first Lys NRPS codon.

[0085]The Module 4 A-domain protein was shown to selectively activate L-Ile (100%), with minor activation of L-Val (9%) and L-Leu (7%). The apparent K_m value for L-Ile was measured at 0.5 mM.

[0086]The Module 2 A-domain protein was found to be quite ambiguous. It activated L-Leu (98%) and L-Met (100%) with nearly equal efficiency, with significant activation of L-Val (67%) and minor activation of L-Ile (19%) and L-Phe (3.5%).

[0087]In general, all purified A-domain proteins were found to selectively activate predicted amino acid substrates in the ATP-PPi exchange assays. These results experimentally confirmed the identity of the BT NRPS operon.

[0088]Synthetic peptides. To further verify the BT peptide sequence as well as the identity of the BT NRPS operon, a synthetic peptide P81 (FIG. 6) was made (by Biomer Technology, Concord, Calif.) and tested for its properties. Since Bmt is not commercially available, we were not able to synthesize a peptide according to the refined BT1583 sequence and we used octanic acid-modified Threonine to synthesize the lipopeptide P81 to mimic BT1583. P81 showed full antibiotic activity and Pronase resistance as BT1583. These results lend support to the refined BT1583 peptide sequence and the identity of the BT NRPS operon.

[0089]To investigate the significance of the C-terminal alcoholic modification, an amide form of P81 (P59) was synthesized. P59 displayed antibiotic activity but no Pronase resistance. These results indicated that the C-terminal alcoholic modification plays a key role in conferring protease resistance to P81 and likely BT1583 as well.

[0090]Since the codon for the first BT NRPS module matches perfectly with known Thr NRPS codons, the possibility of an active BT isomer with an unmodified Thr at Position 1 needed to be investigated. An amide form of P81 (P58) was therefore synthesized, and P58 displayed poor antibiotic activity. This result confirmed that a Thr derivative (rather than unmodified) Thr needs to be at Position 1 to confer antibiotic activity.

[0091]The L- and D-form residues alternate in the middle of BT1583 with the exception of Position 5 (Val). Since the alternating chirality is a key structural feature for the peptide antibiotic Gramicidine A, we decided to investigate whether we missed the coding sequence of an E-domain for Module 5. A D/L alternating version of P59 (P80) was synthesized. P80 displayed no antibiotic activity. The above results confirmed not only the BT1583 peptide structure (with the exception of the N-terminal residue) but also the identity of the BT NRPS operon.

[0092]The BT1583 peptide structure, the BT NRPS operon and the BT NRPS allow us to propose a degenerate formula for isomers of BT1583 (Table 5). Based on the relative substrate selectivity of each module, the BT isomers likely to be produced by E58 in significant amounts were predicted and listed in Table 6. Most of the predicted BT isomers were experimentally verified by MS/MS (data not shown).

[0093]The structure and biosynthesis of the BT peptides was determined using an integrated approach of biochemistry, biophysics and genomics. Amino acid composition and tandem mass spectrometry experiments with purified BT1583 (the main BT isomer) produced a partial peptide structure. The presence of Ornithine and D-form residues in the partial structure indicated that the peptide was synthesized by a non-ribosomal peptide synthase in vivo. The BT NRPS operon was rapidly and accurately identified via a novel in silica gene hunting scheme. Sequence analysis of the BT NRPS operon revealed that it encodes a co-linear modular NRPS. The co-linear nature of the BT NRPS enabled us to use the BT operon genomic information and refine the BT1583 peptide sequence to (CH₃)₂-Bmt-L-_dO-I-V-V-_dK-V-_dL-K-_dY-L-V-OH. Moreover, new NRPS codons for Valine, Lysine, Ornithine and Tyrosine were discovered and are reported here.

[0094]In silica NRPS operon identification. Traditional NRPS gene identification involves probing a genomic cosmid library with a generic probe. Such approach has the inherited shortcoming of causing researchers to chase the wrong gene in a genome with multiple NRPS operons. As shown herein, NRPS gene identification is improved for all NRPS operons in the genome when compared at the same time to find a best fit. Such comparison requires a draft genome. Fortunately, sequencing cost has decreased significantly to allow routine sequencing of microbial genomes. A two-fold coverage was sufficient for accurate NRPS operon identification. In actual BT NRPS operon selection, the following two sets of information are generated and compared to find the best candidate: the NRPS module clustering pattern of according to similarities of the substrate-binding pocket constituents; and positional information such as the positions of D-form residues. The module clustering technique is especially powerful in establishing the candidacy of an operon that involves novel NRPS codons (i.e., in the case of Modules 5, 6 and 8 of the BT NRPS operon). The in silica strategy is particularly useful for NRPS operon identification in strains (such as E58) that have a large number of NRPS operons in the genome.

TABLE-US-00001 TABLE 1 Amino acid composition of purified BT1583 peptide Molar ratios normalized to Residues Amino Acid nmoles Tyr Ile per peptide Tyrosine 1.75 1.00 1.16 1 Valine 4.58 2.62 3.05 3 Isoleucine 1.50 0.86 1.00 1 Leucine 5.32 3.04 3.54 3 Lysine 3.57 2.04 2.38 2 Ornithine 1.2 0.69 0.80 1 Total # of derivatizable residues 10.25 11.93 11

TABLE-US-00002 TABLE 2 Tandem mass spectrometry of BT1583 M/H+ Possible amino acid M/H+ y- Possible amino acid Compiled b-series ΔM residue series ΔM residue (N to C) 198.1 (CH3)₂-Bmt(?) (CH3)₂-Bmt(?) 311.16 113.06 L/I 1386.73 113.12 L/I L/I 425.21 114.05 O 1273.61 114.04 O O 538.28 113.07 L/I 1159.57 113.05 L/I L/I 637.32 99.04 V 1046.52 198.08 V + V V V 864.42 227.10 V + K 848.44 128.07 K K 963.46 99.04 V 720.37 99.04 V V 1076.52 113.06 L/I 621.33 L/I 1204.58 128.06 K K 1367.65 163.07 Y Y 1480.81 113.16 L/I L/I 1583.87 103.06 Val-CH₂OH Val-CH₂OH

TABLE-US-00003 TABLE 3 Predicted BT NRPS module substrate specificities and refinement of the BT1583 peptide structure. The residues were numbered according to the corresponding residues of PheA (Conti, et al., 1997). Predicted Partial Refined PheA Numbering Substrate BT1583 BT1583 Module 235 236 239 278 299 301 322 330 331 517 Specificity Seq. Seq. 1 D F W N I G M V H K Thr/Dht (CH₃)₂-Bmt* (CH₃)₂-Bmt 2 D G F L L G G V F K Ile/Leu Leu/Ile Leu** 3 D S G P S G A V D K Orn* Orn 4 D G F F L G V V Y K Ile* Leu/Ile Ile 5 D G F F V G G V F K Ile/Leu/Val Val* Val 6 D G F F V G G V F K Ile/Leu/Val Val* Val 7 D A G P S G A V D K Lys* Lys 8 D G F F V G G V F K Ile/Leu/Val Val* Val 9 D A W F L G N V V K Leu* Leu/Ile Leu 10 D A G P S G A V G K Lys* Lys 11 D A A A V V G V A K Phe/Trp/Tyr Tyr* Tyr 12 D A W F L G N V W K Leu* Leu/Ile Leu 13 D G F F A G G V F K Ile/Leu/Val Val-CH₂OH* Val-CH₂OH *The information was used for the BT1583 peptide sequence refinement. **The Leu at this position was deduced from the fact that the only Ile had been assigned to Position 4.

TABLE-US-00004 TABLE 4 The BT NRPS operon (see FIGS. 3G-3L). ORF Gene product Start End Length SEQ ID length MW Homology (nt) (nt) (bp) NO.: (amino acid) (kD) to btA 2,889 4,814 1,926 22 641 72.87 NRPS btB 4,817 12,409 7,593 23 2,530 288.99 NRPS btC 12,438 26,291 13,854 24 4,617 526.68 NRPS btD 26,321 33,946 7,626 25 2,541 289.31 NRPS btE 33,976 41,556 7,581 26 2,526 288.45 NRPS btF 41,584 49,059 7,476 27 2,491 284.46 NRPS btG 49,120 49,842 723 28 240 26.95 ABC transporter

TABLE-US-00005 TABLE 5 A degenerate formula for the BT isomers 1 2 3 4 5 6 7 8 9 10 11 12 13 Me₂Bmt L dO I V V dK V dL K dY L V--CH₂OH M V I V L I F Numbers indicate the amino acid residue positions.

TABLE-US-00006 TABLE 6 Summary of BT isomers Peptide sequences of the predicated Name products by the BT NRPS MW SEQ ID NO.: BT1583 Me₂Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1583 1 BT1601 Me₂Bmt-M-dO-I-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1601 2 BT1569V2 Me₂Bmt-V-dO-I-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1569 3 BT1583I2 Me₂Bmt-I-dO-I-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1583 4 BT1617 Me₂Bmt-F-dO-I-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1617 5 BT1597I5 Me₂Bmt-L-dO-I-I-V-dK-V-dL-K-dY-L-V-CH₂OH 1597 6 BT1597L5 Me₂Bmt-L-dO-I-L-V-dK-V-dL-K-dY-L-V-CH₂OH 1597 7 BT1615I5 Me₂Bmt-M-dO-I-I-V-dK-V-dL-K-dY-L-V-CH₂OH 1615 8 BT1615L5 Me₂Bmt-M-dO-I-L-V-dK-V-dL-K-dY-L-V-CH₂OH 1615 9 BT1583V2I5 Me₂Bmt-V-dO-I-I-V-dK-V-dL-K-dY-L-V-CH₂OH 1583 10 BT1583V2L5 Me₂Bmt-V-dO-I-L-V-dK-V-dL-K-dY-L-V-CH₂OH 1583 11 BT1597I2I5 Me₂Bmt-I-dO-I-I-V-dK-V-dL-K-dY-L-V-CH₂OH 1597 12 BT1597I2L5 Me₂Bmt-I-dO-I-L-V-dK-V-dL-K-dY-L-V-CH₂OH 1597 13 BT1631I5 Me₂Bmt-F-dO-I-I-V-dK-V-dL-K-dY-L-V-CH₂OH 1631 14 BT1631L5 Me₂Bmt-F-dO-I-L-V-dK-V-dL-K-dY-L-V-CH₂OH 1631 15 BT1569V4 Me₂Bmt-L-dO-V-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1569 16 BT1587M2V4 Me₂Bmt-M-dO-V-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1587 17 BT1555 Me₂Bmt-V-dO-V-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1555 18 BT1569I2V4 Me₂Bmt-I-dO-V-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1569 19 BT1603 Me₂Bmt-F-dO-V-V-V-dK-V-dL-K-dY-L-V-CH₂OH 1603 20 BT as a Feed Additive. (Semi-purified BT peptides rather than BT1583 were used in chicken growth promotion experiments.)

[0095]Based on the structure of the BT peptides and its biological and biochemical properties the present investigator produced sufficient material to test its use as a feed additive. In summary, the properties of the peptide used were as follows, 13 amino acid residues with numerous potential natural variants or isomers (>8) and derivatives (>30). Biologically, it was found that the BT peptides were a natural product produced by a Gram(+) bacterium. The BT peptide family is synthesized by a non-ribosome peptide synthase (NRPS), the cloning and characterization is disclosed herein. One such peptide, BT1583: Me₂Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH₂OH (SEQ ID NO.: 1) was selected for further studies because it is cationic and likely amphipathic, It contains unusual amino acid residues and/or includes multiple modifications.

[0096]BT1583 was also selected due to its high level of stability. The high stability observed for this peptide included one or more of the following characteristics: (1) no known enzymes can digest it; (2) it is not digested in the mouse or chicken GI track; (3) it can be autoclaved; (4) it survived the feed pelleting process; (5) it can stand extreme pHs (pH 1.0 and pH 13.0); and (6) the only known in vitro method to inactivate it is pH 1.0 plus 100° C. overnight.

[0097]In addition to the remarkable stability of BT1583, it demonstrated, in vitro, antibacterial against Gram-positive bacteria, e.g., for most Gram (+): MIC=1 microgram/ml. For Gram (-) the following were the antimicrobial activities observed: E. coli: MIC>20 microgram/ml; Pseudomonas and Salmonella: MIC>100 microgram/ml. BT1583 also shows antifungal, e.g., S. cerevisiae: MIC=50 microgram/ml. Anti-protozoal activity was also observed for BT1583 against, Tetrahymena: MIC=25 microgram/ml.

[0098]The E58 strain for producing BT1583 was selected because it was a fast growing and high peptide producer strain. Furthermore, in addition to fast peptide biosynthesis the strain is also grows in cheap media, e.g., with medium cost as low as 0.4 cents/L and a yield of, e.g., 0.5 g/L. Growth is generally carried out in an air shaker but may also be fermented. Furthermore, the peptide and the strain may be used without extensive adaptation of well-known procedures to an easy, one-step purification process.

[0099]The following tables and examples show the growth promotion capabilities and characteristics of the BT1583 peptide in Broiler Chicken, e.g., in a 21-day battery study.

TABLE-US-00007 TABLE 7 Summary of Growth Promoting Studies. Feed conversion Additional Study BT1583 concentration improvement (point) weight gain (%) 1-1 10 ppm 7 17 1-2 30 ppm 8 17 2-1 6 ppm 9 6.7 2-2 12 ppm 10 11 3-1 12 ppm 9 16 3-2 12 ppm with Coban 9 5.4 (vs Coban alone) 4-1 24 ppm with direct 13 7.1 coccidial challenge 4-2 48 ppm with direct 17 9.3 coccidial challenge

[0100]Briefly, the peptide was used in a semi-purified form to study the growth and feed conversion of 20-day old straight run broilers in batteries (Studies 2-1 and 2-2). Two amounts were tested against a feed control, peptide at 6 ppm and peptide at 12 ppm, 12 repetitions were carried out per treatment with 4 birds per pen. The diet used in the study was as follows.

TABLE-US-00008 TABLE 8 Basic Feed for Studies 2-1 and 2-2 PERCENT INGREDIENTS (Mash Feed) TAMU Corn 62.91 TAMU Dehulled Soybean Meal 30.67 DL Methionine 0.07 Blended A-V Fat 2.68 Limestone 1.45 Mono-Dicalcium Phosphate 1.58 Salt 0.33 TAMU Trace Minerals 0.05 TAMU Vitamins 0.25 NUTRIENT CONTENT (Calculated) Metabolizable Energy (kcal/kg) 3100 Protein (%) 20.0 Lysine (%) 1.05 Methionine + Cystine (%) 0.72 Threonine (%) 0.75 BT1583 added in 200 grams of corn meal carrier

Table 9 shows the Statistics for a Dependent Variable: 20-day cumulative weight gain.

TABLE-US-00009 Treatment Mean Std. Deviation Number Control 554.8236 38.13395 12 BT1583 @ 12 ppm 618.9340 46.79301 12 BT1583 @ 6 ppm 591.9750 47.93018 12 Total 588.5775 50.77136 36

Table 10 shows the Tests of Between-Subjects EffectsDependent Variable: 20-day cumulative weight gain

TABLE-US-00010 Type III Sum Source of Squares df Mean Square F Sig. Corrected 24868.642(a) 2 12434.321 6.279 .005 Model Intercept 12471247.008 1 12471247.008 6297.459 .000 TRE 24868.642 2 12434.321 .005 Error 65351.939 33 1980.362 6.279 Total 12561467.588 36 Corrected Total 90220.580 35 (a)R Squared = .276 (Adjusted R Squared = .232)

Table 11 shows the estimated marginal means for the study.Dependent Variable: 20-day cumulative weight gain

TABLE-US-00011 95% Confidence Interval Lower Treatment Mean Std. Error Bound Upper Bound Control 554.824 12.846 528.687 580.960 BT1583 @ 618.934 12.846 592.798 645.070 12 ppm BT1583 @ 6 ppm 591.975 12.846 565.839 618.111

Table 12 shows the Post Hoc Tests for Homogeneous SubsetsDependent Variable: 20-day cumulative weight gain Duncan

TABLE-US-00012 Subset Treatment N 1 2 Control 12 554.8236 BT1583 @ 6 ppm 12 591.9750 BT1583 @ 12 ppm 12 618.9340 Sig. 1.000 .147 Means for groups in homogeneous subsets are displayed. Based on Type III Sum of Squares The error term is Mean Square(Error) = 1980.362. a Uses Harmonic Mean Sample Size = 12.000. b Alpha = .05.

Table 13 shows the Descriptive StatisticsDependent Variable: 20-day cumulative feed conversion rate

TABLE-US-00013 Treatment Mean Std. Deviation N Control 1.5922 .13721 12 BT1583 @ 12 ppm 1.4959 .10089 12 BT1583 @ 6 ppm 1.5065 .04795 12 Total 1.5315 .10841 36

Table 14 shows the Tests of Between-Subjects EffectsDependent Variable: 20-day cumulative feed conversion rate

TABLE-US-00014 Type III Sum of Source Squares df Mean Square F Sig. Corrected 6.702E-02(a) 2 3.351E-02 3.212 .005 Model Intercept 84.440 1 84.440 8092.585 .000 TRE 6.702E-02 2 3.351E-02 3.212 .053 Error .344 33 1.043E-02 6.279 Total 84.851 36 Corrected .411 35 Total (a)R Squared = .163 (Adjusted R Squared = .112)

Table 15 shows the Estimated Marginal MeansDependent Variable: 20-day cumulative feed conversion rate

TABLE-US-00015 95% Confidence Interval Treatment Mean Std. Error Lower Bound Upper Bound Control 1.592 .029 1.532 1.652 BT1583 @ 1.496 .029 1.436 1.556 12 ppm BT1583 @ 6 ppm 1.506 .029 1.446 1.566

Table 16 shows the Post Hoc Tests for Homogeneous SubsetsDependent Variable: 20-day cumulative feed conversion rate--Duncan

TABLE-US-00016 Subset Treatment N 1 2 BT1583 @ 12 ppm 12 1.4959 BT1583 @ 6 ppm 12 1.5065 Control 12 1.5922 Sig. .801 1.000 Means for groups in homogeneous subsets are displayed. Based on Type III Sum of Squares The error term is Mean Square (Error) = 1.043E-02. a Uses Harmonic Mean Sample Size = 12.000. b Alpha = .05.

[0101]To evaluate TAMUS BT1583 on growth and feed conversion of 3-wk old straight run broilers fed an industry type pelleted starter feed (in batteries, Studies 3-1 and 3-2). Briefly, the following six treatment regimens were examined: Control, Monensin at 90 ppm, BMD 50 at 50 ppm, BT1583 at 12 ppm, Monensin+BMD 50, Monensin+ and BT1583 at 12 ppm. Eight (8) study repetitions per treatment were used, again with 4 birds per pen.

TABLE-US-00017 TABLE 17 Basic Feed for Studies 3-1 and 3-2. PERCENT INGREDIENTS (Pelleted Feed) TAMU Corn 56.11 TAMU Dehulled Soybean Meal 35.90 DL Methionine 0.22 Blended A-V Fat 4.02 Limestone 1.43 Mono-Dicalcium Phosphate 1.55 Salt 0.46 TAMU Trace Minerals 0.05 TAMU Vitamins 0.25 NUTRIENT CONTENT (Calculated) Metabolizable Energy (kcal/kg) 3100 Protein (%) 22.31 Lysine (%) 1.21 Methionine + Cystine (%) 0.92 Threonine (%) 0.84 BT1583 added via 200 grams of corn meal

Table 18 shows the Descriptive StatisticsDependent Variable: 21-day cumulative weight gain

TABLE-US-00018 Treatment Mean Std. Deviation Number BT1583 @ 12 ppm 831.7396 40.47789 8 BMD @ 50 ppm 832.9688 30.12576 8 COB @ 90 ppm 792.8438 67.05913 8 COB + BT1583 835.7604 62.00447 8 COB + BMD 810.2188 74.64333 8 Control 719.7813 71.97.115 8 Total 803.8854 70.02414 48

Table 19 shows the Tests of Between-Subjects EffectsDependent Variable: 21-day cumulative weight gain

TABLE-US-00019 Type III Sum of Source Squares df Mean Square F Sig. Corrected 78986.007(a) 5 15797.201 4.380 .003 Model Intercept 31019124.630 1 31019124.630 8600.903 .000 TRE 78986.007 5 15797.201 4.380 .003 Error 151472.835 42 3606.496 Total 31249583.472 48 Corrected 230458.842 47 Total (a)R Squared = .343 (Adjusted R Squared = .264)

Table 20 shows the Estimated Marginal MeansDependent Variable: 21-day cumulative weight gain

TABLE-US-00020 95% Confidence Interval Lower Upper Treatment Mean Std. Error Bound Bound BT1583 831.740 21.232 788.891 874.588 BMD 832.969 21.232 790.120 875.817 COB 792.844 21.232 749.995 835.692 COB + BT1583 835.760 21.232 792.912 878.609 COB + BMD 810.219 21.232 767.370 853.067 Control 719.781 21.232 676.933 762.630

Table 21 shows the Post Hoc Tests for Homogeneous SubsetsDependent Variable: 21-day cumulative weight vain--Duncan

TABLE-US-00021 Subset Treatment N 1 2 Control 8 719.7813 COB 8 792.8438 COB + BMD 8 810.2188 BT1583 8 831.7396 BMD 8 832.9688 COB + BT1583 8 835.7604 Sig. 1.000 .211 Means for groups in homogeneous subsets are displayed. Based on Type III Sum of Squares The error term is Mean Square (Error) = 3606.496. a Uses Harmonic Mean Sample Size = 8.000. b Alpha = .05.

Table 22 shows the Descriptive StatisticsDependent Variable: 20-day cumulative feed conversion rate

TABLE-US-00022 Treatment Mean Std. Deviation N BT1583 1.3308 .03340 8 BMD 1.3397 .03132 8 COB 1.3712 .03023 8 COB + BT1583 1.2816 .02680 8 COB + BMD 1.3435 .02477 8 Control 1.4154 .03299 8 Total 1.3470 .04989 48

Table 23 shows the Tests of Between-Subjects EffectsDependent Variable: 21-day cumulative feed conversion rate

TABLE-US-00023 Type III Sum of Source Squares df Mean Square F Sig. Corrected 7.894E-02(a) 5 1.579E-02 17.442 .000 Model Intercept 87.096 1 87.096 96218.356 .000 TRE 7.894E-02 5 1.579E-02 17.442 .000 Error 3.802E-02 42 9.052E-04 Total 87.213 48 Corrected Total .117 47 (a)R Squared = .675 (Adjusted R Squared = .636)

Table 24 shows the Estimated Marginal MeansDependent Variable: 21-day cumulative feed conversion rate

TABLE-US-00024 95% Confidence Interval Treatment Mean Std. Error Lower Bound Upper Bound BT1583 1.331 .011 1.309 1.352 BMD 1.340 .011 1.318 1.361 COB 1.371 .011 1.350 1.393 COB + BT1583 1.282 .011 1.260 1.303 COB + BMD 1.344 .011 1.322 1.365 Control 1.415 .011 1.394 1.437

Table 25 shows the Dependent Variable:

[0102]20-day cumulative feed conversion rate--Duncan

TABLE-US-00025 Subset Treatment N 1 2 3 4 COB + BT1583 8 1.2816 BT1583 8 1.3308 BMD 8 1.3397 1.3397 COB + BMD 8 1.3435 1.3435 COB 8 1.3712 Control 8 1.4154 Sig. 1.000 .432 .053 1.000 Means for groups in homogeneous subsets are displayed. Based on Type III Sum of Squares The error term is Mean Square (Error) = 9.052E-04. a Uses Harmonic Mean Sample Size = 8.000. b Alpha = .05.

[0103]A more complete study to evaluate TAMUS BT1583 on growth and feed conversion of 42 day old straight run broilers in floor pens may be as follows: Treatments of six (6) groups, Control, Monensin at 90 ppm, BT1583 at 12 ppm, Monensin+BMD at 50 ppm, Monensin+BT1583 at 12 ppm and BMD at 50 ppm. 10 study repetitions per treatment were used to evaluate the effect of using the BT1583 peptide as a feed additive, this time with 40 birds per pen.

[0104]When used to promote growth in food-producing animals it was found that the BT1583 peptide provided about 10 points in feed conversion plus extra weight gains. One distinct advantage of the present invention is that no or very little absorption by the chicken GI track, thereby making it useful for widespread use. Furthermore, unlike conventional antibiotics, the present invention may target the bacterial membrane, and there is currently not a drug target that can be altered with one or two mutations to allow development of drug resistance. Furthermore, it was found that growth promotion via possible host immunity modulation is intrinsic to chicken and independent of drug resistance. Alternatively, but in no way limiting the present invention, the present invention may be used as an animal-use only antibiotic for bacterial infections. Also, to date, there has been no observed decrease in the growth promoting activity of the peptide.

[0105]A broiler floor pen trial to compare the performance of a new anti-microbial designated for this project as BT1583 alone and in combination with the widely used coccidiostat monensin (MON) to MON fed alone, MON in combination with the also widely used antimicrobial Bacitracin MD (BMD) and BMD alone.

[0106]The following levels of each treatment were evaluated:

[0107]1: Non-supplemented

[0108]2: Monensin (MON) 99 ppm

[0109]3: BT1583 12 ppm

[0110]4: BT1583 12 ppm+MON 99 ppm

[0111]5: MON 99 ppm+BMD 55 ppm

[0112]6: BMD 55 ppm

[0113]The study design included 10 pens per treatment and 40 birds per pen housed on day of hatch. Two basal corn-soy based diets of decreasing protein (approximately 23 to 20%) and increasing metabolizable energy (approximately 1400 to 1455 kcal/lb) were used from Day 0 to 21 (starter feed) and Days 22 to 42 (grower feed), respectively. Treatment premixes were measured and blended into diets at required levels. Between days 0 and 21 mortality was less than 1% with all birds growing optimally and of high health across all groups.

[0114]Beginning on study day 22, the study director modulated house temperature and air flow to mimic industry conditions conducive to outbreaks of colibacillosis within naive broiler flocks. This was done to stimulate a natural challenge for this study. Mortality climbed to a house average of approximately 10% by Day 42. A majority of these deaths occurred in groups not receiving BT1583 or MON. Lesions were consistent with those of colibacillosis (air sac, pneumonia, peri-hepatitis, peri-carditis and extreme morbidity). All mortality was documented (weight at death and post-mortem observations). All birds and feeds were weighed at 42 days. All remaining birds were euthanized on Day 42 by asphyxiation with the carcasses submitted for rendering.

[0115]All data were analyzed as described below and are displayed in Tables 26 through 30. The following variables were tested: Response Variables: Gain Per Bird, Feed Per Gain, Mortality (%), Adjusted Feed Per Gain. F test from One Way ANOVA with one blocking factor=location, at Day 42 using 0.05 level of significance.

[0116]All Response Variables: LSD T-test procedure for pair-wise comparisons with Type 1 error of means when ANOVA F ratio is significant, overall significance level of 0.05 used. Lines below means (see Table 30, below) indicate groups with insignificant differences in means.

TABLE-US-00026 TABLE 26 Weight gains (in lb) per bird Treatment Day 42 Gain/Bird Non-supplemented 3.900^d Monensin (MON) 99 ppm 4.111^bcd BT1583 12 ppm 4.333^ab MON 99 ppm + BT1583 12 ppm 4.385^a MON 99 ppm + BMD 55 ppm 4.127^c BMD 55 ppm 3.971^cd

[0117]Weight gains were heaviest for the 2 groups of broilers receiving BT1583 measured at 42 days with the MON+BT1583 significantly heavier (p<0.05) than that provided by the MON+BMD and MON groups.

[0118]Feed/Gain: Table 27 shows that MON+BT1583 fed broilers had the feed/gain values which were lower (p<0.05) than all other groups with the exception of the group receiving BT1583 alone.

TABLE-US-00027 TABLE 27 Feed/Gain Treatment Day 42 Feed/Gain Non-supplemented 2.189^c Monensin (MON) 99 ppm 1.854^b BT1583 12 ppm 1.722^ab MON 99 ppm + BT1583 12 ppm 1.689^a MON 99 ppm + BMD 55 ppm 1.885^bc BMD 55 ppm 2.147^c

[0119]Adjusted Feed/Gain: The total weight of mortality in each pen was added to the final live weight, that value reduced by subtracting the initial weight and then dividing that value into the Total feed consumed to calculate the Adjusted Feed/Gain.

[0120]Table 28 demonstrates the effects of the natural challenge on feed/gain values. Even with the adjustments for mortality, MON+BT1583 fed broilers had an adjusted feed/gain value which again was significantly lower (p<0.05) than all other groups with the exception of the group receiving BT1583 alone.

TABLE-US-00028 TABLE 28 Feed/Gain Adjusted Treatment Day 42 Adjusted Feed/Gain Non-supplemented 1.928^c Monensin (MON) 99 ppm 1.761^b BT1583 12 ppm 1.704^ab MON 99 ppm + BT1583 12 ppm 1.654^a MON 99 ppm + BMD 55 ppm 1.725^b BMD 55 ppm 1.838^bc

[0121]Mortality: The majority of the deaths were caused by acute and chronic colibacillosis. Broilers receiving BT1583 or Monensin alone or in combination had lower mortality rates than the non-supplemented controls.

TABLE-US-00029 TABLE 29 Mortality by acute and chronic colibacillosis. Treatment Day 42 Mortality (%) Non-supplemented 17.50^c Monensin (MON) 99 ppm 8.00^a BT1583 2.75^a MON 99 ppm + BT1583 12 ppm 3.50^a MON 99 ppm + BMD 55 ppm 7.75^abc BMD 55 ppm 18.25^bc

[0122]Monensin is a polyether antibiotic that is approved and used as an anti-protozoal agent in the poultry industry. Slight efficacy by monensin and other polyether antibiotics against gram negative bacteria has been documented by many researchers and poultry industry personnel. BT1583 has also been stated to have efficacy against gram negative bacteria. Escherichia coli has been a major problem in the food industries both health wise and financially. Most products highly effective against this pathogen are too costly to use in broiler older than 21 days or have been pulled off the market due to similarities to human health products raising public health concerns. This study demonstrated that BT1583 is highly effective against colibacillosis in 3 to 4 week old naive broiler chickens raised under simulated commercial broiler conditions. The 20+point weight gain advantages and 10+point feed/gain advantages held by BT1583 over monensin and BMD fed alone and in combination observed on this trial is a strong indicator that this product may be an invaluable tool for the future of the poultry industry.

TABLE-US-00030 TABLE 30 P-Values Comparisons Day 42 Day 42 Day 42 Day 42 Day 42 Adjusted Day 42 Wt/Gain Indiv. Bird Gain/Bird Feed/Gain Feed/Gain Mortality WtGn (lb) Wt (lb) (lb) (FdWt/WTGn) (Adj Feed/Gn) (%) Controls vs. MON 0.216456 0.153695 0.001923 0.023562 0.013284 Controls vs. BT1583 0.000028 0.000023 0.000134 0.002449 0.001433 Controls vs. MON + BT1583 0.000421 0.000333 0.000115 0.000147 0.006250 Controls vs. MON + BMD 0.004270 0.002816 0.096440 0.003283 0.179121 Controls vs. BMD 0.523702 0.546456 0.754966 0.231502 0.845776 MON vs. BT1583 0.177311 0.192494 0.159703 0.425156 0.172685 MON vs. MON + BT1583 0.018776 0.022738 0.041045 0.033717 0.234506 MON vs. MON + BMD 0.805698 0.905725 0.840264 0.275135 0.967501 MON vs. BMD 0.521589 0.443090 0.123047 0.453955 0.031824 BT1583 vs. MON + BT1583 0.645444 0.638704 0.472007 0.338775 0.663743 BT1583 vs. MON + BMD 0.014212 0.011862 0.254035 0.712535 0.336982 BT1583 vs. BMD 0.005943 0.005142 0.017789 0.091063 0.006670 MON + BT1583 vs. MON + BMD 0.006085 0.004962 0.145588 0.037877 0.386550 MON + BT1583 vs. BMD 0.006044 0.005385 0.013376 0.034621 0.003991 MON + BMD vs BMD 0.244875 0.214655 0.181891 0.205577 0.134645 (Note: Bold type and underlining indicate comparisons where p-value is less than 0.05)

[0123]It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0124]All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0125]All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

[0126]Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215(3), 403-10. [0127]Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17), 3389-402. [0128]Challis, G. L., Ravel, J. & Townsend, C. A. (2000). Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol 7(3), 211-24. [0129]Conti, E., Stachelhaus, T., Marahiel, M. A. & Brick, P. (1997). Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. Embo J 16(14), 4174-83. [0130]Gaitatzis, N., Kunze, B. & Muller, R. (2001). In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases. Proc Natl Acad Sci USA 98(20), 11136-41. Epub 2001 Sep. 18. [0131]Gonzalez-Pastor, J. E., Hobbs, E. C. & Losick, R. (2003). Cannibalism by sporulating bacteria. Science 301(5632), 510-3. Epub 2003 Jun. 19. [0132]Hopwood, D. A. (1997). Genetic Contributions to Understanding Polyketide Synthases. Chem. Rev 97(7), 2465-2498. [0133]Keating, T. A., Ehmann, D. E., Kohli, R. M., Marshall, C. G., Trauger, J. W. & Walsh, C. T. (2001). Chain termination steps in nonribosomal peptide synthetase assembly lines: directed acyl-5-enzyme breakdown in antibiotic and siderophore biosynthesis. Chembiochem 2(2), 99-107. [0134]Keating, T. A. & Walsh, C. T. (1999). Initiation, elongation, and termination strategies in polyketide and polypeptide antibiotic biosynthesis. Curr Opin Chem Biol 3(5), 598-606. [0135]Kessler, N., Schuhmann, H., Morneweg, S., Linne, U. & Marahiel, M. A. (2004). The linear pentadecapeptide gramicidin is assembled by four multimodular nonribosomal peptide synthetases that comprise 16 modules with 56 catalytic domains. J Biol Chem 279(9), 7413-9. [0136]Konz, D., Doekel, S. & Marahiel, M. A. (1999). Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin. J Bacteriol 181(1), 133-40. [0137]Linne, U. & Marahiel, M. A. (2000). Control of directionality in nonribosomal peptide synthesis: role of the condensation domain in preventing misinitiation and timing of epimerization. Biochemistry 39(34), 10439-47. [0138]Marahiel, M. A. (1997). Protein templates for the biosynthesis of peptide antibiotics. Chem Biol 4(8), 561-7. [0139]Mootz, H. D. & Marahiel, M. A. (1997). The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J Bacteriol 179(21), 6843-50. [0140]Mootz, H. D., Schwarzer, D. & Marahiel, M. A. (2002). Ways of assembling complex natural products on modular nonribosomal peptide synthetases. Chembiochem 3(6), 490-504. [0141]Offenzeller, M., Santer, G., Totschnig, K., Su, Z., Moser, H., Traber, R. & Schneider-Scherzer, E. (1996). Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A: enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway. Biochemistry 35(25), 8401-12. [0142]Offenzeller, M., Su, Z., Santer, G., Moser, H., Traber, R., Memmert, K. & Schneider-Scherzer, E. (1993). Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A. Identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as a key intermediate by enzymatic in vitro synthesis and by in vivo labeling techniques. J Biol Chem 268(35), 26127-34. [0143]Quadri, L. E., Sello, J., Keating, T. A., Weinreb, P. H. & Walsh, C. T. (1998). Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin. Chem Biol 5(11), 631-45. [0144]Sagisaka, S. & Shimura, K. (1959). Enzymic reduction of alpha-amino-adipic acid by yeast enzyme. Nature 184(Suppl 22), 1709-10. [0145]Sanglier, J. J., Traber, R., Buck, R. H., Hofmann, H. & Kobel, H. (1990). Isolation of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine, the characteristic structural element of cyclosporins, from a blocked mutant of Tolypocladium inflatum. J Antibiot (Tokyo) 43(6), 707-14. [0146]Stachelhaus, T., Mootz, H. D., Bergendahl, V. & Marahiel, M. A. (1998). Peptide bond formation in nonribosomal peptide biosynthesis. Catalytic role of the condensation domain. J Biol Chem 273(35), 22773-81. [0147]Stachelhaus, T., Mootz, H. D. & Marahiel, M. A. (1999). The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol 6(8), 493-505. [0148]Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22), 4673-80.

Sequence CWU 1

28113PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 1Xaa Leu Xaa Ile Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10213PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 2Xaa Met Xaa Ile Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10313PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 3Xaa Val Xaa Ile Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10413PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 4Xaa Ile Xaa Ile Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10513PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 5Xaa Phe Xaa Ile Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10613PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 6Xaa Leu Xaa Ile Ile Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10713PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 7Xaa Leu Xaa Ile Leu Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10813PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 8Xaa Met Xaa Ile Ile Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 10913PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 9Xaa Met Xaa Ile Leu Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101013PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 10Xaa Val Xaa Ile Ile Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101113PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 11Xaa Val Xaa Ile Leu Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101213PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 12Xaa Ile Xaa Ile Ile Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101313PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 13Xaa Ile Xaa Ile Leu Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101413PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 14Xaa Phe Xaa Ile Ile Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101513PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 15Xaa Phe Xaa Ile Leu Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101613PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 16Xaa Leu Xaa Val Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101713PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 17Xaa Met Xaa Val Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101813PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 18Xaa Val Xaa Val Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 101913PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 19Xaa Ile Xaa Val Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 102013PRTBrevibacillus texasporusMOD_RES(1)..(1)N,N-methylated (4R)-4-[(E)-2-butenyl]-4-methyl- L-threonine 20Xaa Phe Xaa Val Val Val Xaa Val Xaa Lys Xaa Leu Xaa1 5 102150674DNABrevibacillus texasporus 21attcgttgga tccagtgtgg tggaattcaa accctcagtg ggtaaagata ttgccagagt 60cttgaaatgt accaaacagg gaaatgggta ccttgaaggc gacaaatatg tagtaacctg 120ggcatttggc catctggtta cgctggctga tcctgaagcc tatggagaga catataaagc 180ttggaagctg gaggatttac cactgttgcc gtctcgcctg caattaactg tcatcagaca 240gagctccaag caatatcaga ttgtaaaaaa attattagcg cgtcaggata tttcagaagt 300gattattgct actgatgctg gtcgtgaagg tgagctggtg gcgcgatgga ttttagaaaa 360ggcacatgtg aaaaagccta ttaaacgact atggatttcc tctgtgactg ataaagcaat 420cagtgatggc ttcagaaagc tgcgagatgg caaggaatac gagaatctct atgcttctgc 480tgtagctcgc gctgaagctg actggtttgt cgggatcaat gccactcgtg ctcttacaac 540gaagcataat gcccagctct cctgcgggcg tgtacaaact cctacagtgg caatgattgc 600caaacgtgag gaggagattc aaaggttcgt tcctcgcccc tattatggtg ttcaagcgat 660cacaggtaat ggattaaagc ttacgtggca ggatcagcaa accaaagata tgaagacgtt 720taccaaggag aaggctgaga aaattgtcga aagcagtaaa aacaagcaag ctgaaattat 780tgacatcaag aaggctgaca agaaaagctt cgccccagct ttatatgatc taactgagct 840acaacgtgat gcgaataagc gttttggttt ttcggcaaag gaaaccctct ccattatgca 900agggctatac gaaacacata aggtactcac atacccgcgg acagattctc gctatttgac 960atcagatatt gtcgaaacat tacctgatcg attgagagcg atatctgtta agccatacac 1020tccgtttgca gctaagctgt taaagcaacc gattcgagct ggtaaacatg tagtggataa 1080tagcaaggta tctgatcacc atgcgattat tccaactgag caatctgtgc tgttaaataa 1140gcttagcgat aaggaacgta aaatttatga ccttgtggtt aaacgctttc tggctgtgct 1200atatccgcct tttgaatatg aacaaattag tattcgtgcc cgaattggca acgaggaatt 1260tctagcaaaa gggaagacga taacgcatca gggctggaag gaaatttatg ataatcactt 1320tgatgaagag gatcaaggag atggattaaa agaacagctt ttaccaacgc ttgtacaagg 1380tgagcaacta tctgtacaaa ccgtttcttt tacaaaaggg gaaacaaaac ctccagagcc 1440atttacggag gctacactcc tttcagctat ggagaatcct gtccgttata tgggacaggt 1500tgataagcag ctagccaaaa cattaggtga gacaggtgga ttaggaactg ttgctactcg 1560cgccgatatt attgaaaaat tgtttaatag cttcctcatt gaaaagcgca gcaagcatat 1620tcatattact tctaaaggaa agcaattact tgagcttgta ccagaaggac ttcgctcacc 1680ggctcttact gctgaatggg aagtgaagct tggagcgatc tcgaaaggaa gcctggctaa 1740aaacagcttc atacaggaaa tgaagaagta tgcagagcaa atcgttcaac agatcaaatt 1800tagtgagcaa aagtttcgtc atgacaatct gactcgctcc aaatgcccgg attgtggaaa 1860gctgatgctt gaggtaaatg ggaaaaaagg aaaaatgtta gtttgtcagg atcgtgagtg 1920tggtcaccgt aaggccgtat ccaaggttac aaatgccaga tgcccacaat gtcgaaagaa 1980gatggagatg cgtggggaag gagaaggaaa gatattcgta tgcaaatgcg gacatcgtga 2040aaaactgtcg acgtttaacg acagacgtag caaggagaaa cagacaaatg tttcgaaacg 2100agatgtagcc cagtatatga aaaatcagca acgagatcaa gaaagcttag gtaatccagc 2160cttgatggag gcattaaaga attttaagct agatcagtaa gtcattttat atagaaaaga 2220taggctcgaa aataaggcgc ccgctctctt tattagctaa gagaaagggc gtctatttta 2280gcttctcttt tctatatgga aaaaaggaat taacaaaatc tgaagaaaat ttcagaattt 2340attattttta tgttaagaga ttttgaaaga agtgcatcta tttactttat ccataaatga 2400aagcaaaaat gagattgtta tattccataa aatgtaaatg tttttatatc ttggaaaaca 2460tagagtcgag aacgctgttc actatacaag aagactatag atacgagctt ttacggctat 2520ttttacgatt tgataacgcc ttcttttttt agtctaataa aaaacagtaa cgaatcgaaa 2580aaataacaat aatttgattg gtaaataaca ttgttactaa ttcacagaaa acaaaatcat 2640tatttttaca taataaaaac ataataataa aatgtgatag aaaaatattg aagaaaatgt 2700ccattattgg tagtattgaa aaataaacct gtaattttat gtattataat gcaattattt 2760gtcgcatatt tactagcata aatgttagcg atgatgtgag caaatcgtta cctgttagaa 2820ggaagatcag ctacatatga ttcaaaattt ttcgtttttt tattttaaaa gacaaagagg 2880tggtcctgat ggatttatct acattaaatt ttttgggtga aacagaaaag cataagttat 2940tgaatcaatt caatgatacg gacgctaatt ttcctcagga gatgaccatt catgggctgt 3000ttgaaaagca agtccaagaa agaccgaatc aaactgcggt aatttttaat gaacaaagta 3060tgacgtataa agaaatgaat gaacgagcca atcaagtagc acatagctta cggaagcatg 3120gagctgctcc agatgagatc gttggaattc tagcagatcg caacatggac atgcttattt 3180ccattctcgg cgtattaaag gctggagctg cttatatgcc tattgatcct acatacccta 3240cagaacgtat tctttatatg atccatgata gccagaccaa aattgtctta gctgaacata 3300gagagatggt tccggaaggc tgtaatgcag agctgatcct cttgcacgat agctcccttt 3360taaacgaaga gacatctgat ctagagcatg taaataagcc tgaagatttg gcctatatta 3420tctatacatc aggttctact ggtaaaccaa aaggggttat gattgaacat cgaaatgtca 3480ttcgcttgct atttaatgac agaaacctat ttgattttac tagtgatgat gtctggaccg 3540ttttccattc gttctgtttt gacttctctg tttgggagat gtatggggct ttactgtatg 3600gaggaaaaat cgttctcgtc tcttttgaga tagctagaga tcctcaggcc ttccgagatt 3660tacttcagga gcaaaaggtt acgattttaa atcaaacccc tacagctttt tatcagctct 3720cgtctcaaga gatgcagcac tcagacagca atctatcgat tcgtaaaatc atttttggtg 3780gagaagcgtt gacgccatca cagttgaaag catggaaaca aaaatatcca aatacagcct 3840tgattaatat gtacggtatt acagaaacaa ctgttcatgt gacttataag gagtttcaat 3900tacatgatat ggacagcaca gttagcaata tcggaaagcc tatcccaacg cttagaacct 3960atgttttaga ttccaagaga aacctagctc caattggagt gaaaggtgaa ctgtatgtga 4020gcggcaaggg agtagcccgc ggttatttaa acaaacctga attgacggaa gaacggttta 4080tggataaccc gtttgttgct ggagaaagaa tgtatcgcac aggagaccta gctagatggc 4140tacctgaagg agagctagaa tatctaggca ggattgacca tcaggtaaaa atcagaggct 4200atcgcattga actcggagaa atagaagccg agctattgaa gcaaaaaggg attaaagaag 4260cagtagtttt agttacaaat gataaagatg cacaaccaca attacatgcc tatttaacat 4320ctaaggaaga tttggcagca gcagatcttc gtaatcaact tactacaaca ttaccctctt 4380acatgattcc ggctcatttc atttttgtgt cgcaaatgcc tgttacgcca aatggaaaaa 4440ttgataaaga atcacttcgt aaaatagaac catcacttca agaaagccct acagaagctt 4500atgtagctcc acaaacacct acagaaaagc aattagtcca catatgggaa gaaaatattg 4560gaatgcaacc gatcagcata gacgataatt attttgctct aggtggtgat tccatcaaag 4620cgattaagct attgcatgct ataaataaag agtttcagat tagtttccaa attggagatt 4680tgtataagca tggaaccatt agagaaatgg gacagcaaat cggtgaaaag ggcaagcaat 4740ctagcaatca aaaactgttg aaacttcagg aattggaccg tttaaaagag aaaattttgg 4800gaagtgagaa atagtcatgt cggataagct aagcaacgct aaagacctat ttccaatgag 4860cgatatacag ctagggatgg tctaccattc gttaaaacat gtacacgaag ctgtatacca 4920tgatcaattt gtttatcaag tagatgatga ttcatttgat gttcatgtgc tagagcaagc 4980gatgagaatg atggttgata agcacgacat cttaaaaacc agctttcata ttgaggaatt 5040ttccactcca gttcaagtag tgcaccagga ggtttctgtt cgaattgatg agacagacat 5100tacgcatctg ggagaaaaac aaaaagagta tatccatcag tatttggcac aggatcgtca 5160atcccctttt gatgtaacaa ccgctcctct atggagaatg agcgttttta aactgaatgc 5220aagccaagtt gctttagtct ggatctttca tcatgctatt ttggatggat ggagtgttgc 5280atcttttatt acggaattaa ttgatgttta tttcaaatta aagcacaaaa cttgcacttt 5340ggagcatttg aacacgacct ataaggatta tgtgattgat cagatgctat tatctgagca 5400aaatgagctg cgtgaatatt ggaaagaaga attaaaagat tacaaacggc tacagctccc 5460agtaaaagtg gatgaaaatg gcggtgttca cgttaccgtt gttgagaagc tagaccctga 5520cattataaat aaatgcagag aaattgcaca agctcatcac attccattaa agaccgtatg 5580cctaacagcc tttctttcta tgatgcatat gatttcttat gagagagacc tgactgtggg 5640attgattgag aacaaccgac caattataga agatgctgaa aaggtgttgg gatgttttct 5700taactcagtt ccattccgcg ccattataaa gaaagatatg agctacagag agctattaga 5760gcagacacag caaaagcttg ttgagattaa aacatatgga agactttcct ttgctaagat 5820tattgaagta attggcgata cgggaagcga gcgtaatcca gtttttgact gtctttttaa 5880ctttgtcgac ttccatgtat ttaaagggat aaaggatcat aaagtaaagt tttggttaga 5940tggatatgaa aaaacaaaca ccatgtttga cttttctgtt tcgaccacaa tggatgacta 6000ttttgttcgg gttgtatctg cactgccaga agaagatacg ataaaactaa ttaactatta 6060tcaacgaatt ttagaaaaga ttgctcttca catagatgaa aaaatagata aacaagccaa 6120tcttgatgaa aaggaaagcc acttgctgct agaggaatgg aatcaaacgt cagttgatta 6180tccagacaag caaacattgc ataaacggtt tgaggagcaa gtagccaaaa atgaagatca 6240ggtagcgctg gaatatgagg ataagcagct tacctatagg gaattgaacg ctaaagccaa 6300tcaattggca cgtgttttac agaagcataa tacgctgcca actcaggtag ttggtctaat 6360ggcagagcgt tcactagaga tgataatagg cattcttggg atattaaaag ccggcggagc 6420ttatatgcct attgacccta cgtatcctgc ggagcgtatc caatatatgc tcgaagatag 6480tcgatcctat ctcttacttg tacaaaaagc agaaatgatt ccagccaatt atcaggggga 6540agtacttatc ctcacagagg aactttgggc agatgagaat acagagaacc tggaactagt 6600caatcagccg caggatgttg ccaatatcat gtatacatct gggactacag gaaagccaaa 6660aggtatcctg atcactcatc gaaacattat gactaccata atcaacaatg gctatctcga 6720tattttttca acagatcgaa tattgcaaat atctaactat gcttttgatg gttctacctt 6780tgatatatac agtgctttgc taaacggagc tactctcgtg ctagttccca agcaaacact 6840catgaatacg accgatctgt tagcaatcat caaagatagc aatatcacgg tagctttaat 6900gacaacctct ctattcaata cgttggttga tcttgatgta accagcttcc aacatacacg 6960taaggtttta tttggcgggg aaaaggcttc atgtaagcat gtagaaaaag cattggatta 7020tttgggtgaa gggcgcctag taaatggata tggtccgaca gaaacaacgg tgttcgctac 7080tacctataca gtcgataaca cgattaaaaa gctgggaagt atcccgatcg gacgtccttt 7140gagcaacact tcggtatata tttttggatt agatgatcaa ttacaaccac ttggagtacc 7200aggggagtta tgtgtagcag gagaatgcat ttcgcctgga tatctgaatc gtcccgactt 7260aacggcagac aaatttattg ataatccact taaaccaggt gagagaatgt accgtacagg 7320tgacctagtt cgttggctgc ctgaaggtgt catggaatac atggggcgga ttgatgaaca 7380agtcaagatt cgtggacatc gtatcgagct aggggagatt gaggcaaagc tgcttgagca 7440tccttcgatt cgagaaacag tgctggtggc taaacaggat gcaaatggcc attctttttt 7500aggtgcgtat cttgttacag acaacttctg ccctgtaacg gaattacgga attatctgat 7560ggaaaccttg ccagaatata tggttccttc ttattttatc gagctggata gcctaccgct 7620tacttcaaat ggaaaagtag ataagcgagc attgcccgaa ccggaatctc aggctttaca 7680cgcatatacc atgccggaga atgagacgga agaaaaattg gttcagctat tccaggaagt 7740gatggatgta gagcgtgttg gtactcaaga tagcttttat gaattaggcg gtcattcctt 7800aaaagcaatg cttttggttt cacgaattca taaggatttt ggaataaaga taccgttgaa 7860ggaagtattc agtcgtccga ccgtgaagga attggctgcc tatctgactg ggtcagaaga 7920agcaaactat attgaaattg aagcagcaga agagaaacca tactatccag ttactgccgc 7980ccaaaaacgg atgtatatcg cccagcaatg ggaggatggg gaagccacta gcagttatca 8040catgccgttt atgatggaaa tcacagggcc tcttcaagta gaaaagctac aacaaacagt 8100aaagagtctt gtcgcaaggc acgagtcgtt gcggacatca tttcacatga tcaatgaagt 8160attgatgcaa aagatacatg cagatgtatt gtgggattta gacattgatc tagagtcagt 8220tgtcgcttca gagcaagaaa ttgatgaaaa aatgttccaa ttcctccgca aatttgattt 8280gagtcaagct cctctcttta gagctaagct gattcgtgtc aatgctagtc ggcatgtatt 8340gttattagat atgcaccata ttatttcgga tggattttca taccagatat tttttgatga 8400gcttaccaag ctgtatcagg gcgatgaact gccatctctc aaaatacaat ataaggatta 8460tgccgtttgg cagcattcgg aagaacaaca gaagcgtttg caacagcaag aggattattg 8520gttaggtcaa ttccaagggg aaattcctgt tctggaattg cctacggatt accagcgccc 8580ggttgataaa cagtttgctg gagcattatt cacacacggg ttatctgctg gtctaacaga 8640gaagctgaga aaattagcga ttaaggaaaa aacgacgtta tacaccgtac tgctgacggt 8700ctataacatt ctattgagca aatatacaag tcaagaggac ctcattgtag gtacaccgat 8760tgctggacgt ccacatgctg atttagacag agtatttggg atgtttgtaa acacgctggc 8820catcagaaca gctccaaaag tagagcattc cttcttaacg tatctatctg aggtcaaaga 8880aacagtgcta ggtgcttatc aaaatccaga ctatccattt gaggagctgg ttgaaaaaac 8940gctagttcag cgcgatgtaa gccgtaatcc tttattcgat gtaatgttct ccgtagagaa 9000attaccatct gctgtacagt tcgatgattt acgtttctgc ccacgcttat ttgattggaa 9060gaaggcaaaa tttgacttgg attggacagt ggtggaaggt gaatcattgg aggttttggt 9120tgaatatagc acgagcttgt tcgatcgggc gaccattgag cgcatggcta agcattttga 9180gcatattttg gagcaaatcc ttgatcagcc agacctgtct atttctgaga ttgaactgct 9240gaccgaggca gaaaaacaac aaattttgat tgagtttaat caatcggata aatcctttga 9300cagcgaaaaa acaattcagg agcaatttga agaatgggca gaaaaagccc cgcacagcat 9360tgccttagtc tttaaagaca agcaaatgac ctatcaggaa ttaaatcaac gtgctaacca 9420agttgcgcat ttattacgtg gcaatgggat ttccgcaaat gattttatcg gtttaatggt 9480ggatcgatcg tttgagatga tcattagtat gctaggtatt ttgaaggcgg gtggagccta 9540cctacctatt gatcctgatt atcctgagga ccgtatcgat tatatgttat ctgacagcaa 9600agcgaagatt ctcttaaagc aaagtgacca aactgcacca gcttcctttg aaggtaaagt 9660catcgctatt gatactccag aattgctaga gatggatata gaaaatattc ctaaggtgaa 9720taactcatcc gacttggctt atatcattta tacatctgga tcaaccggaa aaccaaaagg 9780agtattgatt aatcatcgat gcgtgatcaa tatgcagctt acagctgaaa cctttggtat 9840ctatccttcg agtcgtattc tacagtttgc atcctttagt tttgattcat ctgtgggcga 9900gattttttat acattattaa acggagcatg cctgtatttg gtagaaaagg atttgctttt 9960atccggtaat gaattcgtgg catggctaaa gaaaaatcgg attagctcga ttccatttat 10020ttcaccgtcg gctctgcgga tgcttcctta tgaggattta cctgatctcg catatataag 10080tacgggtggg gagacattgc cggctgacct tgttaaagcg tggggagaaa atcgtgtctt 10140cctaaatgca tatggcccga cggaaacaac tgtagatgcc actgtcggtg tatgtacacc 10200agaagggaaa ccgcatatcg gtagacccgt tacgaataaa aaggtgtacg tagtaaatag 10260taacaatcaa ttacagccga ttggtgttcc tggcgagctt tgcattggcg gggaaggggt 10320tgcacttggc tatctaaaca gacctgatct aacccaagaa aaattcgttt ccaatccgtt 10380tgccccgggt gaaagaatgt accgctccgg agacttagtc agatggctac ctgatggaac 10440aattgagtac ttcggaagat tagacgatca agtaaaaatt agaggtcacc gtattgaact 10500aggagagatt gaaacaaggc tactagagca tccatccatt aaagaagcca ttgtcattcc 10560acgttctgat gagtcagagg ctacatattt atgcagctat ttgattgcag aaggatcatg 10620gaatgcggct gacttacgta agtatttgaa ggcttcttta ccggaatata tgataccttc 10680gtattttgtg gagctgcacg agctaccgct aacacctaat ggaaaagtta ataaaaaagc 10740attaccaaaa ccagaaaagc aaatgcagag agggaaggat tatgtagccc ctactaaccc 10800tatccaatcc attttatctc agatttggac tgatgtgctt ggtgttgaaa atataggaat 10860tcacgacaat ttctttgaat taggtggaga ttcaattaaa gccatccaaa tttcagctcg 10920acttaataag cataatctca aggttaaaat gcgggaattg tttaagaacc caacgattgc 10980tgagctaagt ctgcttgtac aacagatcgt tcaggagatc gatcaaggag tagtagaagg 11040aaatattccg cttacaccga tccagcattg gttctttacc caatcattcc cgcaggtcaa 11100ccattacaat caatcggttc ttctttttaa tgcggagggc tgggatgagc agaaagtaga 11160caaagctttt gagatgctaa cccagcacca tgatgcactg cgaatcgtat atagcctcga 11220cgagcaaggg gttgtacagc gtaaccgggg attggaaggc tcgaactatc atttcgaaat 11280cattgatgca agacaagatg gagaagatca gtcgaactgg aaagcagcgg cgaatcggat 11340gcaggcaagt atggatatcg tagaaggacc tttagtgcag atcggattgt tccgtgctaa 11400tgaaggagct tatttgttaa ttgccattca

tcacttagtg gtagatgggg tgtcttggcg 11460tatcctacta gaagacttct atcatttata taacggaaac gactctttgc cattaaaaac 11520gacctcgttc caagcatggt ctcaaaagct ccaagagtac gcccaaagca aggagctaga 11580acatgagctt tcctattggc gccatttaga tgaagctatc acggactata ccttacacaa 11640agatatagaa gccgcaacct caaataagac aacctatgag gaatttttaa ctgtatcgat 11700gtctttatca actgaggaaa cccaacagct agtaacagag gctcataaag cgtaccaaac 11760ggaaataaat gatctgctac tcacggcact ggctttagct ttgaaggaat ggacgaataa 11820agagcagttg ctagttagta tggaggggca tggacgtgaa gaaattctag ataacgtaga 11880tatctcccgt acagttgggt ggtttacatc agagtatccg gttgctattc atctgacgaa 11940aacagacatt tcgtttgcca ttaaacaagt aaaggaaacg ttgcgtcgtg tacctaacaa 12000agggtttggc tatgggattc ttaaatattt ggcaaaagag acgttcaagc ttaagccaga 12060aatcagtttt aactatctag gccaatttac agataaggaa gaggggaact cctctttaat 12120gggtgatctg attagcccgg caaataccag tgagctgtcc ctagatatca atggaagtat 12180agaagctgac agactgcaaa tgcactttag ttataactct cgtgcgtact atccagagac 12240aatcgcaacc cttgttcaaa acttcaaatc ctacttgctt gagattatca atcattgccg 12300ggcgaaagaa ggagtagagc atacaccaag cgactttgat atcaatgatc tcaccatgga 12360agaactagat gatatttttg atgacctgga agaagaggta tacaaataac taggcaaaaa 12420tatggagtga tttagatatg tttagcagaa gtaatgtgca aaatttgtat cgcttatctc 12480ctatgcaaaa agggatctta tttcattcct taaaagataa agaaaatcat gcctattttg 12540atcaactgat cttcactttg gaaggtaagg tagagcttga atatttggaa gaagccttta 12600cccaattaat caaaaagcat gatattttac gaactgtttt tcgttacaaa aaagtaaaag 12660aacctgtaca aatggtatta aaggaaagaa gctccactat ttattttgaa gatatttctc 12720atctggagcc agaagaaaaa gtgaattaca ttaagcagtt taaaatgagg gatcgggaga 12780aggggtttga cctctcccgg gacctcctca tccgaatgtc attatttaag cttgatcagg 12840agcagtatca gttaataatg agtaatcacc atatcattat ggatggttgg tgccttggca 12900ttatccttac tgatttctta cgtatgtata aaggaatcgt gaatcatacc cctgttccat 12960acgagcatgt gacaccttac agtaagcata ttcaatggct agaaaaacag gatcatcagg 13020aagcaaagga tttttatcaa cagctattag agggatacga caaagtaaca ggtgttccac 13080agcaattagt acgggcgaat cacgaagaat atactcacgg acaatgcatc gtgaaattaa 13140atcaagaaac tgccgaccga ttgattgcca tagccaaagc ctaccaggtt acagtcaata 13200ccgtcttcca aacgatttgg gggatattat tacaaaaata taataatacg gatgacatag 13260tatttggatc agttgtctcg gggagaccgg cagagattcc tgatgttgaa aaaatggttg 13320ggctatttat caatacaatt cctgtgcgaa tcaaagctga tcaacaagag cgatttgaca 13380cgctagtagc caaagtacag gaaatggcct tggcttcaga atcatatgat tatctttcgt 13440tggcagatat tcatccagaa gctggcgatt ttatcaatca tattattgcg tttgaaaatt 13500tttatatcga tatggacagc tttaatcagc tagcagataa aaaagagctt ggattctcgc 13560tcgcattcgc cacagatcat cacgagcaaa ccaattatga tctaagtgtg caggcgcaga 13620ttggtgatga atcttccatt aaaattttat ataattccaa gctttataca tcggaataca 13680tagcaaatgt aattgatcat tttgttactg tggctgacat agtggctgct aatcctagca 13740tccctgtaaa ggaaatcgat attttaacaa aagataaaaa agatcagatt ctctatggtt 13800ttaacaatac ctatgcagat tatccaagag agaagaccat ccatcagcta tttgaagaac 13860aagtagataa aaatccgaat cagatcgcac ttgtgtttaa agaagagaag ctgacttacg 13920gtgaggtaaa tgcgaaagca aatcagttgg catacgtgtt aagaaagcaa ggtgtacagc 13980ctaatgatgt aatcggcatc atcaccgaac gctccccaga aatgatcata ggcattttgg 14040cgatttttaa agcaggcgga gcttatatgc caattgatcc ttcttatccg gctgaacgca 14100ttcaatatat gctacaggat aatcaaacga agctattatt agtgcaaaaa caagaaatga 14160taccagccaa ttatcaggga gaggtattgt tcttaaccca agagagttgg atgcatgagg 14220aaacatctaa tccggctcat attactcaag cacaggcttt agcatatgtg atgtatacct 14280ctggttctac aggagagcct aagggcattt tgacaacaca tcaaaatatt atgaagaccg 14340tcattcataa cggttatgtt gagattacgc caggagattg cttgtcgcag ctctccaatt 14400atgcctttga cggctctacc tttgaaatct atggggcatt attgcatgga gctacattac 14460ttttagtaac aaaagaggct gtactcaata tgaatgagct ggcacgtctt attaagaagg 14520agcaagtgac ggtttccttc atgacgactg ctctgtttaa tacactggtg gatttggata 14580taacgtgctt tcaatcgata cgaaaggtgt tgttcggagg agagcttgct tcggttaagc 14640atgtcctgaa agcccttgat tatttaggcg agcaccgggt tatcaatgtg tatggaccaa 14700cggaaactac cgtgtatgct acctattact ctgtagatca ctccatgctg acgagggcat 14760ctgttcctat cggaagaccg attaataaca cgaaagctta cattgtaaat acagatggac 14820agcctcagcc aataggagta gtcggtgagc tatgcattgg cggtgagggg gtagcatgtg 14880gttatcttaa ccgtccagag ctgacaaaga aacatttcgt ggataatccg tttgtcttgg 14940gtgaacgaat gtattgtacc ggagatttag cccgcttttt accagacggc aacatcgaat 15000acatcgggcg gatggatgaa caggtaaaga ttcgtggtca ccggattgag ctgggcgaaa 15060tcgaaaaggt tcttttacag cacccagcta tcagcgagac agtgctttta gcaaaacgag 15120atgagcaagg ccattcctat ctgtgtgcgt atatagtagg tcaggtattt tggactgtta 15180cagagctgcg tcaacacttg atggaatcct tgccagaata catggtgcct tcctacttta 15240tcgagattga gaaactaccg cttacggcaa acgggaaggt agataagcga gcgttgcctg 15300aaccagacag aaaaatgggc agtgcttacg ttgctccaga gaacgaaaca gaggagaagc 15360tggttcaatt tttccaagag attttgggtg ttgagcgagt tggcacgcag gatacatttt 15420tcgagcttgg tggtcactcc cttaaggcaa tgatgctcgt tttacagatt cataaagaaa 15480tgggcattga agtcccgtta aaggagatat ttacacgtcc taccatcaaa gaattagcgg 15540cgtatattca taagatggat cgctctgcct acagcatgat tgagccaact gccaaacaag 15600agtattatcc agtctccttt gcccaaagac gaatgtttgt agtgcagcaa attagagata 15660cgaatacaac cagctacaat atgccgattt tgctagaaat agaaggggct cttgataggg 15720aaaatgtgag acaaactctg aagaaattga tagagcgtca tgaatcaatg agaacgtcat 15780tccatatgat tgacgagacc ttgctacaaa aggtgcatga tgatgtgaca tgggaaatgg 15840aggagatgga agcgtctgag gaagaagttt atgctttgac aaaatccttc attcgtcctt 15900ttgatctcgg tcaagctcca ttgtttagag caggattaat tcgtgttaat tctgagcgtc 15960atttgctgct gctagatacg catcacatta tctcagatgg cgtatctact aacatactct 16020ttcaagattt tacgcaatta tatcgtggac gagagctgcc tgccctgcga attcaataca 16080aggatttcgc cgtctggcaa caaggagagg ctcagcttgc tcgtttgcaa gaacaagaag 16140aatactggct gaaacaattt tcagagagtg tgcctgtact agagcttcct actgattttc 16200cacgtccagc gatgcagcag tttgatggtg acgtattgga ctttgcatta aatcagcaag 16260tatggcagga attacaacag ctcattgtta aagagggctg tacggcttac atgatattgc 16320tggcggctta tcatgtcttg ctttccaagt attcgtcgca aaacgatatt gtgataggtt 16380ccccgatagc aggccgaaca aatgctgatt tgcaatcgat tgtcgggatg tttgttaaca 16440cgctggctat ccgcaccaaa tcagagggaa ctcagacatt ccgcgagttt ctctctacga 16500ttaaacaact ggttcttcaa gctcaatcca atgcagagta tccatttgaa gagctggttg 16560ataaggtaaa tccaagtcgc gatctaagtc gccagccttt atttgacaca atctttgtca 16620tgcaaaacat ggatattacc gaggttgcga tacaaggtct ttcaatcgta acgaaggaca 16680tggaatggaa gcattcaaaa tttgatctta catgggcggc tgtagagaaa gaatccttgc 16740atttttcagt tgaatatagt acccgcttat ttaagaaaga aacaatcgag cggatggcga 16800agcattttgc ccatttgcta aatcaagtgg cggaaaatcc tgacttgagc ctttcagata 16860tggaattggc aacggatgaa gaagtgtacc agcttttgga ggagtttaat aatacagaag 16920ctgattatcc gagtgataaa acgattcacc agcagtttga gcagaaggta gaggaaaacc 16980ctgatcagat agcgttgtta tttaaagata aggaaattac ttacggacag ttgaatgcaa 17040aagcaaatca atttgctcgc gtattaagaa agcatggggt acagccggat caagtggttg 17100gattaatcac tgatcgttcc attgaaatga tgataggaat tttggcaatc ttaaaagctg 17160gcggagccta tttgccaatt gatccttctt atccattaga acggattacc tacatgctag 17220aggatagtca ggcacagctt ttgattgtgc aggaagctgc tatgattcca gaggggtatc 17280agggcaaagt attgcttcta gcagaagagt gttggatgca ggaggaagcg tccaacttag 17340agttgattaa tgatgcccag gatttggcgt atgtgatgta tacctcaggg tctactggta 17400agccaaaggg caatctgacg actcaccaaa acattttgag aaccatcatc aacaatggat 17460ttatcgagat tgtaccagca gaccgtctat tacagctatc gaactacgcc tttgatggct 17520ctaccttcga tatctacagc gcgctattaa atggagccac tcttgtactg gtgccaaaag 17580aggtcatgct aaatccaatg gagctggcga ggatcgtccg cgagcaggat attacggttt 17640cgtttatgac cacgtccctg ttccatacgc tagtggagct tgacgtgact agtatgaaat 17700ccatacgcaa ggttgtattt ggtggggaaa aggcttcata caagcatgta gaaaaggctc 17760tggattatct cggagaaggc cgtttagtaa atggatacgg ccctacagaa acaaccgttt 17820ttgctaccac atacacggtg gattctagta tcaaggaaac gggaattgta ccgattggcc 17880gtccgttaaa caatacgagt gtctatattt tgaatgagaa taatcaacca cagccgattg 17940gagtaccagg ggaattgtgc gttggcggag caggaattgc acgtggatat ttaaaccgtc 18000cagagctgac agcagagcgc tttgtggata atccgtttct tgtaggagat agaatgtatc 18060ggacgggaga tatggctaga ttcttaccag atggcaacat tgagtacatc ggacgaatgg 18120atgaacaagt gaagattcgc ggacatcgaa ttgaactggg cgaaattgaa aaaagtctcc 18180tggagtaccc tgctatcagt gaagcagtac ttgtcgcaaa acgtgatgaa caaggtcatt 18240cctatctgtg cgcttatgtt gtaagcacgg atcaatggac ggtggctaag gtacgtcaac 18300acatactgga ggctctgcca gagtacatgg taccatccta tttcgttgag cttgaaaagc 18360tacctcttac ttctaatggc aaggtagaca agcgtgcatt gcctgaacca gatcgagtga 18420ttaccaatga gtatgtggcg gcagtcaatg agacagagga gaagctagtt cagtttttcc 18480aagagatctt agctgtagac cgagtcggaa cgcaggatac attctttgaa ttgggtggtc 18540attccctaaa agcaatgatg ctggtttcaa gaatacacaa ggaattagaa atagaggttc 18600cgttaaaaga agtattcgcc agacaaaccg ttaaagaatt agcagcctat atcagacagg 18660ctgaacagtc ggattacagc gaaatccaac cggccatgga gcaagaatac tacccggtat 18720ctaatgcaca gcgacggatg tatgtggttc agcaaatgag agatgtagaa acaacaggct 18780acaatatgcc gttctattta gaaatggagg gtgctcttga ggtagaaaag ctatctctag 18840ctttgaaaca actaattgag cgtcatgagt cattgcgaac ctccttccat atggttgaag 18900atgaactgat gcaaaaggta catgcagaag tcgcatggga gatggaaatg attcatgccg 18960tagaggaaga agttcaacag ctgaccgatt cctttatgcg tcctttcgat cttgctaagg 19020cgccattatt ccgagcgaga ctcattcaaa tcaatccgaa gcgacattta ttgatgctgg 19080atatgcatca tatcatctca gatggggtat cgatgaatgt attgttccag gatataacgc 19140agttgtatca agggatagag ctgagtcctc tcaagattca atacaaggat tttgcggtgt 19200ggcaacaagg catcgctcag gttgtccgtt ttcaggagca ggaaaggtat tggttaaacc 19260aattctctgg tgacctacca attttggaaa tggtaactga ttatccacga ccagccatac 19320agcagttcga cggagattcc tggtcatttg aaattgatgc caaagtattg gacagcataa 19380agcaattgtc agctaagcaa ggcactacgt tgtatatgac tctattggcg atttatcaaa 19440tcctgttagc caagtatacc cgtcaagatg acatcattgt cggaactccg atcgcaggaa 19500gacctcatgc agacacagag agcattgtgg ggatgtttgt caatacacta gccctacgtg 19560gtcaaccaaa agaagagcaa tctttcatct cttacttatc agaagtgaaa gaaaacgtac 19620tacaagccta tgccaacgct gattatccat ttgaagagtt ggtagagaag ctgcatttgc 19680aaagagatat gagtcgtcat ccattgtttg atacgatgtt tgttttacaa aacatggata 19740tgtccgatat aaatatttct ggtctaaagc ttcattcgcg tgatttaaac tggaaaaatg 19800caaaatttga tatgacctgg atgatagccg aacaaaataa tctattgatt tcggttgagt 19860acagtaccaa cctgtttaaa catgaaacca ttcaaaggct agaaaagcat ttcacttatt 19920tagtagaaca agtggctaag catccggatt gcttactcag agatttagaa ctcacaacag 19980acgaagaaaa acagcaaata ctgacggtat ttaacgatac tgctactgat gatttacagg 20040atttatccat ttgccatcta ttcgaacaac aagtgcagcg tttttcagat cggccggcac 20100ttgtgtttaa agaaaagcag ctcacataca gtgagttcca tgcaaaagta aatcaattag 20160cccgggtact cagaaagaaa ggtgtgcagc cggatcaagc ggttggatta atcaccgatc 20220gttccattga gatgatgata gggattttcg ccatcctaaa agcaggcgga gcttatatgc 20280caattgatcc ttcctatcca atcgatcgga tcgagcacat gctagaggac agccggacta 20340agttgttatt cgtgcaaaaa acagaaatga tccctgctag ctatcagggg gaggtattac 20400tcctagcgga agagtgctgg atgcatgaag attcatcgaa tttggagctg atcaataaaa 20460cacaggattt ggcatatgtc atgtatacct caggttctac tggtaaacca aagggcaacc 20520tgacaacgca ccaaaacatt ttgaccacca tcatcaacaa tggctatatc gagatcgcgc 20580caacagaccg tctattacag ctatctaact atgcttttga tggctctacc ttcgatatct 20640acagtgcgct attaaatgga gccactcttg tactggtgcc aaaagaggtc atgttaaatc 20700caatggagct ggcgaagatc gtccgcgagc aggatattac ggtttcgttt atgaccacgt 20760ccctgttcca tacgctagtg gagcttgacg tgactagtat gaaatccatg cgcaaggttg 20820tatttggcgg ggaaaaggct tcatacaagc atgtagaaaa ggctctggat tatctcggag 20880aaggccgttt agtaaatgga tacggcccta cagaaacaac cgttttcgct accacataca 20940ccgtggattc tagcatcaag gaaacgggaa tcgtaccgat tggacgtccg ttaaacaata 21000cgagtgtcta tgtcttaaat gagaataatc agcttcagcc gattggagta ccaggggaat 21060tgtgcgttgg cggagcagga attgcacggg gctatttaaa tcgtccagag ctaacagcag 21120agcgctttgt ggaaaatcct ttcgtgtcag gagatagaat gtatcgtacc ggtgatttag 21180cacgttggtt gccggatgga agcatggagt atttaggacg gatggatgag caggttaagg 21240tacgcggtta ccgaattgag ctgggagaaa tagagacaag attattggag catccttcta 21300taagcgcagc ggttttacta gcaaagcaag atgagcaagg gcattcgtac ctatgtgctt 21360acatcgttgc aaatggggta tggacggttg cggaactacg taagcatcta agcgaggctt 21420tgccagaata catggtgcct acttattttg ttgaactaga gcagatacca ttcacttcta 21480atggaaaggt gaacaaacgc gctttaccag agccagaagg acaaatgacc agtgtatatg 21540tggccccaga aacggagaca gaagcaaaag tagcagcgtt attccaagag attttgggtg 21600tcgagagagt tggtacacag gacatgttct ttgagctggg tggtcattcg ctaaaagcga 21660tgatgctcgt tttacgaatg aataaagaac tgggcatcga ggtgcctttg aaagaggtat 21720tcgcccatcc tactgtcaag gaattggcag caacgatcga ccttcttgat cgatcaggcc 21780actcagagat tgagcctgcc ccaaggcagg aattctatcc ggtatcttcc gcgcagagac 21840ggatgtacgt ggtgcagcat ttaggaaatg tccaaacaac cagctacaat atgccgcttt 21900tccttgaagt ggagggagct ttagaaattg ataagcttca tctagcactt gagaaattgg 21960tcgaaagaca cgagtcgcta cgaacctcct ttcatatggt tgacgaagag ctgatgcagc 22020aggtgcatga agaggtggcc tgggatttag agatcatgga tggaacggaa ggagaccttg 22080caagcatcac agcaggattt atacgtccgt ttgatctcag ccaagctcca ttgttccgtg 22140caggcatcgt gcggattagc cctgagagat tccttttcat gctagatatg caccatatca 22200tctcagacgg agtttctacc aatgtattgt tcaaggatat aacgcagctc tatcaaggaa 22260aggacctgcc ccctcttccg atacagtaca aggactacgc tgtgtggcaa caagctgatg 22320ctcaagtgac tcgcttacaa gatcaggaaa gctattggtt acatcaattt gctggagaag 22380cttctgtctt ggaaatgccg acagatttcc cgcgtcctgc agtccagcag ttcgaaggag 22440atgtatggac ctttgagatt gatgctgaca ttctcagcca gttgaaaaaa ttatcagtga 22500gtcagggttc tactctatat atgactttat tggcggttta tcaggtgttg ctggctaagt 22560ataccggtca agatgatatt attgtcggtt caccaattgc cggacgccct catgcggatg 22620tagagagcat cgtcggtatg ttcgtcaaca cgctagcttt acgtggacag cctgtaggag 22680agcagacgtt tattacctat ctggcacaag ttaaggaaca ggttttacaa gcttatgcca 22740atgcagagta tccatttgag aaattggtag agaagctcga tttacaacga gatatgagtc 22800gccatccact cttcgatacg atgtttactt tgcaaaacat ggagatgact gatattgatt 22860tggcaggctt gaccttcaag ccatttgatt ttgaatggaa aaatgccaag tttgacatgg 22920attggacaat gcttgaggaa gaaacactca aggtagctat tgaatacagt acaagcctgt 22980atacaaaaga aaccattagc agaatggctc aacatttcac ctatgtttta caacaaatta 23040ttgagcatcc agccattcgt ttggctgaaa tcaaaattgc tactctacca gaaattgaac 23100agattttaac gcaatttaat gatactaggg ccaattaccc tgataaccaa accattcata 23160gtctattcga gcaacaagtg gagcgtacac cagaacagat agctgttgtc tatcaggatc 23220aatccatcac gtatcgtgag cttaatgaac gtgcaaatag attggcacgt tgcttgatcg 23280acaaagggat acagagaaat caatttgttg caatcatggc ggatcgttcc atagaaaccg 23340ttattggaat gatgggaatt ctcaaagcag gaggagctta tgttccaatt gatcctgatt 23400accctctaga tcgaaagctg tatattcttg aagacagcca tgcatcacta ttattgttcc 23460agcaaaagca tgaggtcccc tcagaattca caggtgatcg gatattaatt gagcagatgc 23520agtggtacca agcggctgat acgaatgtgg ggatcgtcaa tacagctcaa gatttggcgt 23580atatgatcta tacctcaggt tctacaggtc aaccaaaagg ggtaatgatt gatcatcaag 23640cagtatgtaa cctatgctta atggcccaaa cctatggaat ctttgcgaat agtcgcgttc 23700tacagtttgc ctcctttagc tttgacgctt ccgtaggaga ggttttccat acccttacaa 23760atggagccac tctctatctg atggatcgca atttgctcat ggctggcgtt gagtttgttg 23820aatggttacg agtaaatgaa ataacttcta ttccgtttat ctcgccttct gcattgcgtg 23880cattgccgta tgaggattta ccagcattga aatatatcag tacaggtggg gaagcattac 23940ctgtagattt agtcagacta tggggaactg agcgaatctt cttaaatgca tatggcccga 24000ctgaaacaac agtagatgca acgattggct tatgtacgcc agaggataag ccacatattg 24060gtaagcctgt gttgaataaa aaagcctaca ttattaatcc aaattatcaa cttcagccaa 24120ttggggtacc gggtgagtta tgcatcggtg gagtagggat tgctcctgga tattggaacc 24180gccctgaact aactagagag aaatttgtgg ataatccatt tgcccaaggc gaaagaatgt 24240ataagacggg ggacttagta cgttggcttc cagatggaaa tattgagttt ttaggacgta 24300ttgatgatca ggtgaaaatt cgtggacacc gaattgaatt gggtgaaatt gagacgcggc 24360ttcttgagca tgagcaggta atagaggcgg ttgtgctggc gcgtgaagat gaacaaggtc 24420aagcttatct gtgtgcttat ctggtagcag cagatgaatg gacggtagca gaactgcgca 24480aacatctagg aaaaacactg cccgattata tgattcctgc ttattttatc gagcttgagg 24540agtttccttt gacaccaagc gggaaggtga ataaaaaagc tttaccagag cctgatggac 24600aaatacaaac gggagtggag tacgtagagg ctactaccga aagccaaaaa atccttgttg 24660agctttggca agaggtgtta cgtgtcgagc ggatcggtat ttacgataac ttctttgagc 24720tgggcggtga ctccatcaaa gcaattcaaa tcacagcaag attgcgtcgc caccaccgca 24780agctggaaat cagccatctg tttaagcacc caacgattgc agagcttgct ccatggatgc 24840aaaccagtca ggcattactt gaacaaggaa ctgttgaagg cgaagttatg ctcacgccaa 24900ttcaaaaagc attctttgaa gaaaatcagg aacagccgca gcattttaat caggattcgt 24960tactgtacag ctcgaatggc tggaaccaag atgcgatcga gcaggtattt gaaaaaataa 25020cggagcatca cgatgccctg cgaatggtgt atccgcatac cgagggcaag gtgactcaga 25080tcaacagggg acttgaggac aaggcgttca cattgcaggt gttcgatttt acccaagaac 25140caactgatac gcaggcaacg aaaattgagc aaatcgctac tcaattgcaa gcgagctttg 25200atttaaaaaa gggacctctg gtacgacttg gcttatttac caccaaggct ggggattatt 25260tactgatcgt gatccatcac ctagtgattg acggcgtctc ttggcgtata ttgcttgagg 25320attttcataa tgcttatcag caagtcattc aaggtcaagc aattgtactt cctgaaaaaa 25380cgacctcctt taaaacatgg agtgagcgct tgaatgaata tgcaaatagt catgctcttt 25440tacacgagat tccatattgg aagcagatgg aagaaatatc gatcgcccct cttcctaaaa 25500aaggaaacaa tgacggtaga tattatgtga aggacagcga atatgccacg atgagtctaa 25560cagaagaaga aacccaaaat cttcttactc gtgtacatcg agcttatcga acggagatta 25620atgatctgtt gcttgctgca ttaggattag caagtaagga atggacaaaa gagaatcgag 25680tggctatcca cttagagggt catggtcgtg aggaaatagg tgaaggggta gatgtcaacc 25740gcactgttgg atggtttacc tccctgttcc cattcgtgat tgatttagaa aatgacgaat 25800tgcctctcat cattaaatcg gtaaaagaaa ccttgcgccg agttcctaat aaaggcatgg 25860gctacggcat actcaagcat ctgacaagcg atgcgaacaa acaggagata accttctcgc 25920ttcgcccaga gatcagcttt aactatctgg gggtatttga tcaacaagag gaggaaagcg 25980aatctgctgg gattcctact ggtcagccga tcagcccgca atattatgac acgcacctgc 26040tggagtttaa tggagcggtc tcgaataacc agttgcatgt aaattgccga tttgctcctg 26100cagccgttga tcgagcgatt gttgaaattt tgatggagcg cttcaagcac catttacttc 26160taattagtaa gcattgcttg gaaaaggata ccgtagaatt tacacctact gattttacag 26220aaaaggaatt aagccaagaa cagcttgacg atctattaga tgatttgttt gaagacatag 26280atgatctgta atcgcaatga gataggtggt gccacacatc gtgcaaaaaa aagacaagat 26340caaagatatc tattcacttt ctccgttgca aaagggtatg ctatttcatt ccatgaaaga 26400cccgcagagc gatgcctatt tcgagcaggt tacccttttg ctggaggggg ttgtaaaccc 26460aacctatttg gctgaaagta ttcagggact

cgtacaaaaa tacgacatgt tccgaagtgt 26520gttccgctat aaaaaagtag accctgttca ggttgtgctt agtgaacgaa aaatagattt 26580acagattgag gaccttactc aaatcaatga agaagagcaa cggaaattca ttgaggaata 26640tagaaaaaag gaccgggaaa gaggcttcga cctttcccgg gatatcctgc tacgttttac 26700attgtttcaa acagccgcca atcggtatga attactgtgg agtcatcatc atatcctgat 26760ggatggctgg tgtacgggta tcgtttttca ggatttattt caaatgtacc aacgtcgctt 26820gtcaggacag gccttacttc cagaggtggc ccctcaatat agcgaatata tacgctggtt 26880aaagaaacaa gatgaccaac aagcattggc attttggaag gagtatctac aggggtttga 26940aaaccttacg ggaatcccgc gtctaaggtc aggcaatcat ccctacaagc aagaggaatt 27000cattttctcc ttgggagagg aagctacaca aaaactaacg caaacggctc aaaagtatca 27060ggtgacctta aatactgttg tgcaaacaat ttggggagcg ttattgcaaa aatacaataa 27120cacgaatgac gcggcctacg gtgtggttgt ctccggacga cccgccgagg tgccaaatgt 27180tgaacaaatg gtggggttat ttagtaatac cattcctatt cgtattaaaa aagaagcagg 27240aaaaacgttt ggggaagtgc tgaaaaacgt acagcaaaca gcgctggagg cagaaaaata 27300cggatatctt tctttagccg atattcaggc gagcgcagct tatacgcatc aattgcttga 27360tcatatttta gcgtttgaaa atttcccgat ggatcaagaa acatttaatc aagaaaacgt 27420tctcggattt gccgtgaagg atgcccacac gtttgagcag acgcactatg atctgaccgt 27480gctagtcatt cctggcaagg aattaatctt taagtttatg tataacgaaa gtgttcattc 27540aaaagagtac ctcaatcttt tagagctgaa tatgaaaaag ctggtctctt tggttattga 27600gcagcaggat atctttgacc cagctaccga gtttgtatct gatttggaaa aggataagct 27660tttaaccatt tttaatcgta cggatgcaaa gtacccaaga gaaaaaacga ttcatgagct 27720gtttcaagag caggttgaca agaaccctga tcaagtggca ctcgtatttg gcgaggctca 27780actaacatac cgcgagctga acgaaaaggc gaatcaaatg gcccgcggtt tgcgcaaaca 27840aggggtttta cctgatcagg tgatagggtt acttacggat cgttccttag agatgatcat 27900agccattcta gcgatcttta aagctggtgg cgcttatatg cctatcgacc catcttatcc 27960gagtgaacgc attcaataca tgctagcaga tagtcgtacc catttgctat tggtgcaaaa 28020agctgaaatg atcccagcta attatcaggg tgaggtacta ctgttaacag aagatagctg 28080gatggacgag aatacagata atttagattt ggtcaaccaa gcacaagacc ttgcttatgt 28140catgtatacc tcaggttcaa caggtaaacc aaagggaaat ctgacaaccc atcaaaatat 28200cgtcaagacc atcatgaaca atggttacat ggagattacg ccaaatgatc gtcttctcca 28260gttgtccaat tacgcgtttg atggatcaac ctttgatata tacagcgcat tgttaaacgg 28320agcttctctt attttagtac caacgcatgt actgatgaat ccgactgatt tggcatcggt 28380cattcaagac cagcatatta ccgtgtcctt tatgacaaca tctctattta acactctggt 28440tgagctggat gtgactagtc tcaaacacat gcgtaaggtg gtgtttggag gagaaaaggc 28500ttcgatcaag cacgtagaaa aagcgctgga ttatttggga gctggacgtt tggtcaatgg 28560gtatggacca acagaaacta ctgtttttgc cactacctat acggtggacc atacgatcaa 28620ggagacgggg attatgccga taggtcgccc gttgaacaat acgaaggtgt ttattttagg 28680agcagacaat caactacagc cgataggtgc attaggcgag ctatgtgtga gcggggaagg 28740gcttgcccgc gggtatctca atcttccaga gctgactgct gatcgtttcg ttgaaaatcc 28800ttttatgcgg ggagagagaa tgtatcgcac aggggattta gcgcgttggt taccggatgg 28860aagcattgag tacgtaggta gaatagatga acaagttaag attcggggac atcggatcga 28920attaggtgaa attgaagcta gattactaga gcatcctgct attagcgaga ccgttttgct 28980ggcgaagcag gatgagcagg ggcattcctt cctatgtgcc tatctagtga caaatggtgc 29040ctggtcagtc gcagagcttc gcaagcatat caaggaaaca ttgccggatt ctatggtgcc 29100atcttatttt atcgagatag ataaaatgcc gctcacttca aatggcaagg cagacaagcg 29160tgcattgcca gagccagatg ttcaacaagt aagctcttat attgctcctg agaccgaaac 29220agaggaaaag ctggttcaat tatttcaaga aatcctaagt gttgaacaag tcggtacgca 29280ggataatttc ttcgagctgg gcggacattc gttaaaagcg atgatgctgg tttcaagaat 29340gcacaaggaa ttagatatag aagtaccgct caaggacgtg tttgctcgac cttcagtaaa 29400agaattggcc gcatttctta caaacacaga agtgtcggat tatatagcga ttgaaccggc 29460ggcaaaacag gaattttatc cggtttcttc tgcacagcgc cgaatgtatg tagtagagca 29520aatcggtagc agtaatacaa ccagctacaa tatgcctttt ttgcttgaaa taggaggagc 29580cctcgatgta gtagggttac aaaaagcatt aaagaaactg gtcataagac atgaatcgtt 29640gagaacgtcc tttcacatgg ttgatgaggt attaatgcag aagatccatc ctgacgtgga 29700atgggattta atggtcatgg aagcaaaaga cgaggacctt ccgcaaatca ttgatggttt 29760tatccagccg tttgatttaa gtgacgcttc tttatttaga gcgggactcg tacgaatgga 29820agctgatcga catctactga tgcttgatat gcaccatatt atttcagatg gggtatcaac 29880caatgtatta ttccaagacc tgatgcaaat ctatcagggc aaggagctcc cttctcttag 29940aattcaatac aaggattatg ctgtttggca gcaggcagaa gcccaggtta atcgtttacg 30000agaacaggag cagtattggc ttaaccaatt ttcgggagag ttacctgtac tggaaatgcc 30060taccgattac actcgtccat ctattcagca gtcagaaggg gatatatggt catttgaaat 30120tagtgccgag atcataaaca aagtaaagaa actgtcctcc tcgcagggta caaccttgta 30180tatgacattg ctggccgcct accaagtatt attgtcaaaa tatacggggc aagaggacgt 30240tattgtgggt tctcctattg ctggccgacc tcatgcggat gtagaaaaga ttgttggtat 30300gttcgtgaac acgttagcct tcagagggca gccaaaatca actcaaacct ttagtacata 30360tctgtccgag gttaaggagc aggtattgca cgcctatgac aatgcagaat atccgtttga 30420ggaattactt gaaaagcttg atttagaaag agatctaagt cgtcatccac tgtttgatac 30480catgtttgct ttgcagaata tggaaatggc tgaaatcaat atcatggatc tctcctttca 30540gccgcgggat ttaacatgga aaaatgcaaa attcgacctg acatggatga tggcggaagc 30600ggaaaatttg tatgtcacca ttgagtatag tacctcgctc tttaagccag aaacaattga 30660gcgattaggt aaacgattca cccatttact aaaacagatc ggggatgctc ctgaacgttt 30720gattgctgac ttagaagtag cgacggagga tgaaaaacat cagattttat cggtatttaa 30780tttgactcaa tcggattatc cagtaaataa aaccgttcat cagctctttg aggagcaagt 30840gcaaaatatg cctgatcaaa aggcgatagt atttggtgaa gagcaagtaa catacaaaga 30900attaaacgcc aaagccaacc atctggctac cctcttaaaa caaaaaggca taacaaacga 30960gcaacttgtg gctgttatga ttgagccttc catcgagttt tttgtaggca ttctagctgt 31020tctaaaagca ggaggggctt atctaccaat tgacccaact tatccgacgg aacgaattgc 31080ctatattttg gaggatagtc aatcaaaggt tctgttagtg agaggtcatg aacaggtaca 31140gacacaattt gctggggaaa tcttggaaat tgatagcaag aagttgtcta ccgaagagct 31200gaaagacgta cctatgaata acaaagtaac cgatctagcc tatgtcattt atacatcggg 31260ttccactggg caaccaaaag gtgtcatggt ggagcataga tcgttgatga atctttcagc 31320ttggcacgtt cagtattttg gcatcacaaa ggatgatcga agcaccaaat acgcaggggt 31380tggatttgat gcatctgtat gggaggtctt cccttactta atagctggtg caacgattta 31440cgtcatcgat caagagacaa gatacgatgt agaaaaactg aatcagtacg taacagatca 31500agggattacg atcagctttt tacctacgca atttgctgaa cagtttatgc tgacagatca 31560tacggatcat actgccctac gctggttgct tatcggcggt gataaagccc agcaagccgt 31620tcagcagaag cagtatcaga ttgtaaataa ctatgggcct actgagaaca cggttgtaac 31680aaccagctat atagtgagtc ctgaggataa aaaaatcccg atagggcgtc caattgctaa 31740taatcaggta tttatcctga ataaagagaa tcaattacag ccagtaggga ttccaggtga 31800actatgcgtt agcggcgaca gcctagcacg cggctatctg catcgtccag agttaacgag 31860tgagcgtttt gtagctaatc cgtttgtccc tggcgaacgc atgtataaaa ccggagatat 31920tgcccgctgg ttaccagatg gaaatattga gtatctaggt agattggatg atcaaattaa 31980gatcagagga taccgggttg aattaggtga gatagaatcc gctattttgg agcatgaagc 32040aattcatgag acagtagtgc tcgcaagaca agacgatcag aatcagacat atctatgtgc 32100ttatgttgta ccgaaaaaat cttttgatgt agccgagctt cgtcaatatc taggcagaaa 32160gctacctcac tttatgattc cggccttttt tacggaaatg acagagttcc caattacatc 32220gaatgggaaa gtagataaaa aagcactccc actaccggat ttgtccaagc aatcagagat 32280cgattacgtt gccccaacca ccacgttaga agaaacgctg gcggaactat ggacagaagt 32340gctaggagtt tcccaagtgg gaatccatga taacttcttt aaactgggtg gggattcgat 32400caaggctatt cagattgcag caagattaaa tacgaagcaa ttaaaattgg aagttaagga 32460tttattccag gcacaaacga ttgctcaggt tattccatac atcaaaacca aggaaagtaa 32520agctgagcaa ggaattgttc aaggaaaggt agagctaacc cctatacagg aatggttttt 32580ccagcaatcc ttcgatattc cacatcattg gaatcagtcc atgatgtttt atcgaaagga 32640agggtgggat cagcacgttg tacaaagggt gttccaaaaa attgcagaac accatgatgc 32700cttgcgaatg gcttatcagc aggaaaatgg caaaacgatt cagatcaatc gcggagtgga 32760aggcaagttg tttgagctaa gcatttttga ctttaaacaa caggcgaatg tgccagagct 32820gatcgagcaa gcagctaatc gtctacaatc cgcaatgaac ttgcaggacg gtccattggt 32880tcaactggga ctctttcaga catctgaggg ggatcatctt ttgatagcaa ttcatcactt 32940agtggtcgat gccgtttcat ggcgaatcat tacggaggat ttcatgaatg gctatcaaca 33000agatttgcag ggagagccga ttgcatttac gagcaaaaca gactcctacc aaaaatgggc 33060caagagcctg ctagagtacg ctactagtga agaaattcaa tcagagctga aatactggca 33120aagcatgatt gcaaaagggt tacctgcatt gccaagagat tcaaaagtag gtgccccgta 33180tctactcaag gatatacaag aggtcgctat ccaattgaca aaagagcaaa cgaataaact 33240attaacggat gcccataacg cctacaacac acagattaac gatcttttgt tgacagcatt 33300agctctaact attcaggaat gggcacaaac caattcaatc gcaattacac tagaaggaca 33360tggacgcgag gatattgggg tggacattga cattaaccgt acagttggtt ggtttacgtc 33420catgtatcca gtggtatttg atttgcagaa gcaagggatt gcaaatacgg ttaagcaagt 33480aaaagaagag ctgcgacaaa taccgaataa agggattggc tatggggttg ttagatacct 33540atcgaatcaa ggaagtacag agctggatct aagctcccat gcgataaatc cagagattag 33600cttcaattac cttgggcaaa tggatcaatc tggacaggaa gaggagtatc aattgtcccc 33660attgtcttcc ggtcaacaga ttagtcagat gaatcaaggc ttgttcccga taaatgtgag 33720tggaattgta gtggaaaatc agttgtccat tcaaatatct tatgatagcc aagcttatca 33780tgattctact atggaaaagc tgattcaacg ttatcaatat cacttgttgg agattattaa 33840tcattgtgtt cagcagacag aaacagaatt aaccccgagt gatttttcca ccaaagagct 33900ttcgatggag gatttagaat cagtatttga gttactagat gaataaactt tggttatgtc 33960attaggaggc tttatatgtt aagtaaagca aatattaaag acatctatac attatctccg 34020ctacaaaaag gcatgttatt tcagcattta aaagaagaaa gcacggctta ttttgagcaa 34080ttacacttta cgattaaggg acaactatat gtagatagct ttgaagcaag ctttcagcat 34140ctcataaaca aatatgatgt gctacgaacc gtttttctgt ataaaaatat gacccagccc 34200atgcaaatgg ttttaaaaga aagaaaaaca agtgtgcatt ttgaagatat ctcccaccta 34260gattctaaag ccgtgagtga atatgttgaa gagtttaaaa atcaggatcg ggagaaggga 34320tttgaactct cgaaggacat tctcatgcgt tttgctattt tgaaggctgg tgctgagtcc 34380tatcatttaa tttggagctt ccatcatatt ttaatggacg gctggtgcat gggcattgtg 34440ttacaggatt tgttcagaat gtatcagcag catcgtcaaa atataccgat taccgttgag 34500agcgttcctg cctatagcga gtatatccgt tggcttgaga agcagaatgt aacaaaggcg 34560agggattact ggaaaaatta cttagagggc tatgaggaat taacaggtat cattcgtctc 34620gatacgaagc atacgagtca caacaacgag gtacaggaat gcgcctttac actggataag 34680gacataacgg aaggacttac tcagcttgct cgtcattatt cagtgacagt aaatacgctt 34740tttcaaacaa tttggggcat gctgttgcaa aagtataaca ataaggatga tgttgtgttt 34800ggtgcggtcg tatctggccg cccctctgaa atccatggcg tagaaaacat ggttggcttg 34860tttatcaaca ctgtccctat tcgtattcaa aaacaaatga atgatacctt tagccattta 34920ttaaaaagag ttcacgaatc tacgctattg tctaaacagt atgagtttgt atccttggca 34980gatattcaaa ccgatgcagg attttctggt caattgctag atcacatctt agtttttgaa 35040aactatccga taagtgaagg ttcttttgag gaagaagaat ttacgatgga tagtataaaa 35100acctatgaga aaacaagcta tgacctaaac gtgatgattc ggcctaatga ggatcagctt 35160gatattgcct tccaattcaa cgatgacgtg tactcaagcg aaaatgtaaa aagactgttc 35220cagcatatga agcaactggc tctagctgta atcaagaatc cggatgtgcg cttggaagaa 35280atagcaatga tcacagaaga ggaacgctat caaatcttgc acgatttcca aggggagata 35340gttgattttg taacagaaaa aacgcttcct gaactgtttg aagaccaggt gaaacgaact 35400ccagaagcaa ttgcacttcg atttgaagat caacaattga cctatcagga gctaaatcag 35460cgagtaaatc aattagcttg gacactaaga atgaagggct tgcagcaaga agaactcgtt 35520ggaattatgg tgcagcgctc attagaaatg atcgttggtg tgctagccgt tataaaagca 35580ggcggcgcat acgtaccaat tgatccggaa tatccgcttg accgaatcca atatatgctg 35640gaagacagtg gaaccaattg gctgttaacc acgaaacaga gcgaaattcc ttccatctat 35700ctagggcatg tcctgtatct tgaggaagat acggtgtatc acgagcggtc ttcagatgta 35760gagattgtaa atcaatccag cgacttagct tatattatct acacgtccgg ttctactggt 35820cagcctaagg gtgtcatgat tgatcatcgt gctgttcata atttgcattt gtcagcagga 35880atctatggaa tcgcacaggg aagccaggtt ttgcagtttg cctctttaag ctttgatgct 35940tcggtgggtg atatcttcca cagcctatta acgggagcta ccttgcatct tgtaaaaaaa 36000gagcaattgc tatccggaca cgcctttatg gagtggttag acgaagctgg cattacgact 36060attccgttta ttccaccaag cgtcctaaaa gaattaccat atgcaaaact gcctaagctc 36120aaaacaatca gtactggcgg ggaagaatta ccggctgatt tagtaaggat ttggggagca 36180aaccgcacat ttttaaatgc atatggtccg acagaaacga cggttgatgc ttcgattggt 36240aattgtgtag agatgacgga taagccttcg attggtacgc caaccgttaa taagcgagcg 36300tatattttgg atcaatacgg tcatattcag ccaatcggtg ttcccgggga attatgcgta 36360ggtggagaag gcgtagctcg tggatattta catagacctg agcttacaga tgaaaagttc 36420gtgaacgatc cttatgtacc aaacgggaga atgtataaaa cgggagactt agctagatgg 36480ttgccggatg gaacaatcga atttttaggc cgtatggatg gccaagtaaa aattcgtgga 36540tttaggattg agcttggaga aattgaagct cggctaaacc aagccccatc tgtaaagcaa 36600gctgtggttc tagctcgttc aggagaacaa aagcaggtat acctatgcgc atatttggtg 36660acggacaacg atttaaaggt ttctgcccta cgtaaggaat taagtcaaac gttaccagac 36720tatatgattc catcgttttt tataaaagtc gaaaagattc cagtcacagt aaacggcaag 36780atagacaaga aagccttgcc agaaccagaa aaagaagtag agctgcaaac cgaatatgta 36840gctccaacga acccaacaga ggagattctt gtacagattt ggcaaaaggt gctgggaatg 36900gagcgagtag ggatagagga taacttcttt gagctaggtg gtcactctat caaggcaatg 36960atgcttgctt ccaatattta taaggaatta aagattgatc tgcctttgcg tgagattttt 37020aagcatacga cagtaaaaga aatggcgcgt tttatcgacg gtcgggatga ggaagaatac 37080gtcggaattc aacccgcagc caaacaagaa tactaccctg tctcttctgc acaaaaaagg 37140atgtatgtca ttcaatcatt ggaagataag gctcaaggca cgagctataa tatgccgtct 37200ttctataaaa tgaagggctc ggtagatgca gagaaattag agaaggtatt ccaaacatta 37260ttggatcggc acgaatcatt acgaacctcc tttcatatga tcgaggagca gctagttcaa 37320aaggttcacg aacaggtttc atggaaaatg gacatgaaaa ccgtcagcgc caatgatgtt 37380tcaagattaa aggattcgtt tgtccaaccg tttgacatca gtacagctcc tttgttccga 37440gccagtcttc ttacgattca taaagatgag cacattctta tgatggatgt acaccatatt 37500gtaggagacg gtgtttcgac cacgatcttg ttccaggagc ttatccagtt gtatcaaggg 37560caagcgctac ctgaagtgaa ggtacactat aaagattacg ctgtgtggca attgtcccag 37620caggatcgtt tgaaagaaag tgaaaatttc tggttgcagc aattttctgg agagttgccg 37680gtgttggagc tacctactga ttattctcgt cccccaattc gccgattgga aggagaatat 37740gtaagccaaa gcctacgtgg tgatctccat gaaagcgtaa aagccttcat gaaaaatcac 37800gaagtaacgc tatatatggt actgcttgcg acatataacg ttcttctgca caaatacacg 37860aatcagcacg acattattgt tggtacgcct gtttcggacc gaccgcatcc agatgtcatg 37920tccactgtcg gtatgtttgt aaatacgctg gcagtccgaa atcagttgga gtctgagcaa 37980accttcgaaa agtttttagc aaatgtgaaa aataaaatgc tagaggtcta tggtcatcag 38040gagtatccgt ttgaagatgt aattgaaaaa gtaaaggttc aaagggatac aagcagacat 38100ccgctatttg acacaatgtt tggtgtacaa aatctggaga tatcccacgt ggagctaccc 38160gattggggta tagaagcatt ggatattgac tggactaact ccaagtttga tatgagctgg 38220atggtatttg aagcagacgg tctagaaatt ggcgtggagt atagcacaag cctatttgag 38280cgcaatacga ttcagcgaat gatcggacac tttgaacata tcatcgagca gattatggaa 38340aatcctcaaa ttcgtttagc tgatattcag ttgacgacag aagatgagag aatccaaatc 38400ttagaggaat tcaatcatca accaacaaaa ataacctacg atcaggcaat ccaaaacaga 38460tttgaagaac aggctatgaa gacacctgat gcagtggcac ttgtatataa aggtcaggag 38520ttaacctatc gtgagcttaa ccaaagatca aatcagatgg ctcgtacatt aagagagcat 38580ggggtcgggc gtgatcaaat aattgcggtc atgattaatc gttcacatga gctgatcatt 38640agtatcctag ccgtattaaa ggcaggagga gcatacctgc caattgatcc aacgtacccg 38700cttgatcgga ttgaacacat gctagaggat agccagactg caatgctgtt aactcaaaaa 38760gaaatccaaa tacctacagg atattcaggg gaagttctct tcgttgatca agctgatatt 38820tatcatgagg atgctacgga tttatctagt atgaatcagc ctgcggattt ggcctatatt 38880atttacacat caggctctac tggaaagtcc aagggagtaa tgatcgagca tcgttcatta 38940cataatctga ttcatatttc tcacccctat aaaatgggag caggaagcag agtccttcaa 39000tttgcctcta gcagctttga tgcctcggta gcagagatct ttccagctct tttaactgga 39060tcaactttat atatagaaga gaaagaggag ctattaacca atttagttcc ctacttactt 39120gagaatcaaa taacaacagt agcattgccg ccatctttat taagatccgt tccttatagg 39180gaactgccag ctttagagtg catagttagt gtcggagaag cttgcacatt tgacattgta 39240caaacttggg ggcaaaaccg cacctttata aacggatacg gccctacaga atcaactgtt 39300tgcagtgcct ttggtgtggt tacagcagag gacaagcgta tcacgattgg taaaccgttc 39360cctaatcaaa aggtctatat catcaatgaa aatcaacagc tacaaccaat cggggttcca 39420ggtgagcttt gcatagcagg ggctggatta agccgtgggt acttgaatcg tccagagctg 39480acacaggaaa aatttgtaaa caaccccttt gcacctggtg agcgtatgta taaaacagga 39540gacgtagctc gctggttgcc tgatggcaat atcgaatatg ccggtcgtat ggatgatcag 39600gttaaagtac gcggaaatcg ggtcgagctt ggggaggtta ccagccaatt acttacgcat 39660ccttcgatta cagaagctgt tgttgtacca atagtcgata cacatggagc aacgacacta 39720tgcgcctatt tcatcgagga taaagaagtg aaggtcaacg atttgcgcca tcatttggct 39780aaagctctac ctgagtttat gattcctact tactttatta aagtagatca tattccattg 39840acaggaaacg gaaaggtaaa taaacaagca ttacctgacc cttccgaatt catttcagca 39900caaacaggcc atgaaatcgt tgccccttct tctcaggacg aggaaatact ggttcaggta 39960tgggaagaag tcctgcagtt caaaccgatt ggggtagagg acaacttctt tgaacgaggc 40020ggagactcca ttaaggcatt gcaaatcgta gctagactta gtaaatataa tcggaaattg 40080gatagtagac atatttttaa aaatccaacg atttccatgc tggctcctta ccttgaacaa 40140agaggtgctt tgattgaaca agattcaatt gaaggcgaag tgccgcttac accgattcaa 40200tcctggttct ttgaacaacc ctttgtgtat ccacaccact ttaatcaatc tatgcttcta 40260ccaaatgaac aaggctggga tcgtcaacga atagaacaag catttacaac cattgttaga 40320caccatgatg ccttaagaat gaagtaccag tttagagaga agatcattca agaaaatcag 40380ggtatcgagg gagagttttt taccctgcat gaggtggatg taaccaagga aagagactgg 40440caaatgcgca tcgaacaaga agcgaatcaa ctccaagcaa gctttgattt gacaacaggc 40500cctcttgtaa agcttggctt ataccatacg gcatatggcg attatcttct gattgttgta 40560catcatctct taattgatgg tgtctcatgg cgcatcctgc tggaggattt ccagacgctt 40620tatgagcaaa agggtgagtt gccagcgaaa accacttcct ttaaggcgtg ggctgtacaa 40680ctggaggggt atgctcgcag caaaaagcta caagacgagg caagctactg gaaagggttg 40740ttgaataaat cgataagaga gctgcctgcg gataaggaat caagcgatac attcctcttt 40800ggagatacaa aagaagtaca gcttaccttt gatataaatg aaacccaaga cctgcttacg 40860gatgcccacc atgcttataa gacaaaagcg gatgatttat tgctggcagc gttggttctt 40920agcataaatg agtggacgaa gcaaagcgat atcatagtga atttggaagg tcatggccgt 40980gagacgatcg gcgaaggcat tgatttgagc cgtacaattg gctggtttac tacaatttat 41040ccagttctgt ttgaagtaga gaaccatcaa ctttccagcg tgattaaaca tgtaaaagaa 41100acgctgcgca atgtaccgaa taatggtatt ggttttggga tcttacaaca catgtctcat 41160tctgatgtaa gccagagcca attaagttct catcacataa gcttcaacta cctaggtcag 41220atgggagaag attccgctag tcagtctgag acggataatg gagtccttat caatacagga 41280gaccagataa gcccaatgaa cgcaaatccg ggctcgctta atatgacttg ccttgtaatg 41340aataatacgt tgcttgttac ttttgattat aatccgcaac gttacgaaca ggagacaatt 41400caacgtctgg cagatcgtta taagagcaat ttaaaagcag tcctcgatca ttgtgttcaa 41460cgagagcaga cagagcgaac acctagtgat tttagtacga agaagctttc tttagaggac 41520ttagacgacg tgtttgcaac acttaaaaat

ctataaaggt atcctgagga ggagaagatt 41580aacttgatta atacctcaga cgtcaaagac atttatagtt tatccccgat gcaacgagga 41640atgttatttc atacattaaa agacaaagaa aaccttgcct attttgatca gacaactttt 41700caaatagaag gtgacatatg tgtcgaatcc cttgagaaaa gttttaacga gctgattcgc 41760aagtatgatg ttctgcgtac gatcttttta tatcagaaat taaaagagcc gatgcaggtt 41820gtgttaaagg agagaacagc aaacattcat tatgaggatt tctctatgaa gagcgagtcg 41880gataaagcaa aggctcttcg tgtagcaaaa cagagggacc gggacgaggg ctttgacctc 41940tcccgggaca tcctcatgcg gttatcttta ttaaaagtcg cccctaacca atacgaatta 42000gtgatcagta gccaccatat tatcattgat ggatggtgta caggaatttt gtatcaggag 42060ctgttttatt tttatcaatg cttcgtagca aatcaaccta tccctgctga gaaatcgatt 42120ccgtatagca gatatattcg ttggcttgaa gaacaggatg aagaggaagg aaaagcctat 42180tggggtgaat atctacaaga tttcgagggg gcatctgtta tccctaagca aaacgctaag 42240ggagagaagg aagtatgctc cattgataag gtaaccttcc actttgataa aaagctgacg 42300gaggaactgg tgcaggtagc aaaaacttgc caagtaacaa taagtacctt gtttcaaaca 42360atgtggggca tcctgctcca aaagtataat aactcgcagg aagctatatt tggatcggtt 42420atttcaggaa gatcaccaga gattcctgat gtggaaaaaa tagttggaat ttttattaat 42480accattcctg ttcgcattcg tacattggac aagcaaacct tcaaggaatt gctgatccag 42540gttcaggagg catctgtcaa ctctgaaaaa tataattatc taacattggc tgatattcaa 42600gcggttaccg gatcgaatca tgcacttatc catcatattg tggcatttga aaatttcccg 42660attgcctcgg acagcttcgt agattcgagc gattccgatt cagaagaatt gaaagttgtg 42720aacgtcatag acgatcatga aaagaccaac tttgatttta gtgtgcaagt tcagcttgat 42780acagagttac tagtaaaaat ctcttataat caacatcttt atcatagaag ctttattgaa 42840aatatctttc atcacctgca acagattgcc gggtctatca ctcataaccc agatattcaa 42900ataaatgaga tagctattgt ttctaaggaa gagaagaagc aactattacg ctattccact 42960ccagccaagt cagattttcc aatggataaa accattcatc agctatttga ggagcaggta 43020tcacggacac cagagcagat cgcggtcgtt tttaaagggg agtccttcac ctatcgcgag 43080ttaaatgaaa aggcaaatca attggcatgg gtgctaagaa aacgggaggt aagacctaac 43140gagatcgttg cgatcatggc agagcactct ctagagatgc tggttggggt gattgggact 43200ttaaaggcag gtgcggccta tcttcctatt gacccatcct acccagaaaa aagaatcgct 43260catatgctac aagatagcaa agcggagcaa ctacttatcc agcctcattt gaatatgcca 43320caggacttta agggaagtgt cttatggtta acagaagaga gctgggcgaa ggagagtacg 43380accgatctgc cgcttgcaac gagtgcaaat gatctagcat acatgattta tacctcaggc 43440tcaacaggac tgccgaaggg agttatggtt gagcatcaag ccttggttaa tttagttatg 43500tggcataacg aggcatttgg cgtaaccatg actgatcaat gcacgaaatt ggcgggattt 43560ggattcgatg cgtcggtgtg ggagaccttc cctccgctta tacagggagc gacgcttcat 43620gtgttagagg aatcgagacg tggagatatt tatgctctgc atgaatactt tgaaaagaat 43680gcgatcacca ttagcttctt gcctactcaa ttagccgaac aatttatgga gcttacaagc 43740agtacattac gtgtgttact cattggcggt gaccgagccc aaaaggttaa agagacatcg 43800tatcaaatca taaacaacta cggtccaacc gaaaatacag tagtcacgac gagcggtcaa 43860ctgcatcctg agcaggatgt cttccctatt ggaaagccga tcaccaatca cagcgtttat 43920attttagatc agaacagaca tctacagccg atcggaatac ctggcgagct gtgcgtcagt 43980ggtgcagggc ttgctagagg ctaccttaat cagcctgaac tcaccgtaga acgctttgtt 44040gataatccct ttgtacctgg agagagaatg tatcgcacag gggacttagt tcgttggaga 44100atcgatggta gcatcgaata tctgggaagg attgacgagc aagtcaagat tcgaggatac 44160agaattgagt taggtgagat cgaaacaaag cttcttgagc atccttccat tagtgaggcg 44220ctcgtcgtgg ctcgaaatga cgagcaaggt tatacctatc tatgcgctta tgtggtagca 44280actggggcct ggagcgtatc ttcattacgt gagcatttaa tcgaaacatt gcccgaatat 44340atgattccag cttacatgat ggaagtggaa aaaatgccgc ttactgcaaa cggaaagata 44400gataagcgag cgttaccagt gcctgatagg caaagaatga acgaatatgt ggcacctgca 44460acagagacag aggaaaagct agttctactg ttccaagaga ttttaggact tgagcgtatt 44520ggtactaaag atcacttctt tgaattaggg ggacattcgc tgaaggcgat gatgcttgtg 44580tctcgtatgc acaaggagct aggtgtggat gtgcagttaa atgagatgtt tgctcgtcca 44640acggttaaag atctatctgc ttacatagat cagatgaacg gctctgctta cacagcaatt 44700caaccagtgg aggaacagcc ttattatcct gtttcttttg cccaaagaag aatgtatgtt 44760gtacagcaaa tgagagatag tgaaacgacg agctataaca tgccgtttac gtttgagcta 44820aaaggaaagc tacatctgga caagctgcga gaagcgttac agattctggt tctacgacat 44880gaaagtctgc gtacatcctt tcatatgatt gatgaaaatc ttgttcaaaa agtgaataaa 44940gatatttcat gggatttaga agtaatagaa gctcaggagt cagagataga agtaaaactg 45000gaggaattta tcagaccgtt ccatttaagt gaggctccgc ttttcagagc tcgtttaatt 45060tgcttgaatc cacagcatca tcttttgagc ttagacatgc atcatattat ttcagatgga 45120gtatctatga acctgttcct acaggaattc atgacactct atcagggaga agcattgcca 45180gcgctctcta ttcaatacaa ggattacgcc gtatggcaac aatcagacaa gcagcgagct 45240agattaaaag agcaggaaaa atattggtta catcattttt ctggagagct gcctacctta 45300gaattgccaa cagattttcc acgccctgca atacagcaat ttgatggaga tgaatgggcg 45360tttgaaatga atgctgatct tttagcgaag gtcaaacaga tctgctctag ccaaggcacg 45420acgttatata tgacgcttct cgctgcttat caggtgttct tagccagata taccgggcag 45480gaggatatca ttgtaggttc tccaattgct ggacgttctc atgctgattt ggaaaacatg 45540ataggtatgt ttgtcaatac attagctttg cgcggtaagc caaaggcaga tcaatccttc 45600ctctcctatt taaaacaggt aaaagagacc gtattccaag catacgcgaa cgcagaatat 45660ccatttgaag agttgattga gaaactcgat ttagaacgag atatgagccg tcatccgcta 45720tttgatacct tgttctcttt gcaaaatatg gaaatatctg agttccaaat gaataatcta 45780gagatttttc cttatgaaac gggacaaaag aatgcaaaat tcgctcttag ctggttaata 45840gcagaaggag agtcccttta tgtaacaatc gaatacagca ccaaatgctt taagcgagaa 45900accattaaac gcatggcaag tcattttgaa caactgctag cccaaattgt tgagcaaccg 45960gaagcgcgca ttggccaact ggagttagta gcagatgccg aaagaaaaat gttactggaa 46020gactttaatc tgacaaaagt cgactatcca cgggaaaaaa caattcaaga attatttgaa 46080gagcaggtgg acaaaaaccc tgatcaaatc gcgcttatat gtggagagca acagtttacc 46140tacgaacaat taaatgtgaa atttaaccaa ttagctcacg tattaagaag agaaggcgtt 46200caacccaatc aggtaatagg gctaattacg gatcgatcgc tgtcgatgat tgtaggtatt 46260tttggaatta taaaagcagg tgggggctat ctgccaatcg atccgaccta tcctaccgaa 46320agaattgaat acatgcttga agatagtcaa actcacctat tgttggtaca acacagagac 46380atggttccag caggttatca gggagaggtt ttgataatag aggatgagat aagtcgagat 46440gaacaagtag ctaacataga attgatcaat cagccgcaag acttggctta tgtcatgtac 46500acatctggct ctacaggtaa accaaagggg aacctgacta ctcatcgaaa cattatcaaa 46560acggtatgca ataacggata tattgagata acgactgagg atcgtctttt gcagttatct 46620aattatgctt ttgacggctc tacctttgat atattcagct cgttattaca cggagcaacg 46680ctggtactgg taccaaaaga agtgatcttg aatccaacag acttgattac attgatacgc 46740gaacagcaga tcactgtatc gtttatgact acctcattgt ttaatgcatt agtggaactg 46800gatgtaagca gtttccaaaa catgcgcaag atcgcatttg gaggagaaaa ggcttccttt 46860aagcatgtgg aaaaggcatt ggatttcctc ggaaatggac gattggtgaa tggatatggt 46920cccacagaaa caaccgtttt tgctacaacc tacactgtgg atgagcgcat aaaggaatgg 46980gggattatac cgattggtcg accgctacat aatactacgg tccacatttt aagcgctgat 47040gacaagctac agccaattgg agtcattgga gaactgtgcg taagcggtga aggattggca 47100cgcggttacc ttaatctacc agagttgacg atggagcgat ttgttgaaaa tccatttaga 47160cctggtgaaa gaatgtaccg cacaggggac ttggctcgtt ggttaccgga tggggttctt 47220gaatatgtag gacgcaagga tgaacaagtg aaaattcgcg gacatcgcat tgagcttagt 47280gaaattgaaa caaggatatt ggagcatcct gcgatcagtg aaacggttct gctagccaag 47340cgaaatgagc aaggcagctc atacctgtgc gcttatattg ttgcccatgg ccaatggaat 47400atccaagaat tgcgcaaaca tgtaagagat gttttgccag aacacatggt gccttcttat 47460tttattggct tagacaaact tccacttacc tccaatggta aagtcgacaa acgagcattg 47520ccagaaccag agggcagcct gcaactgact agagaaattg ttgctccacg caatgaatct 47580gaaaaacagt tagttgaaat tgttgctgag gttctgggac tagaagctag tgaaataagt 47640attaccgata atctttttga gctaggtgga cattccctaa cgattctgag aatccttgct 47700aaggttcata catgtaactg gaagcttgaa atgaaagact tctataattg caagaacctt 47760gaggaaatag caagcaaggc aactgatatg caggaaaatc aaaatctgtc tggcagtggc 47820tcagtcttta aaaagggtgg gaagaaatca atcccggtag tacccgtcca cgatagacaa 47880aaagaaatgg agcatgtttt attgctcggc tccactggtt tcttaggtat tcatttgcta 47940catgagctgc tacagaaaac agaagcgaca attctttgcg tcattcgtgc agaaaatgat 48000gaggctgcta tgcaacgact acgcaaaaaa attgattttt actttacctc acagtacagt 48060agctctcaaa ttgatgagtg gtttacccgc atccaaatca ttcacggtga tattacgcaa 48120gccaactttg gattagaggc aaaacattac gagtcgctag gagctatcgt tgacactgtc 48180attcatacgg ctgcattggt gaagcactac gggcactatg aagagtttga aagagcaaat 48240gtacatggaa ctcagcaagt agttaccttt tgcttgaaca ataaattacc aatgcactat 48300gtttcaaccc tgagcgtttc gggaaccacc gttgaagaag caacagagct tgtagaattt 48360accgagaagg acttttatgt tggtcaaaac tatgagtcaa atgtatatct gagaagtaaa 48420tttgaagccg aagccgtact tgttggcgga atggaaaacg gactcgatgc acgtatctac 48480cgggttggca atttaacagg acgctttcag gatggatggt tccaggaaaa tatcaatgaa 48540aatatgtttt atctcctatc gaaagccttc cttgagcttg gaggttttga tcaggaaatt 48600atgcagggta tggttgattt aacccctatt gatatatgtg cacaagctat tatacacatc 48660atcaacagca aaggaattga ggaaagagtc ttccatttac agaatccgca cttggtaaca 48720tacgatgata tgtatcgtgt atttgaaggg cttggctttt ctagacgggt acaaagtcga 48780gaagatgtta cacgtgaact agatgtaatg atgtctcagg gtaatgaaaa gctatttttg 48840gctgggattc tgaccacgat gttggatgat gtagagcgtg ctgaacaatt taatgttgca 48900gtcgattcaa gtaggacaat gcagctatta gaggatacct cgtttaccta tcctgttcct 48960gatgatgagt atttgcgcaa gctggctatg catatgatca aagttgggtt tgttactcct 49020aatcatactg ttgctgaaaa gataggaact agtcgttagc gctatgctag cgactggttc 49080ccaacctaaa tgaatagcta aaggaaggag agggaaccca tggcagtcat tgaactaaaa 49140aaccttacga aaaagtataa tgaggtctat gctgttgatc atctaaatat agaagtacct 49200caaggacata tttatgcgtt tttaggtagc aatggggcgg gaaagacaac cacaattaaa 49260atgatgacgg gccaattgaa cccttcagag ggagaggttc tatttctagg gcgcaatatt 49320tggcaggatc gtgaggcaag aagaattgcg ggctatgctc cagacgttcc acttcttcat 49380gaaggattga cagtcagaga aatggtacgc tttgtggggg ctctttatgg tagtgacgaa 49440gatctgaata aacgtgttga cacgttgtta gaacattttg agctggcaga taaagcagac 49500cagcttatta aagaatactc attaggaatg aaacgaaagg tttcgattgc ttgcgcattg 49560attcatcgcc ccaaaatctt gctattagac gaagttacga atgggttgga cccaaaggcg 49620acccgtgaag tgaaaaatta tattcgacat tttgccaaag aagagggtgg tactgttttt 49680attacgaccc atattttgga cattgttgaa gaattagccg ataccatttc catcctgcat 49740aaaggaaaaa tcaaagtgac gggaagcatg gaagaattgc gtcatgtggc aggcaatgaa 49800gaaggtcgat tggaagatat ctttttatcc gctatcgagt agtaggaggt gacagaattg 49860tatgtgggca caaacgaaat ggattagttt cttttacaca agacccttct ttaatcgctt 49920ttttatccat agtccttcta aatggatcat ttatgtgggc ttgggaacca ttgctattgc 49980catgtacttt tcggagaatt ttgggcagct tctcttacat gccagtctca gtgctagatt 50040gatgcttctc ataggggaat gtatttttgt cggtttgctt cgtggcatga atacgttgac 50100acaacaaatg tacgctgatc gattactgac attgttttat gtatcgggag tttctccgtt 50160tcggatgatc cttgggcaat ctacttcaag tctacctctg tacacgtggt catccattat 50220gattgctatt ccattaacga ttggctattc cgccatggaa agagttctgt atgttttgtt 50280attcctagtc gtttctctat tgatgatttg gttaacagac atcttaagcc gatttttaat 50340ggttctgacc atgcggtttt tccctattat tgtcaaaaca ttcgtaggta tctcctcgct 50400tgcctatgtt gctttaattg gcctattggt ttgggcattg attgaggttg aaacaatttc 50460tccagaagct tggcagagct tagagcgttt tatggtatat gttttgtgca ttttcgcggt 50520cggtcttgga gcgttgtttc tattctctga acaaattgga gggttttatt acgaaagctg 50580gctgaaccat gcggagtcgc aagataggac cagaccagaa acacaggaaa atctatcgaa 50640tttggtcaaa aacgctcatg atgccatcgt tttt 5067422641PRTBrevibacillus texasporus 22Met Asp Leu Ser Thr Leu Asn Phe Leu Gly Glu Thr Glu Lys His Lys1 5 10 15Leu Leu Asn Gln Phe Asn Asp Thr Asp Ala Asn Phe Pro Gln Glu Met20 25 30Thr Ile His Gly Leu Phe Glu Lys Gln Val Gln Glu Arg Pro Asn Gln35 40 45Thr Ala Val Ile Phe Asn Glu Gln Ser Met Thr Tyr Lys Glu Met Asn50 55 60Glu Arg Ala Asn Gln Val Ala His Ser Leu Arg Lys His Gly Ala Ala65 70 75 80Pro Asp Glu Ile Val Gly Ile Leu Ala Asp Arg Asn Met Asp Met Leu85 90 95Ile Ser Ile Leu Gly Val Leu Lys Ala Gly Ala Ala Tyr Met Pro Ile100 105 110Asp Pro Thr Tyr Pro Thr Glu Arg Ile Leu Tyr Met Ile His Asp Ser115 120 125Gln Thr Lys Ile Val Leu Ala Glu His Arg Glu Met Val Pro Glu Gly130 135 140Cys Asn Ala Glu Leu Ile Leu Leu His Asp Ser Ser Leu Leu Asn Glu145 150 155 160Glu Thr Ser Asp Leu Glu His Val Asn Lys Pro Glu Asp Leu Ala Tyr165 170 175Ile Ile Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys Gly Val Met Ile180 185 190Glu His Arg Asn Val Ile Arg Leu Leu Phe Asn Asp Arg Asn Leu Phe195 200 205Asp Phe Thr Ser Asp Asp Val Trp Thr Val Phe His Ser Phe Cys Phe210 215 220Asp Phe Ser Val Trp Glu Met Tyr Gly Ala Leu Leu Tyr Gly Gly Lys225 230 235 240Ile Val Leu Val Ser Phe Glu Ile Ala Arg Asp Pro Gln Ala Phe Arg245 250 255Asp Leu Leu Gln Glu Gln Lys Val Thr Ile Leu Asn Gln Thr Pro Thr260 265 270Ala Phe Tyr Gln Leu Ser Ser Gln Glu Met Gln His Ser Asp Ser Asn275 280 285Leu Ser Ile Arg Lys Ile Ile Phe Gly Gly Glu Ala Leu Thr Pro Ser290 295 300Gln Leu Lys Ala Trp Lys Gln Lys Tyr Pro Asn Thr Ala Leu Ile Asn305 310 315 320Met Tyr Gly Ile Thr Glu Thr Thr Val His Val Thr Tyr Lys Glu Phe325 330 335Gln Leu His Asp Met Asp Ser Thr Val Ser Asn Ile Gly Lys Pro Ile340 345 350Pro Thr Leu Arg Thr Tyr Val Leu Asp Ser Lys Arg Asn Leu Ala Pro355 360 365Ile Gly Val Lys Gly Glu Leu Tyr Val Ser Gly Lys Gly Val Ala Arg370 375 380Gly Tyr Leu Asn Lys Pro Glu Leu Thr Glu Glu Arg Phe Met Asp Asn385 390 395 400Pro Phe Val Ala Gly Glu Arg Met Tyr Arg Thr Gly Asp Leu Ala Arg405 410 415Trp Leu Pro Glu Gly Glu Leu Glu Tyr Leu Gly Arg Ile Asp His Gln420 425 430Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu Ile Glu Ala Glu435 440 445Leu Leu Lys Gln Lys Gly Ile Lys Glu Ala Val Val Leu Val Thr Asn450 455 460Asp Lys Asp Ala Gln Pro Gln Leu His Ala Tyr Leu Thr Ser Lys Glu465 470 475 480Asp Leu Ala Ala Ala Asp Leu Arg Asn Gln Leu Thr Thr Thr Leu Pro485 490 495Ser Tyr Met Ile Pro Ala His Phe Ile Phe Val Ser Gln Met Pro Val500 505 510Thr Pro Asn Gly Lys Ile Asp Lys Glu Ser Leu Arg Lys Ile Glu Pro515 520 525Ser Leu Gln Glu Ser Pro Thr Glu Ala Tyr Val Ala Pro Gln Thr Pro530 535 540Thr Glu Lys Gln Leu Val His Ile Trp Glu Glu Asn Ile Gly Met Gln545 550 555 560Pro Ile Ser Ile Asp Asp Asn Tyr Phe Ala Leu Gly Gly Asp Ser Ile565 570 575Lys Ala Ile Lys Leu Leu His Ala Ile Asn Lys Glu Phe Gln Ile Ser580 585 590Phe Gln Ile Gly Asp Leu Tyr Lys His Gly Thr Ile Arg Glu Met Gly595 600 605Gln Gln Ile Gly Glu Lys Gly Lys Gln Ser Ser Asn Gln Lys Leu Leu610 615 620Lys Leu Gln Glu Leu Asp Arg Leu Lys Glu Lys Ile Leu Gly Ser Glu625 630 635 640Lys232530PRTBrevibacillus texasporus 23Met Ser Asp Lys Leu Ser Asn Ala Lys Asp Leu Phe Pro Met Ser Asp1 5 10 15Ile Gln Leu Gly Met Val Tyr His Ser Leu Lys His Val His Glu Ala20 25 30Val Tyr His Asp Gln Phe Val Tyr Gln Val Asp Asp Asp Ser Phe Asp35 40 45Val His Val Leu Glu Gln Ala Met Arg Met Met Val Asp Lys His Asp50 55 60Ile Leu Lys Thr Ser Phe His Ile Glu Glu Phe Ser Thr Pro Val Gln65 70 75 80Val Val His Gln Glu Val Ser Val Arg Ile Asp Glu Thr Asp Ile Thr85 90 95His Leu Gly Glu Lys Gln Lys Glu Tyr Ile His Gln Tyr Leu Ala Gln100 105 110Asp Arg Gln Ser Pro Phe Asp Val Thr Thr Ala Pro Leu Trp Arg Met115 120 125Ser Val Phe Lys Leu Asn Ala Ser Gln Val Ala Leu Val Trp Ile Phe130 135 140His His Ala Ile Leu Asp Gly Trp Ser Val Ala Ser Phe Ile Thr Glu145 150 155 160Leu Ile Asp Val Tyr Phe Lys Leu Lys His Lys Thr Cys Thr Leu Glu165 170 175His Leu Asn Thr Thr Tyr Lys Asp Tyr Val Ile Asp Gln Met Leu Leu180 185 190Ser Glu Gln Asn Glu Leu Arg Glu Tyr Trp Lys Glu Glu Leu Lys Asp195 200 205Tyr Lys Arg Leu Gln Leu Pro Val Lys Val Asp Glu Asn Gly Gly Val210 215 220His Val Thr Val Val Glu Lys Leu Asp Pro Asp Ile Ile Asn Lys Cys225 230 235 240Arg Glu Ile Ala Gln Ala His His Ile Pro Leu Lys Thr Val Cys Leu245 250 255Thr Ala Phe Leu Ser Met Met His Met Ile Ser Tyr Glu Arg Asp Leu260 265 270Thr Val Gly Leu Ile Glu Asn Asn Arg Pro Ile Ile Glu Asp Ala Glu275 280 285Lys Val Leu Gly Cys Phe Leu Asn Ser Val Pro Phe Arg Ala Ile Ile290 295 300Lys Lys Asp Met Ser Tyr Arg Glu Leu Leu Glu Gln Thr Gln Gln Lys305 310 315 320Leu Val Glu Ile Lys Thr Tyr Gly Arg Leu Ser Phe Ala Lys Ile Ile325 330 335Glu Val Ile Gly Asp Thr Gly Ser Glu Arg Asn Pro Val Phe Asp Cys340 345 350Leu Phe Asn Phe Val Asp Phe His Val Phe Lys Gly Ile Lys Asp His355 360 365Lys Val Lys Phe Trp Leu Asp Gly Tyr Glu Lys Thr Asn Thr Met Phe370 375 380Asp Phe Ser Val Ser Thr Thr Met Asp Asp Tyr Phe Val Arg Val

Val385 390 395 400Ser Ala Leu Pro Glu Glu Asp Thr Ile Lys Leu Ile Asn Tyr Tyr Gln405 410 415Arg Ile Leu Glu Lys Ile Ala Leu His Ile Asp Glu Lys Ile Asp Lys420 425 430Gln Ala Asn Leu Asp Glu Lys Glu Ser His Leu Leu Leu Glu Glu Trp435 440 445Asn Gln Thr Ser Val Asp Tyr Pro Asp Lys Gln Thr Leu His Lys Arg450 455 460Phe Glu Glu Gln Val Ala Lys Asn Glu Asp Gln Val Ala Leu Glu Tyr465 470 475 480Glu Asp Lys Gln Leu Thr Tyr Arg Glu Leu Asn Ala Lys Ala Asn Gln485 490 495Leu Ala Arg Val Leu Gln Lys His Asn Thr Leu Pro Thr Gln Val Val500 505 510Gly Leu Met Ala Glu Arg Ser Leu Glu Met Ile Ile Gly Ile Leu Gly515 520 525Ile Leu Lys Ala Gly Gly Ala Tyr Met Pro Ile Asp Pro Thr Tyr Pro530 535 540Ala Glu Arg Ile Gln Tyr Met Leu Glu Asp Ser Arg Ser Tyr Leu Leu545 550 555 560Leu Val Gln Lys Ala Glu Met Ile Pro Ala Asn Tyr Gln Gly Glu Val565 570 575Leu Ile Leu Thr Glu Glu Leu Trp Ala Asp Glu Asn Thr Glu Asn Leu580 585 590Glu Leu Val Asn Gln Pro Gln Asp Val Ala Asn Ile Met Tyr Thr Ser595 600 605Gly Thr Thr Gly Lys Pro Lys Gly Ile Leu Ile Thr His Arg Asn Ile610 615 620Met Thr Thr Ile Ile Asn Asn Gly Tyr Leu Asp Ile Phe Ser Thr Asp625 630 635 640Arg Ile Leu Gln Ile Ser Asn Tyr Ala Phe Asp Gly Ser Thr Phe Asp645 650 655Ile Tyr Ser Ala Leu Leu Asn Gly Ala Thr Leu Val Leu Val Pro Lys660 665 670Gln Thr Leu Met Asn Thr Thr Asp Leu Leu Ala Ile Ile Lys Asp Ser675 680 685Asn Ile Thr Val Ala Leu Met Thr Thr Ser Leu Phe Asn Thr Leu Val690 695 700Asp Leu Asp Val Thr Ser Phe Gln His Thr Arg Lys Val Leu Phe Gly705 710 715 720Gly Glu Lys Ala Ser Cys Lys His Val Glu Lys Ala Leu Asp Tyr Leu725 730 735Gly Glu Gly Arg Leu Val Asn Gly Tyr Gly Pro Thr Glu Thr Thr Val740 745 750Phe Ala Thr Thr Tyr Thr Val Asp Asn Thr Ile Lys Lys Leu Gly Ser755 760 765Ile Pro Ile Gly Arg Pro Leu Ser Asn Thr Ser Val Tyr Ile Phe Gly770 775 780Leu Asp Asp Gln Leu Gln Pro Leu Gly Val Pro Gly Glu Leu Cys Val785 790 795 800Ala Gly Glu Cys Ile Ser Pro Gly Tyr Leu Asn Arg Pro Asp Leu Thr805 810 815Ala Asp Lys Phe Ile Asp Asn Pro Leu Lys Pro Gly Glu Arg Met Tyr820 825 830Arg Thr Gly Asp Leu Val Arg Trp Leu Pro Glu Gly Val Met Glu Tyr835 840 845Met Gly Arg Ile Asp Glu Gln Val Lys Ile Arg Gly His Arg Ile Glu850 855 860Leu Gly Glu Ile Glu Ala Lys Leu Leu Glu His Pro Ser Ile Arg Glu865 870 875 880Thr Val Leu Val Ala Lys Gln Asp Ala Asn Gly His Ser Phe Leu Gly885 890 895Ala Tyr Leu Val Thr Asp Asn Phe Cys Pro Val Thr Glu Leu Arg Asn900 905 910Tyr Leu Met Glu Thr Leu Pro Glu Tyr Met Val Pro Ser Tyr Phe Ile915 920 925Glu Leu Asp Ser Leu Pro Leu Thr Ser Asn Gly Lys Val Asp Lys Arg930 935 940Ala Leu Pro Glu Pro Glu Ser Gln Ala Leu His Ala Tyr Thr Met Pro945 950 955 960Glu Asn Glu Thr Glu Glu Lys Leu Val Gln Leu Phe Gln Glu Val Met965 970 975Asp Val Glu Arg Val Gly Thr Gln Asp Ser Phe Tyr Glu Leu Gly Gly980 985 990His Ser Leu Lys Ala Met Leu Leu Val Ser Arg Ile His Lys Asp Phe995 1000 1005Gly Ile Lys Ile Pro Leu Lys Glu Val Phe Ser Arg Pro Thr Val1010 1015 1020Lys Glu Leu Ala Ala Tyr Leu Thr Gly Ser Glu Glu Ala Asn Tyr1025 1030 1035Ile Glu Ile Glu Ala Ala Glu Glu Lys Pro Tyr Tyr Pro Val Thr1040 1045 1050Ala Ala Gln Lys Arg Met Tyr Ile Ala Gln Gln Trp Glu Asp Gly1055 1060 1065Glu Ala Thr Ser Ser Tyr His Met Pro Phe Met Met Glu Ile Thr1070 1075 1080Gly Pro Leu Gln Val Glu Lys Leu Gln Gln Thr Val Lys Ser Leu1085 1090 1095Val Ala Arg His Glu Ser Leu Arg Thr Ser Phe His Met Ile Asn1100 1105 1110Glu Val Leu Met Gln Lys Ile His Ala Asp Val Leu Trp Asp Leu1115 1120 1125Asp Ile Asp Leu Glu Ser Val Val Ala Ser Glu Gln Glu Ile Asp1130 1135 1140Glu Lys Met Phe Gln Phe Leu Arg Lys Phe Asp Leu Ser Gln Ala1145 1150 1155Pro Leu Phe Arg Ala Lys Leu Ile Arg Val Asn Ala Ser Arg His1160 1165 1170Val Leu Leu Leu Asp Met His His Ile Ile Ser Asp Gly Phe Ser1175 1180 1185Tyr Gln Ile Phe Phe Asp Glu Leu Thr Lys Leu Tyr Gln Gly Asp1190 1195 1200Glu Leu Pro Ser Leu Lys Ile Gln Tyr Lys Asp Tyr Ala Val Trp1205 1210 1215Gln His Ser Glu Glu Gln Gln Lys Arg Leu Gln Gln Gln Glu Asp1220 1225 1230Tyr Trp Leu Gly Gln Phe Gln Gly Glu Ile Pro Val Leu Glu Leu1235 1240 1245Pro Thr Asp Tyr Gln Arg Pro Val Asp Lys Gln Phe Ala Gly Ala1250 1255 1260Leu Phe Thr His Gly Leu Ser Ala Gly Leu Thr Glu Lys Leu Arg1265 1270 1275Lys Leu Ala Ile Lys Glu Lys Thr Thr Leu Tyr Thr Val Leu Leu1280 1285 1290Thr Val Tyr Asn Ile Leu Leu Ser Lys Tyr Thr Ser Gln Glu Asp1295 1300 1305Leu Ile Val Gly Thr Pro Ile Ala Gly Arg Pro His Ala Asp Leu1310 1315 1320Asp Arg Val Phe Gly Met Phe Val Asn Thr Leu Ala Ile Arg Thr1325 1330 1335Ala Pro Lys Val Glu His Ser Phe Leu Thr Tyr Leu Ser Glu Val1340 1345 1350Lys Glu Thr Val Leu Gly Ala Tyr Gln Asn Pro Asp Tyr Pro Phe1355 1360 1365Glu Glu Leu Val Glu Lys Thr Leu Val Gln Arg Asp Val Ser Arg1370 1375 1380Asn Pro Leu Phe Asp Val Met Phe Ser Val Glu Lys Leu Pro Ser1385 1390 1395Ala Val Gln Phe Asp Asp Leu Arg Phe Cys Pro Arg Leu Phe Asp1400 1405 1410Trp Lys Lys Ala Lys Phe Asp Leu Asp Trp Thr Val Val Glu Gly1415 1420 1425Glu Ser Leu Glu Val Leu Val Glu Tyr Ser Thr Ser Leu Phe Asp1430 1435 1440Arg Ala Thr Ile Glu Arg Met Ala Lys His Phe Glu His Ile Leu1445 1450 1455Glu Gln Ile Leu Asp Gln Pro Asp Leu Ser Ile Ser Glu Ile Glu1460 1465 1470Leu Leu Thr Glu Ala Glu Lys Gln Gln Ile Leu Ile Glu Phe Asn1475 1480 1485Gln Ser Asp Lys Ser Phe Asp Ser Glu Lys Thr Ile Gln Glu Gln1490 1495 1500Phe Glu Glu Trp Ala Glu Lys Ala Pro His Ser Ile Ala Leu Val1505 1510 1515Phe Lys Asp Lys Gln Met Thr Tyr Gln Glu Leu Asn Gln Arg Ala1520 1525 1530Asn Gln Val Ala His Leu Leu Arg Gly Asn Gly Ile Ser Ala Asn1535 1540 1545Asp Phe Ile Gly Leu Met Val Asp Arg Ser Phe Glu Met Ile Ile1550 1555 1560Ser Met Leu Gly Ile Leu Lys Ala Gly Gly Ala Tyr Leu Pro Ile1565 1570 1575Asp Pro Asp Tyr Pro Glu Asp Arg Ile Asp Tyr Met Leu Ser Asp1580 1585 1590Ser Lys Ala Lys Ile Leu Leu Lys Gln Ser Asp Gln Thr Ala Pro1595 1600 1605Ala Ser Phe Glu Gly Lys Val Ile Ala Ile Asp Thr Pro Glu Leu1610 1615 1620Leu Glu Met Asp Ile Glu Asn Ile Pro Lys Val Asn Asn Ser Ser1625 1630 1635Asp Leu Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr Gly Lys Pro1640 1645 1650Lys Gly Val Leu Ile Asn His Arg Cys Val Ile Asn Met Gln Leu1655 1660 1665Thr Ala Glu Thr Phe Gly Ile Tyr Pro Ser Ser Arg Ile Leu Gln1670 1675 1680Phe Ala Ser Phe Ser Phe Asp Ser Ser Val Gly Glu Ile Phe Tyr1685 1690 1695Thr Leu Leu Asn Gly Ala Cys Leu Tyr Leu Val Glu Lys Asp Leu1700 1705 1710Leu Leu Ser Gly Asn Glu Phe Val Ala Trp Leu Lys Lys Asn Arg1715 1720 1725Ile Ser Ser Ile Pro Phe Ile Ser Pro Ser Ala Leu Arg Met Leu1730 1735 1740Pro Tyr Glu Asp Leu Pro Asp Leu Ala Tyr Ile Ser Thr Gly Gly1745 1750 1755Glu Thr Leu Pro Ala Asp Leu Val Lys Ala Trp Gly Glu Asn Arg1760 1765 1770Val Phe Leu Asn Ala Tyr Gly Pro Thr Glu Thr Thr Val Asp Ala1775 1780 1785Thr Val Gly Val Cys Thr Pro Glu Gly Lys Pro His Ile Gly Arg1790 1795 1800Pro Val Thr Asn Lys Lys Val Tyr Val Val Asn Ser Asn Asn Gln1805 1810 1815Leu Gln Pro Ile Gly Val Pro Gly Glu Leu Cys Ile Gly Gly Glu1820 1825 1830Gly Val Ala Leu Gly Tyr Leu Asn Arg Pro Asp Leu Thr Gln Glu1835 1840 1845Lys Phe Val Ser Asn Pro Phe Ala Pro Gly Glu Arg Met Tyr Arg1850 1855 1860Ser Gly Asp Leu Val Arg Trp Leu Pro Asp Gly Thr Ile Glu Tyr1865 1870 1875Phe Gly Arg Leu Asp Asp Gln Val Lys Ile Arg Gly His Arg Ile1880 1885 1890Glu Leu Gly Glu Ile Glu Thr Arg Leu Leu Glu His Pro Ser Ile1895 1900 1905Lys Glu Ala Ile Val Ile Pro Arg Ser Asp Glu Ser Glu Ala Thr1910 1915 1920Tyr Leu Cys Ser Tyr Leu Ile Ala Glu Gly Ser Trp Asn Ala Ala1925 1930 1935Asp Leu Arg Lys Tyr Leu Lys Ala Ser Leu Pro Glu Tyr Met Ile1940 1945 1950Pro Ser Tyr Phe Val Glu Leu His Glu Leu Pro Leu Thr Pro Asn1955 1960 1965Gly Lys Val Asn Lys Lys Ala Leu Pro Lys Pro Glu Lys Gln Met1970 1975 1980Gln Arg Gly Lys Asp Tyr Val Ala Pro Thr Asn Pro Ile Gln Ser1985 1990 1995Ile Leu Ser Gln Ile Trp Thr Asp Val Leu Gly Val Glu Asn Ile2000 2005 2010Gly Ile His Asp Asn Phe Phe Glu Leu Gly Gly Asp Ser Ile Lys2015 2020 2025Ala Ile Gln Ile Ser Ala Arg Leu Asn Lys His Asn Leu Lys Val2030 2035 2040Lys Met Arg Glu Leu Phe Lys Asn Pro Thr Ile Ala Glu Leu Ser2045 2050 2055Leu Leu Val Gln Gln Ile Val Gln Glu Ile Asp Gln Gly Val Val2060 2065 2070Glu Gly Asn Ile Pro Leu Thr Pro Ile Gln His Trp Phe Phe Thr2075 2080 2085Gln Ser Phe Pro Gln Val Asn His Tyr Asn Gln Ser Val Leu Leu2090 2095 2100Phe Asn Ala Glu Gly Trp Asp Glu Gln Lys Val Asp Lys Ala Phe2105 2110 2115Glu Met Leu Thr Gln His His Asp Ala Leu Arg Ile Val Tyr Ser2120 2125 2130Leu Asp Glu Gln Gly Val Val Gln Arg Asn Arg Gly Leu Glu Gly2135 2140 2145Ser Asn Tyr His Phe Glu Ile Ile Asp Ala Arg Gln Asp Gly Glu2150 2155 2160Asp Gln Ser Asn Trp Lys Ala Ala Ala Asn Arg Met Gln Ala Ser2165 2170 2175Met Asp Ile Val Glu Gly Pro Leu Val Gln Ile Gly Leu Phe Arg2180 2185 2190Ala Asn Glu Gly Ala Tyr Leu Leu Ile Ala Ile His His Leu Val2195 2200 2205Val Asp Gly Val Ser Trp Arg Ile Leu Leu Glu Asp Phe Tyr His2210 2215 2220Leu Tyr Asn Gly Asn Asp Ser Leu Pro Leu Lys Thr Thr Ser Phe2225 2230 2235Gln Ala Trp Ser Gln Lys Leu Gln Glu Tyr Ala Gln Ser Lys Glu2240 2245 2250Leu Glu His Glu Leu Ser Tyr Trp Arg His Leu Asp Glu Ala Ile2255 2260 2265Thr Asp Tyr Thr Leu His Lys Asp Ile Glu Ala Ala Thr Ser Asn2270 2275 2280Lys Thr Thr Tyr Glu Glu Phe Leu Thr Val Ser Met Ser Leu Ser2285 2290 2295Thr Glu Glu Thr Gln Gln Leu Val Thr Glu Ala His Lys Ala Tyr2300 2305 2310Gln Thr Glu Ile Asn Asp Leu Leu Leu Thr Ala Leu Ala Leu Ala2315 2320 2325Leu Lys Glu Trp Thr Asn Lys Glu Gln Leu Leu Val Ser Met Glu2330 2335 2340Gly His Gly Arg Glu Glu Ile Leu Asp Asn Val Asp Ile Ser Arg2345 2350 2355Thr Val Gly Trp Phe Thr Ser Glu Tyr Pro Val Ala Ile His Leu2360 2365 2370Thr Lys Thr Asp Ile Ser Phe Ala Ile Lys Gln Val Lys Glu Thr2375 2380 2385Leu Arg Arg Val Pro Asn Lys Gly Phe Gly Tyr Gly Ile Leu Lys2390 2395 2400Tyr Leu Ala Lys Glu Thr Phe Lys Leu Lys Pro Glu Ile Ser Phe2405 2410 2415Asn Tyr Leu Gly Gln Phe Thr Asp Lys Glu Glu Gly Asn Ser Ser2420 2425 2430Leu Met Gly Asp Leu Ile Ser Pro Ala Asn Thr Ser Glu Leu Ser2435 2440 2445Leu Asp Ile Asn Gly Ser Ile Glu Ala Asp Arg Leu Gln Met His2450 2455 2460Phe Ser Tyr Asn Ser Arg Ala Tyr Tyr Pro Glu Thr Ile Ala Thr2465 2470 2475Leu Val Gln Asn Phe Lys Ser Tyr Leu Leu Glu Ile Ile Asn His2480 2485 2490Cys Arg Ala Lys Glu Gly Val Glu His Thr Pro Ser Asp Phe Asp2495 2500 2505Ile Asn Asp Leu Thr Met Glu Glu Leu Asp Asp Ile Phe Asp Asp2510 2515 2520Leu Glu Glu Glu Val Tyr Lys2525 2530244617PRTBrevibacillus texasporus 24Met Phe Ser Arg Ser Asn Val Gln Asn Leu Tyr Arg Leu Ser Pro Met1 5 10 15Gln Lys Gly Ile Leu Phe His Ser Leu Lys Asp Lys Glu Asn His Ala20 25 30Tyr Phe Asp Gln Leu Ile Phe Thr Leu Glu Gly Lys Val Glu Leu Glu35 40 45Tyr Leu Glu Glu Ala Phe Thr Gln Leu Ile Lys Lys His Asp Ile Leu50 55 60Arg Thr Val Phe Arg Tyr Lys Lys Val Lys Glu Pro Val Gln Met Val65 70 75 80Leu Lys Glu Arg Ser Ser Thr Ile Tyr Phe Glu Asp Ile Ser His Leu85 90 95Glu Pro Glu Glu Lys Val Asn Tyr Ile Lys Gln Phe Lys Met Arg Asp100 105 110Arg Glu Lys Gly Phe Asp Leu Ser Arg Asp Leu Leu Ile Arg Met Ser115 120 125Leu Phe Lys Leu Asp Gln Glu Gln Tyr Gln Leu Ile Met Ser Asn His130 135 140His Ile Ile Met Asp Gly Trp Cys Leu Gly Ile Ile Leu Thr Asp Phe145 150 155 160Leu Arg Met Tyr Lys Gly Ile Val Asn His Thr Pro Val Pro Tyr Glu165 170 175His Val Thr Pro Tyr Ser Lys His Ile Gln Trp Leu Glu Lys Gln Asp180 185 190His Gln Glu Ala Lys Asp Phe Tyr Gln Gln Leu Leu Glu Gly Tyr Asp195 200 205Lys Val Thr Gly Val Pro Gln Gln Leu Val Arg Ala Asn His Glu Glu210 215 220Tyr Thr His Gly Gln Cys Ile Val Lys Leu Asn Gln Glu Thr Ala Asp225 230 235 240Arg Leu Ile Ala Ile Ala Lys Ala Tyr Gln Val Thr Val Asn Thr Val245 250 255Phe Gln Thr Ile Trp Gly Ile Leu Leu Gln Lys Tyr Asn Asn Thr Asp260 265 270Asp Ile Val Phe Gly Ser Val Val Ser Gly Arg Pro Ala Glu Ile Pro275 280 285Asp Val Glu Lys Met Val Gly Leu Phe Ile Asn Thr Ile Pro Val Arg290 295 300Ile Lys Ala Asp Gln Gln Glu Arg Phe Asp Thr Leu Val Ala Lys Val305 310 315 320Gln Glu Met Ala Leu Ala Ser Glu Ser Tyr Asp Tyr Leu Ser Leu Ala325 330 335Asp Ile His Pro Glu Ala Gly Asp Phe Ile Asn His Ile Ile Ala Phe340 345 350Glu Asn Phe Tyr Ile Asp Met Asp Ser Phe Asn Gln Leu Ala Asp Lys355 360 365Lys Glu Leu Gly Phe Ser Leu Ala Phe Ala Thr Asp His His Glu Gln370 375 380Thr Asn Tyr Asp Leu Ser Val Gln Ala Gln Ile Gly Asp Glu Ser Ser385 390 395 400Ile Lys Ile Leu Tyr Asn Ser Lys Leu Tyr Thr Ser Glu Tyr Ile Ala405 410 415Asn Val Ile Asp His Phe Val Thr Val Ala Asp Ile Val Ala Ala Asn420

425 430Pro Ser Ile Pro Val Lys Glu Ile Asp Ile Leu Thr Lys Asp Lys Lys435 440 445Asp Gln Ile Leu Tyr Gly Phe Asn Asn Thr Tyr Ala Asp Tyr Pro Arg450 455 460Glu Lys Thr Ile His Gln Leu Phe Glu Glu Gln Val Asp Lys Asn Pro465 470 475 480Asn Gln Ile Ala Leu Val Phe Lys Glu Glu Lys Leu Thr Tyr Gly Glu485 490 495Val Asn Ala Lys Ala Asn Gln Leu Ala Tyr Val Leu Arg Lys Gln Gly500 505 510Val Gln Pro Asn Asp Val Ile Gly Ile Ile Thr Glu Arg Ser Pro Glu515 520 525Met Ile Ile Gly Ile Leu Ala Ile Phe Lys Ala Gly Gly Ala Tyr Met530 535 540Pro Ile Asp Pro Ser Tyr Pro Ala Glu Arg Ile Gln Tyr Met Leu Gln545 550 555 560Asp Asn Gln Thr Lys Leu Leu Leu Val Gln Lys Gln Glu Met Ile Pro565 570 575Ala Asn Tyr Gln Gly Glu Val Leu Phe Leu Thr Gln Glu Ser Trp Met580 585 590His Glu Glu Thr Ser Asn Pro Ala His Ile Thr Gln Ala Gln Ala Leu595 600 605Ala Tyr Val Met Tyr Thr Ser Gly Ser Thr Gly Glu Pro Lys Gly Ile610 615 620Leu Thr Thr His Gln Asn Ile Met Lys Thr Val Ile His Asn Gly Tyr625 630 635 640Val Glu Ile Thr Pro Gly Asp Cys Leu Ser Gln Leu Ser Asn Tyr Ala645 650 655Phe Asp Gly Ser Thr Phe Glu Ile Tyr Gly Ala Leu Leu His Gly Ala660 665 670Thr Leu Leu Leu Val Thr Lys Glu Ala Val Leu Asn Met Asn Glu Leu675 680 685Ala Arg Leu Ile Lys Lys Glu Gln Val Thr Val Ser Phe Met Thr Thr690 695 700Ala Leu Phe Asn Thr Leu Val Asp Leu Asp Ile Thr Cys Phe Gln Ser705 710 715 720Ile Arg Lys Val Leu Phe Gly Gly Glu Leu Ala Ser Val Lys His Val725 730 735Leu Lys Ala Leu Asp Tyr Leu Gly Glu His Arg Val Ile Asn Val Tyr740 745 750Gly Pro Thr Glu Thr Thr Val Tyr Ala Thr Tyr Tyr Ser Val Asp His755 760 765Ser Met Leu Thr Arg Ala Ser Val Pro Ile Gly Arg Pro Ile Asn Asn770 775 780Thr Lys Ala Tyr Ile Val Asn Thr Asp Gly Gln Pro Gln Pro Ile Gly785 790 795 800Val Val Gly Glu Leu Cys Ile Gly Gly Glu Gly Val Ala Cys Gly Tyr805 810 815Leu Asn Arg Pro Glu Leu Thr Lys Lys His Phe Val Asp Asn Pro Phe820 825 830Val Leu Gly Glu Arg Met Tyr Cys Thr Gly Asp Leu Ala Arg Phe Leu835 840 845Pro Asp Gly Asn Ile Glu Tyr Ile Gly Arg Met Asp Glu Gln Val Lys850 855 860Ile Arg Gly His Arg Ile Glu Leu Gly Glu Ile Glu Lys Val Leu Leu865 870 875 880Gln His Pro Ala Ile Ser Glu Thr Val Leu Leu Ala Lys Arg Asp Glu885 890 895Gln Gly His Ser Tyr Leu Cys Ala Tyr Ile Val Gly Gln Val Phe Trp900 905 910Thr Val Thr Glu Leu Arg Gln His Leu Met Glu Ser Leu Pro Glu Tyr915 920 925Met Val Pro Ser Tyr Phe Ile Glu Ile Glu Lys Leu Pro Leu Thr Ala930 935 940Asn Gly Lys Val Asp Lys Arg Ala Leu Pro Glu Pro Asp Arg Lys Met945 950 955 960Gly Ser Ala Tyr Val Ala Pro Glu Asn Glu Thr Glu Glu Lys Leu Val965 970 975Gln Phe Phe Gln Glu Ile Leu Gly Val Glu Arg Val Gly Thr Gln Asp980 985 990Thr Phe Phe Glu Leu Gly Gly His Ser Leu Lys Ala Met Met Leu Val995 1000 1005Leu Gln Ile His Lys Glu Met Gly Ile Glu Val Pro Leu Lys Glu1010 1015 1020Ile Phe Thr Arg Pro Thr Ile Lys Glu Leu Ala Ala Tyr Ile His1025 1030 1035Lys Met Asp Arg Ser Ala Tyr Ser Met Ile Glu Pro Thr Ala Lys1040 1045 1050Gln Glu Tyr Tyr Pro Val Ser Phe Ala Gln Arg Arg Met Phe Val1055 1060 1065Val Gln Gln Ile Arg Asp Thr Asn Thr Thr Ser Tyr Asn Met Pro1070 1075 1080Ile Leu Leu Glu Ile Glu Gly Ala Leu Asp Arg Glu Asn Val Arg1085 1090 1095Gln Thr Leu Lys Lys Leu Ile Glu Arg His Glu Ser Met Arg Thr1100 1105 1110Ser Phe His Met Ile Asp Glu Thr Leu Leu Gln Lys Val His Asp1115 1120 1125Asp Val Thr Trp Glu Met Glu Glu Met Glu Ala Ser Glu Glu Glu1130 1135 1140Val Tyr Ala Leu Thr Lys Ser Phe Ile Arg Pro Phe Asp Leu Gly1145 1150 1155Gln Ala Pro Leu Phe Arg Ala Gly Leu Ile Arg Val Asn Ser Glu1160 1165 1170Arg His Leu Leu Leu Leu Asp Thr His His Ile Ile Ser Asp Gly1175 1180 1185Val Ser Thr Asn Ile Leu Phe Gln Asp Phe Thr Gln Leu Tyr Arg1190 1195 1200Gly Arg Glu Leu Pro Ala Leu Arg Ile Gln Tyr Lys Asp Phe Ala1205 1210 1215Val Trp Gln Gln Gly Glu Ala Gln Leu Ala Arg Leu Gln Glu Gln1220 1225 1230Glu Glu Tyr Trp Leu Lys Gln Phe Ser Glu Ser Val Pro Val Leu1235 1240 1245Glu Leu Pro Thr Asp Phe Pro Arg Pro Ala Met Gln Gln Phe Asp1250 1255 1260Gly Asp Val Leu Asp Phe Ala Leu Asn Gln Gln Val Trp Gln Glu1265 1270 1275Leu Gln Gln Leu Ile Val Lys Glu Gly Cys Thr Ala Tyr Met Ile1280 1285 1290Leu Leu Ala Ala Tyr His Val Leu Leu Ser Lys Tyr Ser Ser Gln1295 1300 1305Asn Asp Ile Val Ile Gly Ser Pro Ile Ala Gly Arg Thr Asn Ala1310 1315 1320Asp Leu Gln Ser Ile Val Gly Met Phe Val Asn Thr Leu Ala Ile1325 1330 1335Arg Thr Lys Ser Glu Gly Thr Gln Thr Phe Arg Glu Phe Leu Ser1340 1345 1350Thr Ile Lys Gln Leu Val Leu Gln Ala Gln Ser Asn Ala Glu Tyr1355 1360 1365Pro Phe Glu Glu Leu Val Asp Lys Val Asn Pro Ser Arg Asp Leu1370 1375 1380Ser Arg Gln Pro Leu Phe Asp Thr Ile Phe Val Met Gln Asn Met1385 1390 1395Asp Ile Thr Glu Val Ala Ile Gln Gly Leu Ser Ile Val Thr Lys1400 1405 1410Asp Met Glu Trp Lys His Ser Lys Phe Asp Leu Thr Trp Ala Ala1415 1420 1425Val Glu Lys Glu Ser Leu His Phe Ser Val Glu Tyr Ser Thr Arg1430 1435 1440Leu Phe Lys Lys Glu Thr Ile Glu Arg Met Ala Lys His Phe Ala1445 1450 1455His Leu Leu Asn Gln Val Ala Glu Asn Pro Asp Leu Ser Leu Ser1460 1465 1470Asp Met Glu Leu Ala Thr Asp Glu Glu Val Tyr Gln Leu Leu Glu1475 1480 1485Glu Phe Asn Asn Thr Glu Ala Asp Tyr Pro Ser Asp Lys Thr Ile1490 1495 1500His Gln Gln Phe Glu Gln Lys Val Glu Glu Asn Pro Asp Gln Ile1505 1510 1515Ala Leu Leu Phe Lys Asp Lys Glu Ile Thr Tyr Gly Gln Leu Asn1520 1525 1530Ala Lys Ala Asn Gln Phe Ala Arg Val Leu Arg Lys His Gly Val1535 1540 1545Gln Pro Asp Gln Val Val Gly Leu Ile Thr Asp Arg Ser Ile Glu1550 1555 1560Met Met Ile Gly Ile Leu Ala Ile Leu Lys Ala Gly Gly Ala Tyr1565 1570 1575Leu Pro Ile Asp Pro Ser Tyr Pro Leu Glu Arg Ile Thr Tyr Met1580 1585 1590Leu Glu Asp Ser Gln Ala Gln Leu Leu Ile Val Gln Glu Ala Ala1595 1600 1605Met Ile Pro Glu Gly Tyr Gln Gly Lys Val Leu Leu Leu Ala Glu1610 1615 1620Glu Cys Trp Met Gln Glu Glu Ala Ser Asn Leu Glu Leu Ile Asn1625 1630 1635Asp Ala Gln Asp Leu Ala Tyr Val Met Tyr Thr Ser Gly Ser Thr1640 1645 1650Gly Lys Pro Lys Gly Asn Leu Thr Thr His Gln Asn Ile Leu Arg1655 1660 1665Thr Ile Ile Asn Asn Gly Phe Ile Glu Ile Val Pro Ala Asp Arg1670 1675 1680Leu Leu Gln Leu Ser Asn Tyr Ala Phe Asp Gly Ser Thr Phe Asp1685 1690 1695Ile Tyr Ser Ala Leu Leu Asn Gly Ala Thr Leu Val Leu Val Pro1700 1705 1710Lys Glu Val Met Leu Asn Pro Met Glu Leu Ala Arg Ile Val Arg1715 1720 1725Glu Gln Asp Ile Thr Val Ser Phe Met Thr Thr Ser Leu Phe His1730 1735 1740Thr Leu Val Glu Leu Asp Val Thr Ser Met Lys Ser Ile Arg Lys1745 1750 1755Val Val Phe Gly Gly Glu Lys Ala Ser Tyr Lys His Val Glu Lys1760 1765 1770Ala Leu Asp Tyr Leu Gly Glu Gly Arg Leu Val Asn Gly Tyr Gly1775 1780 1785Pro Thr Glu Thr Thr Val Phe Ala Thr Thr Tyr Thr Val Asp Ser1790 1795 1800Ser Ile Lys Glu Thr Gly Ile Val Pro Ile Gly Arg Pro Leu Asn1805 1810 1815Asn Thr Ser Val Tyr Ile Leu Asn Glu Asn Asn Gln Pro Gln Pro1820 1825 1830Ile Gly Val Pro Gly Glu Leu Cys Val Gly Gly Ala Gly Ile Ala1835 1840 1845Arg Gly Tyr Leu Asn Arg Pro Glu Leu Thr Ala Glu Arg Phe Val1850 1855 1860Asp Asn Pro Phe Leu Val Gly Asp Arg Met Tyr Arg Thr Gly Asp1865 1870 1875Met Ala Arg Phe Leu Pro Asp Gly Asn Ile Glu Tyr Ile Gly Arg1880 1885 1890Met Asp Glu Gln Val Lys Ile Arg Gly His Arg Ile Glu Leu Gly1895 1900 1905Glu Ile Glu Lys Ser Leu Leu Glu Tyr Pro Ala Ile Ser Glu Ala1910 1915 1920Val Leu Val Ala Lys Arg Asp Glu Gln Gly His Ser Tyr Leu Cys1925 1930 1935Ala Tyr Val Val Ser Thr Asp Gln Trp Thr Val Ala Lys Val Arg1940 1945 1950Gln His Ile Leu Glu Ala Leu Pro Glu Tyr Met Val Pro Ser Tyr1955 1960 1965Phe Val Glu Leu Glu Lys Leu Pro Leu Thr Ser Asn Gly Lys Val1970 1975 1980Asp Lys Arg Ala Leu Pro Glu Pro Asp Arg Val Ile Thr Asn Glu1985 1990 1995Tyr Val Ala Ala Val Asn Glu Thr Glu Glu Lys Leu Val Gln Phe2000 2005 2010Phe Gln Glu Ile Leu Ala Val Asp Arg Val Gly Thr Gln Asp Thr2015 2020 2025Phe Phe Glu Leu Gly Gly His Ser Leu Lys Ala Met Met Leu Val2030 2035 2040Ser Arg Ile His Lys Glu Leu Glu Ile Glu Val Pro Leu Lys Glu2045 2050 2055Val Phe Ala Arg Gln Thr Val Lys Glu Leu Ala Ala Tyr Ile Arg2060 2065 2070Gln Ala Glu Gln Ser Asp Tyr Ser Glu Ile Gln Pro Ala Met Glu2075 2080 2085Gln Glu Tyr Tyr Pro Val Ser Asn Ala Gln Arg Arg Met Tyr Val2090 2095 2100Val Gln Gln Met Arg Asp Val Glu Thr Thr Gly Tyr Asn Met Pro2105 2110 2115Phe Tyr Leu Glu Met Glu Gly Ala Leu Glu Val Glu Lys Leu Ser2120 2125 2130Leu Ala Leu Lys Gln Leu Ile Glu Arg His Glu Ser Leu Arg Thr2135 2140 2145Ser Phe His Met Val Glu Asp Glu Leu Met Gln Lys Val His Ala2150 2155 2160Glu Val Ala Trp Glu Met Glu Met Ile His Ala Val Glu Glu Glu2165 2170 2175Val Gln Gln Leu Thr Asp Ser Phe Met Arg Pro Phe Asp Leu Ala2180 2185 2190Lys Ala Pro Leu Phe Arg Ala Arg Leu Ile Gln Ile Asn Pro Lys2195 2200 2205Arg His Leu Leu Met Leu Asp Met His His Ile Ile Ser Asp Gly2210 2215 2220Val Ser Met Asn Val Leu Phe Gln Asp Ile Thr Gln Leu Tyr Gln2225 2230 2235Gly Ile Glu Leu Ser Pro Leu Lys Ile Gln Tyr Lys Asp Phe Ala2240 2245 2250Val Trp Gln Gln Gly Ile Ala Gln Val Val Arg Phe Gln Glu Gln2255 2260 2265Glu Arg Tyr Trp Leu Asn Gln Phe Ser Gly Asp Leu Pro Ile Leu2270 2275 2280Glu Met Val Thr Asp Tyr Pro Arg Pro Ala Ile Gln Gln Phe Asp2285 2290 2295Gly Asp Ser Trp Ser Phe Glu Ile Asp Ala Lys Val Leu Asp Ser2300 2305 2310Ile Lys Gln Leu Ser Ala Lys Gln Gly Thr Thr Leu Tyr Met Thr2315 2320 2325Leu Leu Ala Ile Tyr Gln Ile Leu Leu Ala Lys Tyr Thr Arg Gln2330 2335 2340Asp Asp Ile Ile Val Gly Thr Pro Ile Ala Gly Arg Pro His Ala2345 2350 2355Asp Thr Glu Ser Ile Val Gly Met Phe Val Asn Thr Leu Ala Leu2360 2365 2370Arg Gly Gln Pro Lys Glu Glu Gln Ser Phe Ile Ser Tyr Leu Ser2375 2380 2385Glu Val Lys Glu Asn Val Leu Gln Ala Tyr Ala Asn Ala Asp Tyr2390 2395 2400Pro Phe Glu Glu Leu Val Glu Lys Leu His Leu Gln Arg Asp Met2405 2410 2415Ser Arg His Pro Leu Phe Asp Thr Met Phe Val Leu Gln Asn Met2420 2425 2430Asp Met Ser Asp Ile Asn Ile Ser Gly Leu Lys Leu His Ser Arg2435 2440 2445Asp Leu Asn Trp Lys Asn Ala Lys Phe Asp Met Thr Trp Met Ile2450 2455 2460Ala Glu Gln Asn Asn Leu Leu Ile Ser Val Glu Tyr Ser Thr Asn2465 2470 2475Leu Phe Lys His Glu Thr Ile Gln Arg Leu Glu Lys His Phe Thr2480 2485 2490Tyr Leu Val Glu Gln Val Ala Lys His Pro Asp Cys Leu Leu Arg2495 2500 2505Asp Leu Glu Leu Thr Thr Asp Glu Glu Lys Gln Gln Ile Leu Thr2510 2515 2520Val Phe Asn Asp Thr Ala Thr Asp Asp Leu Gln Asp Leu Ser Ile2525 2530 2535Cys His Leu Phe Glu Gln Gln Val Gln Arg Phe Ser Asp Arg Pro2540 2545 2550Ala Leu Val Phe Lys Glu Lys Gln Leu Thr Tyr Ser Glu Phe His2555 2560 2565Ala Lys Val Asn Gln Leu Ala Arg Val Leu Arg Lys Lys Gly Val2570 2575 2580Gln Pro Asp Gln Ala Val Gly Leu Ile Thr Asp Arg Ser Ile Glu2585 2590 2595Met Met Ile Gly Ile Phe Ala Ile Leu Lys Ala Gly Gly Ala Tyr2600 2605 2610Met Pro Ile Asp Pro Ser Tyr Pro Ile Asp Arg Ile Glu His Met2615 2620 2625Leu Glu Asp Ser Arg Thr Lys Leu Leu Phe Val Gln Lys Thr Glu2630 2635 2640Met Ile Pro Ala Ser Tyr Gln Gly Glu Val Leu Leu Leu Ala Glu2645 2650 2655Glu Cys Trp Met His Glu Asp Ser Ser Asn Leu Glu Leu Ile Asn2660 2665 2670Lys Thr Gln Asp Leu Ala Tyr Val Met Tyr Thr Ser Gly Ser Thr2675 2680 2685Gly Lys Pro Lys Gly Asn Leu Thr Thr His Gln Asn Ile Leu Thr2690 2695 2700Thr Ile Ile Asn Asn Gly Tyr Ile Glu Ile Ala Pro Thr Asp Arg2705 2710 2715Leu Leu Gln Leu Ser Asn Tyr Ala Phe Asp Gly Ser Thr Phe Asp2720 2725 2730Ile Tyr Ser Ala Leu Leu Asn Gly Ala Thr Leu Val Leu Val Pro2735 2740 2745Lys Glu Val Met Leu Asn Pro Met Glu Leu Ala Lys Ile Val Arg2750 2755 2760Glu Gln Asp Ile Thr Val Ser Phe Met Thr Thr Ser Leu Phe His2765 2770 2775Thr Leu Val Glu Leu Asp Val Thr Ser Met Lys Ser Met Arg Lys2780 2785 2790Val Val Phe Gly Gly Glu Lys Ala Ser Tyr Lys His Val Glu Lys2795 2800 2805Ala Leu Asp Tyr Leu Gly Glu Gly Arg Leu Val Asn Gly Tyr Gly2810 2815 2820Pro Thr Glu Thr Thr Val Phe Ala Thr Thr Tyr Thr Val Asp Ser2825 2830 2835Ser Ile Lys Glu Thr Gly Ile Val Pro Ile Gly Arg Pro Leu Asn2840 2845 2850Asn Thr Ser Val Tyr Val Leu Asn Glu Asn Asn Gln Leu Gln Pro2855 2860 2865Ile Gly Val Pro Gly Glu Leu Cys Val Gly Gly Ala Gly Ile Ala2870 2875 2880Arg Gly Tyr Leu Asn Arg Pro Glu Leu Thr Ala Glu Arg Phe Val2885 2890 2895Glu Asn Pro Phe Val Ser Gly Asp Arg Met Tyr Arg Thr Gly Asp2900 2905 2910Leu Ala Arg Trp Leu Pro Asp Gly Ser Met Glu Tyr Leu Gly Arg2915 2920 2925Met Asp Glu Gln Val Lys Val Arg Gly Tyr Arg Ile Glu Leu Gly2930 2935 2940Glu Ile Glu Thr Arg Leu Leu Glu His Pro Ser Ile Ser Ala Ala2945 2950 2955Val Leu Leu Ala Lys Gln Asp Glu Gln Gly His Ser Tyr Leu Cys2960

2965 2970Ala Tyr Ile Val Ala Asn Gly Val Trp Thr Val Ala Glu Leu Arg2975 2980 2985Lys His Leu Ser Glu Ala Leu Pro Glu Tyr Met Val Pro Thr Tyr2990 2995 3000Phe Val Glu Leu Glu Gln Ile Pro Phe Thr Ser Asn Gly Lys Val3005 3010 3015Asn Lys Arg Ala Leu Pro Glu Pro Glu Gly Gln Met Thr Ser Val3020 3025 3030Tyr Val Ala Pro Glu Thr Glu Thr Glu Ala Lys Val Ala Ala Leu3035 3040 3045Phe Gln Glu Ile Leu Gly Val Glu Arg Val Gly Thr Gln Asp Met3050 3055 3060Phe Phe Glu Leu Gly Gly His Ser Leu Lys Ala Met Met Leu Val3065 3070 3075Leu Arg Met Asn Lys Glu Leu Gly Ile Glu Val Pro Leu Lys Glu3080 3085 3090Val Phe Ala His Pro Thr Val Lys Glu Leu Ala Ala Thr Ile Asp3095 3100 3105Leu Leu Asp Arg Ser Gly His Ser Glu Ile Glu Pro Ala Pro Arg3110 3115 3120Gln Glu Phe Tyr Pro Val Ser Ser Ala Gln Arg Arg Met Tyr Val3125 3130 3135Val Gln His Leu Gly Asn Val Gln Thr Thr Ser Tyr Asn Met Pro3140 3145 3150Leu Phe Leu Glu Val Glu Gly Ala Leu Glu Ile Asp Lys Leu His3155 3160 3165Leu Ala Leu Glu Lys Leu Val Glu Arg His Glu Ser Leu Arg Thr3170 3175 3180Ser Phe His Met Val Asp Glu Glu Leu Met Gln Gln Val His Glu3185 3190 3195Glu Val Ala Trp Asp Leu Glu Ile Met Asp Gly Thr Glu Gly Asp3200 3205 3210Leu Ala Ser Ile Thr Ala Gly Phe Ile Arg Pro Phe Asp Leu Ser3215 3220 3225Gln Ala Pro Leu Phe Arg Ala Gly Ile Val Arg Ile Ser Pro Glu3230 3235 3240Arg Phe Leu Phe Met Leu Asp Met His His Ile Ile Ser Asp Gly3245 3250 3255Val Ser Thr Asn Val Leu Phe Lys Asp Ile Thr Gln Leu Tyr Gln3260 3265 3270Gly Lys Asp Leu Pro Pro Leu Pro Ile Gln Tyr Lys Asp Tyr Ala3275 3280 3285Val Trp Gln Gln Ala Asp Ala Gln Val Thr Arg Leu Gln Asp Gln3290 3295 3300Glu Ser Tyr Trp Leu His Gln Phe Ala Gly Glu Ala Ser Val Leu3305 3310 3315Glu Met Pro Thr Asp Phe Pro Arg Pro Ala Val Gln Gln Phe Glu3320 3325 3330Gly Asp Val Trp Thr Phe Glu Ile Asp Ala Asp Ile Leu Ser Gln3335 3340 3345Leu Lys Lys Leu Ser Val Ser Gln Gly Ser Thr Leu Tyr Met Thr3350 3355 3360Leu Leu Ala Val Tyr Gln Val Leu Leu Ala Lys Tyr Thr Gly Gln3365 3370 3375Asp Asp Ile Ile Val Gly Ser Pro Ile Ala Gly Arg Pro His Ala3380 3385 3390Asp Val Glu Ser Ile Val Gly Met Phe Val Asn Thr Leu Ala Leu3395 3400 3405Arg Gly Gln Pro Val Gly Glu Gln Thr Phe Ile Thr Tyr Leu Ala3410 3415 3420Gln Val Lys Glu Gln Val Leu Gln Ala Tyr Ala Asn Ala Glu Tyr3425 3430 3435Pro Phe Glu Lys Leu Val Glu Lys Leu Asp Leu Gln Arg Asp Met3440 3445 3450Ser Arg His Pro Leu Phe Asp Thr Met Phe Thr Leu Gln Asn Met3455 3460 3465Glu Met Thr Asp Ile Asp Leu Ala Gly Leu Thr Phe Lys Pro Phe3470 3475 3480Asp Phe Glu Trp Lys Asn Ala Lys Phe Asp Met Asp Trp Thr Met3485 3490 3495Leu Glu Glu Glu Thr Leu Lys Val Ala Ile Glu Tyr Ser Thr Ser3500 3505 3510Leu Tyr Thr Lys Glu Thr Ile Ser Arg Met Ala Gln His Phe Thr3515 3520 3525Tyr Val Leu Gln Gln Ile Ile Glu His Pro Ala Ile Arg Leu Ala3530 3535 3540Glu Ile Lys Ile Ala Thr Leu Pro Glu Ile Glu Gln Ile Leu Thr3545 3550 3555Gln Phe Asn Asp Thr Arg Ala Asn Tyr Pro Asp Asn Gln Thr Ile3560 3565 3570His Ser Leu Phe Glu Gln Gln Val Glu Arg Thr Pro Glu Gln Ile3575 3580 3585Ala Val Val Tyr Gln Asp Gln Ser Ile Thr Tyr Arg Glu Leu Asn3590 3595 3600Glu Arg Ala Asn Arg Leu Ala Arg Cys Leu Ile Asp Lys Gly Ile3605 3610 3615Gln Arg Asn Gln Phe Val Ala Ile Met Ala Asp Arg Ser Ile Glu3620 3625 3630Thr Val Ile Gly Met Met Gly Ile Leu Lys Ala Gly Gly Ala Tyr3635 3640 3645Val Pro Ile Asp Pro Asp Tyr Pro Leu Asp Arg Lys Leu Tyr Ile3650 3655 3660Leu Glu Asp Ser His Ala Ser Leu Leu Leu Phe Gln Gln Lys His3665 3670 3675Glu Val Pro Ser Glu Phe Thr Gly Asp Arg Ile Leu Ile Glu Gln3680 3685 3690Met Gln Trp Tyr Gln Ala Ala Asp Thr Asn Val Gly Ile Val Asn3695 3700 3705Thr Ala Gln Asp Leu Ala Tyr Met Ile Tyr Thr Ser Gly Ser Thr3710 3715 3720Gly Gln Pro Lys Gly Val Met Ile Asp His Gln Ala Val Cys Asn3725 3730 3735Leu Cys Leu Met Ala Gln Thr Tyr Gly Ile Phe Ala Asn Ser Arg3740 3745 3750Val Leu Gln Phe Ala Ser Phe Ser Phe Asp Ala Ser Val Gly Glu3755 3760 3765Val Phe His Thr Leu Thr Asn Gly Ala Thr Leu Tyr Leu Met Asp3770 3775 3780Arg Asn Leu Leu Met Ala Gly Val Glu Phe Val Glu Trp Leu Arg3785 3790 3795Val Asn Glu Ile Thr Ser Ile Pro Phe Ile Ser Pro Ser Ala Leu3800 3805 3810Arg Ala Leu Pro Tyr Glu Asp Leu Pro Ala Leu Lys Tyr Ile Ser3815 3820 3825Thr Gly Gly Glu Ala Leu Pro Val Asp Leu Val Arg Leu Trp Gly3830 3835 3840Thr Glu Arg Ile Phe Leu Asn Ala Tyr Gly Pro Thr Glu Thr Thr3845 3850 3855Val Asp Ala Thr Ile Gly Leu Cys Thr Pro Glu Asp Lys Pro His3860 3865 3870Ile Gly Lys Pro Val Leu Asn Lys Lys Ala Tyr Ile Ile Asn Pro3875 3880 3885Asn Tyr Gln Leu Gln Pro Ile Gly Val Pro Gly Glu Leu Cys Ile3890 3895 3900Gly Gly Val Gly Ile Ala Pro Gly Tyr Trp Asn Arg Pro Glu Leu3905 3910 3915Thr Arg Glu Lys Phe Val Asp Asn Pro Phe Ala Gln Gly Glu Arg3920 3925 3930Met Tyr Lys Thr Gly Asp Leu Val Arg Trp Leu Pro Asp Gly Asn3935 3940 3945Ile Glu Phe Leu Gly Arg Ile Asp Asp Gln Val Lys Ile Arg Gly3950 3955 3960His Arg Ile Glu Leu Gly Glu Ile Glu Thr Arg Leu Leu Glu His3965 3970 3975Glu Gln Val Ile Glu Ala Val Val Leu Ala Arg Glu Asp Glu Gln3980 3985 3990Gly Gln Ala Tyr Leu Cys Ala Tyr Leu Val Ala Ala Asp Glu Trp3995 4000 4005Thr Val Ala Glu Leu Arg Lys His Leu Gly Lys Thr Leu Pro Asp4010 4015 4020Tyr Met Ile Pro Ala Tyr Phe Ile Glu Leu Glu Glu Phe Pro Leu4025 4030 4035Thr Pro Ser Gly Lys Val Asn Lys Lys Ala Leu Pro Glu Pro Asp4040 4045 4050Gly Gln Ile Gln Thr Gly Val Glu Tyr Val Glu Ala Thr Thr Glu4055 4060 4065Ser Gln Lys Ile Leu Val Glu Leu Trp Gln Glu Val Leu Arg Val4070 4075 4080Glu Arg Ile Gly Ile Tyr Asp Asn Phe Phe Glu Leu Gly Gly Asp4085 4090 4095Ser Ile Lys Ala Ile Gln Ile Thr Ala Arg Leu Arg Arg His His4100 4105 4110Arg Lys Leu Glu Ile Ser His Leu Phe Lys His Pro Thr Ile Ala4115 4120 4125Glu Leu Ala Pro Trp Met Gln Thr Ser Gln Ala Leu Leu Glu Gln4130 4135 4140Gly Thr Val Glu Gly Glu Val Met Leu Thr Pro Ile Gln Lys Ala4145 4150 4155Phe Phe Glu Glu Asn Gln Glu Gln Pro Gln His Phe Asn Gln Asp4160 4165 4170Ser Leu Leu Tyr Ser Ser Asn Gly Trp Asn Gln Asp Ala Ile Glu4175 4180 4185Gln Val Phe Glu Lys Ile Thr Glu His His Asp Ala Leu Arg Met4190 4195 4200Val Tyr Pro His Thr Glu Gly Lys Val Thr Gln Ile Asn Arg Gly4205 4210 4215Leu Glu Asp Lys Ala Phe Thr Leu Gln Val Phe Asp Phe Thr Gln4220 4225 4230Glu Pro Thr Asp Thr Gln Ala Thr Lys Ile Glu Gln Ile Ala Thr4235 4240 4245Gln Leu Gln Ala Ser Phe Asp Leu Lys Lys Gly Pro Leu Val Arg4250 4255 4260Leu Gly Leu Phe Thr Thr Lys Ala Gly Asp Tyr Leu Leu Ile Val4265 4270 4275Ile His His Leu Val Ile Asp Gly Val Ser Trp Arg Ile Leu Leu4280 4285 4290Glu Asp Phe His Asn Ala Tyr Gln Gln Val Ile Gln Gly Gln Ala4295 4300 4305Ile Val Leu Pro Glu Lys Thr Thr Ser Phe Lys Thr Trp Ser Glu4310 4315 4320Arg Leu Asn Glu Tyr Ala Asn Ser His Ala Leu Leu His Glu Ile4325 4330 4335Pro Tyr Trp Lys Gln Met Glu Glu Ile Ser Ile Ala Pro Leu Pro4340 4345 4350Lys Lys Gly Asn Asn Asp Gly Arg Tyr Tyr Val Lys Asp Ser Glu4355 4360 4365Tyr Ala Thr Met Ser Leu Thr Glu Glu Glu Thr Gln Asn Leu Leu4370 4375 4380Thr Arg Val His Arg Ala Tyr Arg Thr Glu Ile Asn Asp Leu Leu4385 4390 4395Leu Ala Ala Leu Gly Leu Ala Ser Lys Glu Trp Thr Lys Glu Asn4400 4405 4410Arg Val Ala Ile His Leu Glu Gly His Gly Arg Glu Glu Ile Gly4415 4420 4425Glu Gly Val Asp Val Asn Arg Thr Val Gly Trp Phe Thr Ser Leu4430 4435 4440Phe Pro Phe Val Ile Asp Leu Glu Asn Asp Glu Leu Pro Leu Ile4445 4450 4455Ile Lys Ser Val Lys Glu Thr Leu Arg Arg Val Pro Asn Lys Gly4460 4465 4470Met Gly Tyr Gly Ile Leu Lys His Leu Thr Ser Asp Ala Asn Lys4475 4480 4485Gln Glu Ile Thr Phe Ser Leu Arg Pro Glu Ile Ser Phe Asn Tyr4490 4495 4500Leu Gly Val Phe Asp Gln Gln Glu Glu Glu Ser Glu Ser Ala Gly4505 4510 4515Ile Pro Thr Gly Gln Pro Ile Ser Pro Gln Tyr Tyr Asp Thr His4520 4525 4530Leu Leu Glu Phe Asn Gly Ala Val Ser Asn Asn Gln Leu His Val4535 4540 4545Asn Cys Arg Phe Ala Pro Ala Ala Val Asp Arg Ala Ile Val Glu4550 4555 4560Ile Leu Met Glu Arg Phe Lys His His Leu Leu Leu Ile Ser Lys4565 4570 4575His Cys Leu Glu Lys Asp Thr Val Glu Phe Thr Pro Thr Asp Phe4580 4585 4590Thr Glu Lys Glu Leu Ser Gln Glu Gln Leu Asp Asp Leu Leu Asp4595 4600 4605Asp Leu Phe Glu Asp Ile Asp Asp Leu4610 4615252541PRTBrevibacillus texasporus 25Met Gln Lys Lys Asp Lys Ile Lys Asp Ile Tyr Ser Leu Ser Pro Leu1 5 10 15Gln Lys Gly Met Leu Phe His Ser Met Lys Asp Pro Gln Ser Asp Ala20 25 30Tyr Phe Glu Gln Val Thr Leu Leu Leu Glu Gly Val Val Asn Pro Thr35 40 45Tyr Leu Ala Glu Ser Ile Gln Gly Leu Val Gln Lys Tyr Asp Met Phe50 55 60Arg Ser Val Phe Arg Tyr Lys Lys Val Asp Pro Val Gln Val Val Leu65 70 75 80Ser Glu Arg Lys Ile Asp Leu Gln Ile Glu Asp Leu Thr Gln Ile Asn85 90 95Glu Glu Glu Gln Arg Lys Phe Ile Glu Glu Tyr Arg Lys Lys Asp Arg100 105 110Glu Arg Gly Phe Asp Leu Ser Arg Asp Ile Leu Leu Arg Phe Thr Leu115 120 125Phe Gln Thr Ala Ala Asn Arg Tyr Glu Leu Leu Trp Ser His His His130 135 140Ile Leu Met Asp Gly Trp Cys Thr Gly Ile Val Phe Gln Asp Leu Phe145 150 155 160Gln Met Tyr Gln Arg Arg Leu Ser Gly Gln Ala Leu Leu Pro Glu Val165 170 175Ala Pro Gln Tyr Ser Glu Tyr Ile Arg Trp Leu Lys Lys Gln Asp Asp180 185 190Gln Gln Ala Leu Ala Phe Trp Lys Glu Tyr Leu Gln Gly Phe Glu Asn195 200 205Leu Thr Gly Ile Pro Arg Leu Arg Ser Gly Asn His Pro Tyr Lys Gln210 215 220Glu Glu Phe Ile Phe Ser Leu Gly Glu Glu Ala Thr Gln Lys Leu Thr225 230 235 240Gln Thr Ala Gln Lys Tyr Gln Val Thr Leu Asn Thr Val Val Gln Thr245 250 255Ile Trp Gly Ala Leu Leu Gln Lys Tyr Asn Asn Thr Asn Asp Ala Ala260 265 270Tyr Gly Val Val Val Ser Gly Arg Pro Ala Glu Val Pro Asn Val Glu275 280 285Gln Met Val Gly Leu Phe Ser Asn Thr Ile Pro Ile Arg Ile Lys Lys290 295 300Glu Ala Gly Lys Thr Phe Gly Glu Val Leu Lys Asn Val Gln Gln Thr305 310 315 320Ala Leu Glu Ala Glu Lys Tyr Gly Tyr Leu Ser Leu Ala Asp Ile Gln325 330 335Ala Ser Ala Ala Tyr Thr His Gln Leu Leu Asp His Ile Leu Ala Phe340 345 350Glu Asn Phe Pro Met Asp Gln Glu Thr Phe Asn Gln Glu Asn Val Leu355 360 365Gly Phe Ala Val Lys Asp Ala His Thr Phe Glu Gln Thr His Tyr Asp370 375 380Leu Thr Val Leu Val Ile Pro Gly Lys Glu Leu Ile Phe Lys Phe Met385 390 395 400Tyr Asn Glu Ser Val His Ser Lys Glu Tyr Leu Asn Leu Leu Glu Leu405 410 415Asn Met Lys Lys Leu Val Ser Leu Val Ile Glu Gln Gln Asp Ile Phe420 425 430Asp Pro Ala Thr Glu Phe Val Ser Asp Leu Glu Lys Asp Lys Leu Leu435 440 445Thr Ile Phe Asn Arg Thr Asp Ala Lys Tyr Pro Arg Glu Lys Thr Ile450 455 460His Glu Leu Phe Gln Glu Gln Val Asp Lys Asn Pro Asp Gln Val Ala465 470 475 480Leu Val Phe Gly Glu Ala Gln Leu Thr Tyr Arg Glu Leu Asn Glu Lys485 490 495Ala Asn Gln Met Ala Arg Gly Leu Arg Lys Gln Gly Val Leu Pro Asp500 505 510Gln Val Ile Gly Leu Leu Thr Asp Arg Ser Leu Glu Met Ile Ile Ala515 520 525Ile Leu Ala Ile Phe Lys Ala Gly Gly Ala Tyr Met Pro Ile Asp Pro530 535 540Ser Tyr Pro Ser Glu Arg Ile Gln Tyr Met Leu Ala Asp Ser Arg Thr545 550 555 560His Leu Leu Leu Val Gln Lys Ala Glu Met Ile Pro Ala Asn Tyr Gln565 570 575Gly Glu Val Leu Leu Leu Thr Glu Asp Ser Trp Met Asp Glu Asn Thr580 585 590Asp Asn Leu Asp Leu Val Asn Gln Ala Gln Asp Leu Ala Tyr Val Met595 600 605Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys Gly Asn Leu Thr Thr His610 615 620Gln Asn Ile Val Lys Thr Ile Met Asn Asn Gly Tyr Met Glu Ile Thr625 630 635 640Pro Asn Asp Arg Leu Leu Gln Leu Ser Asn Tyr Ala Phe Asp Gly Ser645 650 655Thr Phe Asp Ile Tyr Ser Ala Leu Leu Asn Gly Ala Ser Leu Ile Leu660 665 670Val Pro Thr His Val Leu Met Asn Pro Thr Asp Leu Ala Ser Val Ile675 680 685Gln Asp Gln His Ile Thr Val Ser Phe Met Thr Thr Ser Leu Phe Asn690 695 700Thr Leu Val Glu Leu Asp Val Thr Ser Leu Lys His Met Arg Lys Val705 710 715 720Val Phe Gly Gly Glu Lys Ala Ser Ile Lys His Val Glu Lys Ala Leu725 730 735Asp Tyr Leu Gly Ala Gly Arg Leu Val Asn Gly Tyr Gly Pro Thr Glu740 745 750Thr Thr Val Phe Ala Thr Thr Tyr Thr Val Asp His Thr Ile Lys Glu755 760 765Thr Gly Ile Met Pro Ile Gly Arg Pro Leu Asn Asn Thr Lys Val Phe770 775 780Ile Leu Gly Ala Asp Asn Gln Leu Gln Pro Ile Gly Ala Leu Gly Glu785 790 795 800Leu Cys Val Ser Gly Glu Gly Leu Ala Arg Gly Tyr Leu Asn Leu Pro805 810 815Glu Leu Thr Ala Asp Arg Phe Val Glu Asn Pro Phe Met Arg Gly Glu820 825 830Arg Met Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Ser835 840 845Ile Glu Tyr Val Gly Arg Ile Asp Glu Gln Val Lys Ile Arg Gly His850 855 860Arg Ile Glu Leu Gly Glu Ile Glu Ala Arg Leu Leu Glu His Pro Ala865 870 875 880Ile Ser Glu Thr Val Leu Leu Ala Lys Gln Asp Glu Gln Gly His Ser885 890 895Phe Leu Cys Ala Tyr Leu Val Thr Asn Gly Ala Trp Ser Val Ala

Glu900 905 910Leu Arg Lys His Ile Lys Glu Thr Leu Pro Asp Ser Met Val Pro Ser915 920 925Tyr Phe Ile Glu Ile Asp Lys Met Pro Leu Thr Ser Asn Gly Lys Ala930 935 940Asp Lys Arg Ala Leu Pro Glu Pro Asp Val Gln Gln Val Ser Ser Tyr945 950 955 960Ile Ala Pro Glu Thr Glu Thr Glu Glu Lys Leu Val Gln Leu Phe Gln965 970 975Glu Ile Leu Ser Val Glu Gln Val Gly Thr Gln Asp Asn Phe Phe Glu980 985 990Leu Gly Gly His Ser Leu Lys Ala Met Met Leu Val Ser Arg Met His995 1000 1005Lys Glu Leu Asp Ile Glu Val Pro Leu Lys Asp Val Phe Ala Arg1010 1015 1020Pro Ser Val Lys Glu Leu Ala Ala Phe Leu Thr Asn Thr Glu Val1025 1030 1035Ser Asp Tyr Ile Ala Ile Glu Pro Ala Ala Lys Gln Glu Phe Tyr1040 1045 1050Pro Val Ser Ser Ala Gln Arg Arg Met Tyr Val Val Glu Gln Ile1055 1060 1065Gly Ser Ser Asn Thr Thr Ser Tyr Asn Met Pro Phe Leu Leu Glu1070 1075 1080Ile Gly Gly Ala Leu Asp Val Val Gly Leu Gln Lys Ala Leu Lys1085 1090 1095Lys Leu Val Ile Arg His Glu Ser Leu Arg Thr Ser Phe His Met1100 1105 1110Val Asp Glu Val Leu Met Gln Lys Ile His Pro Asp Val Glu Trp1115 1120 1125Asp Leu Met Val Met Glu Ala Lys Asp Glu Asp Leu Pro Gln Ile1130 1135 1140Ile Asp Gly Phe Ile Gln Pro Phe Asp Leu Ser Asp Ala Ser Leu1145 1150 1155Phe Arg Ala Gly Leu Val Arg Met Glu Ala Asp Arg His Leu Leu1160 1165 1170Met Leu Asp Met His His Ile Ile Ser Asp Gly Val Ser Thr Asn1175 1180 1185Val Leu Phe Gln Asp Leu Met Gln Ile Tyr Gln Gly Lys Glu Leu1190 1195 1200Pro Ser Leu Arg Ile Gln Tyr Lys Asp Tyr Ala Val Trp Gln Gln1205 1210 1215Ala Glu Ala Gln Val Asn Arg Leu Arg Glu Gln Glu Gln Tyr Trp1220 1225 1230Leu Asn Gln Phe Ser Gly Glu Leu Pro Val Leu Glu Met Pro Thr1235 1240 1245Asp Tyr Thr Arg Pro Ser Ile Gln Gln Ser Glu Gly Asp Ile Trp1250 1255 1260Ser Phe Glu Ile Ser Ala Glu Ile Ile Asn Lys Val Lys Lys Leu1265 1270 1275Ser Ser Ser Gln Gly Thr Thr Leu Tyr Met Thr Leu Leu Ala Ala1280 1285 1290Tyr Gln Val Leu Leu Ser Lys Tyr Thr Gly Gln Glu Asp Val Ile1295 1300 1305Val Gly Ser Pro Ile Ala Gly Arg Pro His Ala Asp Val Glu Lys1310 1315 1320Ile Val Gly Met Phe Val Asn Thr Leu Ala Phe Arg Gly Gln Pro1325 1330 1335Lys Ser Thr Gln Thr Phe Ser Thr Tyr Leu Ser Glu Val Lys Glu1340 1345 1350Gln Val Leu His Ala Tyr Asp Asn Ala Glu Tyr Pro Phe Glu Glu1355 1360 1365Leu Leu Glu Lys Leu Asp Leu Glu Arg Asp Leu Ser Arg His Pro1370 1375 1380Leu Phe Asp Thr Met Phe Ala Leu Gln Asn Met Glu Met Ala Glu1385 1390 1395Ile Asn Ile Met Asp Leu Ser Phe Gln Pro Arg Asp Leu Thr Trp1400 1405 1410Lys Asn Ala Lys Phe Asp Leu Thr Trp Met Met Ala Glu Ala Glu1415 1420 1425Asn Leu Tyr Val Thr Ile Glu Tyr Ser Thr Ser Leu Phe Lys Pro1430 1435 1440Glu Thr Ile Glu Arg Leu Gly Lys Arg Phe Thr His Leu Leu Lys1445 1450 1455Gln Ile Gly Asp Ala Pro Glu Arg Leu Ile Ala Asp Leu Glu Val1460 1465 1470Ala Thr Glu Asp Glu Lys His Gln Ile Leu Ser Val Phe Asn Leu1475 1480 1485Thr Gln Ser Asp Tyr Pro Val Asn Lys Thr Val His Gln Leu Phe1490 1495 1500Glu Glu Gln Val Gln Asn Met Pro Asp Gln Lys Ala Ile Val Phe1505 1510 1515Gly Glu Glu Gln Val Thr Tyr Lys Glu Leu Asn Ala Lys Ala Asn1520 1525 1530His Leu Ala Thr Leu Leu Lys Gln Lys Gly Ile Thr Asn Glu Gln1535 1540 1545Leu Val Ala Val Met Ile Glu Pro Ser Ile Glu Phe Phe Val Gly1550 1555 1560Ile Leu Ala Val Leu Lys Ala Gly Gly Ala Tyr Leu Pro Ile Asp1565 1570 1575Pro Thr Tyr Pro Thr Glu Arg Ile Ala Tyr Ile Leu Glu Asp Ser1580 1585 1590Gln Ser Lys Val Leu Leu Val Arg Gly His Glu Gln Val Gln Thr1595 1600 1605Gln Phe Ala Gly Glu Ile Leu Glu Ile Asp Ser Lys Lys Leu Ser1610 1615 1620Thr Glu Glu Leu Lys Asp Val Pro Met Asn Asn Lys Val Thr Asp1625 1630 1635Leu Ala Tyr Val Ile Tyr Thr Ser Gly Ser Thr Gly Gln Pro Lys1640 1645 1650Gly Val Met Val Glu His Arg Ser Leu Met Asn Leu Ser Ala Trp1655 1660 1665His Val Gln Tyr Phe Gly Ile Thr Lys Asp Asp Arg Ser Thr Lys1670 1675 1680Tyr Ala Gly Val Gly Phe Asp Ala Ser Val Trp Glu Val Phe Pro1685 1690 1695Tyr Leu Ile Ala Gly Ala Thr Ile Tyr Val Ile Asp Gln Glu Thr1700 1705 1710Arg Tyr Asp Val Glu Lys Leu Asn Gln Tyr Val Thr Asp Gln Gly1715 1720 1725Ile Thr Ile Ser Phe Leu Pro Thr Gln Phe Ala Glu Gln Phe Met1730 1735 1740Leu Thr Asp His Thr Asp His Thr Ala Leu Arg Trp Leu Leu Ile1745 1750 1755Gly Gly Asp Lys Ala Gln Gln Ala Val Gln Gln Lys Gln Tyr Gln1760 1765 1770Ile Val Asn Asn Tyr Gly Pro Thr Glu Asn Thr Val Val Thr Thr1775 1780 1785Ser Tyr Ile Val Ser Pro Glu Asp Lys Lys Ile Pro Ile Gly Arg1790 1795 1800Pro Ile Ala Asn Asn Gln Val Phe Ile Leu Asn Lys Glu Asn Gln1805 1810 1815Leu Gln Pro Val Gly Ile Pro Gly Glu Leu Cys Val Ser Gly Asp1820 1825 1830Ser Leu Ala Arg Gly Tyr Leu His Arg Pro Glu Leu Thr Ser Glu1835 1840 1845Arg Phe Val Ala Asn Pro Phe Val Pro Gly Glu Arg Met Tyr Lys1850 1855 1860Thr Gly Asp Ile Ala Arg Trp Leu Pro Asp Gly Asn Ile Glu Tyr1865 1870 1875Leu Gly Arg Leu Asp Asp Gln Ile Lys Ile Arg Gly Tyr Arg Val1880 1885 1890Glu Leu Gly Glu Ile Glu Ser Ala Ile Leu Glu His Glu Ala Ile1895 1900 1905His Glu Thr Val Val Leu Ala Arg Gln Asp Asp Gln Asn Gln Thr1910 1915 1920Tyr Leu Cys Ala Tyr Val Val Pro Lys Lys Ser Phe Asp Val Ala1925 1930 1935Glu Leu Arg Gln Tyr Leu Gly Arg Lys Leu Pro His Phe Met Ile1940 1945 1950Pro Ala Phe Phe Thr Glu Met Thr Glu Phe Pro Ile Thr Ser Asn1955 1960 1965Gly Lys Val Asp Lys Lys Ala Leu Pro Leu Pro Asp Leu Ser Lys1970 1975 1980Gln Ser Glu Ile Asp Tyr Val Ala Pro Thr Thr Thr Leu Glu Glu1985 1990 1995Thr Leu Ala Glu Leu Trp Thr Glu Val Leu Gly Val Ser Gln Val2000 2005 2010Gly Ile His Asp Asn Phe Phe Lys Leu Gly Gly Asp Ser Ile Lys2015 2020 2025Ala Ile Gln Ile Ala Ala Arg Leu Asn Thr Lys Gln Leu Lys Leu2030 2035 2040Glu Val Lys Asp Leu Phe Gln Ala Gln Thr Ile Ala Gln Val Ile2045 2050 2055Pro Tyr Ile Lys Thr Lys Glu Ser Lys Ala Glu Gln Gly Ile Val2060 2065 2070Gln Gly Lys Val Glu Leu Thr Pro Ile Gln Glu Trp Phe Phe Gln2075 2080 2085Gln Ser Phe Asp Ile Pro His His Trp Asn Gln Ser Met Met Phe2090 2095 2100Tyr Arg Lys Glu Gly Trp Asp Gln His Val Val Gln Arg Val Phe2105 2110 2115Gln Lys Ile Ala Glu His His Asp Ala Leu Arg Met Ala Tyr Gln2120 2125 2130Gln Glu Asn Gly Lys Thr Ile Gln Ile Asn Arg Gly Val Glu Gly2135 2140 2145Lys Leu Phe Glu Leu Ser Ile Phe Asp Phe Lys Gln Gln Ala Asn2150 2155 2160Val Pro Glu Leu Ile Glu Gln Ala Ala Asn Arg Leu Gln Ser Ala2165 2170 2175Met Asn Leu Gln Asp Gly Pro Leu Val Gln Leu Gly Leu Phe Gln2180 2185 2190Thr Ser Glu Gly Asp His Leu Leu Ile Ala Ile His His Leu Val2195 2200 2205Val Asp Ala Val Ser Trp Arg Ile Ile Thr Glu Asp Phe Met Asn2210 2215 2220Gly Tyr Gln Gln Asp Leu Gln Gly Glu Pro Ile Ala Phe Thr Ser2225 2230 2235Lys Thr Asp Ser Tyr Gln Lys Trp Ala Lys Ser Leu Leu Glu Tyr2240 2245 2250Ala Thr Ser Glu Glu Ile Gln Ser Glu Leu Lys Tyr Trp Gln Ser2255 2260 2265Met Ile Ala Lys Gly Leu Pro Ala Leu Pro Arg Asp Ser Lys Val2270 2275 2280Gly Ala Pro Tyr Leu Leu Lys Asp Ile Gln Glu Val Ala Ile Gln2285 2290 2295Leu Thr Lys Glu Gln Thr Asn Lys Leu Leu Thr Asp Ala His Asn2300 2305 2310Ala Tyr Asn Thr Gln Ile Asn Asp Leu Leu Leu Thr Ala Leu Ala2315 2320 2325Leu Thr Ile Gln Glu Trp Ala Gln Thr Asn Ser Ile Ala Ile Thr2330 2335 2340Leu Glu Gly His Gly Arg Glu Asp Ile Gly Val Asp Ile Asp Ile2345 2350 2355Asn Arg Thr Val Gly Trp Phe Thr Ser Met Tyr Pro Val Val Phe2360 2365 2370Asp Leu Gln Lys Gln Gly Ile Ala Asn Thr Val Lys Gln Val Lys2375 2380 2385Glu Glu Leu Arg Gln Ile Pro Asn Lys Gly Ile Gly Tyr Gly Val2390 2395 2400Val Arg Tyr Leu Ser Asn Gln Gly Ser Thr Glu Leu Asp Leu Ser2405 2410 2415Ser His Ala Ile Asn Pro Glu Ile Ser Phe Asn Tyr Leu Gly Gln2420 2425 2430Met Asp Gln Ser Gly Gln Glu Glu Glu Tyr Gln Leu Ser Pro Leu2435 2440 2445Ser Ser Gly Gln Gln Ile Ser Gln Met Asn Gln Gly Leu Phe Pro2450 2455 2460Ile Asn Val Ser Gly Ile Val Val Glu Asn Gln Leu Ser Ile Gln2465 2470 2475Ile Ser Tyr Asp Ser Gln Ala Tyr His Asp Ser Thr Met Glu Lys2480 2485 2490Leu Ile Gln Arg Tyr Gln Tyr His Leu Leu Glu Ile Ile Asn His2495 2500 2505Cys Val Gln Gln Thr Glu Thr Glu Leu Thr Pro Ser Asp Phe Ser2510 2515 2520Thr Lys Glu Leu Ser Met Glu Asp Leu Glu Ser Val Phe Glu Leu2525 2530 2535Leu Asp Glu2540262526PRTBrevibacillus texasporus 26Met Leu Ser Lys Ala Asn Ile Lys Asp Ile Tyr Thr Leu Ser Pro Leu1 5 10 15Gln Lys Gly Met Leu Phe Gln His Leu Lys Glu Glu Ser Thr Ala Tyr20 25 30Phe Glu Gln Leu His Phe Thr Ile Lys Gly Gln Leu Tyr Val Asp Ser35 40 45Phe Glu Ala Ser Phe Gln His Leu Ile Asn Lys Tyr Asp Val Leu Arg50 55 60Thr Val Phe Leu Tyr Lys Asn Met Thr Gln Pro Met Gln Met Val Leu65 70 75 80Lys Glu Arg Lys Thr Ser Val His Phe Glu Asp Ile Ser His Leu Asp85 90 95Ser Lys Ala Val Ser Glu Tyr Val Glu Glu Phe Lys Asn Gln Asp Arg100 105 110Glu Lys Gly Phe Glu Leu Ser Lys Asp Ile Leu Met Arg Phe Ala Ile115 120 125Leu Lys Ala Gly Ala Glu Ser Tyr His Leu Ile Trp Ser Phe His His130 135 140Ile Leu Met Asp Gly Trp Cys Met Gly Ile Val Leu Gln Asp Leu Phe145 150 155 160Arg Met Tyr Gln Gln His Arg Gln Asn Ile Pro Ile Thr Val Glu Ser165 170 175Val Pro Ala Tyr Ser Glu Tyr Ile Arg Trp Leu Glu Lys Gln Asn Val180 185 190Thr Lys Ala Arg Asp Tyr Trp Lys Asn Tyr Leu Glu Gly Tyr Glu Glu195 200 205Leu Thr Gly Ile Ile Arg Leu Asp Thr Lys His Thr Ser His Asn Asn210 215 220Glu Val Gln Glu Cys Ala Phe Thr Leu Asp Lys Asp Ile Thr Glu Gly225 230 235 240Leu Thr Gln Leu Ala Arg His Tyr Ser Val Thr Val Asn Thr Leu Phe245 250 255Gln Thr Ile Trp Gly Met Leu Leu Gln Lys Tyr Asn Asn Lys Asp Asp260 265 270Val Val Phe Gly Ala Val Val Ser Gly Arg Pro Ser Glu Ile His Gly275 280 285Val Glu Asn Met Val Gly Leu Phe Ile Asn Thr Val Pro Ile Arg Ile290 295 300Gln Lys Gln Met Asn Asp Thr Phe Ser His Leu Leu Lys Arg Val His305 310 315 320Glu Ser Thr Leu Leu Ser Lys Gln Tyr Glu Phe Val Ser Leu Ala Asp325 330 335Ile Gln Thr Asp Ala Gly Phe Ser Gly Gln Leu Leu Asp His Ile Leu340 345 350Val Phe Glu Asn Tyr Pro Ile Ser Glu Gly Ser Phe Glu Glu Glu Glu355 360 365Phe Thr Met Asp Ser Ile Lys Thr Tyr Glu Lys Thr Ser Tyr Asp Leu370 375 380Asn Val Met Ile Arg Pro Asn Glu Asp Gln Leu Asp Ile Ala Phe Gln385 390 395 400Phe Asn Asp Asp Val Tyr Ser Ser Glu Asn Val Lys Arg Leu Phe Gln405 410 415His Met Lys Gln Leu Ala Leu Ala Val Ile Lys Asn Pro Asp Val Arg420 425 430Leu Glu Glu Ile Ala Met Ile Thr Glu Glu Glu Arg Tyr Gln Ile Leu435 440 445His Asp Phe Gln Gly Glu Ile Val Asp Phe Val Thr Glu Lys Thr Leu450 455 460Pro Glu Leu Phe Glu Asp Gln Val Lys Arg Thr Pro Glu Ala Ile Ala465 470 475 480Leu Arg Phe Glu Asp Gln Gln Leu Thr Tyr Gln Glu Leu Asn Gln Arg485 490 495Val Asn Gln Leu Ala Trp Thr Leu Arg Met Lys Gly Leu Gln Gln Glu500 505 510Glu Leu Val Gly Ile Met Val Gln Arg Ser Leu Glu Met Ile Val Gly515 520 525Val Leu Ala Val Ile Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro530 535 540Glu Tyr Pro Leu Asp Arg Ile Gln Tyr Met Leu Glu Asp Ser Gly Thr545 550 555 560Asn Trp Leu Leu Thr Thr Lys Gln Ser Glu Ile Pro Ser Ile Tyr Leu565 570 575Gly His Val Leu Tyr Leu Glu Glu Asp Thr Val Tyr His Glu Arg Ser580 585 590Ser Asp Val Glu Ile Val Asn Gln Ser Ser Asp Leu Ala Tyr Ile Ile595 600 605Tyr Thr Ser Gly Ser Thr Gly Gln Pro Lys Gly Val Met Ile Asp His610 615 620Arg Ala Val His Asn Leu His Leu Ser Ala Gly Ile Tyr Gly Ile Ala625 630 635 640Gln Gly Ser Gln Val Leu Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser645 650 655Val Gly Asp Ile Phe His Ser Leu Leu Thr Gly Ala Thr Leu His Leu660 665 670Val Lys Lys Glu Gln Leu Leu Ser Gly His Ala Phe Met Glu Trp Leu675 680 685Asp Glu Ala Gly Ile Thr Thr Ile Pro Phe Ile Pro Pro Ser Val Leu690 695 700Lys Glu Leu Pro Tyr Ala Lys Leu Pro Lys Leu Lys Thr Ile Ser Thr705 710 715 720Gly Gly Glu Glu Leu Pro Ala Asp Leu Val Arg Ile Trp Gly Ala Asn725 730 735Arg Thr Phe Leu Asn Ala Tyr Gly Pro Thr Glu Thr Thr Val Asp Ala740 745 750Ser Ile Gly Asn Cys Val Glu Met Thr Asp Lys Pro Ser Ile Gly Thr755 760 765Pro Thr Val Asn Lys Arg Ala Tyr Ile Leu Asp Gln Tyr Gly His Ile770 775 780Gln Pro Ile Gly Val Pro Gly Glu Leu Cys Val Gly Gly Glu Gly Val785 790 795 800Ala Arg Gly Tyr Leu His Arg Pro Glu Leu Thr Asp Glu Lys Phe Val805 810 815Asn Asp Pro Tyr Val Pro Asn Gly Arg Met Tyr Lys Thr Gly Asp Leu820 825 830Ala Arg Trp Leu Pro Asp Gly Thr Ile Glu Phe Leu Gly Arg Met Asp835 840 845Gly Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Gly Glu Ile Glu850 855 860Ala Arg Leu Asn Gln Ala Pro Ser Val Lys Gln Ala Val Val Leu Ala865 870 875 880Arg Ser Gly Glu Gln Lys Gln Val Tyr Leu Cys Ala Tyr Leu Val Thr885 890 895Asp Asn Asp Leu Lys Val Ser Ala Leu Arg Lys Glu Leu Ser Gln Thr900 905 910Leu Pro Asp Tyr Met Ile Pro Ser Phe Phe Ile Lys Val Glu Lys Ile915 920

925Pro Val Thr Val Asn Gly Lys Ile Asp Lys Lys Ala Leu Pro Glu Pro930 935 940Glu Lys Glu Val Glu Leu Gln Thr Glu Tyr Val Ala Pro Thr Asn Pro945 950 955 960Thr Glu Glu Ile Leu Val Gln Ile Trp Gln Lys Val Leu Gly Met Glu965 970 975Arg Val Gly Ile Glu Asp Asn Phe Phe Glu Leu Gly Gly His Ser Ile980 985 990Lys Ala Met Met Leu Ala Ser Asn Ile Tyr Lys Glu Leu Lys Ile Asp995 1000 1005Leu Pro Leu Arg Glu Ile Phe Lys His Thr Thr Val Lys Glu Met1010 1015 1020Ala Arg Phe Ile Asp Gly Arg Asp Glu Glu Glu Tyr Val Gly Ile1025 1030 1035Gln Pro Ala Ala Lys Gln Glu Tyr Tyr Pro Val Ser Ser Ala Gln1040 1045 1050Lys Arg Met Tyr Val Ile Gln Ser Leu Glu Asp Lys Ala Gln Gly1055 1060 1065Thr Ser Tyr Asn Met Pro Ser Phe Tyr Lys Met Lys Gly Ser Val1070 1075 1080Asp Ala Glu Lys Leu Glu Lys Val Phe Gln Thr Leu Leu Asp Arg1085 1090 1095His Glu Ser Leu Arg Thr Ser Phe His Met Ile Glu Glu Gln Leu1100 1105 1110Val Gln Lys Val His Glu Gln Val Ser Trp Lys Met Asp Met Lys1115 1120 1125Thr Val Ser Ala Asn Asp Val Ser Arg Leu Lys Asp Ser Phe Val1130 1135 1140Gln Pro Phe Asp Ile Ser Thr Ala Pro Leu Phe Arg Ala Ser Leu1145 1150 1155Leu Thr Ile His Lys Asp Glu His Ile Leu Met Met Asp Val His1160 1165 1170His Ile Val Gly Asp Gly Val Ser Thr Thr Ile Leu Phe Gln Glu1175 1180 1185Leu Ile Gln Leu Tyr Gln Gly Gln Ala Leu Pro Glu Val Lys Val1190 1195 1200His Tyr Lys Asp Tyr Ala Val Trp Gln Leu Ser Gln Gln Asp Arg1205 1210 1215Leu Lys Glu Ser Glu Asn Phe Trp Leu Gln Gln Phe Ser Gly Glu1220 1225 1230Leu Pro Val Leu Glu Leu Pro Thr Asp Tyr Ser Arg Pro Pro Ile1235 1240 1245Arg Arg Leu Glu Gly Glu Tyr Val Ser Gln Ser Leu Arg Gly Asp1250 1255 1260Leu His Glu Ser Val Lys Ala Phe Met Lys Asn His Glu Val Thr1265 1270 1275Leu Tyr Met Val Leu Leu Ala Thr Tyr Asn Val Leu Leu His Lys1280 1285 1290Tyr Thr Asn Gln His Asp Ile Ile Val Gly Thr Pro Val Ser Asp1295 1300 1305Arg Pro His Pro Asp Val Met Ser Thr Val Gly Met Phe Val Asn1310 1315 1320Thr Leu Ala Val Arg Asn Gln Leu Glu Ser Glu Gln Thr Phe Glu1325 1330 1335Lys Phe Leu Ala Asn Val Lys Asn Lys Met Leu Glu Val Tyr Gly1340 1345 1350His Gln Glu Tyr Pro Phe Glu Asp Val Ile Glu Lys Val Lys Val1355 1360 1365Gln Arg Asp Thr Ser Arg His Pro Leu Phe Asp Thr Met Phe Gly1370 1375 1380Val Gln Asn Leu Glu Ile Ser His Val Glu Leu Pro Asp Trp Gly1385 1390 1395Ile Glu Ala Leu Asp Ile Asp Trp Thr Asn Ser Lys Phe Asp Met1400 1405 1410Ser Trp Met Val Phe Glu Ala Asp Gly Leu Glu Ile Gly Val Glu1415 1420 1425Tyr Ser Thr Ser Leu Phe Glu Arg Asn Thr Ile Gln Arg Met Ile1430 1435 1440Gly His Phe Glu His Ile Ile Glu Gln Ile Met Glu Asn Pro Gln1445 1450 1455Ile Arg Leu Ala Asp Ile Gln Leu Thr Thr Glu Asp Glu Arg Ile1460 1465 1470Gln Ile Leu Glu Glu Phe Asn His Gln Pro Thr Lys Ile Thr Tyr1475 1480 1485Asp Gln Ala Ile Gln Asn Arg Phe Glu Glu Gln Ala Met Lys Thr1490 1495 1500Pro Asp Ala Val Ala Leu Val Tyr Lys Gly Gln Glu Leu Thr Tyr1505 1510 1515Arg Glu Leu Asn Gln Arg Ser Asn Gln Met Ala Arg Thr Leu Arg1520 1525 1530Glu His Gly Val Gly Arg Asp Gln Ile Ile Ala Val Met Ile Asn1535 1540 1545Arg Ser His Glu Leu Ile Ile Ser Ile Leu Ala Val Leu Lys Ala1550 1555 1560Gly Gly Ala Tyr Leu Pro Ile Asp Pro Thr Tyr Pro Leu Asp Arg1565 1570 1575Ile Glu His Met Leu Glu Asp Ser Gln Thr Ala Met Leu Leu Thr1580 1585 1590Gln Lys Glu Ile Gln Ile Pro Thr Gly Tyr Ser Gly Glu Val Leu1595 1600 1605Phe Val Asp Gln Ala Asp Ile Tyr His Glu Asp Ala Thr Asp Leu1610 1615 1620Ser Ser Met Asn Gln Pro Ala Asp Leu Ala Tyr Ile Ile Tyr Thr1625 1630 1635Ser Gly Ser Thr Gly Lys Ser Lys Gly Val Met Ile Glu His Arg1640 1645 1650Ser Leu His Asn Leu Ile His Ile Ser His Pro Tyr Lys Met Gly1655 1660 1665Ala Gly Ser Arg Val Leu Gln Phe Ala Ser Ser Ser Phe Asp Ala1670 1675 1680Ser Val Ala Glu Ile Phe Pro Ala Leu Leu Thr Gly Ser Thr Leu1685 1690 1695Tyr Ile Glu Glu Lys Glu Glu Leu Leu Thr Asn Leu Val Pro Tyr1700 1705 1710Leu Leu Glu Asn Gln Ile Thr Thr Val Ala Leu Pro Pro Ser Leu1715 1720 1725Leu Arg Ser Val Pro Tyr Arg Glu Leu Pro Ala Leu Glu Cys Ile1730 1735 1740Val Ser Val Gly Glu Ala Cys Thr Phe Asp Ile Val Gln Thr Trp1745 1750 1755Gly Gln Asn Arg Thr Phe Ile Asn Gly Tyr Gly Pro Thr Glu Ser1760 1765 1770Thr Val Cys Ser Ala Phe Gly Val Val Thr Ala Glu Asp Lys Arg1775 1780 1785Ile Thr Ile Gly Lys Pro Phe Pro Asn Gln Lys Val Tyr Ile Ile1790 1795 1800Asn Glu Asn Gln Gln Leu Gln Pro Ile Gly Val Pro Gly Glu Leu1805 1810 1815Cys Ile Ala Gly Ala Gly Leu Ser Arg Gly Tyr Leu Asn Arg Pro1820 1825 1830Glu Leu Thr Gln Glu Lys Phe Val Asn Asn Pro Phe Ala Pro Gly1835 1840 1845Glu Arg Met Tyr Lys Thr Gly Asp Val Ala Arg Trp Leu Pro Asp1850 1855 1860Gly Asn Ile Glu Tyr Ala Gly Arg Met Asp Asp Gln Val Lys Val1865 1870 1875Arg Gly Asn Arg Val Glu Leu Gly Glu Val Thr Ser Gln Leu Leu1880 1885 1890Thr His Pro Ser Ile Thr Glu Ala Val Val Val Pro Ile Val Asp1895 1900 1905Thr His Gly Ala Thr Thr Leu Cys Ala Tyr Phe Ile Glu Asp Lys1910 1915 1920Glu Val Lys Val Asn Asp Leu Arg His His Leu Ala Lys Ala Leu1925 1930 1935Pro Glu Phe Met Ile Pro Thr Tyr Phe Ile Lys Val Asp His Ile1940 1945 1950Pro Leu Thr Gly Asn Gly Lys Val Asn Lys Gln Ala Leu Pro Asp1955 1960 1965Pro Ser Glu Phe Ile Ser Ala Gln Thr Gly His Glu Ile Val Ala1970 1975 1980Pro Ser Ser Gln Asp Glu Glu Ile Leu Val Gln Val Trp Glu Glu1985 1990 1995Val Leu Gln Phe Lys Pro Ile Gly Val Glu Asp Asn Phe Phe Glu2000 2005 2010Arg Gly Gly Asp Ser Ile Lys Ala Leu Gln Ile Val Ala Arg Leu2015 2020 2025Ser Lys Tyr Asn Arg Lys Leu Asp Ser Arg His Ile Phe Lys Asn2030 2035 2040Pro Thr Ile Ser Met Leu Ala Pro Tyr Leu Glu Gln Arg Gly Ala2045 2050 2055Leu Ile Glu Gln Asp Ser Ile Glu Gly Glu Val Pro Leu Thr Pro2060 2065 2070Ile Gln Ser Trp Phe Phe Glu Gln Pro Phe Val Tyr Pro His His2075 2080 2085Phe Asn Gln Ser Met Leu Leu Pro Asn Glu Gln Gly Trp Asp Arg2090 2095 2100Gln Arg Ile Glu Gln Ala Phe Thr Thr Ile Val Arg His His Asp2105 2110 2115Ala Leu Arg Met Lys Tyr Gln Phe Arg Glu Lys Ile Ile Gln Glu2120 2125 2130Asn Gln Gly Ile Glu Gly Glu Phe Phe Thr Leu His Glu Val Asp2135 2140 2145Val Thr Lys Glu Arg Asp Trp Gln Met Arg Ile Glu Gln Glu Ala2150 2155 2160Asn Gln Leu Gln Ala Ser Phe Asp Leu Thr Thr Gly Pro Leu Val2165 2170 2175Lys Leu Gly Leu Tyr His Thr Ala Tyr Gly Asp Tyr Leu Leu Ile2180 2185 2190Val Val His His Leu Leu Ile Asp Gly Val Ser Trp Arg Ile Leu2195 2200 2205Leu Glu Asp Phe Gln Thr Leu Tyr Glu Gln Lys Gly Glu Leu Pro2210 2215 2220Ala Lys Thr Thr Ser Phe Lys Ala Trp Ala Val Gln Leu Glu Gly2225 2230 2235Tyr Ala Arg Ser Lys Lys Leu Gln Asp Glu Ala Ser Tyr Trp Lys2240 2245 2250Gly Leu Leu Asn Lys Ser Ile Arg Glu Leu Pro Ala Asp Lys Glu2255 2260 2265Ser Ser Asp Thr Phe Leu Phe Gly Asp Thr Lys Glu Val Gln Leu2270 2275 2280Thr Phe Asp Ile Asn Glu Thr Gln Asp Leu Leu Thr Asp Ala His2285 2290 2295His Ala Tyr Lys Thr Lys Ala Asp Asp Leu Leu Leu Ala Ala Leu2300 2305 2310Val Leu Ser Ile Asn Glu Trp Thr Lys Gln Ser Asp Ile Ile Val2315 2320 2325Asn Leu Glu Gly His Gly Arg Glu Thr Ile Gly Glu Gly Ile Asp2330 2335 2340Leu Ser Arg Thr Ile Gly Trp Phe Thr Thr Ile Tyr Pro Val Leu2345 2350 2355Phe Glu Val Glu Asn His Gln Leu Ser Ser Val Ile Lys His Val2360 2365 2370Lys Glu Thr Leu Arg Asn Val Pro Asn Asn Gly Ile Gly Phe Gly2375 2380 2385Ile Leu Gln His Met Ser His Ser Asp Val Ser Gln Ser Gln Leu2390 2395 2400Ser Ser His His Ile Ser Phe Asn Tyr Leu Gly Gln Met Gly Glu2405 2410 2415Asp Ser Ala Ser Gln Ser Glu Thr Asp Asn Gly Val Leu Ile Asn2420 2425 2430Thr Gly Asp Gln Ile Ser Pro Met Asn Ala Asn Pro Gly Ser Leu2435 2440 2445Asn Met Thr Cys Leu Val Met Asn Asn Thr Leu Leu Val Thr Phe2450 2455 2460Asp Tyr Asn Pro Gln Arg Tyr Glu Gln Glu Thr Ile Gln Arg Leu2465 2470 2475Ala Asp Arg Tyr Lys Ser Asn Leu Lys Ala Val Leu Asp His Cys2480 2485 2490Val Gln Arg Glu Gln Thr Glu Arg Thr Pro Ser Asp Phe Ser Thr2495 2500 2505Lys Lys Leu Ser Leu Glu Asp Leu Asp Asp Val Phe Ala Thr Leu2510 2515 2520Lys Asn Leu2525272491PRTBrevibacillus texasporus 27Met Ile Asn Thr Ser Asp Val Lys Asp Ile Tyr Ser Leu Ser Pro Met1 5 10 15Gln Arg Gly Met Leu Phe His Thr Leu Lys Asp Lys Glu Asn Leu Ala20 25 30Tyr Phe Asp Gln Thr Thr Phe Gln Ile Glu Gly Asp Ile Cys Val Glu35 40 45Ser Leu Glu Lys Ser Phe Asn Glu Leu Ile Arg Lys Tyr Asp Val Leu50 55 60Arg Thr Ile Phe Leu Tyr Gln Lys Leu Lys Glu Pro Met Gln Val Val65 70 75 80Leu Lys Glu Arg Thr Ala Asn Ile His Tyr Glu Asp Phe Ser Met Lys85 90 95Ser Glu Ser Asp Lys Ala Lys Ala Leu Arg Val Ala Lys Gln Arg Asp100 105 110Arg Asp Glu Gly Phe Asp Leu Ser Arg Asp Ile Leu Met Arg Leu Ser115 120 125Leu Leu Lys Val Ala Pro Asn Gln Tyr Glu Leu Val Ile Ser Ser His130 135 140His Ile Ile Ile Asp Gly Trp Cys Thr Gly Ile Leu Tyr Gln Glu Leu145 150 155 160Phe Tyr Phe Tyr Gln Cys Phe Val Ala Asn Gln Pro Ile Pro Ala Glu165 170 175Lys Ser Ile Pro Tyr Ser Arg Tyr Ile Arg Trp Leu Glu Glu Gln Asp180 185 190Glu Glu Glu Gly Lys Ala Tyr Trp Gly Glu Tyr Leu Gln Asp Phe Glu195 200 205Gly Ala Ser Val Ile Pro Lys Gln Asn Ala Lys Gly Glu Lys Glu Val210 215 220Cys Ser Ile Asp Lys Val Thr Phe His Phe Asp Lys Lys Leu Thr Glu225 230 235 240Glu Leu Val Gln Val Ala Lys Thr Cys Gln Val Thr Ile Ser Thr Leu245 250 255Phe Gln Thr Met Trp Gly Ile Leu Leu Gln Lys Tyr Asn Asn Ser Gln260 265 270Glu Ala Ile Phe Gly Ser Val Ile Ser Gly Arg Ser Pro Glu Ile Pro275 280 285Asp Val Glu Lys Ile Val Gly Ile Phe Ile Asn Thr Ile Pro Val Arg290 295 300Ile Arg Thr Leu Asp Lys Gln Thr Phe Lys Glu Leu Leu Ile Gln Val305 310 315 320Gln Glu Ala Ser Val Asn Ser Glu Lys Tyr Asn Tyr Leu Thr Leu Ala325 330 335Asp Ile Gln Ala Val Thr Gly Ser Asn His Ala Leu Ile His His Ile340 345 350Val Ala Phe Glu Asn Phe Pro Ile Ala Ser Asp Ser Phe Val Asp Ser355 360 365Ser Asp Ser Asp Ser Glu Glu Leu Lys Val Val Asn Val Ile Asp Asp370 375 380His Glu Lys Thr Asn Phe Asp Phe Ser Val Gln Val Gln Leu Asp Thr385 390 395 400Glu Leu Leu Val Lys Ile Ser Tyr Asn Gln His Leu Tyr His Arg Ser405 410 415Phe Ile Glu Asn Ile Phe His His Leu Gln Gln Ile Ala Gly Ser Ile420 425 430Thr His Asn Pro Asp Ile Gln Ile Asn Glu Ile Ala Ile Val Ser Lys435 440 445Glu Glu Lys Lys Gln Leu Leu Arg Tyr Ser Thr Pro Ala Lys Ser Asp450 455 460Phe Pro Met Asp Lys Thr Ile His Gln Leu Phe Glu Glu Gln Val Ser465 470 475 480Arg Thr Pro Glu Gln Ile Ala Val Val Phe Lys Gly Glu Ser Phe Thr485 490 495Tyr Arg Glu Leu Asn Glu Lys Ala Asn Gln Leu Ala Trp Val Leu Arg500 505 510Lys Arg Glu Val Arg Pro Asn Glu Ile Val Ala Ile Met Ala Glu His515 520 525Ser Leu Glu Met Leu Val Gly Val Ile Gly Thr Leu Lys Ala Gly Ala530 535 540Ala Tyr Leu Pro Ile Asp Pro Ser Tyr Pro Glu Lys Arg Ile Ala His545 550 555 560Met Leu Gln Asp Ser Lys Ala Glu Gln Leu Leu Ile Gln Pro His Leu565 570 575Asn Met Pro Gln Asp Phe Lys Gly Ser Val Leu Trp Leu Thr Glu Glu580 585 590Ser Trp Ala Lys Glu Ser Thr Thr Asp Leu Pro Leu Ala Thr Ser Ala595 600 605Asn Asp Leu Ala Tyr Met Ile Tyr Thr Ser Gly Ser Thr Gly Leu Pro610 615 620Lys Gly Val Met Val Glu His Gln Ala Leu Val Asn Leu Val Met Trp625 630 635 640His Asn Glu Ala Phe Gly Val Thr Met Thr Asp Gln Cys Thr Lys Leu645 650 655Ala Gly Phe Gly Phe Asp Ala Ser Val Trp Glu Thr Phe Pro Pro Leu660 665 670Ile Gln Gly Ala Thr Leu His Val Leu Glu Glu Ser Arg Arg Gly Asp675 680 685Ile Tyr Ala Leu His Glu Tyr Phe Glu Lys Asn Ala Ile Thr Ile Ser690 695 700Phe Leu Pro Thr Gln Leu Ala Glu Gln Phe Met Glu Leu Thr Ser Ser705 710 715 720Thr Leu Arg Val Leu Leu Ile Gly Gly Asp Arg Ala Gln Lys Val Lys725 730 735Glu Thr Ser Tyr Gln Ile Ile Asn Asn Tyr Gly Pro Thr Glu Asn Thr740 745 750Val Val Thr Thr Ser Gly Gln Leu His Pro Glu Gln Asp Val Phe Pro755 760 765Ile Gly Lys Pro Ile Thr Asn His Ser Val Tyr Ile Leu Asp Gln Asn770 775 780Arg His Leu Gln Pro Ile Gly Ile Pro Gly Glu Leu Cys Val Ser Gly785 790 795 800Ala Gly Leu Ala Arg Gly Tyr Leu Asn Gln Pro Glu Leu Thr Val Glu805 810 815Arg Phe Val Asp Asn Pro Phe Val Pro Gly Glu Arg Met Tyr Arg Thr820 825 830Gly Asp Leu Val Arg Trp Arg Ile Asp Gly Ser Ile Glu Tyr Leu Gly835 840 845Arg Ile Asp Glu Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly850 855 860Glu Ile Glu Thr Lys Leu Leu Glu His Pro Ser Ile Ser Glu Ala Leu865 870 875 880Val Val Ala Arg Asn Asp Glu Gln Gly Tyr Thr Tyr Leu Cys Ala Tyr885 890 895Val Val Ala Thr Gly Ala Trp Ser Val Ser Ser Leu Arg Glu His Leu900 905 910Ile Glu Thr Leu Pro Glu Tyr Met Ile Pro Ala Tyr Met Met Glu Val915 920 925Glu Lys Met Pro Leu Thr Ala Asn Gly Lys Ile Asp Lys Arg Ala Leu930 935 940Pro Val Pro Asp Arg Gln Arg Met Asn Glu Tyr Val Ala Pro Ala Thr945 950 955 960Glu Thr Glu Glu Lys Leu Val Leu Leu

Phe Gln Glu Ile Leu Gly Leu965 970 975Glu Arg Ile Gly Thr Lys Asp His Phe Phe Glu Leu Gly Gly His Ser980 985 990Leu Lys Ala Met Met Leu Val Ser Arg Met His Lys Glu Leu Gly Val995 1000 1005Asp Val Gln Leu Asn Glu Met Phe Ala Arg Pro Thr Val Lys Asp1010 1015 1020Leu Ser Ala Tyr Ile Asp Gln Met Asn Gly Ser Ala Tyr Thr Ala1025 1030 1035Ile Gln Pro Val Glu Glu Gln Pro Tyr Tyr Pro Val Ser Phe Ala1040 1045 1050Gln Arg Arg Met Tyr Val Val Gln Gln Met Arg Asp Ser Glu Thr1055 1060 1065Thr Ser Tyr Asn Met Pro Phe Thr Phe Glu Leu Lys Gly Lys Leu1070 1075 1080His Leu Asp Lys Leu Arg Glu Ala Leu Gln Ile Leu Val Leu Arg1085 1090 1095His Glu Ser Leu Arg Thr Ser Phe His Met Ile Asp Glu Asn Leu1100 1105 1110Val Gln Lys Val Asn Lys Asp Ile Ser Trp Asp Leu Glu Val Ile1115 1120 1125Glu Ala Gln Glu Ser Glu Ile Glu Val Lys Leu Glu Glu Phe Ile1130 1135 1140Arg Pro Phe His Leu Ser Glu Ala Pro Leu Phe Arg Ala Arg Leu1145 1150 1155Ile Cys Leu Asn Pro Gln His His Leu Leu Ser Leu Asp Met His1160 1165 1170His Ile Ile Ser Asp Gly Val Ser Met Asn Leu Phe Leu Gln Glu1175 1180 1185Phe Met Thr Leu Tyr Gln Gly Glu Ala Leu Pro Ala Leu Ser Ile1190 1195 1200Gln Tyr Lys Asp Tyr Ala Val Trp Gln Gln Ser Asp Lys Gln Arg1205 1210 1215Ala Arg Leu Lys Glu Gln Glu Lys Tyr Trp Leu His His Phe Ser1220 1225 1230Gly Glu Leu Pro Thr Leu Glu Leu Pro Thr Asp Phe Pro Arg Pro1235 1240 1245Ala Ile Gln Gln Phe Asp Gly Asp Glu Trp Ala Phe Glu Met Asn1250 1255 1260Ala Asp Leu Leu Ala Lys Val Lys Gln Ile Cys Ser Ser Gln Gly1265 1270 1275Thr Thr Leu Tyr Met Thr Leu Leu Ala Ala Tyr Gln Val Phe Leu1280 1285 1290Ala Arg Tyr Thr Gly Gln Glu Asp Ile Ile Val Gly Ser Pro Ile1295 1300 1305Ala Gly Arg Ser His Ala Asp Leu Glu Asn Met Ile Gly Met Phe1310 1315 1320Val Asn Thr Leu Ala Leu Arg Gly Lys Pro Lys Ala Asp Gln Ser1325 1330 1335Phe Leu Ser Tyr Leu Lys Gln Val Lys Glu Thr Val Phe Gln Ala1340 1345 1350Tyr Ala Asn Ala Glu Tyr Pro Phe Glu Glu Leu Ile Glu Lys Leu1355 1360 1365Asp Leu Glu Arg Asp Met Ser Arg His Pro Leu Phe Asp Thr Leu1370 1375 1380Phe Ser Leu Gln Asn Met Glu Ile Ser Glu Phe Gln Met Asn Asn1385 1390 1395Leu Glu Ile Phe Pro Tyr Glu Thr Gly Gln Lys Asn Ala Lys Phe1400 1405 1410Ala Leu Ser Trp Leu Ile Ala Glu Gly Glu Ser Leu Tyr Val Thr1415 1420 1425Ile Glu Tyr Ser Thr Lys Cys Phe Lys Arg Glu Thr Ile Lys Arg1430 1435 1440Met Ala Ser His Phe Glu Gln Leu Leu Ala Gln Ile Val Glu Gln1445 1450 1455Pro Glu Ala Arg Ile Gly Gln Leu Glu Leu Val Ala Asp Ala Glu1460 1465 1470Arg Lys Met Leu Leu Glu Asp Phe Asn Leu Thr Lys Val Asp Tyr1475 1480 1485Pro Arg Glu Lys Thr Ile Gln Glu Leu Phe Glu Glu Gln Val Asp1490 1495 1500Lys Asn Pro Asp Gln Ile Ala Leu Ile Cys Gly Glu Gln Gln Phe1505 1510 1515Thr Tyr Glu Gln Leu Asn Val Lys Phe Asn Gln Leu Ala His Val1520 1525 1530Leu Arg Arg Glu Gly Val Gln Pro Asn Gln Val Ile Gly Leu Ile1535 1540 1545Thr Asp Arg Ser Leu Ser Met Ile Val Gly Ile Phe Gly Ile Ile1550 1555 1560Lys Ala Gly Gly Gly Tyr Leu Pro Ile Asp Pro Thr Tyr Pro Thr1565 1570 1575Glu Arg Ile Glu Tyr Met Leu Glu Asp Ser Gln Thr His Leu Leu1580 1585 1590Leu Val Gln His Arg Asp Met Val Pro Ala Gly Tyr Gln Gly Glu1595 1600 1605Val Leu Ile Ile Glu Asp Glu Ile Ser Arg Asp Glu Gln Val Ala1610 1615 1620Asn Ile Glu Leu Ile Asn Gln Pro Gln Asp Leu Ala Tyr Val Met1625 1630 1635Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys Gly Asn Leu Thr Thr1640 1645 1650His Arg Asn Ile Ile Lys Thr Val Cys Asn Asn Gly Tyr Ile Glu1655 1660 1665Ile Thr Thr Glu Asp Arg Leu Leu Gln Leu Ser Asn Tyr Ala Phe1670 1675 1680Asp Gly Ser Thr Phe Asp Ile Phe Ser Ser Leu Leu His Gly Ala1685 1690 1695Thr Leu Val Leu Val Pro Lys Glu Val Ile Leu Asn Pro Thr Asp1700 1705 1710Leu Ile Thr Leu Ile Arg Glu Gln Gln Ile Thr Val Ser Phe Met1715 1720 1725Thr Thr Ser Leu Phe Asn Ala Leu Val Glu Leu Asp Val Ser Ser1730 1735 1740Phe Gln Asn Met Arg Lys Ile Ala Phe Gly Gly Glu Lys Ala Ser1745 1750 1755Phe Lys His Val Glu Lys Ala Leu Asp Phe Leu Gly Asn Gly Arg1760 1765 1770Leu Val Asn Gly Tyr Gly Pro Thr Glu Thr Thr Val Phe Ala Thr1775 1780 1785Thr Tyr Thr Val Asp Glu Arg Ile Lys Glu Trp Gly Ile Ile Pro1790 1795 1800Ile Gly Arg Pro Leu His Asn Thr Thr Val His Ile Leu Ser Ala1805 1810 1815Asp Asp Lys Leu Gln Pro Ile Gly Val Ile Gly Glu Leu Cys Val1820 1825 1830Ser Gly Glu Gly Leu Ala Arg Gly Tyr Leu Asn Leu Pro Glu Leu1835 1840 1845Thr Met Glu Arg Phe Val Glu Asn Pro Phe Arg Pro Gly Glu Arg1850 1855 1860Met Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Val1865 1870 1875Leu Glu Tyr Val Gly Arg Lys Asp Glu Gln Val Lys Ile Arg Gly1880 1885 1890His Arg Ile Glu Leu Ser Glu Ile Glu Thr Arg Ile Leu Glu His1895 1900 1905Pro Ala Ile Ser Glu Thr Val Leu Leu Ala Lys Arg Asn Glu Gln1910 1915 1920Gly Ser Ser Tyr Leu Cys Ala Tyr Ile Val Ala His Gly Gln Trp1925 1930 1935Asn Ile Gln Glu Leu Arg Lys His Val Arg Asp Val Leu Pro Glu1940 1945 1950His Met Val Pro Ser Tyr Phe Ile Gly Leu Asp Lys Leu Pro Leu1955 1960 1965Thr Ser Asn Gly Lys Val Asp Lys Arg Ala Leu Pro Glu Pro Glu1970 1975 1980Gly Ser Leu Gln Leu Thr Arg Glu Ile Val Ala Pro Arg Asn Glu1985 1990 1995Ser Glu Lys Gln Leu Val Glu Ile Val Ala Glu Val Leu Gly Leu2000 2005 2010Glu Ala Ser Glu Ile Ser Ile Thr Asp Asn Leu Phe Glu Leu Gly2015 2020 2025Gly His Ser Leu Thr Ile Leu Arg Ile Leu Ala Lys Val His Thr2030 2035 2040Cys Asn Trp Lys Leu Glu Met Lys Asp Phe Tyr Asn Cys Lys Asn2045 2050 2055Leu Glu Glu Ile Ala Ser Lys Ala Thr Asp Met Gln Glu Asn Gln2060 2065 2070Asn Leu Ser Gly Ser Gly Ser Val Phe Lys Lys Gly Gly Lys Lys2075 2080 2085Ser Ile Pro Val Val Pro Val His Asp Arg Gln Lys Glu Met Glu2090 2095 2100His Val Leu Leu Leu Gly Ser Thr Gly Phe Leu Gly Ile His Leu2105 2110 2115Leu His Glu Leu Leu Gln Lys Thr Glu Ala Thr Ile Leu Cys Val2120 2125 2130Ile Arg Ala Glu Asn Asp Glu Ala Ala Met Gln Arg Leu Arg Lys2135 2140 2145Lys Ile Asp Phe Tyr Phe Thr Ser Gln Tyr Ser Ser Ser Gln Ile2150 2155 2160Asp Glu Trp Phe Thr Arg Ile Gln Ile Ile His Gly Asp Ile Thr2165 2170 2175Gln Ala Asn Phe Gly Leu Glu Ala Lys His Tyr Glu Ser Leu Gly2180 2185 2190Ala Ile Val Asp Thr Val Ile His Thr Ala Ala Leu Val Lys His2195 2200 2205Tyr Gly His Tyr Glu Glu Phe Glu Arg Ala Asn Val His Gly Thr2210 2215 2220Gln Gln Val Val Thr Phe Cys Leu Asn Asn Lys Leu Pro Met His2225 2230 2235Tyr Val Ser Thr Leu Ser Val Ser Gly Thr Thr Val Glu Glu Ala2240 2245 2250Thr Glu Leu Val Glu Phe Thr Glu Lys Asp Phe Tyr Val Gly Gln2255 2260 2265Asn Tyr Glu Ser Asn Val Tyr Leu Arg Ser Lys Phe Glu Ala Glu2270 2275 2280Ala Val Leu Val Gly Gly Met Glu Asn Gly Leu Asp Ala Arg Ile2285 2290 2295Tyr Arg Val Gly Asn Leu Thr Gly Arg Phe Gln Asp Gly Trp Phe2300 2305 2310Gln Glu Asn Ile Asn Glu Asn Met Phe Tyr Leu Leu Ser Lys Ala2315 2320 2325Phe Leu Glu Leu Gly Gly Phe Asp Gln Glu Ile Met Gln Gly Met2330 2335 2340Val Asp Leu Thr Pro Ile Asp Ile Cys Ala Gln Ala Ile Ile His2345 2350 2355Ile Ile Asn Ser Lys Gly Ile Glu Glu Arg Val Phe His Leu Gln2360 2365 2370Asn Pro His Leu Val Thr Tyr Asp Asp Met Tyr Arg Val Phe Glu2375 2380 2385Gly Leu Gly Phe Ser Arg Arg Val Gln Ser Arg Glu Asp Val Thr2390 2395 2400Arg Glu Leu Asp Val Met Met Ser Gln Gly Asn Glu Lys Leu Phe2405 2410 2415Leu Ala Gly Ile Leu Thr Thr Met Leu Asp Asp Val Glu Arg Ala2420 2425 2430Glu Gln Phe Asn Val Ala Val Asp Ser Ser Arg Thr Met Gln Leu2435 2440 2445Leu Glu Asp Thr Ser Phe Thr Tyr Pro Val Pro Asp Asp Glu Tyr2450 2455 2460Leu Arg Lys Leu Ala Met His Met Ile Lys Val Gly Phe Val Thr2465 2470 2475Pro Asn His Thr Val Ala Glu Lys Ile Gly Thr Ser Arg2480 2485 249028240PRTBrevibacillus texasporus 28Met Ala Val Ile Glu Leu Lys Asn Leu Thr Lys Lys Tyr Asn Glu Val1 5 10 15Tyr Ala Val Asp His Leu Asn Ile Glu Val Pro Gln Gly His Ile Tyr20 25 30Ala Phe Leu Gly Ser Asn Gly Ala Gly Lys Thr Thr Thr Ile Lys Met35 40 45Met Thr Gly Gln Leu Asn Pro Ser Glu Gly Glu Val Leu Phe Leu Gly50 55 60Arg Asn Ile Trp Gln Asp Arg Glu Ala Arg Arg Ile Ala Gly Tyr Ala65 70 75 80Pro Asp Val Pro Leu Leu His Glu Gly Leu Thr Val Arg Glu Met Val85 90 95Arg Phe Val Gly Ala Leu Tyr Gly Ser Asp Glu Asp Leu Asn Lys Arg100 105 110Val Asp Thr Leu Leu Glu His Phe Glu Leu Ala Asp Lys Ala Asp Gln115 120 125Leu Ile Lys Glu Tyr Ser Leu Gly Met Lys Arg Lys Val Ser Ile Ala130 135 140Cys Ala Leu Ile His Arg Pro Lys Ile Leu Leu Leu Asp Glu Val Thr145 150 155 160Asn Gly Leu Asp Pro Lys Ala Thr Arg Glu Val Lys Asn Tyr Ile Arg165 170 175His Phe Ala Lys Glu Glu Gly Gly Thr Val Phe Ile Thr Thr His Ile180 185 190Leu Asp Ile Val Glu Glu Leu Ala Asp Thr Ile Ser Ile Leu His Lys195 200 205Gly Lys Ile Lys Val Thr Gly Ser Met Glu Glu Leu Arg His Val Ala210 215 220Gly Asn Glu Glu Gly Arg Leu Glu Asp Ile Phe Leu Ser Ala Ile Glu225 230 235 240

Claims

1. A feed additive comprising: an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus sp., wherein the peptide is selected from at least one of SEQ ID NOS: 6-20.

2. The additive of claim 1, wherein the Vol is valine alcohol produced by reducing --COOH group of the C-terminal Valine to --CH₂OH.

3. The additive of claim 1, wherein the carboxy-terminus --COOH group of the C-terminal Valine is reduced to --CH₂OH and confers protease resistance to the peptide.

4. The additive of claim 1, wherein the peptide is stable at a pH of 1.0, at a pH 13.0, resistant to proteases or combinations thereof.

5. (canceled)

6. The additive of claim 1, wherein the peptide kills, gram positive bacteria, gram negative bacteria, fungi, protozoa or combinations thereof.

7. (canceled)

8. The additive of claim 1, wherein the peptide is added at between about 0.5 and about 100 ppm.

9. The additive of claim 1, wherein the peptide is added at between about 6 and about 12 ppm.

10. The additive of claim 1, wherein the peptide is added to a feed adapted for use by one or more of poultry, livestock, farm-raised fish, crabs, shrimp and fresh-water turtles.

11. The feed additive of claim 1, wherein the additive is added to a cereal-based animal feed comprising: at least one cereal selected from barley, soya, wheat, triticale, rye and maize; and an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus sp., wherein the peptide comprises SEQ ID NOS: 2-20.

12-13. (canceled)

14. A biologically pure culture of microorganism Brevibacillus texasporus deposit No. ATCC PTA-5854) that produces an antimicrobial peptide that is carboxy-terminus reduced heat stable, amino terminus-methylated peptide and comprises two or more D-amino acids, wherein the peptide is selected from at least one of SEQ ID NOS: 1-20.

15. The microorganism of claim 14, wherein the microorganism is added to a feed.

16. The microorganism of claim 14, wherein the microorganism is mixed with a feed for livestock selected from the group consisting of a milk replacer, a grower feed, a finisher feed, a pre-starter feed and a starter feed.

17. A method for increasing body weight gain efficiency and feed efficiency in animals, comprising mixing the composition of claim 1 with an animal feed.

18. The method of claim 17, wherein the animal feed is adapted for feeding livestock selected from the group consisting of a cattle, a swine, a chicken, a horse, a turkey, a sheep, a goat, a farm-raised fish, crab, shrimp and a turtle.

19. The method of claim 17, wherein the animal feed is adapted for feeding birds selected from the group consisting of chicken, turkey, duck, quail, Cornish hens, and pigeon.

20. The method of claim 17, wherein said feed is selected from the group consisting of a cereal, soybean meal, isolated soybean protein, isolated soybean oil, isolated soybean fat, skimmed milk, fish meal, meat meal, bone meal, blood meal, blood plasma protein, whey, rice bran, wheat bran, a sweetener, a mineral, a vitamin, salt, and grass.

21. The method of claim 17, wherein the daily dose of the peptide ranges from about 1 milligram to about 10 grams per kg body weight of the animal.

22-23. (canceled)

24. An isolated and purified nucleic acid having the sequence of the BT operon (SEQ ID NO.: 21), or portions thereof, that express proteins that produce a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids.

25. The isolated and purified nucleic acid of claim 24, wherein the nucleic acid encodes one or more polypeptide sequences for BT operon proteins (SEQ ID NOS.: 22 to 28) that comprise one or more enzymes used to make a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids, wherein the peptide is selected from at least one of SEQ ID NOS: 1-20, and the nucleic acid has been transformed into a bacteria.

26-28. (canceled)

29. The microorganism of claim 14, wherein the organism is an isolated bacterial comprising Brevibacillus texasporus E58.

30-35. (canceled)

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