LUCIGEN YELLOW (LucY), A YELLOW FLUORESCENT PROTEIN

Abstract

Described herein are isolated polynucleotides that encode a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74. Also described are expression constructs containing the polynucleotides, transformed host cells containing the expression constructions, the encoded fluorescent proteins themselves, and methods of using the nucleotides and encoded fluorescent proteins for bioanalytical research.

Description

BACKGROUND

[0001] Fluorescent proteins (FPs) make it possible to visualize biological processes through in vivo imaging and in vitro fluorescence labeling. Processes like protein expression, localization, degradation, and interaction can be observed through fusion of a protein of interest with a FP. Green Fluorescent Protein (GFP) from Aequorea victoria and its varied derivatives constitute a multi-colored toolbox ranging from blue to yellow, with red-shifted FPs (RFPs) originating mostly from the sea anemone Discosoma striata (Day and Davidson, 2009). Following the discovery and subsequent explosion of available FPs, came the development of their use in techniques like Fluorescence or Forster Resonance Energy Transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET), and Bimolecular Fluorescence Complementation (BiFC), all of which capitalize on the variety of choices in excitation and emission maxima characteristic to each FP.

[0002] In GFP-like and RFP-like fluorophores, fluorescence emanates from a chromophore developed by the formation of an imidazolinone ring system between two centrally located residues. However, complete maturation of the chromophore requires oxidation of an adjacent tyrosine residue, making molecular oxygen a strict requirement for these systems (Tsien, 1998). For example, the chromophore of GFP itself is formed by the cyclization of the tripeptide Ser65-Tyr66-Gly67.

[0003] Bacterial MurB enzymes are a family of flavoproteins that non-covalently bind flavin adenine dinucleotide (FAD). The MurB enzyme family catalyzes a step in peptidoglycan biosynthesis. Because peptidoglycans are cell wall components, enzymes in this pathway have been targets for developing antimicrobial compounds. In a physiological setting, MurB flavoprotein enzymes catalyze a hydride transfer from NADPH to the substrate through FAD to produce the final reduced product, UDP-N-acetylmuramic acid (El Zoelby et al., 2003). A byproduct of FAD binding is fluorescence due to FAD's intrinsic fluorescent properties. Free FAD fluorescence is rather weak (.PHI..sub.F=0.032) due the quenching effects of the adenine moiety. However, sequestration within a protein environment can enhance its fluorescence (Munro and Noble, 1999).

SUMMARY OF THE INVENTION

[0004] Disclosed and claimed herein is a novel fluorescent protein (FP) initially identified in and isolated from a metagenomic library. The FP has been cloned and expressed in a variety of hosts. Nucleotides encoding the novel FP and variations thereof are also disclosed herein. The FP has been given the name "LucY" for Lucigen Yellow. LucY can be used in the same methods that employ conventional FPs. For example, the LucY protein and its derivatives are useful as fluorescent markers in the many ways that such markers are already in use by those of ordinary skill in the art. Such uses include determining subcellular localization and coupling the FP to antibodies, nucleic acids or other receptors for use in detection assays (for example immunoassays or hybridization assays). Further, LucY can be used to track the movement of proteins in cells by expressing the FP in an expression vector. For another example, the FP can be useful in systems to detect induction of transcription. The FP's described herein are also useful as a visualization tool to judge solubility of fusion proteins.

[0005] Additionally, LucY and its derivatives can be used in a novel method to facilitate positive identification of membrane protein crystals grown in lipidic cubic phase.

[0006] Additionally, LucY has been developed into a split-fluorescence system by which protein-protein interactions can be determined. Due to the non-covalent binding of the FAD molecule, LucY fluorescence is reversible, distinguishing it from other available FPs. This reversibility may allow the LucY split-fluorescent system disclosed herein to be used in drug-screening platforms when looking for inhibitors of protein-protein interaction. To evaluate the reversibility of the split-LucY system, the dissociation of split-LucY fragments can be experimentally controlled through a well characterized chemically reversible dimerization strategy. Inducible dimerization of the FK506 binding protein (FKBP) has been used to evaluate BiFC complex formation and signal development (Robida and Kerppola 2009). Typically FKBP and a truncated version of its binding partner, FRB, are used in a chemically induced dimerization strategy in which a ligand promotes dimerization (Chen et al., 1995). FKBP variants (F.sub.M) have since been developed that form constitutive homodimers which dissociate upon addition of drug, thus providing a reversible protein interaction model (Rollins et al., 2000). Although ligand-reversible F.sub.M dimerization has been used successfully, it is theoretically possible that FKBP specific ligands may interfere with LucY reassembly or fluorescence. If necessary an alternative drug-inducible dimerization/dissociation system may be used. The gyrase B N-terminal domain (GyrB NTD) contains binding sites for coumermycin and novobiocin (Gilbert et al., 1994). Coumermycin is a bivalent drug that binds simultaneously to two GyrB NTD monomers and promotes formation of parallel homodimers, while novobiocin is a monovalent analog that can displace coumermycin and thereby drive dimer dissociation. Fusions to the GyrB NTD have been used for demonstration of dimerization-dependent activation of Raf1 kinase activity (Farrar et al., 1996), and as a part of a dimerization-dependent transcriptional activation strategy for controlled gene expression (Zhao et al., 2003).

[0007] Nucleotides that encode LucY and its variants are also described herein. The FP may be introduced into a host cell by direct delivery or may be expressed by the host cell, e.g., by a vector. In addition, the FP expressed in bacterial, eukaryotic, insect, mammalian and in vitro systems can be used directly to monitor the interactions with fused partners in cell lysates, at the extracellular spaces, or in tissue samples. The FP's disclosed herein are very useful for high-throughput screening in drug discovery and identification procedures, and for new target validations of diseases.

[0008] Also disclosed herein are kits containing one or more compositions comprising the fluorescent proteins, which can be a portion of a fusion protein, or one or more polynucleotides that encode the fluorescent proteins. The kits may also can contain one or more recombinant nucleic acid molecules, which encode, in part, fluorescent proteins, which can be the same or different, and may further include, for example, an operatively linked second polynucleotide containing or encoding a restriction endonuclease recognition site or a recombinase recognition site, or any polypeptide of interest.

[0009] Thus, specifically disclosed herein is an isolated polynucleotide comprising a nucleotide sequence encoding a fluorescent protein which is at least 80%, 85%, 90%, 95%, and/or 97% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, as well as circular permutations thereof. Also disclosed are these same polynucleotides further comprising an in-frame subsequence encoding a poly-His sequence at a terminus of the encoded fluorescent protein. These His-tagged polynucleotides include polynucleotides selected from the group consisting of SEQ. ID NOS: 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, and 75, and polynucleotides having at least 80% 85%, 90%, 95%, and/or 97% sequence identity to them, as well as circular permutations thereof, exclusive of the poly-His subsequence.

[0010] Also disclosed are isolated polynucleotides as described in the immediately preceding paragraph, wherein the encoded fluorescent protein has absorbance maxima at about 274 nm, about 376 nm and about 460 nm, and an emission maxima at about 530 nm. Preferably, although not required, the isolated polynucleotide as described herein encodes between 279 and 347 amino acid residues. The polynucleotide sequence may optionally further encode at least one additional polypeptide of interest in-frame with the encoded fluorescent protein.

[0011] Also disclosed herein is an expression construct comprising any of the isolated polynucleotides as described herein. Also disclosed is a host cell comprising such an expression construct. Any and all suitable host cells are within the scope of the present disclosure. The host cell, for example, may be selected from the group consisting of unicellular prokaryote cells, unicellular eukaryote cells, insect cells, and mammalian cells. Also disclosed herein is a method for making a fluorescent protein comprising cultivating the transformed host cell.

[0012] Also encompassed by the present disclosure are novel fluorescent proteins having at least 80%, 85%, 90%, 95%, and/or 97% sequence identity to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, as well as circular permutations thereof. Also disclosed herein are proteins comprising a polypeptide of interest operationally linked to fluorescent protein which is at least 80%, 85%, 90%, 95%, and/or 97% sequence identity to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or circular permutations thereof.

[0013] Also disclosed herein are methods for detecting molecular interactions. One such method comprises fragmenting a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, so that the fragmentation results in two or more non-fluorescent protein fragments and a reversible loss of protein fluorescence. The non-fluorescent protein fragments are separately fused or attached to other molecules. The non-fluorescent protein fragments are then re-associated through interactions of the molecules that are fused or attached to the non-fluorescent protein fragments. A resulting fluorescent signal is then detected. In the preferred version of this invention, the fluorescent protein is fragmented into two non-fluorescent protein fragments.

[0014] As noted previously, the fluorescent protein may optionally comprise a poly-His sequence at a terminus of the protein. The fluorescent protein may optionally comprise between 279 and 347 amino acid residues.

[0015] Another method for detecting molecular interactions disclosed herein comprises providing a first reagent comprising a first compound of interest linked to a first non-fluorescent protein fragment; and providing a second reagent comprising a second compound of interest linked to a second non-fluorescent protein fragment. Here, the first and second non-fluorescent protein fragments comprise complementary fragments of a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, and wherein the first and second non-fluorescent protein fragments generate a fluorescent detectable signal when associated. "Complementary" when used in this context means "serving to fill out or complete" or to be "complements" in the sense of "something that completes something else." See "Merriam-Webster".RTM. Online Dictionary (http://www.merriam-webster.com, .COPYRGT. 2014, Merriam-Webster, Inc., Springfield, Mass., USA). The first and second non-fluorescent protein fragments are associated through interactions of the first and second compounds of interest, and any resulting fluorescent signal is detected. The first compound of interest, the second compound of interest, or both the first and second compounds of interest may comprise polypeptides.

[0016] Also disclosed herein are kits. A first kit comprises, in combination, a first non-fluorescent protein fragment in a first container; and a second non-fluorescent protein fragment in a second container. The first and second non-fluorescent protein fragments comprise complementary fragments of a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, and wherein the first and second non-fluorescent protein fragments generate a fluorescent detectable signal when associated.

[0017] Another kit disclosed herein comprises, in combination, a first isolated polynucleotide that encodes a first non-fluorescent protein fragment in a first container; and a second isolated polynucleotide that encodes a second non-fluorescent protein fragment in a second container; and wherein the first and second isolated polynucleotides encode complementary, non-fluorescent protein fragments of a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a fluorescent circular permutation thereof, and wherein the encoded first and second non-fluorescent protein fragments generate a fluorescent detectable signal when associated.

[0018] Also disclosed herein is a method of increasing the aqueous solubility of a protein of interest. The method comprises fusing or attaching to the protein of interest a protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74, or a circular permutation thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1F. LucY expression. FIG. 1A depicts cell pellets (1 OD) from cultures expressing LucY from the rhaP.sub.BAD promoter in response to rhamnose at the indicated concentrations. FIG. 1B is a graph depicting fluorescence response to rhamnose induction measured with a plate reader using excitation at 485 nm and emission at 528 nm. FIG. 1C is a photograph depicting induction of LucY expression in E. coli on a plate containing 0.2% rhamnose. FIG. 1D is a photograph depicting expression of LucY in the mammalian cells COS-7. FIG. 1E is a photograph depicting expression of LucY in the mammalian cells CHO-K1. FIG. 1F is a photograph depicting expression of LucY in the mammalian cells HeLa. All mammalian transfections were carried out with "TransIT-2020"-brand transfection-reagent (Mirus Bio LLC, Madison, Wis.) and imaged 24 h post-transfection with an inverted fluorescence microscope fitted with a GFP excitation/emission filter.

[0020] FIG. 2 is a photograph depicting the purification of LucY-His6 from E. coli. LucY was expressed with a carboxyl terminal His6 tag using Lucigen's Expresso.TM.-brand T7 cloning and expression system. LucY-His6 was purified by metal affinity chromatography. Fractions from purification including whole cell pellet (lane 1), insoluble pellet after lysis (lane 2), soluble supernatant (lane 3), flowthrough (lane 4), wash (lane 5) and elution fractions (lanes 6-10) with imidazole were photographed under UV light (upper panel) and are aligned with corresponding gel lanes.

[0021] FIG. 3 is a graph depicting the superimposed excitation and emission spectra of LucY. The excitation scan (blue) was performed with emission at 528 nm and the emission scan (green) was performed with excitation at 465 nm.

[0022] FIG. 4A and FIG. 4B are photographs depicting E. coli cell pellets expressing candidate thermo-tolerant homologs of LucY and visualized under a hand-held UV lamp. In FIG. 4A: Sye=Sulfurihydrogenibium yellowstonense; Y4.1 MC1=Geobacillus sp. ((strain Y4.1MC1); Dtu=Dictyoglomus turgidum; Rma=Rhodothermus marinus; Aae=Aquifex aeolicus; Aac=Alicyclobacillus acidocaldarius; Ace1=Acidothermus cellulolyticus; Tma=Thermotoga maritima; Chy=Calsicellulosiruptor hydrothermalis; Tye=Thermodesulfovibrio yellowstonii. In FIG. 4B, Pma=Persephonella marina; Saz=Sulfurihydrogenibium azorense; Hth=Hydrogenobacter thermophilus; SspY=Sulfurihydrogenibium sp. (strain YO3AOP1); Tit=Thermoanaerobacter italicus; Tal=Thermocrinis albus; Hsp1=Hydrogenivirga sp. 128-5-R1-1.

[0023] FIG. 4C is a plot of fluorescence following 45 min incubation at temperatures ranging from 30.degree. C. to 80.degree. C. Fluorescence is expressed as a percentage of highest fluorescence recorded across temperature range. The temperature at which fluorescence diminished to 10% is listed. The abbreviations are the same as listed for FIGS. 4A and 4B.

[0024] FIGS. 5A and 5B are photographs depicting subcellular localization via LucY fluorescence. FIG. 5A is a photograph depicting E. coli cell pellets following illumination with UV light showing expression of LucY fused to the membrane GPCR protein, A1a, with growth at 25.degree. C. and at 30.degree. C. LSP=low-speed pellet; HSP=high-speed pellet. FIG. 5B depicts LucY fused to ATPb following induction with IPTG or arabinose.

[0025] FIG. 5C is a coomassie stained SDS-PAGE of fractions from E. coli expressing the LucY-ATPb fusion shown in FIG. 5B. GPCR-containing extracellular membranes separate with the high-speed pellet (HSP). ATPb separates with intracellular membranes in the low-speed pellet (LSP). HSS=high-speed supernatant; LSS=low-speed supernatant.

[0026] FIG. 6 is a photograph depicting expression of the COR-kinase domain of the LRRK2 enzyme as a C-terminal fusion with LucY from E. coli. Five different clones are shown. Each clone is yellow fluorescent, indicating positive fusion protein expression.

[0027] FIGS. 7A, 7B, 7C, and 7D are three-part photographs depicting expression of LucY as an intracellular loop fusion in mammalian cells. Each three-part series shows the fusion illuminated with a bright field (left), using a GFP filter (middle), and a merged bright field/GFP image (right). Each set of photos represents fusion at a different site within the intracellular loop 3 of a GPCR. FIG. 7A illustrates replacement of the GPCR intracellular loop positions 245-271 with LucY. FIG. 7B shows replacement of the GPCR intracellular loop positions 245-271 with LucY and GSG linker sequence. FIG. 7C illustrates replacement of the GPCR intracellular loop positions 245-274 with LucY and GSG linker sequence. FIG. 7D illustrates replacement of the GPCR intracellular loop positions 245-276 with LucY and GSG linker sequence. GSG linker sequence lies between LucY C-terminus and the GPCR.

[0028] FIG. 8 is a photograph depicting insect cells ("Hi 5"-brand; Life Technologies, Carlsbad, Calif., USA; generically BTI-TN-5B1-4 cells) expressing turkey .beta.1-adrenergic receptor-LucY fusions. The far left tube is negative control, containing only unmodified "Hi 5"-brand cells. The other four tubes contain "Hi 5"-brand cells expressing LucY fused within the intracellular loop 3 of turkey .beta.1-adrenergic receptor at four different junction points.

[0029] FIG. 9A is a photograph showing the utility of LucY as an indicator of detergent solubilization. ATPb was solubilized with 31 different detergents and the fluorescence was visualized by a UV light. FIG. 9B is a corresponding histogram of the fluorescence exhibited by the tubes in FIG. 9A as quantified with a Biotek Synergy 2 microplate fluorometer (BioTek Instruments, Inc., Winooski, Vt., USA).

[0030] FIGS. 10A, 10B, and 10C together demonstrate the utility of LucY as an indicator of protein expression and solubility. FIG. 10A is a photograph depicting Fisuc 1793-LucY expression on plates containing 0.2% rhamnose, with (right) or without (left) an amino-terminal SUMO tag. FIG. 10B is a photograph depicting cleared lysates from .about.5 ml cultures of cells expressing the indicated Fibrobacter genes as LucY fusions, with (right) or without (left) a SUMO tag. FIG. 10C is a series of Coomassie blue-stained polyacrylamide gels showing expression of LucY fusion proteins in total cell lysate (T), soluble fraction (S), and pellet fraction (P) after centrifugation at 12,000.times.G for 5 minutes. The asterisks indicate the induced fusion protein. No band was detected corresponding to the 1793-LucY protein without the SUMO tag.

[0031] FIGS. 11A, 11B, and 11C demonstrate insoluble expression of ABV-LucY and 4110-LucY as "solubility trap" fusion proteins. FIG. 11A is a photograph depicting weak fluorescence due to expression of SUMO-ABV-LucY fusion protein from rhaP.sub.BAD. Plates contained no rhamnose (left), or 0.2% rhamnose (right). FIG. 11B depicts gel analysis of ABV-LucY protein expressed from rhaP.sub.BAD, with or without an amino-terminal SUMO tag. FIG. 11C depicts gel analysis of 4110-LucY protein expressed from rhaP.sub.BAD, with or without a SUMO solubility tag. The right and left portions of FIG. 11C are from the same gel. In FIGS. 11B and 11C, T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation.

[0032] FIGS. 12A and 12B are photos demonstrating the utility of LucY to select fluorescent colonies from a random fusion library in a "solubility trap" experiment. FIG. 12A is a photograph of a section of a primary selection plate containing 0.2% rhamnose. Several fluorescent colonies are indicated by arrows. FIG. 12B is a photograph of individual fluorescent colonies from the initial library screen depicted in FIG. 12A, re-streaked onto a plate containing 0.2% rhamnose.

[0033] FIGS. 13A and 13B demonstrate enhanced soluble expression of 4110-LucY fusion protein with amino-terminal fusion tags derived from shotgun library screens. FIG. 13A is a gel analysis of soluble expression of Geobacillus-4110-LucY library clone 11. The Control sample is 4110-LucY with no amino-terminal fusion. T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation. FIG. 13B is a photograph depicting increased partitioning of yellow fluorescence to the soluble fraction with Geobacillus library clone 11.

[0034] FIG. 14 is a topology diagram of LucY. LucY includes three domains as indicated by circles. Domains 1 and 2 are separated by a short loop; Domains 2 and 3 are connected by an approximately 20 amino-acid span.

[0035] FIGS. 15A, 15B, and 15C are schematic diagrams of various split LucY systems. FIG. 15A is a schematic depicting conventional bimolecular fluorescence complementation (BiFC). Here, a reporter protein is split into two sub-fragments, each of which is fused to a different protein. The two proteins (Protein X and Protein Y in the figure) have the potential to interact. Only after a positive interaction of Protein X and Protein Y is achieved will the two fragments combine and elicit a signal. FIG. 15B is a schematic diagram showing the three distinct domains (colored red, blue, and green) of LucY. A five-residue loop connects domains 1 and 2 and an approximately 18-residue loop connects domains 2 and 3. Five points within each of these loops were chosen as Split Points (SP), as indicated by underlined residues. FIG. 15C is a schematic diagram showing synthetic antiparallel leucine zippers (light green), which were used as protein interaction models to test reconstitution of split LucY fragments (right) and were compared to whole LucY fusions (left). LucY fused at the N-terminus of the leucine zipper is designated NZ; LucY fused at the C-terminus of the leucine zipper is designated CZ. Split LucY fragments fused to the N-terminus and C-terminus of the leucine zippers are referred to as SPNZ and SPCZ, respectively.

[0036] FIGS. 16A, 16B, 16C, and 16D are a series of histograms of fluorescence which demonstrate LucY reassembly. The image above each histogram shows whole cell pellets of the corresponding sample photographed under UV light. Fluorescence is represented as a percentage of CZ. Error bars represent standard deviation from the mean (n=3). FIG. 16A: Five split points between domains 1 and 2, referred to as SPa1-5, were made and their fusions with leucine zippers were coexpressed. FIG. 16B: The same conditions as in FIG. 16A, except the five split points were between domains 2 and 3 and are referred to as SPb1-5. FIG. 16C: SPbNZ5 paired with other split points to determine the pair with highest fluorescence. FIG. 16D: SPbNZ5 paired with either SPbCZ1 or SPbCZ5 was tested for fluorescence independently and without the leucine zipper (.DELTA.Zip).

[0037] FIGS. 17A, 17B, 17C, and 17D are a series of Coomassie-stained SDS-PAGE gels depicting various split point coexpressions of LucY. In each gel, the NZ and CZ bands are adjacent to the dot. FIG. 17A shows expression of NZ, CZ, and SPa1-5, corresponding to the data in FIG. 16A. FIG. 17B shows expression of NZ, CZ, and SPb1-5, corresponding to the data in FIG. 16B. FIG. 17C shows expression of SPbNZ5 and SPbCZ5 paired with a non-continuous CZ or NZ partner in whole cells, corresponding to the data in FIG. 16C. FIG. 17D shows expression of SPbNZ5, SPbCZ1 or 5 alone, in combination, or with their leucine zippers removed in whole cells, corresponding to data in FIG. 16D.

[0038] FIGS. 18A and 18B are Coomassie-stained SDS-PAGE gels depicting split point solubility and expression of LucY. FIG. 18A shows insoluble (P) and soluble (S) fractions of SPbNZ5 and SPbCZ4, alone, coexpressed, with leucine zippers, and without leucine zippers. FIG. 18B shows expression of the C-terminal fragments of LucY without leucine zipper, uninduced (UI), and induced (I).

[0039] FIG. 19A is a schematic diagram illustrating circular permutation of LucY. LucY was circularly permutated such that domains 1 and 3 were connected with a small linker (dashed line) and new N and C-termini were created between domains (red loops). Break points (BP) were introduced between domains 1 and 2 (BPs1-5) or domains 2 and 3 (BPs6-10). FIG. 19B is a histogram comparing the fluorescence of the circularized permutations to wild-type LucY. FIG. 19C is a histogram showing fluorescence of LucY permutations in which split points were made from a circular permuted LucY such that domains 3 and 1 make up one half and domain 2 makes up the other. In FIGS. 19B and 19C, fluorescence is represented as a percentage of wild-type LucY and CZ, respectively. The image above each histogram is a photograph of the whole cell pellets of the corresponding sample under UV light. Error bars represent standard deviation from the mean (n=3).

[0040] FIGS. 20A and 20B are Commassie-stained SDS-PAGE gels showing expression of circular permutations of LucY and splits made from them. FIG. 20A is a gel showing expression of each circular permutation, designated BP-1 through BP-10. The gel shown in FIG. 20A corresponds to the data presented in FIG. 19B. FIG. 20B is a gel showing split point trials derived from circularly permuted LucY. The gel shown in FIG. 20B corresponds to the data in FIG. 19C.

[0041] FIGS. 21A-21J make up a series of photographs showing split LucY systems expressed in HEK 293T cells. FIG. 21A is a photograph of NZ-transformed cells viewed with a band pass filter. FIG. 21B is a photograph of CZ-transformed cells viewed with a band pass filter. FIG. 21C is a photograph of SPbNZ5-transformed cells viewed with a band pass filter. FIG. 21D is a photograph of SPbCZ4-transformed cells viewed with a band pass filter. FIG. 21E is a photograph of SPbNZ5+SPbCZ4-transformed cells viewed with a band pass filter. FIG. 21F is a photograph of NZ-transformed cells viewed with bright field and band pass filters merged. FIG. 21G is a photograph of CZ-transformed cells viewed with bright field and band pass filters merged. FIG. 21H is a photograph of SPbNZ5-transformed cells viewed with bright field and band pass filters merged. FIG. 21I is a photograph of SPbCZ4-transformed cells viewed with bright field and band pass filters merged. FIG. 21J is a photograph of SPbNZ5+SPbCZ4-transformed cells viewed with bright field and band pass filters merged. FIG. 21K is a photograph of an immunoblot gel using anti-HA to detect HA-tagged leucine zipper/LucY fusions either whole, NZ and CZ, or splits SPbNZ5 and SPbCZ4.

[0042] FIGS. 22A-22E are a series of fluorescent photomicrographs demonstrating the use of LucY to visualize protein crystals. FIG. 22A is a photomicrograph of LucY crystals visualized under UV light. FIG. 22B, FIG. 22C, FIG. 22D, and FIG. 22E are photomicrographs of putative crystals of a .beta.1-AR-IL3-LucY fusion proteins in lipidic cubic phase (LCP) visualized under UV light.

[0043] FIG. 23A is a gel depicting enhanced soluble expression of TEV protease as a fusion to C-terminal LucY. E. coli cultures harboring plasmids encoding TEV protease with a C-terminal His6 fusion or a C-terminal LucY-His6 fusion were grown at 37.degree. C. and induced with 0.2% rhamnose. The induced cells were harvested and lysed by sonication, and the lysates were separated into soluble and insoluble fractions by centrifugation. Samples of the total lysate (T), soluble (S), and insoluble (I) fractions were run on SDS PAGE gel (4-20%) and proteins were stained with Coomassie blue stain. FIG. 23B depicts soluble and insoluble fractions of TEV-LucY-His6 lysate photographed under long-wavelength UV light. FIG. 23C is a gel depicting sequence-specific protease activity of the TEV-LucY-His6 fusion protein.

DETAILED DESCRIPTION

Abbreviations and Definitions

[0044] ABV=Acidianus bottle-shaped virus. ATPb=ATP synthase (part of the Fo complex; subunit a). .beta.1-AR-IL3=cardiac .beta.1-adrenergic receptor+interleukin 3 fusion. BiFC=bimolecular fluorescence complementation. DNAP=DNA polymerase. FP=fluorescent protein. GFP=green fluorescent protein. GPCR=G protein-coupled receptor. HA tag=human influenza hemagglutinin tag. IPTG=isopropyl .beta.-D-1-thiogalactopyranoside. LRRK2=leucine-rich repeat kinase 2. Ni-NTA=nickel-nitrilotriacetic acid resin. PCR=polymerase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202). RFP=red-shifted fluorescent protein. SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis. SUMO=small ubiquitin-like modifier protein.

[0045] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in biochemistry and genetic engineering. For purposes of the present disclosure, the following terms are specifically defined.

[0046] The term "nucleic acid molecule" or "polynucleotide" refers to a deoxyribonucleotide or ribonucleotide polymer in either single-stranded or double-stranded form, and, unless specifically indicated otherwise, encompasses polynucleotides containing known analogs of naturally occurring nucleotides that can function in a similar manner as naturally occurring nucleotides. For example, this term can refer to single and double stranded forms of DNA and/or RNA.

[0047] The term "recombinant nucleic acid molecule" refers to a non-naturally occurring polynucleotide containing two or more linked polynucleotide sequences. A recombinant nucleic acid molecule can be produced by recombination methods, particularly genetic engineering techniques, or can be produced by a chemical synthesis method. A recombinant nucleic acid molecule can encode a fusion protein, for example, a fluorescent protein as disclosed herein linked to a polypeptide of interest. The term "recombinant host cell" refers to a cell that contains or can express a recombinant nucleic acid molecule.

[0048] The term "encoding" when referring to a polypeptide or protein (the terms are used synonymously herein) refers to the transcription of a corresponding polynucleotide and translation of the mRNA produced therefrom to yield the polypeptide. The encoding polynucleotide is considered to include both the coding strand, whose nucleotide sequence can be identical to an mRNA, as well as its complementary strand. Encoding polynucleotides explicitly include degenerate codons which encode the same amino acid residues or functionally equivalent amino acid residues. Nucleotide sequences encoding a polypeptide or protein can include polynucleotides containing introns and exons.

[0049] The term "expression construct" refers to a polynucleotide molecule containing at least one sub-sequence encoding a protein of interest which is operationally linked to one or more regulatory sub-sequences which drive expression of the encoded protein when the construct is transformed into a suitable host cell. Such constructs may also contain sub-sequences encoding proteins for selecting host cells transformed to contain the construct, such as sub-sequences which confer antibiotic resistance or dietary limitations to transformed cells. An expression construct may also include one or more of the FP's disclosed herein.

[0050] The terms "control sequences (or sub-sequences)," "regulatory sequences (or sub-sequences)," and the like refer to polynucleotide sequences that are necessary to effect the expression of coding and non-coding sequences in a host cell. Such control sequences can include promoters, ribosomal binding sites, transcription termination sequences, and the like. These terms are used synonymously herein and include, at a minimum, components whose presence can influence expression and also include additional components whose presence is advantageous, such as leader sequences. Fusion partner sequences may sometimes also be control sequences.

[0051] The term "operationally linked" when referring to joined polynucleotide sequences denotes that the sequences are in the same reading frame and upstream regulatory sequences will perform as such in relation to downstream structural sequences. Polynucleotide sequences which are operationally linked are not necessarily physically linked directly to one another but may be separated by intervening nucleotides which do not interfere with the operational relationship of the linked sequences. Similarly, when referring to joined polypeptide sequences, operationally linked means that the functionality of the individual joined segments are substantially identical as compared to their functionality prior to being operationally linked. For example, a fluorescent protein can be fused to a polypeptide of interest and in the fused state retain its fluorescence, while the fused polypeptide of interest also retains its original biological activity.

[0052] As used herein, the term "brightness," with reference to a fluorescent protein, is measured as the product of the extinction coefficient (c) at a given wavelength and the fluorescence quantum yield (.PHI..sub.F).

[0053] The term "probe" refers to a substance that specifically binds to another substance (a "target"). Probes include, for example, antibodies, polynucleotides, receptors and their ligands, and may (or may not) be labeled so as to provide a means to identify or isolate a molecule to which the probe has specifically bound.

[0054] The term "label" refers to a composition that is detectable with or without instrumentation, for example, by visual inspection, spectroscopy, or a photochemical, biochemical, immunochemical or chemical reaction. Exemplary labels (non-limiting) include .sup.32P, fluorescent dyes and proteins, electron-dense reagents, enzymes (such as those commonly used in an ELISA), and binding labels or tags, such as biotin, digoxigenin, or other haptens or peptides for an antiserum or antibody. For example, a label can generate a measurable signal such as fluorescent light in a sample.

[0055] The terms "polypeptide" and "protein" refer to a polymer of two or more amino acid residues. For purposes of this disclosure, the two terms are synonymous. "Polypeptides" and "proteins" are polymers of amino acid residues that are connected through amide bonds. As defined herein, the term "amino acid" includes natural .alpha.-amino acids and unnatural .alpha.-amino acids (e.g. beta-alanine, phenylglycine, homoarginine, N-alkyl .alpha.-amino acids and the like). All optical isomers are included within the definition of "amino acid."

[0056] The term "isolated" or "purified" refers to a material that is substantially or essentially free from components that normally accompany the material in its native state in nature. Purity generally can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, high performance liquid chromatography, and the like. A polynucleotide or a polypeptide is considered to be isolated when it is the most predominant species present in a preparation.

[0057] The term "naturally occurring" refers to a protein, nucleic acid molecule, cell, or other material that exists in nature. A naturally occurring material can be in its "native" form, that is as it exists in nature. Naturally-occurring materials may also be modified by human intervention so that they are in an isolated or purified form.

[0058] Two or more amino acid sequences or two or more nucleotide sequences are considered to be "substantially identical" or "substantially similar" if the amino acid sequences or the nucleotide sequences share at least 80% sequence identity with one another, or with a reference sequence over a given comparison window. Thus, substantially similar sequences include those having, for example, at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity. The terms "sequence identity" or "sequence identical" are defined to mean sequence identity as measured using the cluster database at high identity with tolerance method (i.e., CD-HIT). In simplified terms, terminal gaps are ignored and identity is calculated from the remaining aligned columns. An "identity" or "match" is a column having the same amino acid residues or nucleotide bases; a "mismatch" is a column with two different amino acid residues or nucleotide bases. An "indel" is a consecutive series of gaps in one sequence. Percent identity is then calculated by dividing the number of matches by the length of the shorter of the two sequences being compared. CD-HIT is well known in the field and will not be discussed in any detail herein. For a full description, see "Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences," Weizhong Li & Adam Godzik Bioinformatics, (2006) 22:1658-9; and Limin Fu, Beifang Niu, Zhengwei Zhu, Sitao Wu and Weizhong Li, "CD-HIT: accelerated for clustering the next generation sequencing data," Bioinformatics, (2012), 28(23):3150-3152.

[0059] Additionally, two or more amino acid sequences or two or more nucleotide sequences are considered to be "substantially identical" or "substantially similar" if the amino acid sequences or the nucleotide sequences are circular permutations of each other. Not all sequence-based algorithms for determining percent sequence identity will detect circular permutations. However, several well known programs that require an input consisting only of a linear sequence of amino acids or nucleotides will detect circular permutations of the protein (or encoded protein in the case of nucleotides). Such programs include SHEBA (Jung, J.; Lee, B. (2001). "Circularly permuted proteins in the protein structure database," Protein Science 10(9):1881-1886); Multiprot (Shatsky, M.; Nussinov, R.; Wolfson, H. J. (2004). "A method for simultaneous alignment of multiple protein structures," Proteins: Structure, Function, and Bioinformatics 56(1):143-156); RASPODOM (Weiner, J.; Thomas, G.; Bornberg-Bauer, E. (2005). "Rapid motif-based prediction of circular permutations in multi-domain proteins," Bioinformatics 21(7):932-937); CPSARST (Lo, W. C.; Lyu, P. C. (2008). "CPSARST: An efficient circular permutation search tool applied to the detection of novel protein structural relationships," Genome Biology 9(1):R11); GANGSTA+(Schmidt-Goenner, T.; Guerler, A.; Kolbeck, B.; Knapp, E. W. (2010). "Circular permuted proteins in the universe of protein folds," Proteins: Structure, Function, and Bioinformatics 78(7):1618-1630.); SANA (Wang, L.; Wu, L. Y.; Wang, Y.; Zhang, X. S.; Chen, L. (2010). "SANA: An algorithm for sequential and non-sequential protein structure alignment," Amino Acids 39(2):417-425); and CE-CP (Prlic, A.; Bliven, S.; Rose, P. W.; Bluhm, W. F.; Bizon, C.; Godzik, A.; Bourne, P. E. (2010). "Pre-calculated protein structure alignments at the RCSB PDB website," Bioinformatics 26(23):2983-2985).

[0060] The term "fluorescent properties" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy.

[0061] The term "fluorescent protein" refers to any protein capable of light emission when excited with an appropriate electromagnetic energy. Fluorescent proteins include proteins having amino acid sequences that are either natural or engineered, such as the fluorescent proteins derived from Aequorea victoria fluorescent proteins.

[0062] The term "mutant" or "variant" also is used herein in reference to a fluorescent protein that contains a mutation with respect to a corresponding wild-type fluorescent protein. In addition, reference is made herein to a "spectral variant" or "spectral mutant" of a fluorescent protein to indicate a mutant fluorescent protein that has a different fluorescence characteristic with respect to the corresponding wild-type fluorescent protein. Similarly, thermo-tolerant mutants or variants are fluorescent protein that display fluorescent characteristics at elevated temperatures as compared to the corresponding wild-type.

[0063] The recombinant polynucleotides described herein are incorporated into a suitable host cell. The host cell may be any host cell now known or developed in the future which is amenable to transformation, including, but not limited to prokaryotic and eukaryotic unicellular host cells, e.g., bacteria, yeast, and the like, such as unicellular microbes of the genera Saccharomyces, Bacillus, Aspergillus, Pichia, Kluyveromyces, Escherichia and the like, or isolated, high-order cells from multi-cellular organisms, such as insect and mammalian host cells.

[0064] Many of the steps noted below for the manipulation of polynucleotides and proteins, including digesting with restriction endonucleases, amplifying by PCR, hybridizing, ligating, separating and isolating by gel electrophoresis, transforming cells with heterologous DNA, selecting successful transformants, and the like, are well known and widely practiced by those skilled in the art and are not extensively elaborated upon herein. Unless otherwise noted, the standard protocols utilized herein are described extensively in Michael R. Green & Joseph Sambrook, "Molecular Cloning: A Laboratory Manual (Fourth Edition)," .COPYRGT. 2012, Cold Spring Harbor Laboratory Press: New York, N.Y., ISBN 978-1-936113-42-2.

[0065] Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0066] All references to singular characteristics or limitations described herein shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

[0067] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0068] The methods, compositions, and kits described herein can comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, preparatory steps, subsequence steps, or limitations described herein or otherwise useful or desired.

Overview:

[0069] Disclosed herein are nucleic acid sequences and amino acid sequences of a novel yellow fluorescent protein designated LucY. The nucleic acid sequences and amino acid sequences encoding the FP are useful for monitoring and visualizing physiological processes, such as protein localization, expression of genes, solubility of protein, etc. The LucY protein, its sequence variants, and homologues, have broad applicability in characterizing cells and organisms and in detecting or measuring various cellular parameters, notably in thermo-tolerant organisms.

[0070] LucY, its sequence variants, and homologs may also be used to identify protein binding peptide partners (such as protein-protein or protein-peptide interactions) at the cellular level. More particularly, the split-protein approach described below can be used to determine peptide partners, even if previously unknown, in bacteria and eukaryotes and can be used to visualize cellular and sub-cellular protein localization in multicellular organisms. The FP also can be used to monitor signaling processes and molecular interactions in conjunction with other fluorescent entities such as other fluorescent proteins via FRET. LucY, its sequence variants, and homologues can be used in these protocols, as well as in other conventional protocols that use fluorescent markers.

[0071] Thus, LucY, its sequence variants, and homologues can be used for coupling fluorescent protein variants to antibodies, polynucleotides or other receptors for use in detection assays such as immunoassays or hybridization assays, or to track the movement of proteins in cells. For intracellular tracking studies, a first polynucleotide encoding LucY, a LucY sequence variant, or a LucY homologue is fused to at least a second polynucleotide encoding a protein of interest. The construct, if desired, can be inserted into an expression vector. Upon expression inside a heterologous host cell, the protein of interest can be localized based on fluorescence.

[0072] LucY, its sequence variants, and its homologs are also useful in systems to detect induction of transcription. For example, a nucleotide sequence encoding a non-fluorescent protein can be fused to nucleotide sequence encoding LucY, and further linked to a promoter or other expression control sequence of interest, which can be contained in an expression vector. The construct is then transfected into a host cell. Induction of the promoter (or other regulatory element) is then measured by detecting the presence or amount of fluorescence, thereby enabling the responsiveness of a signaling pathway to be evaluated. These and other methods of using the LucY protein and its corresponding polynucleotide are described in greater detail below.

Kits:

[0073] A kit for use in transfecting host cells may be assembled using the nucleic acid molecules encoding the FPs, or for labeling target polypeptides with the FP. Host cell transfection kits may comprise at least one container containing one or more of the nucleic acid molecules encoding a FP (or a composition comprising one or more of the nucleic acid molecules or plasmids described herein), which nucleic acid molecule preferably comprises plasmid. These kits optionally may comprise at least one additional container that may contain, for example, a reagent for delivering the FP nucleic acid molecule into a host cell.

[0074] Further, kits may contain chemical reagents (e.g., polypeptides or polynucleotides) as well as other components. For example, kits may include apparatus and reagents for sample collection and/or purification, apparatus and reagents for product collection and/or purification, reagents for host cell transformation, reagents for eukaryotic cell transfection, previously transformed or transfected host cells, sample tubes, holders, trays, racks, dishes, plates, instructions to the kit user, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples. Kits may also be packaged for convenient storage and safe shipping. In some versions, the kits might include a FP as disclosed herein, a polynucleotide vector (e.g., a plasmid) encoding a FP as disclosed herein, bacterial cell strains suitable for propagating the vector, reagents for purifying the expressed fusion proteins, and the like. The FPs and kits using such proteins and/or their corresponding nucleotides may be configured or optimized to carry out one or more of the analytical methods described herein.

Samples Useful with LucY:

[0075] The samples that can be assayed or analyzed using LucY, its sequence variants, and its homologs include biological samples, environmental samples, or any other samples for which it is desired to determine whether a particular molecule is present therein. With some embodiments, the sample can include a cell or a cell extract from any source, without limitation (prokaryotic, eukaryotic, single-celled, multi-celled, etc.).

[0076] Further, the cells may be obtained from a culture of such cells, for example, a cell line, tissue line, or can be isolated from an organism. As such, the cell can be contained in a tissue sample, which can be obtained from an organism by any means commonly used to obtain a tissue sample, for example, by biopsy of a human or other organism. Where the method is performed using an intact living cell or a freshly isolated tissue or organ sample, the presence of a molecule of interest in living cells can be identified, thus providing a means to determine, for example, the intracellular compartmentalization of the molecule.

Measuring Fluorescence:

[0077] Methods for detecting the FP or of a cell expressing a FP may comprise, for example, illuminating the FP or cell expressing the FP with an illumination source such that the FP or cell expressing the FP emits radiation. Such detection methods may use an illumination source such as an incandescent light source, a fluorescent light source, a halogen light source, a laser light source, sunlight, and other equivalent sources. When illuminated by such an illumination source, the FP will emit fluorescent light that may be detected by unaided observation or by other qualitative or quantitative methods. Suitable methods for measuring fluorescence of samples are known and understood by those with ordinary skill in the art. Alternatively, the fluorescence signal and absorbance may be measured directly from the FP. The native LucY protein has strong absorbance maxima at 247, 376, and 460 nm. Thus the absorption at any of these wavelengths may be used to detect the FP via absorption spectroscopy. Further, the native LucY protein may be detected directly from a fluorescence emission at 530 nm.

[0078] Suitable methods for measuring fluorescence of samples are known and understood by those with ordinary skill in the art. They will not be described in any detail herein. Representative known methods of performing assays on fluorescent materials are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, (Plenum Press 1983); Herman, B., Resonance Energy Transfer Microscopy, Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, pp. 219-243 (ed. Taylor, D. L. & Wang, Y.-L., Academic Press 1989); Turro, N.J., Modern Molecular Photochemistry, pp. 296-361 (Benjamin/Cummings Publishing, Inc. 1978). There are numerous commercial suppliers of suitable fluorimeter, fluorescence spectroscopy, fluorescence microscopy, and confocal laser scanning microscopy equipment, such as Agilent Technologies (Santa Clara, Calif., USA; maker of Cary Eclipse-branded instruments) and PerkinElmer, Inc. (Waltham, Mass., USA).

[0079] One method for measuring fluorescence in samples is through the use of fluorimeters. Radiation is passed through the sample under controlled conditions. As the radiation passes through the sample at an excitation wavelength, the FP in the sample emits radiation at distinct wavelength(s), which then are captured as data by the fluorimeter. Both excitation and emission spectra are taken to determine the excitation and emission maxima for optimal fluorescence signals under any given set of conditions. The data is saved on a computer and or it can be further analyzed by computer. The scanned data is typically compared to control samples, such as calibration samples, negative and positive controls, and the like. The analyte concentration may be determined by extrapolating the fluorescence of the sample with a calibration curve.

LucY Identification and Isolation:

[0080] A 10 g sample of corn stalks collected from a field outside Middleton, Wis. in the fall of 2007 was added to YTP-2 medium containing (per liter) 2.0 g yeast extract, 2.0 g tryptone, 2.0 g sodium pyruvate, 1.0 g KCl, 2.0 g KNO.sub.3, 2.0 g Na.sub.2HPO.sub.4.7H.sub.2O, 0.1 g MgSO.sub.4, 0.03 g CaCl.sub.2, and 2.0 ml clarified tomato juice. (See Gao et al. (2011) Biotechnol Biofuels 4:5.) The sample was grown at 55.degree. C. and 200 rpm in a 2 L flask containing 1 L of medium. After 2 weeks the media was filtered through a Miracloth-brand filter (EMD Millipore, Philadelphia, Pa.; pore size: 22-25 .mu.m) and the remaining material was centrifuged to pellet the microbial cells. The cells from the enrichment culture were resuspended in lysis buffer and high molecular weight genomic DNA was purified using the Qiagen (Valencia, Calif.) Genomic-tip kit. The DNA was randomly fragmented to 3-6 kb using a Hydroshear Apparatus (Digilab, Marlborough, Mass.). The ends of the DNA were made blunt using the DNATerminator kit (Lucigen, Middleton, Wis.). The sheared, end-repaired DNA was gel purified and ligated to the pEZSeq vector (Lucigen, Middleton, Wis.). The ligation reaction was transformed into E. coli 10 G cells (Lucigen, Middleton, Wis.). The cells were plated on LB media containing 30 m/ml kanamycin and grown overnight at 37.degree. C. The plates containing several hundred colonies each were moved to a dark room and checked for fluorescence using a 365 nm long wavelength hand-held UV lamp (UVP, Upland, Calif.). A single colony out of approximately 1000 was a fluorescent yellow color under the UV lamp. The single yellow colony was grown overnight in terrific broth. The plasmid DNA was purified using standard procedures and the nucleotide sequence of the entire 3684 bp recombinant insert was determined by Sanger sequencing biochemistry on an Applied Biosystems 3100 Genetic Analyzer (Foster City, Calif.).

[0081] The DNA sequence of the yellow fluorescent recombinant insert was compared to the GenBank database using BLASTN (Altschul, et al., 1990) and close homology (83% sequence identity) was found to a segment of the Bacillus licheniformis ATCC 14580 genome. Analysis of the cloned region revealed that it contains homologues of bacterial murG and murB genes and portions of the spoVE and divIB genes. The murB gene encodes UDP-N-acetylenolpyruvoylglucosamine reductase, a flavoprotein that functions in the synthesis of the peptidoglycan cell wall (El Zoelby et al., 2003). The novel metagenomic clone encodes a MurB homologue with 95% amino acid sequence identity to the uncharacterized Bacillus licheniformis MurB protein. A lower level of homology to the well-studied MurB proteins from Staphylococcus aureus (39% identity; 63% similarity) and Escherichia coli (26% identity; 43% similarity) was noted. Previous studies have reported that the purified MurB protein from E. coli exhibits a yellow color (Benson et al., 1993). Quenching of the fluorescence of the tightly bound FAD cofactor has been used as the basis for evaluating binding of compounds to S. aureus MurB protein (Yang et al., 2006).

[0082] To confirm that the novel MurB protein is responsible for the observed fluorescence, its coding region (SEQ. ID. NO. 1) was cloned into a bacterial expression plasmid under the control of a bacteriophage T7 promoter. When this plasmid was introduced into BL21(DE3) cells expressing bacteriophage T7 RNA polymerase, colonies exhibited bright yellow fluorescence. Accordingly, the name LucY (Lucigen Yellow) was chosen for the novel MurB homologue. The amino acid sequence of the wild-type LucY protein is given in SEQ. ID. NO. 2.

Expression in Different Hosts:

[0083] E. coli: LucY has been expressed in bacterial cells under the control of the IPTG-inducible T7 promoter and the rhamnose-inducible promoter. Strong fluorescence was observed for both, as evidenced by FIGS. 1A, 1B, and 1C, and FIG. 2. FIG. 1A depicts cell pellets (1 OD) from cultures expressing LucY from the rhaP.sub.BAD promoter in response to rhamnose at the indicated concentrations. FIG. 1B is a graph that maps fluorescence response to rhamnose induction measured with a plate reader using excitation at 485 nm and emission at 528 nm. FIG. 1C is a photograph of LucY-transformed, fluorescent E. coli colonies on a plate containing 0.2% rhamnose.

[0084] Referring to the tubes and corresponding gel lanes in FIG. 2, LucY was expressed with a carboxyl terminal His6 tag using Lucigen's Expresso.TM.-brand T7 cloning and expression system. LucY-His6 was purified by metal affinity chromatography. Fractions from purification including whole cell pellet (lane 1), insoluble pellet after lysis (lane 2), soluble supernatant (lane 3), flow through (lane 4), wash (lane 5) and elution fractions (lanes 6-10) with imidazole were photographed under UV light (upper panel) and are aligned with the corresponding gel lanes.

[0085] HI-Control BL21(DE3) cells harboring the LucY gene under the control of the T7 promoter in Lucigen's pETite C-His vector were induced with 1 mM IPTG for 4 hours. For expression under the control of the rhamnose-inducible promoter, LucY was cloned into pRham (Lucigen), transformed into 10 G cells and expression was induced with varying percentages of rhamnose. Again see FIGS. 1A and 1B.

[0086] Mammalian cell culture: LucY was cloned into Lucigen's mammalian expression plasmid, pME, which contains the constitutively active cytomegalovirus (CMV) promoter. Various mammalian cell lines have been transfected with this construct, including CHO-K1, COS-7, and HeLa cell lines. Cells exhibiting strong yellow fluorescence were observed when visualized with Nikon Eclipse TE2000-S epifluorescence microscope, fitted with a Diagnostic Instruments 11.2 color camera and long pass GFP filter cube. See FIGS. 1D (expression of LucY in the mammalian cells COS-7), 1E (expression of LucY in the mammalian cells CHO-K1), and 1F (expression of LucY in the mammalian cells HeLa).

LucY Purification:

[0087] LucY was expressed in HI-Control BL21(DE3) E. coli cells with a C-terminal 6.times. histidine tag and purified by nickel affinity chromatography. See FIG. 2 (described previously). Clarified lysate was loaded onto an equilibrated Ni-NTA column and then washed with 30 column volumes wash buffer final pH 8.0; 10 mM Imidazole, 1.times.PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na phosphate dibasic, heptahydrate, and 1.9 mM K phosphate, monobasic) and eluted in 5 mL fractions with 5 column volumes of elution buffer final pH 8.0; 500 mM imidazole, 1.times.PBS pH 7.4. Fluorescent elution fractions visualized under UV light were pooled and dialyzed overnight in 2 liters 150 mM NaCl, 50 mM Tris pH 7.5 at 4.degree. C. LucY contained minimal aggregates after dialysis and was spun for 10 minutes at 10,000 rpm (12,062.times.g) to clarify prior to storage at -80.degree. C. Final protein concentration was determined to be 8 mg/mL by Bradford assay.

LucY Fluorescence Properties:

[0088] The fluorescence characteristics of LucY and related MurB proteins from S. aureus, E. coli, and T. thermophilus were determined through measurements on a Tecan Infinite M1000 monochromator-based plate reader (Tecan Group AG, Mannedorf, Switzerland). See FIG. 3 and Table 1. Excitation scans from 230 nm to 540 nm show maximal excitation at three wavelengths, 274 nm, 376 nm, and 460 nm.

TABLE-US-00001 TABLE 1 Fluorescence characteristics of LucY and other MurB homologs. Excitation Emission .PHI..sub.F .PHI..sub.F Brightness Max Max .epsilon. based on based on (% of (nm) (nm) (M.sup.-1cm.sup.-1) FAD FMN LucY) FAD 270/375/452 522 11,900 -- 0.032 9 FMN 268/376/448 525 12,500 0.273 -- 82 LucY 276/377/460 528 11,662 0.356 0.351 100 S. aureus 271/377/460 527 13,511 0.224 0.221 73 E. coli 276/369/460 527 12,008 0.109 0.108 32 T. thermophilus 276/375/460 522 13,745 0.051 0.051 17 .epsilon., extinction coefficient .PHI..sub.F, quantum yield Published .PHI..sub.F for FAD and FMN are 0.032 and 0.27, respectively.

[0089] Quantum yield estimates for LucY and other MurB family members were ascertained via the comparative method in relation to the well characterized FAD and flavin mononucleotide (FMN) cofactors. The quantum yield (.PHI..sub.F) of LucY was determined to be 0.349 and 0.357 using FAD or FMN as a reference standard, respectively. Brightness values were also ascertained by taking the product of the extinction coefficient and quantum yields for each fluorophore and are expressed as a percentage of LucY. Brightness values show that LucY enhances FAD brightness by 10-fold and that it is the brightest of the MurB homologs.

LucY Thermo-Tolerant Homologs:

[0090] Because of their extreme growth conditions, few fluorescent biological reporters are viable in thermophilic organisms. Thus, what are otherwise routine techniques using GFP are not possible in many thermophilic organisms. Enhanced thermo-tolerant fluorescent proteins also have great potential in the thermo-stabilization of GPCRs suitable for crystallization. Thus, a search was conducted for thermo-tolerant LucY homologs using the BLAST function of UniProt (www.uniprot.org). Candidate homologs that originated from thermophilic organisms were selected for further investigation. Seventeen of these genes (Table 2) were synthesized de novo with their coding sequences optimized for expression in E. coli (SEQ. ID. NOS. 5-76) and cloned under the control of an inducible promoter (DNA2.0-brand; DNA 2.0, Inc., Menlo Park, Calif., USA). Cultures were grown at 37.degree. C. in LB medium and expression was induced with IPTG. Induced cells were harvested by centrifugation and cell pellets were visualized with a hand-held UV lamp. See FIG. 4A. Proteins whose expression resulted in substantial fluorescence were purified by nickel affinity chromatography. The effect of temperature on fluorescence output was evaluated by incubating known quantities of protein at temperatures ranging from 30.degree. C. to 80.degree. C. and recording fluorescence every 15 minutes for 45 minutes in a Biotek Synergy 2 microplate fluorometer. LucY homologs from Y4.1MC1 and Thermoanaerobacter italicus (Thit) were the most thermostable LucY homologs in terms of fluorescence, with fluorescence diminishing to approximately 10% at 75.2.degree. C. and 79.0.degree. C. respectively. See FIG. 4B. Because Thit and Pma (Persephonella marina) were visually the brightest appearing homologs, quantum yield measurements were taken as stated above and are approximately, 0.485 and 0.406, respectively. FIG. 4C is a plot of percent fluorescence versus temperature following the 45 min incubation at temperatures ranging from 30.degree. C. to 80.degree. C. Fluorescence is expressed as a percentage of highest fluorescence recorded across temperature range.

TABLE-US-00002 TABLE 2 Thermo-tolerant LucY homologs UniProt Organism Abbreviation A0LRK5 Acidothermus cellulolyticus Acel F8IH59 Alicyclobacillus acidocaldarius Aac O66805 Aquifex aeolicus Aae E4Q8N4 Caldicellulosiruptor hydrothermalis Chy B8E323 Dictyoglomus turgidum Dtu E3IC15 Geobacillus strain Y4.1MC1 Y4.1MC1 G2SID5 SG0.5JP17-172 Rma C4FHX3 Sulfurihydrogenibium yellowstonense SS-5 Sye B5YFT2 Thermodesulfovibrio yellowstonii strain Tye ATCC 51303 Q9X239 Thermotoga maritima strain ATCC 43589 Tma B2V7Y9 Sulfurihydrogenibium sp. (strain YO3AOP1) SspY C1DVM7 Sulfurihydrogenibium azorense Saz C0QUP5 Persephonella marina Pma A8UZI2 Hydrogenivirga sp. 128-5-R1-1 Hsp1 D3DK91 Hydrogenobacter thermophilus Hth D3SPD6 Thermocrinis albus Tal D3T3U7 Thermoanaerobacter italicus Thit B4U6R2 Hydrogenobaculum sp. (strain Y04AAS1) HspY

LucY as a Fluorescent Protein Fusion Partner and as an Indicator of Protein Expression:

[0091] LucY has been used successfully as a reporter of protein expression. Fusing the LucY nucleotide sequence (and its variants and homologs) to various proteins of interest at different junction points have resulted in bright fluorescence.

[0092] Expression in bacterial cells: C-terminal fusions to G-protein coupled receptor (GPCR), specifically A1a, have been visualized by bright yellow fluorescence at both 25.degree. C. and 30.degree. C. See FIG. 5A. In the figure, LSP=low-speed pellet; HSP=high-speed pellet. FIG. 5B corresponds to FIG. 5A and depicts LucY fused to ATPb following induction with IPTG or arabinose. FIG. 5C is a coomassie stained SDS-PAGE of fractions from E. coli expressing the LucY-ATPb fusion shown in FIG. 5B. GPCR-containing extracellular membranes separate with the high-speed pellet (HSP). ATPb separates with intracellular membranes in the low-speed pellet (LSP). HSS=high-speed supernatant; LSS=low-speed supernatant. Similar data have been obtained when LucY was fused to the COR-kinase domain of the Parkinson's disease related protein LRRK2. See FIG. 6, which is a photograph under UV light of the E. coli host cells transformed to contain and express the COR-kinase-LucY fusion protein.

[0093] Expression in mammalian cells: Turkey .beta.1-adrenergic receptor fused to LucY in the 3.sup.rd intracellular loop has been expressed in mammalian cells (human embryonic kidney cells; HEK-293T) under the control of the CMV promoter. The recombinant receptor is expressed as indicated by visible yellow fluorescence. Varying the junction points of insertion did not alter the fluorescence of the fusion protein. See FIGS. 7A, 7B, 7C, and 7D. Each figure is three-part photo series depicting expression of LucY as an intracellular loop fusion in mammalian cells. Each three-part series shows the fusion illuminated with a bright field (left), using a GFP filter (middle), and a merged bright field/GFP image (right). Each set of photos represents fusion at a different site within the intracellular loop 3 of a GPCR. FIG. 7A illustrates fusion at positions 244-272; FIG. 7B at positions 244-GSG-272; FIG. 7C at positions 244-GSG-275; and FIG. 7D at positions 244-GSG-278. GSG is the linker sequence between LucY C-terminus and the GPCR.

[0094] Expression in insect cells: The same intracellular loop constructs used for expression in HEK-293T were also used to express LucY as a fusion protein in insect cells. When expressed in baculovirus-infected insect cells ("Hi 5"-brand cells), the cells exhibited positive protein expression as evidenced by the yellow fluorescence in the cell pellets. See FIG. 8, which is a photograph of the Hi-5 host cells transformed to express the LucY fusion protein.

LucY as a Label to Determine Subcellular Localization:

[0095] A genetically encoded fluorescent marker is extremely useful for determining cellular localization. LucY is a soluble, highly expressed protein amenable to fusion to a variety of proteins. Its fluorescence can be used to track the location of its fusion partner. The GPCR A1a protein preferentially fractionates with extracellular membranes, which can be separated by centrifugation at a low speed and visualized by fusion to LucY. See FIG. 5A, discussed earlier. Alternatively, the single-pass transmembrane protein ATPb fractionates with internal membranes following centrifugation at high speeds and can likewise be visualized by fusion to LucY. See FIGS. 5B and 5C, described previously.

LucY as an Indicator of Detergent Solubilization:

[0096] Tracking the fluorescence in the soluble fraction of a detergent solubilized sample for integral membrane proteins offers an easy and quick solution for high throughput screening of different solubilization combinations. ATPb (a single-pass membrane protein) was used as a typical example of a membrane protein to demonstrate the effectiveness of different screening conditions. Thirty-one (31) different detergents belonging to various classes were tested. Solubilization was performed over night at 4.degree. C. in an end-over rotor. The solubilized fraction was clarified with a high-speed centrifugation step and fluorescence was measured (FIG. 9A) as well as visualized (FIG. 9B) under UV light.

LucY as an Indicator of Protein Expression and Solubility.

[0097] Fluorescence enables straightforward visual evaluation of the expression and solubility of proteins fused to LucY. This visual readout can be exploited to identify expression conditions that improve protein expression, and to screen a variety of fusion partners that may promote soluble expression.

[0098] A large-scale effort to identify hydrolytic enzymes from Fibrobacter succinogenes identified several enzymes that were initially poorly expressed or insoluble in E. coli using a T7 bacteriophage polymerase expression system. Several of these recalcitrant proteins were cloned under control of the rhaP.sub.BAD promoter with carboxyl terminal fusions to LucY, with or without an amino terminal SUMO solubility tag. Expression of the fusion proteins was induced by inclusion of 0.2% rhamnose in plates or in liquid media. Fluorescence was monitored in colonies, in cell pellets from liquid cultures, and in soluble and insoluble fractions of induced cell lysates.

[0099] Results are shown in FIGS. 10A, 10B, and 10C for three different proteins exhibiting different expression levels. In the case of Fisuc_1793, no yellow fluorescence was observed in the absence of SUMO (FIG. 10A, left), and fusion to an amino-terminal SUMO tag led to a dramatic increase in yellow fluorescence (FIG. 10A, right). See also the top pair of tubes in FIG. 10B. A second example, Fisuc_2201, produced moderate yellow fluorescence in the absence of SUMO, but increased fluorescence with the SUMO tag (FIG. 10B, middle pair of tubes). A third case, Fisuc_2442, produced strong fluorescence that was not significantly enhanced by the SUMO tag (FIG. 10B, bottom pair of tubes).

[0100] Lysates from the induced cells were centrifuged to separate soluble and insoluble fractions, and fluorescence, when present, was found primarily in the supernatant (soluble) fraction in each case. Gel analysis of fusion protein expression by SDS-PAGE correlated well with the fluorescence results. See FIG. 10C (top panel is for 1793; middle panel for 2201; bottom panel for 2442). No detectable Fisuc_1793-LucY fusion protein was present in either the soluble or insoluble fraction when expressed without the SUMO tag, while the SUMO tagged counterpart was expressed well and in a mostly soluble form. Results with Fisuc_2201 and Fisuc_2442 similarly recapitulated the fluorescence data, with the SUMO tag enhancing the relatively weak expression of Fisucc_2201, but not significantly increasing the already strong expression of Fisuc_2442.

[0101] A potential limitation of whole cell fluorescence as an indicator of soluble expression is the possibility of false positives. Fluorescence may be observed, for example, if the proper folding of LucY is allowed despite formation of insoluble aggregates via a fusion partner. Importantly, the stable fluorescence of LucY allows the preparation and fractionation of lysates to evaluate the partitioning of fluorescence between soluble and insoluble fractions. Fluorescent fusion proteins that are found exclusively in the insoluble fraction can be eliminated from further consideration, potentially saving the time and expense of analysis by gel electrophoresis. Alternatively, the fluorescence can be used to screen for conditions that allow solubilization of the protein. For example, the use of nonionic detergents, or different buffer conditions (salt concentrations or pH) may allow dispersal of fluorescent aggregates and recovery of soluble protein.

[0102] Fluorescence can also be used as the basis for a solubility "trap" screen, in which an insoluble protein fused to LucY is used to identify for novel fusion partners that impart greater levels soluble expression. Two different insoluble proteins were used to evaluate this screening strategy. In both cases LucY was fused to the C terminus of an insoluble DNA polymerase. The DNA polymerase (DNAP) genes were derived from Acidianus bottle-shaped virus (ABV DNAP), or from a screen for novel thermostable DNAPs ("4110" DNAP). These DNAP-LucY fusions were cloned under the rhaP.sub.BAD promoter. Fluorescence development and expression were then monitored both on plates and in liquid media with and without 0.2% rhamnose. The effect of an amino-terminal SUMO fusion on the fluorescence development and solubility of each DNAP-LucY fusion protein was also tested. The ABV-LucY fusion protein was poorly soluble, and its solubility was not enhanced by the amino-terminal SUMO tag. The low-level solubility of the SUMO-ABV-LucY fusion resulted in weak yellow fluorescence on plates containing 0.2% rhamnose. See FIG. 11A. The 4110-LucY fusion was also largely insoluble, but fusion to SUMO partially rescued solubility. Both ABV-LucY and 4110-LucY were exploited as "solubility traps" in a screen for solubility-enhancing tags. FIG. 11B depicts the gel analysis of ABV-LucY protein expressed from rhaP.sub.BAD, with or without an amino-terminal SUMO tag. FIG. 11C depicts the gel analysis of 4110-LucY protein expressed from rhaP.sub.BAD, with or without a SUMO solubility tag. In FIGS. 11B and 11C, T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation.

[0103] For each DNAP-LucY fusion, separate libraries were constructed using genomic fragments from several sources including E. coli, bacteriophage lambda, and a thermophilic Geobacillus species. In each case, genomic DNA was physically sheared by nebulization to generate random fragments ranging in size from .about.100-700 bp. The DNA fragment ends were made blunt and phosphorylated using Lucigen's DNA terminator kit, and the fragments were cloned into the rhaP.sub.BAD expression vector between the ATG start codon and the second residue of the ABV-LucY or 4110-LucY solubility trap fusion. Library transformants were plated on media containing kanamycin and 0.2% rhamnose. In these small-scale test screens, approximately 5,000 to 10,000 clones were plated on each of 6 to 10 large-diameter (13 cm) plates. Plates were observed under illumination from a hand-held long-wavelength UV lamp, and colonies with varying degrees of fluorescence were detected. See FIG. 12A for an example of a primary screening plate and FIG. 12B for an example a secondary screen plate with re-streaked candidates. The 4110-LucY fusion was screened with bacteriophage .lamda., and Geobacillus genomic inserts, and the ABV-LucY solubility trap was screened with E. coli genomic inserts.

[0104] The ABV-LucY fusion was first screened with genomic inserts from E. coli. In this screen, bright fluorescent colonies were observed at a frequency of <0.1%. Twenty-two (22) of these bright fluorescent colonies were chosen for further analysis. All 22 were found to have significant deletions removing large portions of the ABV sequence, re-creating an in-frame fusion that presumably resulted in expression of an ABV-LucY fragment with increased solubility. While these results illustrate the utility of LucY fusions to map soluble domains of poorly-soluble proteins, the objective of the screen is to identify novel solubility tags. These deletion clones were not analyzed further.

[0105] Screening of the 4110-LucY solubility trap yielded several candidate solubility-enhancing tags. Small-scale screens of the 4110-LucY construct were conducted with two different genomic insert libraries derived from bacteriophage .lamda., and from a thermophilic Geobacillus species. Partial deletions of the 4110 DNAP coding region were obtained far less frequently than with the ABV-LucY trap construct. For the .lamda., insert library, 24 fluorescent clones were selected for analysis. Sequences were obtained for 23 of these clones, and all 23 were found to contain inserts of .lamda., genomic DNA. Twenty-two (22) clones were found to have fusions that restored the correct frame between the ATG initiation codon and the 4110-LucY coding region. A single clone contained a fragment that included a promoter and the amino-terminal portion of a protein coding region, fused in-frame to 4110-LucY. The genomic inserts ranged in size from 93 to 669 base-pairs, and encoded peptides of 31 to 223 residues. Interestingly, two different polypeptide regions were each represented twice by non-identical clones. The repeated isolation of these regions among only 23 clones of 100-700 base-pairs from the 48 kb lambda genome strongly suggests non-randomness in the screen results. Clones were grown in liquid LB media and induced with 0.2% rhamnose for preparation of lysates to evaluate solubility. An example of a library fusion showing a significant increase in solubility of the 4110-LucY protein is presented in FIGS. 13A and 13B. These two figures demonstrate the enhanced soluble expression of 4110-LucY fusion protein with amino-terminal fusion tags derived from the shotgun library screens. FIG. 13A is a gel analysis of soluble expression of Geobacillus-4110-LucY library clone 11. The Control sample is 4110-LucY with no amino-terminal fusion. T represents total cell lysate; S represents the soluble fraction after centrifugation of the lysate; and P represents the insoluble pellet fraction after centrifugation. FIG. 13B is a photograph depicting increased partitioning of yellow fluorescence to the soluble fraction with Geobacillus library clone 11. Again, S represents the soluble fraction after centrifugation of the lysate and P represents the insoluble pellet fraction after centrifugation.

The Split-LucY System for Protein-Protein Interaction Studies:

[0106] The ability of a monomeric protein to be split and reassembled was first demonstrated with ubiquitin (Johnsson and Varshavasky, 1994) and has since been adopted for use with reporter proteins like GFP (Ghosh et al., 2000) and luciferase (Paulmurugan and Gambhir, 2003). LucY is a 32.7 kDa protein made up of three discrete domains, suggesting candidate split points lie between domains. FIG. 14 presents a schematic diagram of the three domains of the native LucY. In practice, a reporter protein is split in half and each half is expressed as a translational fusion to two proteins of interest for which protein-protein interaction is a possibility and their interaction is assayed in living cells. The reporter protein provides an output signal, like fluorescence, only if its two halves are brought together in a complementary manner due to the interaction of the fusions. This is illustrated schematically in FIG. 15A. The yellow fluorescence emitted by the FAD-binding capacity of LucY is an ideal reporter of protein-protein interaction.

[0107] Five points were tested between the short loop between domains 1 and 2 (residues 84 to 88) and within the long loop between domains 2 and 3 (residues 217-234) of LucY. See FIG. 15B, which depicts these split points schematically. Antiparallel leucine zippers were used as an idealized model for interacting protein partners (Ghosh et al., 2000). Because these leucine zippers interact in an antiparallel fashion, one fragment of LucY was fused at the amino-terminus of a leucine zipper (Split Points SPaNZ1-5 and SPbNZ1-5) while the complementary fragment was fused to the carboxy-terminus of the partner leucine zipper (SPaCZ1-5 and SPbCZ1-5). This is depicted schematically in FIG. 15C. The letters a and b and numbers 1 through 5 indicate the location of the split; see Table 3. Matching split point numbers indicates that the CZ split point immediately follows the residue at the carboxy end of the NZ split point. Fusion pairs containing complementary LucY fragments (e.g. SPaNZ1+SPaCZ1; SPbNZ4+SPbCZ4, etc.) were tested for protein expression and for fluorescence complementation. Fluorescence produced by coexpressed fragment pairs fused to leucine zippers was compared to fluorescence of fully intact LucY fused to either the N- or C-terminus of one of the zippers (NZ and CZ). Fluorescence was determined visually in whole cell pellets and quantitatively by a fluorometer.

TABLE-US-00003 TABLE 3 Start and end residues for each split point (SP). NZ constructs start with a 6x-His tag. CZ constructs end with the linker sequence SLSTPPTPSTPPT, followed by an Avi-tag. Constructs destined for expression in mammalian cells instead contain an HA-tag. Split Pairs c and d were constructed from a circular permutation construct. Start End includes domains: SPaNZ1 D2 G84 1 SPaNZ2 D2 A85 1 SPaNZ3 D2 G86 1 SPaNZ4 D2 L87 1 SPaNZ5 D2 D88 1 SPaCZ1 A85 R303 2&3 SPaCZ2 G86 R303 2&3 SPaCZ3 L87 R303 2&3 SPaCZ4 D88 R303 2&3 SPaCZ5 H89 R303 2&3 SPbNZ1 D2 P217 1&2 SPbNZ2 D2 P221 1&2 SPbNZ3 D2 S225 1&2 SPbNZ4 D2 N229 1&2 SPbNZ5 D2 H234 1&2 SPbCZ1 V218 R303 3 SPbCZ2 C222 R303 3 SPbCZ3 I226 R303 3 SPbCZ4 P230 R303 3 SPbCZ5 A235 R303 3 SPcNZ1 L87 P217 2 SPcCZ1 V218 G86 3&1 SPcNZ2 L87 H234 2 SPcCZ2 A235 G86 3&1 SPdNZ1 P217 G86 3&1 SPdCZ1 L87 Q216 2

[0108] Fragmenting LucY at any of the five residues between domains 1 and 2 did not result in fluorescence when fused to leucine pairs and coexpressed (FIG. 16A). A contributing factor may have been the low expression of the CZ half of each of the split points in comparison to the complementary NZ half. See the gel shown in FIG. 17A. However, bright fluorescence was seen in four out of the five pairs split between domains 2 and 3, with highest fluorescence seen with the SPbNZ5 (ending at His 234) and SPbCZ5 partners (beginning at A1a 235). See FIG. 16B. All fusions expressed well and expression did not correlate with fluorescence. See the gel shown in FIG. 17B. Interestingly, the one split point combination between domains 2 and 3 that did not show fluorescence was at Ser 225, a residue previously shown to be important for catalysis in other MurB proteins (Benson et al., 1995).

[0109] Because the SPbNZ5 and SPbCZ5 when reconstituted showed the highest fluorescence, each was tested with other split points between domains 2 and 3 to determine if overlap, or alternatively gaps, in the amino acid sequence would affect fluorescence. Pairing SPbNZ5 with SPbCZ4 was the most successful reconstituted pair and contains a four-residue overlap with SPbNZ5 ending at residue 234 and SPbCZ4 beginning at residue 230. Interestingly, the opposite pair (SPbNZ4 and SPbCZ5) did not show substantial fluorescence. See FIG. 16C. All pairs showed some level of expression. See FIG. 17C.

[0110] To verify that neither LucY fragment was fluorescent on its own and that the leucine zippers were driving the interaction, SPbNZ5 and SPbCZ4 were tested independently and without their leucine zipper fusions. Neither SPbNZ5 nor SPbCZ4 were fluorescent on their own. However, when expressed on its own, SPbNZ5 was less soluble than when coexpressed with SPbCZ4. Removal of the leucine zipper from SPbNZ5 did not affect expression, while removal of the leucine zipper from SPbCZ4 caused a loss of expression. See FIG. 18A. Because the zipperless SPbC4 was not expressed, it was not possible to fully obtain the background fluorescence level of the SPb:NZ5+CZ4 pair. Therefore, the fluorescence of the remaining SPbCZ fragments that exhibited fluorescence were used as a measure when coexpressed with SPbNZ5. Only SPbCZ1 and SPbCZ5 expressed when their zippers were removed. See FIG. 18B. Therefore these were chosen for further study. It was found that the SPb:NZ5+CZ5 pair possessed the highest level of fluorescence (33% of CZ control) with the least background (8% of CZ control). See FIG. 16D, and the corresponding gel showing expression in FIG. 17D.

[0111] Because LucY fluorescence is dependent on the binding of a non-covalently attached small molecule, fluorescence emission will dissipate following separation of the interacting pairs and release of the small molecule. Thus, the split-LucY system is reversible making it possible to test for inhibitors of protein interaction.

Circular Permutation of LucY:

[0112] Reorganization of a polypeptide chain, called circular permutation, has been used as a means to introduce change into a protein scaffold without amino acid substitutions (Yu and Lutz, 2011) and has been successfully investigated with GFP (Baird et al., 1999). A welcome consequence to domain reorganization of LucY would be an increase in fluorescence due to increased binding affinity to FAD. Novel termini also offer variation in points of attachment to fusion partners, as well as new possibilities in split points. The wild-type LucY amino- and carboxy-termini are 16.7 .ANG. apart and thus require a greater than four-residue linker to span the intervening space. To build a circularly permuted LucY, two tandem repeats were constructed with a 6-residue linker connecting the C-terminus of one copy to the N-terminus of the second. Deletions within this construct were performed such that new N and C termini were introduced between either domains 1 and 2 or between domains 2 and 3. This is shown schematically in FIG. 19A. The same residues used as split points above were used as breakpoints (BPs) here. Ten (10) circularly permuted LucY proteins were made. BP1-5 comprised a 2-3-1 domain arrangement, while BP6-10 comprised a 3-1-2 domain arrangement. See Table 4.

TABLE-US-00004 TABLE 4 N- and C-terminal residues of LucY circular permutations, with order of domain occurrence listed. Amino acid numbering is that of wild-type. N-term C-term Domain arrangement BP1 G84 L83 2-3-1 BP2 A85 G84 2-3-1 BP3 G86 A85 2-3-1 BP4 L87 G86 2-3-1 BP5 D88 L87 2-3-1 BP6 P217 Q216 3-1-2 BP7 P221 N220 3-1-2 BP8 S225 G224 3-1-2 BP9 N229 R228 3-1-2 BP10 H234 D233 3-1-2

[0113] Circularly permuted LucY proteins all showed some degree of fluorescence. See the histogram of FIG. 19B. However, none of the circularly permuted LucY variations were any brighter than the wild-type. The best candidate was BP6, which has a 3-1-2 domain arrangement beginning at the long loop connecting domains 2 and 3 in wild-type. Four out of the 10 circular permutation trials (BP4, BP7, BP9 and BP10) resulted in poorly expressed variants. See the gel of FIG. 20A. New split points became available by reorganizing the domain architecture of LucY, such that domains 3 and 1 could make up one LucY fragment and domain 2 could make up the other. To determine if a split-LucY system of this type was more viable then previous trials, three (3) additional split pairs were made: SPcNZ1/CZ1, SPcNZ2/CZ2, and SPdNZ1/CZ1. SPc pairs comprise domain 2 as the SPNZ fragment and domains 3 and 1 as the SPCZ fragment, with the numbers indicating different start and end locations. SPd comprises the opposing pairing, with domains 3 and 1 as the SPNZ fragment and domain 2 as the SPCZ fragment. See Table 3. These new split pairings did not exhibit fluorescence when reconstituted despite high levels of expression. See FIG. 19C and FIG. 20B, which further show that a domain 1+2/domain 3 pair (as in SPb fusions) offers the best split-LucY system.

[0114] Protein complementation assays like BiFC are often used in higher order systems, such as mammalian cells. Therefore, the best leucine zipper split-LucY pair from the above testing (SPbNZ5+SPbCZ4) was tested in HEK 293-T cells (a mammalian host cell). Only when each fragment was present did fluorescence occur. The results are depicted in the photographic series of FIGS. 21A through 21J. FIGS. 21A, 21B, 21C, 21D, and 21E are band pass filter photos of NZ-transformed cells, CZ-transformed cells, SPbNZ5-transformed cells, SPbCZ4-transformed cells, and SPbNZ5+SPbCZ4-transformed cells, respectively. FIGS. 21F, 21G, 21H, 21I, and 21J are merged band pass and bright field filter photos of NZ-transformed cells, CZ-transformed cells, SPbNZ5-transformed cells, SPbCZ4-transformed cells, and SPbNZ5+SPbCZ4-transformed cells, respectively. FIG. 21K is a photograph of an immunoblot gel using anti-HA to detect HA-tagged leucine zipper/LucY fusions either whole, NZ and CZ, or splits SPbNZ5 and SPbCZ4.

LucY as a Visualization Tool for Crystallization:

[0115] Typically protein crystals are visualized using a standard light microscope. When LucY was crystallized for the first time, bright fluorescent yellow crystals were visualized. Observation of the fluorescent crystals opens up a new avenue for the application of LucY in which crystal detection is problematic due to small crystal size, murky mother liquor, and/or excessive precipitation. One such challenging application is growth of membrane crystals in Lipidic Cubic Phase (LCP) (Caffrey, 2009), which mimics the lipid environment in which membrane proteins are most stable. Fusion of LucY to a membrane protein of interest provides a fluorescent beacon for successful crystallization. LucY crystals themselves form within 24 hours in a variety of conditions using a conventional hanging drop method and were easily visualized using a UV light source and light microscope. FIG. 22A is an exemplary photograph. LucY was fused to the GPCR, .beta.1-adrenergic receptor and the resulting fusion protein crystallized using LCP. Screening for crystals within a LCP matrix was visualized with Nikon Eclipse TE2000-S epifluorescence microscope, fitted with a Diagnostic Instruments 11.2 color camera and long pass GFP filter cube. Exemplary photographs of the fusion protein (LucY-.beta.1-adrenergic receptor) in a LCP matrix are shown in FIGS. 22B, 22C, 22D, and 22E.

LucY as a Solubility-Enhancing Fusion Partner:

[0116] Solubility-enhancing fusion partners such as maltose binding protein (MBP), thioredoxin (TRX), small ubiquitin-like modifier (SUMO), glutathione S-transferase (GST), NusA, and others are most frequently employed as fusions to the amino terminus of the protein of interest. In this context, fusion of LucY to the carboxyl terminus of the target protein provides a useful visualization tag to evaluate solubility of the fusion protein. Examples presented above illustrate this application of LucY.

In addition to functioning as a visual indicator of soluble expression, it has been found that fusion to LucY at the carboxyl terminus of a target protein can also enhance the soluble expression of the protein, regardless of fusion to an amino-terminal partner. FIGS. 23A, 23B, and 23C present an example in which the Tobacco Etch Virus (TEV) protease was expressed in E. coli from the rhamnose-inducible rhaP.sub.BAD promoter. Consistent with previous studies (Kapust and Waugh 1999; van den Berg et al. 2006), TEV protease expressed with a C-terminal 6.times.His tag was found almost exclusively in the insoluble (pellet) fraction after centrifugation of a cell lysate. In contrast, expression of TEV protease with LucY fused to its C-terminus resulted in expression of a large proportion of the TEV-LucY fusion protein in a soluble form. See FIGS. 23A and 23B. The proportion of TEV-LucY fusion protein found in the soluble fraction is comparable to that reported with an amino-terminal MBP-TEV fusion protein, and greater than that observed with amino-terminal GST or TRX (Kapust and Waugh 1999). Thus, LucY effectively promotes the soluble expression of TEV protease when fused to the protease as a C-terminal partner.

[0117] The TEV-LucY-His6 fusion protein was purified by nickel-affinity chromatography and assayed for sequence-specific protease activity using a purified substrate consisting of two similar-sized fluorescent proteins joined by a linker containing the TEV protease recognition sequence, ENLYFQ/G. FIG. 23C shows that incubation of the TEV-LucY-His6 fusion protein with the substrate protein results in generation of products. Thus the TEV-LucY-His6 fusion protein exhibits the sequence-specific proteolytic activity of TEV protease.

REFERENCES CITED

[0118] The following documents are incorporated herein by reference.

[0119] Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215:403-410 (1990).

[0120] Baird, G. S., Zacharias, D. A. & Tsien, R. Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 96, 11241-11246 (1999).

[0121] Benson, T. E., Filman, D. J., Walsh, C. T. & Hogle, J. M. An enzyme-substrate complex involved in bacterial cell wall biosynthesis. Nat Struct Biol 2, 644-653 (1995).

[0122] Caffrey M. Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu Rev Biophys 38, 29-51 (June 2009).

[0123] Chen J, Zheng X F, Brown E J, Schreiber S L (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA. 92:4947-4951.

[0124] Day, R. N. & Davidson, M. W. The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38, 2887-2921 (2009).

[0125] El Zoeiby, A., Sanschagrin, F. & Levesque, R. C. Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol 47, 1-12 (2003).

[0126] Farrar M A, Alberol-Ila J, Perlmutter R M (1996) Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization. Nature. 383:178-181.

[0127] Ghosh, I., Hamilton, A. & Regan, L. Antiparallel leucine zipper-directed protein reassembly: Application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658-5659 (2000).

[0128] Gilbert E J, Maxwell A (1994) The 24 kDa N-terminal sub-domain of the DNA gyrase B protein binds coumarin drugs. Mol Microbiol. 12:365-373.

[0129] Johnsson, N. & Varshaysky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci USA 91, 10340-10344 (1994).

[0130] Munro, A. W. & Noble, M. A. Fluorescence analysis of flavoproteins. Methods Mol Biol 131, 25-48 (1999).

[0131] Paulmurugan, R. & Gambhir, S. S. Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Anal Chem 75, 1584-1589 (2003).

[0132] Robida A M, Kerppola T K (2009) Bimolecular fluorescence complementation analysis of inducible protein interactions: effects of factors affecting protein folding on fluorescent protein fragment association. J Mol Biol. 394:391-409.

[0133] Rollins C T, Rivera V M, Woolfson D N, Keenan T, Hatada M, Adams S E, Andrade L J, Yaeger D, van Schravendijk M R, Holt D A, Gilman M, Clackson T (2000) A ligand-reversible dimerization system for controlling protein-protein interactions. Proc Natl Acad Sci USA. 97:7096-8101.

[0134] Tsien, R. Y. The green fluorescent protein. Annu Rev Biochem 67, 509-544 (1998).

[0135] Yu, Y. & Lutz, S. Circular permutation: a different way to engineer enzyme structure and function. Trends Biotechnol 29, 18-25 (2011).

[0136] Zhao H F, Boyd J, Jolicoeur N, Shen S H (2003) A coumermycin/novobiocin-regulated gene expression system. Hum Gene Ther. 14:1619-1629.

Sequence CWU 1

1

761909DNAUnknownNucleotide sequence was isolated from a metagenomic enrichment culture of unknown microbes living on corn stover and grown at 55 degrees C. 1atg gat aag gtg ata caa gaa tta aaa gat ctt caa gtc ggc aaa gtt 48Met Asp Lys Val Ile Gln Glu Leu Lys Asp Leu Gln Val Gly Lys Val 1 5 10 15 ctg gaa aat gaa ccg ctc gca aat cat acg acg att aaa atc ggc ggc 96Leu Glu Asn Glu Pro Leu Ala Asn His Thr Thr Ile Lys Ile Gly Gly 20 25 30 cct gcc gat tgt ctc gtc att cca aag gac att cag gcc gtc cgg gat 144Pro Ala Asp Cys Leu Val Ile Pro Lys Asp Ile Gln Ala Val Arg Asp 35 40 45 acg atg gaa gtc gtg aaa aag cat ggc gtc caa tgg agg gcg atc ggc 192Thr Met Glu Val Val Lys Lys His Gly Val Gln Trp Arg Ala Ile Gly 50 55 60 aga ggc tca aac ctt ctc gtt ctt gat gaa ggc att agg ggc gtc gtc 240Arg Gly Ser Asn Leu Leu Val Leu Asp Glu Gly Ile Arg Gly Val Val 65 70 75 80 atc aag ctc gga gca ggg ctc gat cat atg gaa atc gac ggc gag cag 288Ile Lys Leu Gly Ala Gly Leu Asp His Met Glu Ile Asp Gly Glu Gln 85 90 95 gtg acg gtc ggc gga ggc tat tcc gtc gtg cgg ctg tct act ggc atc 336Val Thr Val Gly Gly Gly Tyr Ser Val Val Arg Leu Ser Thr Gly Ile 100 105 110 agc aaa aaa ggg ctt tca ggc ctt gaa ttt gca tca ggc att ccg gga 384Ser Lys Lys Gly Leu Ser Gly Leu Glu Phe Ala Ser Gly Ile Pro Gly 115 120 125 tct gtc ggg gga gcg gtg tac atg aac gcc ggc gcc cac ggc tca gat 432Ser Val Gly Gly Ala Val Tyr Met Asn Ala Gly Ala His Gly Ser Asp 130 135 140 atc agc cgg att ttg gtt aaa gct tta att ctc ttt gaa gac ggg acg 480Ile Ser Arg Ile Leu Val Lys Ala Leu Ile Leu Phe Glu Asp Gly Thr 145 150 155 160 atg gaa tgg ctg acg aac gaa gag atg gaa ttc agc tac cgc aca tca 528Met Glu Trp Leu Thr Asn Glu Glu Met Glu Phe Ser Tyr Arg Thr Ser 165 170 175 atc ctg cag aac aag cgg ccg ggc att tgc ctt gaa gcg gtt ctg cag 576Ile Leu Gln Asn Lys Arg Pro Gly Ile Cys Leu Glu Ala Val Leu Gln 180 185 190 ctc gaa caa aaa gag cgc gac gcg atc gtc gca caa atg caa aaa aac 624Leu Glu Gln Lys Glu Arg Asp Ala Ile Val Ala Gln Met Gln Lys Asn 195 200 205 aaa gac tac cgg aag gaa acg cag cct gtc tca aac cct tgc gcc gga 672Lys Asp Tyr Arg Lys Glu Thr Gln Pro Val Ser Asn Pro Cys Ala Gly 210 215 220 agc atc ttc aga aat ccc ctc ccg gat cac gcc gga aga ctc gtt gaa 720Ser Ile Phe Arg Asn Pro Leu Pro Asp His Ala Gly Arg Leu Val Glu 225 230 235 240 cag gcc ggg ctg aaa ggg cat cgg atc ggc gga gca aag gtt tcc gaa 768Gln Ala Gly Leu Lys Gly His Arg Ile Gly Gly Ala Lys Val Ser Glu 245 250 255 atg cac ggc aac ttc atc gtc aac gca ggc ggc gca acc gca aaa gac 816Met His Gly Asn Phe Ile Val Asn Ala Gly Gly Ala Thr Ala Lys Asp 260 265 270 gtt ctt gat ttg att gcg ttt atc caa aaa acg att aaa gaa aaa tac 864Val Leu Asp Leu Ile Ala Phe Ile Gln Lys Thr Ile Lys Glu Lys Tyr 275 280 285 gat atc gac atg cac acg gaa gtt gaa atc gtc gga gaa aaa cgg 909Asp Ile Asp Met His Thr Glu Val Glu Ile Val Gly Glu Lys Arg 290 295 300 2303PRTUnknownSynthetic Construct 2Met Asp Lys Val Ile Gln Glu Leu Lys Asp Leu Gln Val Gly Lys Val 1 5 10 15 Leu Glu Asn Glu Pro Leu Ala Asn His Thr Thr Ile Lys Ile Gly Gly 20 25 30 Pro Ala Asp Cys Leu Val Ile Pro Lys Asp Ile Gln Ala Val Arg Asp 35 40 45 Thr Met Glu Val Val Lys Lys His Gly Val Gln Trp Arg Ala Ile Gly 50 55 60 Arg Gly Ser Asn Leu Leu Val Leu Asp Glu Gly Ile Arg Gly Val Val 65 70 75 80 Ile Lys Leu Gly Ala Gly Leu Asp His Met Glu Ile Asp Gly Glu Gln 85 90 95 Val Thr Val Gly Gly Gly Tyr Ser Val Val Arg Leu Ser Thr Gly Ile 100 105 110 Ser Lys Lys Gly Leu Ser Gly Leu Glu Phe Ala Ser Gly Ile Pro Gly 115 120 125 Ser Val Gly Gly Ala Val Tyr Met Asn Ala Gly Ala His Gly Ser Asp 130 135 140 Ile Ser Arg Ile Leu Val Lys Ala Leu Ile Leu Phe Glu Asp Gly Thr 145 150 155 160 Met Glu Trp Leu Thr Asn Glu Glu Met Glu Phe Ser Tyr Arg Thr Ser 165 170 175 Ile Leu Gln Asn Lys Arg Pro Gly Ile Cys Leu Glu Ala Val Leu Gln 180 185 190 Leu Glu Gln Lys Glu Arg Asp Ala Ile Val Ala Gln Met Gln Lys Asn 195 200 205 Lys Asp Tyr Arg Lys Glu Thr Gln Pro Val Ser Asn Pro Cys Ala Gly 210 215 220 Ser Ile Phe Arg Asn Pro Leu Pro Asp His Ala Gly Arg Leu Val Glu 225 230 235 240 Gln Ala Gly Leu Lys Gly His Arg Ile Gly Gly Ala Lys Val Ser Glu 245 250 255 Met His Gly Asn Phe Ile Val Asn Ala Gly Gly Ala Thr Ala Lys Asp 260 265 270 Val Leu Asp Leu Ile Ala Phe Ile Gln Lys Thr Ile Lys Glu Lys Tyr 275 280 285 Asp Ile Asp Met His Thr Glu Val Glu Ile Val Gly Glu Lys Arg 290 295 300 3912DNAArtificial SequenceNucleotide sequence was isolated from a metagenomic enrichment culture of unknown microbes living on corn stover and grown at 55 degrees C and then optimized for expression in a heterologous host. 3atg gat aaa gtc att caa gag ctt aag gac ctc caa gtg gga aaa gtg 48Met Asp Lys Val Ile Gln Glu Leu Lys Asp Leu Gln Val Gly Lys Val 1 5 10 15 ctt gag aac gaa ccg ctc gca aac cac aca acg atc aag att ggc gga 96Leu Glu Asn Glu Pro Leu Ala Asn His Thr Thr Ile Lys Ile Gly Gly 20 25 30 ccg gct gat tgt ctc gtg atc ccc aag gac att cag gcc gtc aga gac 144Pro Ala Asp Cys Leu Val Ile Pro Lys Asp Ile Gln Ala Val Arg Asp 35 40 45 acg atg gag gtc gtg aag aag cat ggg gtc cag tgg cgc gca atc gga 192Thr Met Glu Val Val Lys Lys His Gly Val Gln Trp Arg Ala Ile Gly 50 55 60 agg ggc agc aac ttg ttg gtg ctt gac gag ggt atc cgg ggt gta gtg 240Arg Gly Ser Asn Leu Leu Val Leu Asp Glu Gly Ile Arg Gly Val Val 65 70 75 80 att aag ctg gga gct ggg ttg gat cac atg gaa atc gac ggt gag cag 288Ile Lys Leu Gly Ala Gly Leu Asp His Met Glu Ile Asp Gly Glu Gln 85 90 95 gtg acc gtg gga gga ggg tat tcc gta gta cgc ctg tca aca ggg att 336Val Thr Val Gly Gly Gly Tyr Ser Val Val Arg Leu Ser Thr Gly Ile 100 105 110 tca aag aaa ggg ttg tcg ggg ttg gag ttc gca agc ggg att cct ggt 384Ser Lys Lys Gly Leu Ser Gly Leu Glu Phe Ala Ser Gly Ile Pro Gly 115 120 125 tca gta ggt ggt gca gtg tat atg aac gcg ggt gcc cat ggg tcc gac 432Ser Val Gly Gly Ala Val Tyr Met Asn Ala Gly Ala His Gly Ser Asp 130 135 140 att tcg cgg atc ctt gtc aaa gcc ctc att ctg ttc gaa gat ggt aca 480Ile Ser Arg Ile Leu Val Lys Ala Leu Ile Leu Phe Glu Asp Gly Thr 145 150 155 160 atg gaa tgg ctc act aac gaa gag atg gag ttt tca tac cga acg tcg 528Met Glu Trp Leu Thr Asn Glu Glu Met Glu Phe Ser Tyr Arg Thr Ser 165 170 175 atc ctc caa aac aaa agg cca gga atc tgc ttg gaa gcg gta ttg cag 576Ile Leu Gln Asn Lys Arg Pro Gly Ile Cys Leu Glu Ala Val Leu Gln 180 185 190 ctg gaa cag aaa gag cga gat gcc atc gtg gca cag atg cag aag aac 624Leu Glu Gln Lys Glu Arg Asp Ala Ile Val Ala Gln Met Gln Lys Asn 195 200 205 aaa gac tac cgg aaa gaa acc cag ccg gtg tcg aat ccg tgc gcg ggt 672Lys Asp Tyr Arg Lys Glu Thr Gln Pro Val Ser Asn Pro Cys Ala Gly 210 215 220 agc atc ttt cgc aat ccc ctg ccc gat cat gcg gga aga ttg gtg gaa 720Ser Ile Phe Arg Asn Pro Leu Pro Asp His Ala Gly Arg Leu Val Glu 225 230 235 240 caa gcc ggc ctt aag gga cac cag atc gga ggg gcc aag gta tcg gag 768Gln Ala Gly Leu Lys Gly His Gln Ile Gly Gly Ala Lys Val Ser Glu 245 250 255 atg cac ggg aat ttc atc gtc aat gcg gga ggg gcg act gcg aag gat 816Met His Gly Asn Phe Ile Val Asn Ala Gly Gly Ala Thr Ala Lys Asp 260 265 270 gtc ctc gac ctg atc gcg ttt atc caa aag acg atc aag gag aag tac 864Val Leu Asp Leu Ile Ala Phe Ile Gln Lys Thr Ile Lys Glu Lys Tyr 275 280 285 gac att gat atg cat acc gaa gtc gag att gtc ggc gag aaa agg tga 912Asp Ile Asp Met His Thr Glu Val Glu Ile Val Gly Glu Lys Arg 290 295 300 4303PRTArtificial SequenceSynthetic Construct 4Met Asp Lys Val Ile Gln Glu Leu Lys Asp Leu Gln Val Gly Lys Val 1 5 10 15 Leu Glu Asn Glu Pro Leu Ala Asn His Thr Thr Ile Lys Ile Gly Gly 20 25 30 Pro Ala Asp Cys Leu Val Ile Pro Lys Asp Ile Gln Ala Val Arg Asp 35 40 45 Thr Met Glu Val Val Lys Lys His Gly Val Gln Trp Arg Ala Ile Gly 50 55 60 Arg Gly Ser Asn Leu Leu Val Leu Asp Glu Gly Ile Arg Gly Val Val 65 70 75 80 Ile Lys Leu Gly Ala Gly Leu Asp His Met Glu Ile Asp Gly Glu Gln 85 90 95 Val Thr Val Gly Gly Gly Tyr Ser Val Val Arg Leu Ser Thr Gly Ile 100 105 110 Ser Lys Lys Gly Leu Ser Gly Leu Glu Phe Ala Ser Gly Ile Pro Gly 115 120 125 Ser Val Gly Gly Ala Val Tyr Met Asn Ala Gly Ala His Gly Ser Asp 130 135 140 Ile Ser Arg Ile Leu Val Lys Ala Leu Ile Leu Phe Glu Asp Gly Thr 145 150 155 160 Met Glu Trp Leu Thr Asn Glu Glu Met Glu Phe Ser Tyr Arg Thr Ser 165 170 175 Ile Leu Gln Asn Lys Arg Pro Gly Ile Cys Leu Glu Ala Val Leu Gln 180 185 190 Leu Glu Gln Lys Glu Arg Asp Ala Ile Val Ala Gln Met Gln Lys Asn 195 200 205 Lys Asp Tyr Arg Lys Glu Thr Gln Pro Val Ser Asn Pro Cys Ala Gly 210 215 220 Ser Ile Phe Arg Asn Pro Leu Pro Asp His Ala Gly Arg Leu Val Glu 225 230 235 240 Gln Ala Gly Leu Lys Gly His Gln Ile Gly Gly Ala Lys Val Ser Glu 245 250 255 Met His Gly Asn Phe Ile Val Asn Ala Gly Gly Ala Thr Ala Lys Asp 260 265 270 Val Leu Asp Leu Ile Ala Phe Ile Gln Lys Thr Ile Lys Glu Lys Tyr 275 280 285 Asp Ile Asp Met His Thr Glu Val Glu Ile Val Gly Glu Lys Arg 290 295 300 5894DNAAquifex aeolicusCDS(1)..(894) 5atg ctc ttc cta aag aac gtt ccc ctt cag aac ctt acg act ata aaa 48Met Leu Phe Leu Lys Asn Val Pro Leu Gln Asn Leu Thr Thr Ile Lys 1 5 10 15 ata ggg gga agg gta tcc ttt tac gca gag cct tcc gat cta aag gaa 96Ile Gly Gly Arg Val Ser Phe Tyr Ala Glu Pro Ser Asp Leu Lys Glu 20 25 30 att tcc cta tgt att gat ttt tca aaa tcc cga gac att cct ctt ttt 144Ile Ser Leu Cys Ile Asp Phe Ser Lys Ser Arg Asp Ile Pro Leu Phe 35 40 45 gtt ttg ggt aac ggt tct aat act att ttc ggt gac gta aga ggg ctc 192Val Leu Gly Asn Gly Ser Asn Thr Ile Phe Gly Asp Val Arg Gly Leu 50 55 60 gtt gta aat tta aaa aac tta aaa ggt ttt aaa gta aaa gaa att aaa 240Val Val Asn Leu Lys Asn Leu Lys Gly Phe Lys Val Lys Glu Ile Lys 65 70 75 80 ggg aaa ttt ttt gta gaa gct ttt tcc gga acg cct tta aag gat tta 288Gly Lys Phe Phe Val Glu Ala Phe Ser Gly Thr Pro Leu Lys Asp Leu 85 90 95 ata agg ttt agt gta aag gaa aat gta aag agt ttt tac aaa ctt ctc 336Ile Arg Phe Ser Val Lys Glu Asn Val Lys Ser Phe Tyr Lys Leu Leu 100 105 110 ggt ttt ccg gca agt gtc ggg gga gcg gtt agt atg aac gcc ggg gct 384Gly Phe Pro Ala Ser Val Gly Gly Ala Val Ser Met Asn Ala Gly Ala 115 120 125 ttt ggg gtt gag ata tcg gat ttt tta aag gaa gtt tac ttc gta gat 432Phe Gly Val Glu Ile Ser Asp Phe Leu Lys Glu Val Tyr Phe Val Asp 130 135 140 tgg gag ggg aaa ctc caa aaa gca aaa agg gat gaa ctg aat ttt tct 480Trp Glu Gly Lys Leu Gln Lys Ala Lys Arg Asp Glu Leu Asn Phe Ser 145 150 155 160 tac aga aaa tcg cct ttt cca aaa ctt gga ata gtt ttc aaa gta gtt 528Tyr Arg Lys Ser Pro Phe Pro Lys Leu Gly Ile Val Phe Lys Val Val 165 170 175 ttt gag ttt gaa aga agt aaa gaa aat ata ctt ccc aag tac gaa aaa 576Phe Glu Phe Glu Arg Ser Lys Glu Asn Ile Leu Pro Lys Tyr Glu Lys 180 185 190 ata aga aga ata agg aaa gaa aag caa cct ata aac ctt cca acc agc 624Ile Arg Arg Ile Arg Lys Glu Lys Gln Pro Ile Asn Leu Pro Thr Ser 195 200 205 ggt tct acc ttc aaa aat ccg gag ggt aat ttc gcg gga aag ctt ctg 672Gly Ser Thr Phe Lys Asn Pro Glu Gly Asn Phe Ala Gly Lys Leu Leu 210 215 220 gaa aaa gca ggt tta aaa ggt ttt aga ctt aaa aac gta gga ttt tcc 720Glu Lys Ala Gly Leu Lys Gly Phe Arg Leu Lys Asn Val Gly Phe Ser 225 230 235 240 gaa aaa cac gct aac ttc ctt gta aac tac gga ggt gga act ttt tcg 768Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Gly Gly Thr Phe Ser 245 250 255 gaa gtg gta gat tta ata aat att gca aag gaa agg gtt tac gaa aac 816Glu Val Val Asp Leu Ile Asn Ile Ala Lys Glu Arg Val Tyr Glu Asn 260 265 270 ttc ggt ata gta ttg gag gag gag gta aag ctg att gag agt agt ggt 864Phe Gly Ile Val Leu Glu Glu Glu Val Lys Leu Ile Glu Ser Ser Gly 275 280 285 tct gat ggg tgg aag gtc ctc gga gcg tga 894Ser Asp Gly Trp Lys Val Leu Gly Ala 290 295 6297PRTAquifex aeolicus 6Met Leu Phe Leu Lys Asn Val Pro Leu Gln Asn Leu Thr Thr Ile Lys 1 5 10 15 Ile Gly Gly Arg Val Ser Phe Tyr Ala Glu Pro Ser Asp Leu Lys Glu 20 25 30 Ile Ser Leu Cys Ile Asp Phe Ser Lys Ser Arg Asp Ile Pro Leu Phe 35 40 45 Val Leu Gly Asn Gly Ser Asn Thr Ile Phe Gly Asp Val Arg Gly Leu 50 55

60 Val Val Asn Leu Lys Asn Leu Lys Gly Phe Lys Val Lys Glu Ile Lys 65 70 75 80 Gly Lys Phe Phe Val Glu Ala Phe Ser Gly Thr Pro Leu Lys Asp Leu 85 90 95 Ile Arg Phe Ser Val Lys Glu Asn Val Lys Ser Phe Tyr Lys Leu Leu 100 105 110 Gly Phe Pro Ala Ser Val Gly Gly Ala Val Ser Met Asn Ala Gly Ala 115 120 125 Phe Gly Val Glu Ile Ser Asp Phe Leu Lys Glu Val Tyr Phe Val Asp 130 135 140 Trp Glu Gly Lys Leu Gln Lys Ala Lys Arg Asp Glu Leu Asn Phe Ser 145 150 155 160 Tyr Arg Lys Ser Pro Phe Pro Lys Leu Gly Ile Val Phe Lys Val Val 165 170 175 Phe Glu Phe Glu Arg Ser Lys Glu Asn Ile Leu Pro Lys Tyr Glu Lys 180 185 190 Ile Arg Arg Ile Arg Lys Glu Lys Gln Pro Ile Asn Leu Pro Thr Ser 195 200 205 Gly Ser Thr Phe Lys Asn Pro Glu Gly Asn Phe Ala Gly Lys Leu Leu 210 215 220 Glu Lys Ala Gly Leu Lys Gly Phe Arg Leu Lys Asn Val Gly Phe Ser 225 230 235 240 Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Gly Gly Thr Phe Ser 245 250 255 Glu Val Val Asp Leu Ile Asn Ile Ala Lys Glu Arg Val Tyr Glu Asn 260 265 270 Phe Gly Ile Val Leu Glu Glu Glu Val Lys Leu Ile Glu Ser Ser Gly 275 280 285 Ser Asp Gly Trp Lys Val Leu Gly Ala 290 295 7932DNAAquifex aeolicusCDS(15)..(926) 7aggagataaa acat atg ttg ttc ttg aaa aac gtt cca ttg caa aat ctg 50 Met Leu Phe Leu Lys Asn Val Pro Leu Gln Asn Leu 1 5 10 act acg atc aaa att ggt ggc cgt gtg agc ttt tat gcg gag ccg agc 98Thr Thr Ile Lys Ile Gly Gly Arg Val Ser Phe Tyr Ala Glu Pro Ser 15 20 25 gat ctg aaa gaa att agc ctg tgc atc gac ttc agc aag tct cgc gac 146Asp Leu Lys Glu Ile Ser Leu Cys Ile Asp Phe Ser Lys Ser Arg Asp 30 35 40 atc ccg ctg ttc gtg ttg ggc aat ggt agc aat acc atc ttc ggc gat 194Ile Pro Leu Phe Val Leu Gly Asn Gly Ser Asn Thr Ile Phe Gly Asp 45 50 55 60 gtg cgt ggt ctg gtt gtc aat ctg aaa aac ctg aag ggc ttc aaa gtt 242Val Arg Gly Leu Val Val Asn Leu Lys Asn Leu Lys Gly Phe Lys Val 65 70 75 aaa gag atc aag ggc aag ttc ttc gtc gaa gct ttt tcc ggt acc ccg 290Lys Glu Ile Lys Gly Lys Phe Phe Val Glu Ala Phe Ser Gly Thr Pro 80 85 90 ctg aag gat ctg atc cgt ttc agc gtg aaa gag aac gtc aag agc ttc 338Leu Lys Asp Leu Ile Arg Phe Ser Val Lys Glu Asn Val Lys Ser Phe 95 100 105 tac aag ctg ctg ggt ttt ccg gcg agc gtt ggc ggt gca gtc tcg atg 386Tyr Lys Leu Leu Gly Phe Pro Ala Ser Val Gly Gly Ala Val Ser Met 110 115 120 aac gcc ggt gcg ttt ggt gtg gag att agc gac ttt ctg aaa gag gtg 434Asn Ala Gly Ala Phe Gly Val Glu Ile Ser Asp Phe Leu Lys Glu Val 125 130 135 140 tac ttt gtc gat tgg gaa ggt aaa ctg cag aaa gcg aag cgt gac gaa 482Tyr Phe Val Asp Trp Glu Gly Lys Leu Gln Lys Ala Lys Arg Asp Glu 145 150 155 ctg aat ttc tcc tac cgt aaa agc ccg ttc ccg aaa ctg ggt att gta 530Leu Asn Phe Ser Tyr Arg Lys Ser Pro Phe Pro Lys Leu Gly Ile Val 160 165 170 ttc aag gtg gtg ttt gag ttt gag cgc tct aaa gag aac att ctg ccg 578Phe Lys Val Val Phe Glu Phe Glu Arg Ser Lys Glu Asn Ile Leu Pro 175 180 185 aaa tat gaa aag atc cgc cgt att cgt aaa gag aag cag ccg atc aat 626Lys Tyr Glu Lys Ile Arg Arg Ile Arg Lys Glu Lys Gln Pro Ile Asn 190 195 200 ctg cct acc agc ggc tct acg ttt aag aat ccg gaa ggc aac ttc gca 674Leu Pro Thr Ser Gly Ser Thr Phe Lys Asn Pro Glu Gly Asn Phe Ala 205 210 215 220 ggc aaa ctg ctg gag aag gcc ggt ctg aag ggt ttc cgt ctg aag aac 722Gly Lys Leu Leu Glu Lys Ala Gly Leu Lys Gly Phe Arg Leu Lys Asn 225 230 235 gtg ggc ttc agc gag aaa cac gct aac ttt ctg gtc aac tac ggt ggt 770Val Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Gly 240 245 250 ggt acc ttt agc gag gtt gtc gac ctg att aac atc gca aaa gaa cgc 818Gly Thr Phe Ser Glu Val Val Asp Leu Ile Asn Ile Ala Lys Glu Arg 255 260 265 gtt tat gag aat ttt ggt att gtt ttg gaa gaa gaa gtt aag ctg att 866Val Tyr Glu Asn Phe Gly Ile Val Leu Glu Glu Glu Val Lys Leu Ile 270 275 280 gag agc agc ggc tcc gat ggt tgg aag gtt ctg ggt gcg cat cac cac 914Glu Ser Ser Gly Ser Asp Gly Trp Lys Val Leu Gly Ala His His His 285 290 295 300 cat cac cat taa ctcgag 932His His His 8303PRTAquifex aeolicus 8Met Leu Phe Leu Lys Asn Val Pro Leu Gln Asn Leu Thr Thr Ile Lys 1 5 10 15 Ile Gly Gly Arg Val Ser Phe Tyr Ala Glu Pro Ser Asp Leu Lys Glu 20 25 30 Ile Ser Leu Cys Ile Asp Phe Ser Lys Ser Arg Asp Ile Pro Leu Phe 35 40 45 Val Leu Gly Asn Gly Ser Asn Thr Ile Phe Gly Asp Val Arg Gly Leu 50 55 60 Val Val Asn Leu Lys Asn Leu Lys Gly Phe Lys Val Lys Glu Ile Lys 65 70 75 80 Gly Lys Phe Phe Val Glu Ala Phe Ser Gly Thr Pro Leu Lys Asp Leu 85 90 95 Ile Arg Phe Ser Val Lys Glu Asn Val Lys Ser Phe Tyr Lys Leu Leu 100 105 110 Gly Phe Pro Ala Ser Val Gly Gly Ala Val Ser Met Asn Ala Gly Ala 115 120 125 Phe Gly Val Glu Ile Ser Asp Phe Leu Lys Glu Val Tyr Phe Val Asp 130 135 140 Trp Glu Gly Lys Leu Gln Lys Ala Lys Arg Asp Glu Leu Asn Phe Ser 145 150 155 160 Tyr Arg Lys Ser Pro Phe Pro Lys Leu Gly Ile Val Phe Lys Val Val 165 170 175 Phe Glu Phe Glu Arg Ser Lys Glu Asn Ile Leu Pro Lys Tyr Glu Lys 180 185 190 Ile Arg Arg Ile Arg Lys Glu Lys Gln Pro Ile Asn Leu Pro Thr Ser 195 200 205 Gly Ser Thr Phe Lys Asn Pro Glu Gly Asn Phe Ala Gly Lys Leu Leu 210 215 220 Glu Lys Ala Gly Leu Lys Gly Phe Arg Leu Lys Asn Val Gly Phe Ser 225 230 235 240 Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Gly Gly Thr Phe Ser 245 250 255 Glu Val Val Asp Leu Ile Asn Ile Ala Lys Glu Arg Val Tyr Glu Asn 260 265 270 Phe Gly Ile Val Leu Glu Glu Glu Val Lys Leu Ile Glu Ser Ser Gly 275 280 285 Ser Asp Gly Trp Lys Val Leu Gly Ala His His His His His His 290 295 300 9951DNAAlicyclobacillus acidocaldariusCDS(1)..(951) 9atg caa gac gat ccg ttg gtg aac atc ctg atg gag caa ggg gtg aag 48Met Gln Asp Asp Pro Leu Val Asn Ile Leu Met Glu Gln Gly Val Lys 1 5 10 15 gtg atc cgg ggc gaa ccg atg aag cgg cat acg acg tgg cgg atc ggc 96Val Ile Arg Gly Glu Pro Met Lys Arg His Thr Thr Trp Arg Ile Gly 20 25 30 gga cct gcg gat tac ttt gtc gag ccg gac tcg gtg gac gcc ctg cgc 144Gly Pro Ala Asp Tyr Phe Val Glu Pro Asp Ser Val Asp Ala Leu Arg 35 40 45 gcg tgc gtt tgc gcc gcg cga gac cac ggc cta cct atc acc gtc atc 192Ala Cys Val Cys Ala Ala Arg Asp His Gly Leu Pro Ile Thr Val Ile 50 55 60 ggc cgc ggc tcc aac acc ctg gtg ttg gac ggc gga att cgc ggc ctc 240Gly Arg Gly Ser Asn Thr Leu Val Leu Asp Gly Gly Ile Arg Gly Leu 65 70 75 80 gtc atc aag ctg cac gac gcc ttc gct tcg tgc gac gtg aga gag gac 288Val Ile Lys Leu His Asp Ala Phe Ala Ser Cys Asp Val Arg Glu Asp 85 90 95 gag tgc gcg gtc tac gca atg gcg ggc cgt tcg tac gtc gcg ctg gcg 336Glu Cys Ala Val Tyr Ala Met Ala Gly Arg Ser Tyr Val Ala Leu Ala 100 105 110 aat ttg gcc atc cgc cac ggt ctc tca ggc ctc gaa ttt gcg acc ggc 384Asn Leu Ala Ile Arg His Gly Leu Ser Gly Leu Glu Phe Ala Thr Gly 115 120 125 att ccc ggg tcc gtc ggc ggc gcg gtc atg atg aac gca ggc gcg tac 432Ile Pro Gly Ser Val Gly Gly Ala Val Met Met Asn Ala Gly Ala Tyr 130 135 140 ggc cgc gag aca tgc gag gtg ctc gcg tgg gcc gag gtg atg gac gag 480Gly Arg Glu Thr Cys Glu Val Leu Ala Trp Ala Glu Val Met Asp Glu 145 150 155 160 acc gga gcc atc gcg cgg ctt tcg aac gaa gag ctg cgc ttt ggc tac 528Thr Gly Ala Ile Ala Arg Leu Ser Asn Glu Glu Leu Arg Phe Gly Tyr 165 170 175 cgc tac agc gtg ctc aag gat cgc ttc ggg att gtg aca cgg gcc aag 576Arg Tyr Ser Val Leu Lys Asp Arg Phe Gly Ile Val Thr Arg Ala Lys 180 185 190 ttt cag ctg gag cca ggc aat cgc gac gag atg cgg cgc ctc gtt cgc 624Phe Gln Leu Glu Pro Gly Asn Arg Asp Glu Met Arg Arg Leu Val Arg 195 200 205 gaa tgg tcc cag cgg cgc att gcg act cag cca ctc agc ttt ccg aac 672Glu Trp Ser Gln Arg Arg Ile Ala Thr Gln Pro Leu Ser Phe Pro Asn 210 215 220 tgc ggt tcc gtg ttt cga aat ccc gag ggc acc cac gcg gcc cga ctt 720Cys Gly Ser Val Phe Arg Asn Pro Glu Gly Thr His Ala Ala Arg Leu 225 230 235 240 atc gag gaa gcg ggg ctc aag gga ctg cgc cgc ggc cag gcg atg atc 768Ile Glu Glu Ala Gly Leu Lys Gly Leu Arg Arg Gly Gln Ala Met Ile 245 250 255 agc gac aag cac gcg aac ttc atc atc aac ctg ggg aac gct tcg gcg 816Ser Asp Lys His Ala Asn Phe Ile Ile Asn Leu Gly Asn Ala Ser Ala 260 265 270 agc gat gtg ctg tgg ctg att cgg cac gcg cag tcg gtg gtt cga gag 864Ser Asp Val Leu Trp Leu Ile Arg His Ala Gln Ser Val Val Arg Glu 275 280 285 cgg ttc ggc atc gcc ctc gaa acc gag gtc cgc gtg ctt ggc gaa ccc 912Arg Phe Gly Ile Ala Leu Glu Thr Glu Val Arg Val Leu Gly Glu Pro 290 295 300 ttg tcg gga ggt gcg gac gat gga att gct gcg gat tga 951Leu Ser Gly Gly Ala Asp Asp Gly Ile Ala Ala Asp 305 310 315 10316PRTAlicyclobacillus acidocaldarius 10Met Gln Asp Asp Pro Leu Val Asn Ile Leu Met Glu Gln Gly Val Lys 1 5 10 15 Val Ile Arg Gly Glu Pro Met Lys Arg His Thr Thr Trp Arg Ile Gly 20 25 30 Gly Pro Ala Asp Tyr Phe Val Glu Pro Asp Ser Val Asp Ala Leu Arg 35 40 45 Ala Cys Val Cys Ala Ala Arg Asp His Gly Leu Pro Ile Thr Val Ile 50 55 60 Gly Arg Gly Ser Asn Thr Leu Val Leu Asp Gly Gly Ile Arg Gly Leu 65 70 75 80 Val Ile Lys Leu His Asp Ala Phe Ala Ser Cys Asp Val Arg Glu Asp 85 90 95 Glu Cys Ala Val Tyr Ala Met Ala Gly Arg Ser Tyr Val Ala Leu Ala 100 105 110 Asn Leu Ala Ile Arg His Gly Leu Ser Gly Leu Glu Phe Ala Thr Gly 115 120 125 Ile Pro Gly Ser Val Gly Gly Ala Val Met Met Asn Ala Gly Ala Tyr 130 135 140 Gly Arg Glu Thr Cys Glu Val Leu Ala Trp Ala Glu Val Met Asp Glu 145 150 155 160 Thr Gly Ala Ile Ala Arg Leu Ser Asn Glu Glu Leu Arg Phe Gly Tyr 165 170 175 Arg Tyr Ser Val Leu Lys Asp Arg Phe Gly Ile Val Thr Arg Ala Lys 180 185 190 Phe Gln Leu Glu Pro Gly Asn Arg Asp Glu Met Arg Arg Leu Val Arg 195 200 205 Glu Trp Ser Gln Arg Arg Ile Ala Thr Gln Pro Leu Ser Phe Pro Asn 210 215 220 Cys Gly Ser Val Phe Arg Asn Pro Glu Gly Thr His Ala Ala Arg Leu 225 230 235 240 Ile Glu Glu Ala Gly Leu Lys Gly Leu Arg Arg Gly Gln Ala Met Ile 245 250 255 Ser Asp Lys His Ala Asn Phe Ile Ile Asn Leu Gly Asn Ala Ser Ala 260 265 270 Ser Asp Val Leu Trp Leu Ile Arg His Ala Gln Ser Val Val Arg Glu 275 280 285 Arg Phe Gly Ile Ala Leu Glu Thr Glu Val Arg Val Leu Gly Glu Pro 290 295 300 Leu Ser Gly Gly Ala Asp Asp Gly Ile Ala Ala Asp 305 310 315 11989DNAAlicyclobacillus acidocaldariusCDS(15)..(983) 11aggagataaa acat atg caa gac gat cct ttg gta aac att ttg atg gaa 50 Met Gln Asp Asp Pro Leu Val Asn Ile Leu Met Glu 1 5 10 cag ggc gta aaa gta att cgt ggt gag ccg atg aaa cgt cac acg acc 98Gln Gly Val Lys Val Ile Arg Gly Glu Pro Met Lys Arg His Thr Thr 15 20 25 tgg cgc atc ggt ggt ccg gca gac tat ttc gtg gag ccg gat tcc gtt 146Trp Arg Ile Gly Gly Pro Ala Asp Tyr Phe Val Glu Pro Asp Ser Val 30 35 40 gat gcg ctg cgt gcg tgt gtg tgc gca gcg cgc gac cac ggc ctg cca 194Asp Ala Leu Arg Ala Cys Val Cys Ala Ala Arg Asp His Gly Leu Pro 45 50 55 60 atc acc gtc att ggt cgc ggt agc aac act ctg gtc ctg gac ggt ggt 242Ile Thr Val Ile Gly Arg Gly Ser Asn Thr Leu Val Leu Asp Gly Gly 65 70 75 att cgt ggt ctg gtg atc aag ctg cac gat gct ttc gcc agc tgc gac 290Ile Arg Gly Leu Val Ile Lys Leu His Asp Ala Phe Ala Ser Cys Asp 80 85 90 gtt cgc gag gat gag tgt gcg gtg tat gcg atg gcc ggt cgt agc tat 338Val Arg Glu Asp Glu Cys Ala Val Tyr Ala Met Ala Gly Arg Ser Tyr 95 100 105 gtt gct ctg gca aat ctg gcc att cgt cac ggc ctg agc ggc ctg gag 386Val Ala Leu Ala Asn Leu Ala Ile Arg His Gly Leu Ser Gly Leu Glu 110 115 120 ttt gcg acc ggc att ccg ggt agc gtg ggc ggt gcc gtt atg atg aac 434Phe Ala Thr Gly Ile Pro Gly Ser Val Gly Gly Ala Val Met Met Asn 125 130 135 140 gcc ggt gcg tac ggc cgt gaa acg tgt gag gtc ctg gcg tgg gca gaa 482Ala Gly Ala Tyr Gly Arg Glu Thr Cys Glu Val Leu Ala Trp Ala Glu 145 150 155 gtt atg gac gaa acc ggc gct atc gca cgt ctg tcg aac gaa gaa ctg 530Val Met Asp Glu Thr Gly Ala Ile Ala Arg Leu Ser Asn Glu Glu Leu 160 165 170 cgt ttc ggt tac cgt tac tct gtc ctg aaa gat cgc ttt ggc atc gtg 578Arg Phe Gly Tyr Arg Tyr Ser Val Leu Lys Asp Arg Phe Gly Ile Val 175 180 185 acc cgt gca aag ttt cag ctg gag ccg ggt aat cgt

gac gag atg cgt 626Thr Arg Ala Lys Phe Gln Leu Glu Pro Gly Asn Arg Asp Glu Met Arg 190 195 200 cgt ctg gtt cgc gag tgg agc cag cgc cgc att gcg acg caa ccg ttg 674Arg Leu Val Arg Glu Trp Ser Gln Arg Arg Ile Ala Thr Gln Pro Leu 205 210 215 220 tct ttt ccg aat tgc ggt agc gtt ttc cgt aac ccg gaa ggt acg cat 722Ser Phe Pro Asn Cys Gly Ser Val Phe Arg Asn Pro Glu Gly Thr His 225 230 235 gcg gca cgt ttg atc gaa gag gcc ggt ttg aag ggt ctg cgc cgt ggc 770Ala Ala Arg Leu Ile Glu Glu Ala Gly Leu Lys Gly Leu Arg Arg Gly 240 245 250 cag gca atg atc agc gac aaa cat gcc aat ttc atc atc aac ctg ggc 818Gln Ala Met Ile Ser Asp Lys His Ala Asn Phe Ile Ile Asn Leu Gly 255 260 265 aat gcg agc gca tcc gac gtg ctg tgg ctg att cgc cat gcg caa agc 866Asn Ala Ser Ala Ser Asp Val Leu Trp Leu Ile Arg His Ala Gln Ser 270 275 280 gtc gtt cgt gaa cgt ttt ggc att gcg ctg gaa acc gag gtc cgc gtg 914Val Val Arg Glu Arg Phe Gly Ile Ala Leu Glu Thr Glu Val Arg Val 285 290 295 300 ctg ggt gag ccg ctg agc ggt ggc gcg gat gat ggt atc gct gcg gac 962Leu Gly Glu Pro Leu Ser Gly Gly Ala Asp Asp Gly Ile Ala Ala Asp 305 310 315 cat cac cac cac cat cac taa ctcgag 989His His His His His His 320 12322PRTAlicyclobacillus acidocaldarius 12Met Gln Asp Asp Pro Leu Val Asn Ile Leu Met Glu Gln Gly Val Lys 1 5 10 15 Val Ile Arg Gly Glu Pro Met Lys Arg His Thr Thr Trp Arg Ile Gly 20 25 30 Gly Pro Ala Asp Tyr Phe Val Glu Pro Asp Ser Val Asp Ala Leu Arg 35 40 45 Ala Cys Val Cys Ala Ala Arg Asp His Gly Leu Pro Ile Thr Val Ile 50 55 60 Gly Arg Gly Ser Asn Thr Leu Val Leu Asp Gly Gly Ile Arg Gly Leu 65 70 75 80 Val Ile Lys Leu His Asp Ala Phe Ala Ser Cys Asp Val Arg Glu Asp 85 90 95 Glu Cys Ala Val Tyr Ala Met Ala Gly Arg Ser Tyr Val Ala Leu Ala 100 105 110 Asn Leu Ala Ile Arg His Gly Leu Ser Gly Leu Glu Phe Ala Thr Gly 115 120 125 Ile Pro Gly Ser Val Gly Gly Ala Val Met Met Asn Ala Gly Ala Tyr 130 135 140 Gly Arg Glu Thr Cys Glu Val Leu Ala Trp Ala Glu Val Met Asp Glu 145 150 155 160 Thr Gly Ala Ile Ala Arg Leu Ser Asn Glu Glu Leu Arg Phe Gly Tyr 165 170 175 Arg Tyr Ser Val Leu Lys Asp Arg Phe Gly Ile Val Thr Arg Ala Lys 180 185 190 Phe Gln Leu Glu Pro Gly Asn Arg Asp Glu Met Arg Arg Leu Val Arg 195 200 205 Glu Trp Ser Gln Arg Arg Ile Ala Thr Gln Pro Leu Ser Phe Pro Asn 210 215 220 Cys Gly Ser Val Phe Arg Asn Pro Glu Gly Thr His Ala Ala Arg Leu 225 230 235 240 Ile Glu Glu Ala Gly Leu Lys Gly Leu Arg Arg Gly Gln Ala Met Ile 245 250 255 Ser Asp Lys His Ala Asn Phe Ile Ile Asn Leu Gly Asn Ala Ser Ala 260 265 270 Ser Asp Val Leu Trp Leu Ile Arg His Ala Gln Ser Val Val Arg Glu 275 280 285 Arg Phe Gly Ile Ala Leu Glu Thr Glu Val Arg Val Leu Gly Glu Pro 290 295 300 Leu Ser Gly Gly Ala Asp Asp Gly Ile Ala Ala Asp His His His His 305 310 315 320 His His 131026DNAAcidothermus cellulolyticusCDS(1)..(1026) 13gtg ccc gtg ctc gac ccc ccg gtc tgc ctc gcc gag tgc acc acg ttg 48Val Pro Val Leu Asp Pro Pro Val Cys Leu Ala Glu Cys Thr Thr Leu 1 5 10 15 cgt ctc ggc ggt ccg gcc gcg cgg ttc gtt gac gcc cac gac gag gcc 96Arg Leu Gly Gly Pro Ala Ala Arg Phe Val Asp Ala His Asp Glu Ala 20 25 30 gag ctg ctc gac gag atc cgc cag gcc gac gac aac ggc gag ccg ctg 144Glu Leu Leu Asp Glu Ile Arg Gln Ala Asp Asp Asn Gly Glu Pro Leu 35 40 45 ctc gtc atc ggt gcc ggc agc aac ctc gtg gtt gcc gac gcg ggc ttc 192Leu Val Ile Gly Ala Gly Ser Asn Leu Val Val Ala Asp Ala Gly Phe 50 55 60 ccg gga acc gtg ctg cgg gtc gca ttc cgc ggc atc cgc tgg tca agc 240Pro Gly Thr Val Leu Arg Val Ala Phe Arg Gly Ile Arg Trp Ser Ser 65 70 75 80 gac ggc gac cgg ctg ctg gtc gat atc gcg gcc ggt cag gtg tgg gac 288Asp Gly Asp Arg Leu Leu Val Asp Ile Ala Ala Gly Gln Val Trp Asp 85 90 95 gac gtc gtc acc gcg gcc atc gcc gaa ggg tgc gcg gga ttg gaa tgc 336Asp Val Val Thr Ala Ala Ile Ala Glu Gly Cys Ala Gly Leu Glu Cys 100 105 110 ctc tcc gga att ccc gga ctt gcc ggg gcc acg ccg gtt cag aac gtc 384Leu Ser Gly Ile Pro Gly Leu Ala Gly Ala Thr Pro Val Gln Asn Val 115 120 125 ggc gcg tac ggg gcg gaa atc gcc gat gtc tgt gtc ggc gtc cgc gtc 432Gly Ala Tyr Gly Ala Glu Ile Ala Asp Val Cys Val Gly Val Arg Val 130 135 140 tac gac cgg ctg gca cgc cgg gtg cgg tgg ttg gcc ggg tca gag tgc 480Tyr Asp Arg Leu Ala Arg Arg Val Arg Trp Leu Ala Gly Ser Glu Cys 145 150 155 160 cga ttc ggt tac cgg cac agc atc ctg aaa aac gac gac cga tac gtc 528Arg Phe Gly Tyr Arg His Ser Ile Leu Lys Asn Asp Asp Arg Tyr Val 165 170 175 gtg ctc acc gtc cgg ctt tcg ctg cgg cgc agc cgt ttg tcg acg ccg 576Val Leu Thr Val Arg Leu Ser Leu Arg Arg Ser Arg Leu Ser Thr Pro 180 185 190 atc cgc tat cag cag ctt gcc gat gcc ctc ggc gtt ccg ctg gga gac 624Ile Arg Tyr Gln Gln Leu Ala Asp Ala Leu Gly Val Pro Leu Gly Asp 195 200 205 tgc gct ccg gtg gac gcc gtc cgc aac gct gtg ctc gaa tta cgg gcc 672Cys Ala Pro Val Asp Ala Val Arg Asn Ala Val Leu Glu Leu Arg Ala 210 215 220 gcc aaa gga atg ctg ctc gac ccg ggt gac ccg gac acg gtg agc gcg 720Ala Lys Gly Met Leu Leu Asp Pro Gly Asp Pro Asp Thr Val Ser Ala 225 230 235 240 gga tcg ttt ttc acc aat ccc att gtc ccg gac tcc cag gcg cct ccc 768Gly Ser Phe Phe Thr Asn Pro Ile Val Pro Asp Ser Gln Ala Pro Pro 245 250 255 gag gcg ccg cgt ttt ccg gcc cat gct ccg ggg ctg gtg aaa atc ccg 816Glu Ala Pro Arg Phe Pro Ala His Ala Pro Gly Leu Val Lys Ile Pro 260 265 270 gcc gcc tgg ctc atc gaa caa gcc ggt ttc gcc aaa gga cac cgc ttg 864Ala Ala Trp Leu Ile Glu Gln Ala Gly Phe Ala Lys Gly His Arg Leu 275 280 285 gac ggc gtc gga att tcc agc aag cac gcg ctc gcg ctg gtg aac cgc 912Asp Gly Val Gly Ile Ser Ser Lys His Ala Leu Ala Leu Val Asn Arg 290 295 300 ggc gga agc acg gcg gac ctc ctg gaa ttg gcg cgc cgc atc cgg gcg 960Gly Gly Ser Thr Ala Asp Leu Leu Glu Leu Ala Arg Arg Ile Arg Ala 305 310 315 320 gcg gtt cag gag aaa ttc gga att ctg ctg gac gtc gaa ccc cgg ttg 1008Ala Val Gln Glu Lys Phe Gly Ile Leu Leu Asp Val Glu Pro Arg Leu 325 330 335 gtc ggc gtc cga ctc tga 1026Val Gly Val Arg Leu 340 14341PRTAcidothermus cellulolyticus 14Val Pro Val Leu Asp Pro Pro Val Cys Leu Ala Glu Cys Thr Thr Leu 1 5 10 15 Arg Leu Gly Gly Pro Ala Ala Arg Phe Val Asp Ala His Asp Glu Ala 20 25 30 Glu Leu Leu Asp Glu Ile Arg Gln Ala Asp Asp Asn Gly Glu Pro Leu 35 40 45 Leu Val Ile Gly Ala Gly Ser Asn Leu Val Val Ala Asp Ala Gly Phe 50 55 60 Pro Gly Thr Val Leu Arg Val Ala Phe Arg Gly Ile Arg Trp Ser Ser 65 70 75 80 Asp Gly Asp Arg Leu Leu Val Asp Ile Ala Ala Gly Gln Val Trp Asp 85 90 95 Asp Val Val Thr Ala Ala Ile Ala Glu Gly Cys Ala Gly Leu Glu Cys 100 105 110 Leu Ser Gly Ile Pro Gly Leu Ala Gly Ala Thr Pro Val Gln Asn Val 115 120 125 Gly Ala Tyr Gly Ala Glu Ile Ala Asp Val Cys Val Gly Val Arg Val 130 135 140 Tyr Asp Arg Leu Ala Arg Arg Val Arg Trp Leu Ala Gly Ser Glu Cys 145 150 155 160 Arg Phe Gly Tyr Arg His Ser Ile Leu Lys Asn Asp Asp Arg Tyr Val 165 170 175 Val Leu Thr Val Arg Leu Ser Leu Arg Arg Ser Arg Leu Ser Thr Pro 180 185 190 Ile Arg Tyr Gln Gln Leu Ala Asp Ala Leu Gly Val Pro Leu Gly Asp 195 200 205 Cys Ala Pro Val Asp Ala Val Arg Asn Ala Val Leu Glu Leu Arg Ala 210 215 220 Ala Lys Gly Met Leu Leu Asp Pro Gly Asp Pro Asp Thr Val Ser Ala 225 230 235 240 Gly Ser Phe Phe Thr Asn Pro Ile Val Pro Asp Ser Gln Ala Pro Pro 245 250 255 Glu Ala Pro Arg Phe Pro Ala His Ala Pro Gly Leu Val Lys Ile Pro 260 265 270 Ala Ala Trp Leu Ile Glu Gln Ala Gly Phe Ala Lys Gly His Arg Leu 275 280 285 Asp Gly Val Gly Ile Ser Ser Lys His Ala Leu Ala Leu Val Asn Arg 290 295 300 Gly Gly Ser Thr Ala Asp Leu Leu Glu Leu Ala Arg Arg Ile Arg Ala 305 310 315 320 Ala Val Gln Glu Lys Phe Gly Ile Leu Leu Asp Val Glu Pro Arg Leu 325 330 335 Val Gly Val Arg Leu 340 151064DNAAcidothermus cellulolyticusCDS(15)..(1058) 15aggagataaa acat atg cca gta ctt gat cca cct gtt tgt tta gct gaa 50 Met Pro Val Leu Asp Pro Pro Val Cys Leu Ala Glu 1 5 10 tgt acg act ttg aga ttg ggc ggc ccg gca gca cgt ttt gtt gac gcg 98Cys Thr Thr Leu Arg Leu Gly Gly Pro Ala Ala Arg Phe Val Asp Ala 15 20 25 cat gac gag gcg gag ctg ctg gat gag atc cgt caa gcg gac gat aat 146His Asp Glu Ala Glu Leu Leu Asp Glu Ile Arg Gln Ala Asp Asp Asn 30 35 40 ggt gaa ccg ttg ctg gtc att ggc gca ggc agc aac ctg gtg gtc gcg 194Gly Glu Pro Leu Leu Val Ile Gly Ala Gly Ser Asn Leu Val Val Ala 45 50 55 60 gac gcg ggt ttc ccg ggt acc gtg ctg cgc gtt gcc ttc cgt ggc att 242Asp Ala Gly Phe Pro Gly Thr Val Leu Arg Val Ala Phe Arg Gly Ile 65 70 75 cgc tgg agc agc gat ggt gac cgc ctg ctg gtc gat att gca gcg ggt 290Arg Trp Ser Ser Asp Gly Asp Arg Leu Leu Val Asp Ile Ala Ala Gly 80 85 90 cag gtc tgg gac gat gtc gtg acg gcg gct att gct gag ggc tgc gca 338Gln Val Trp Asp Asp Val Val Thr Ala Ala Ile Ala Glu Gly Cys Ala 95 100 105 ggc ctg gag tgc ctg agc ggt atc ccg ggt ctg gcg ggt gcg acc ccg 386Gly Leu Glu Cys Leu Ser Gly Ile Pro Gly Leu Ala Gly Ala Thr Pro 110 115 120 gtt cag aac gtg ggt gcc tac ggt gcg gaa att gcc gat gtt tgc gtg 434Val Gln Asn Val Gly Ala Tyr Gly Ala Glu Ile Ala Asp Val Cys Val 125 130 135 140 ggt gtg cgt gtg tat gat cgt ctg gcg cgt cgc gtt cgc tgg ctg gcg 482Gly Val Arg Val Tyr Asp Arg Leu Ala Arg Arg Val Arg Trp Leu Ala 145 150 155 ggc tcg gag tgc cgt ttc ggt tac cgc cac tcc atc ctg aag aac gac 530Gly Ser Glu Cys Arg Phe Gly Tyr Arg His Ser Ile Leu Lys Asn Asp 160 165 170 gac cgt tat gtg gtt ctg acc gtg cgt ctg tcc ctg cgc cgt tct cgc 578Asp Arg Tyr Val Val Leu Thr Val Arg Leu Ser Leu Arg Arg Ser Arg 175 180 185 ctg agc acc ccg atc cgt tac caa cag ctg gca gac gcg ctg ggt gtt 626Leu Ser Thr Pro Ile Arg Tyr Gln Gln Leu Ala Asp Ala Leu Gly Val 190 195 200 ccg ctg ggt gac tgt gcg ccg gtg gac gct gtc cgt aat gcg gtg ttg 674Pro Leu Gly Asp Cys Ala Pro Val Asp Ala Val Arg Asn Ala Val Leu 205 210 215 220 gaa ctg cgt gcc gcc aaa ggt atg ctg ctg gac ccg ggt gat ccg gac 722Glu Leu Arg Ala Ala Lys Gly Met Leu Leu Asp Pro Gly Asp Pro Asp 225 230 235 acc gtg tct gcg ggt agc ttt ttc acg aac ccg atc gtc ccg gat agc 770Thr Val Ser Ala Gly Ser Phe Phe Thr Asn Pro Ile Val Pro Asp Ser 240 245 250 cag gct ccg cct gaa gct ccg cgt ttt ccg gca cac gca ccg ggc ctg 818Gln Ala Pro Pro Glu Ala Pro Arg Phe Pro Ala His Ala Pro Gly Leu 255 260 265 gtt aag atc ccg gca gcc tgg ctg att gaa caa gca ggc ttc gcg aaa 866Val Lys Ile Pro Ala Ala Trp Leu Ile Glu Gln Ala Gly Phe Ala Lys 270 275 280 ggc cac cgt ctg gat ggc gtc ggt atc agc agc aag cac gcg ttg gcg 914Gly His Arg Leu Asp Gly Val Gly Ile Ser Ser Lys His Ala Leu Ala 285 290 295 300 ttg gtc aat cgt ggc ggt agc acc gcc gat ctg ctg gag ctg gcc cgt 962Leu Val Asn Arg Gly Gly Ser Thr Ala Asp Leu Leu Glu Leu Ala Arg 305 310 315 cgc atc cgt gcg gca gtc caa gag aaa ttt ggc att ctg ctg gac gtt 1010Arg Ile Arg Ala Ala Val Gln Glu Lys Phe Gly Ile Leu Leu Asp Val 320 325 330 gaa ccg cgt ctg gtt ggt gtt cgc ctg cat cat cac cat cac cac taa 1058Glu Pro Arg Leu Val Gly Val Arg Leu His His His His His His 335 340 345 ctcgag 1064 16347PRTAcidothermus cellulolyticus 16Met Pro Val Leu Asp Pro Pro Val Cys Leu Ala Glu Cys Thr Thr Leu 1 5 10 15 Arg Leu Gly Gly Pro Ala Ala Arg Phe Val Asp Ala His Asp Glu Ala 20 25 30 Glu Leu Leu Asp Glu Ile Arg Gln Ala Asp Asp Asn Gly Glu Pro Leu 35 40 45 Leu Val Ile Gly Ala Gly Ser Asn Leu Val Val Ala Asp Ala Gly Phe 50 55 60 Pro Gly Thr Val Leu Arg Val Ala Phe Arg Gly Ile Arg Trp Ser Ser 65 70 75 80 Asp Gly Asp Arg Leu Leu Val Asp Ile Ala Ala Gly Gln Val Trp Asp 85 90 95 Asp Val Val Thr Ala Ala Ile Ala Glu Gly Cys Ala Gly Leu Glu Cys 100 105 110 Leu Ser Gly Ile Pro Gly Leu Ala Gly Ala Thr Pro Val Gln Asn Val 115 120 125 Gly Ala Tyr Gly Ala Glu Ile Ala Asp Val Cys Val Gly Val Arg Val 130 135 140 Tyr Asp Arg Leu Ala Arg Arg Val Arg Trp Leu Ala Gly Ser Glu Cys 145 150 155

160 Arg Phe Gly Tyr Arg His Ser Ile Leu Lys Asn Asp Asp Arg Tyr Val 165 170 175 Val Leu Thr Val Arg Leu Ser Leu Arg Arg Ser Arg Leu Ser Thr Pro 180 185 190 Ile Arg Tyr Gln Gln Leu Ala Asp Ala Leu Gly Val Pro Leu Gly Asp 195 200 205 Cys Ala Pro Val Asp Ala Val Arg Asn Ala Val Leu Glu Leu Arg Ala 210 215 220 Ala Lys Gly Met Leu Leu Asp Pro Gly Asp Pro Asp Thr Val Ser Ala 225 230 235 240 Gly Ser Phe Phe Thr Asn Pro Ile Val Pro Asp Ser Gln Ala Pro Pro 245 250 255 Glu Ala Pro Arg Phe Pro Ala His Ala Pro Gly Leu Val Lys Ile Pro 260 265 270 Ala Ala Trp Leu Ile Glu Gln Ala Gly Phe Ala Lys Gly His Arg Leu 275 280 285 Asp Gly Val Gly Ile Ser Ser Lys His Ala Leu Ala Leu Val Asn Arg 290 295 300 Gly Gly Ser Thr Ala Asp Leu Leu Glu Leu Ala Arg Arg Ile Arg Ala 305 310 315 320 Ala Val Gln Glu Lys Phe Gly Ile Leu Leu Asp Val Glu Pro Arg Leu 325 330 335 Val Gly Val Arg Leu His His His His His His 340 345 17933DNACaldicellulosiruptor hydrothermalisCDS(1)..(933) 17atg tat tta aaa aat tca ggt att gag ttt cta aaa gac cat ccg cta 48Met Tyr Leu Lys Asn Ser Gly Ile Glu Phe Leu Lys Asp His Pro Leu 1 5 10 15 aaa gac cta aca aca ttc aag ata ggt gga aaa gca aga tat ata ata 96Lys Asp Leu Thr Thr Phe Lys Ile Gly Gly Lys Ala Arg Tyr Ile Ile 20 25 30 ttt ccc aaa agt acc gag gag ctt gtc aag gta ttg act tta gca aaa 144Phe Pro Lys Ser Thr Glu Glu Leu Val Lys Val Leu Thr Leu Ala Lys 35 40 45 gat aaa gcg ata aac cac gtt gtt gtt gga aac tgc tca aat att ctt 192Asp Lys Ala Ile Asn His Val Val Val Gly Asn Cys Ser Asn Ile Leu 50 55 60 gtc tct gac aaa ggt ttt gat ggt acg ata atc gct aca gtt aag ata 240Val Ser Asp Lys Gly Phe Asp Gly Thr Ile Ile Ala Thr Val Lys Ile 65 70 75 80 gac ttt ttt aag ata gat gga aat gtg att gaa gca gag tgt gga gct 288Asp Phe Phe Lys Ile Asp Gly Asn Val Ile Glu Ala Glu Cys Gly Ala 85 90 95 atg ctc tct cag gtt gca aga aaa gcg tgc gaa gca ggt cta aaa ggt 336Met Leu Ser Gln Val Ala Arg Lys Ala Cys Glu Ala Gly Leu Lys Gly 100 105 110 ttt gag ttt gcg gta gga att cct ggc act gtc ggt ggt gct gtg tac 384Phe Glu Phe Ala Val Gly Ile Pro Gly Thr Val Gly Gly Ala Val Tyr 115 120 125 atg aac gct ggt gca tac gat ggc gag ata aaa gat gtt ttt gaa tgg 432Met Asn Ala Gly Ala Tyr Asp Gly Glu Ile Lys Asp Val Phe Glu Trp 130 135 140 gca gag gtt ttg gat gag aac ttg aac cca gta gaa ctt ggt agg gca 480Ala Glu Val Leu Asp Glu Asn Leu Asn Pro Val Glu Leu Gly Arg Ala 145 150 155 160 gat atg agg ttt tca tac agg cac agc cga ctg aaa gaa gaa aaa atg 528Asp Met Arg Phe Ser Tyr Arg His Ser Arg Leu Lys Glu Glu Lys Met 165 170 175 gtg ctt ctc aga gca gca ttt tgc ctc aag ttt gcc gac aaa gaa gat 576Val Leu Leu Arg Ala Ala Phe Cys Leu Lys Phe Ala Asp Lys Glu Asp 180 185 190 ata tcc cct ttg caa aaa gca aat gaa ttt tca aaa aga cgg cga gaa 624Ile Ser Pro Leu Gln Lys Ala Asn Glu Phe Ser Lys Arg Arg Arg Glu 195 200 205 aaa cag cct ctt tct tat ccg agt gca ggt tct gtg ttt aaa aga ccg 672Lys Gln Pro Leu Ser Tyr Pro Ser Ala Gly Ser Val Phe Lys Arg Pro 210 215 220 cca aac aac tat gca gga aag ctt att gaa gat gca ggc ttg aaa gga 720Pro Asn Asn Tyr Ala Gly Lys Leu Ile Glu Asp Ala Gly Leu Lys Gly 225 230 235 240 tat aga ata ggt gat gcg tgt ata tca gaa aaa cat gca ggg ttt atc 768Tyr Arg Ile Gly Asp Ala Cys Ile Ser Glu Lys His Ala Gly Phe Ile 245 250 255 ata aac tta gga gat gct aaa gct gag gat gtg aga aag ctc atc tat 816Ile Asn Leu Gly Asp Ala Lys Ala Glu Asp Val Arg Lys Leu Ile Tyr 260 265 270 ctt gct cag aag act gtg tac gaa aaa ttt gga att ttg ctt gaa cct 864Leu Ala Gln Lys Thr Val Tyr Glu Lys Phe Gly Ile Leu Leu Glu Pro 275 280 285 gag att cag ttc ata ggc gag ttt gaa aca ccg ctt ttt gtg ccc gaa 912Glu Ile Gln Phe Ile Gly Glu Phe Glu Thr Pro Leu Phe Val Pro Glu 290 295 300 aat gtc caa aat aga aga tga 933Asn Val Gln Asn Arg Arg 305 310 18310PRTCaldicellulosiruptor hydrothermalis 18Met Tyr Leu Lys Asn Ser Gly Ile Glu Phe Leu Lys Asp His Pro Leu 1 5 10 15 Lys Asp Leu Thr Thr Phe Lys Ile Gly Gly Lys Ala Arg Tyr Ile Ile 20 25 30 Phe Pro Lys Ser Thr Glu Glu Leu Val Lys Val Leu Thr Leu Ala Lys 35 40 45 Asp Lys Ala Ile Asn His Val Val Val Gly Asn Cys Ser Asn Ile Leu 50 55 60 Val Ser Asp Lys Gly Phe Asp Gly Thr Ile Ile Ala Thr Val Lys Ile 65 70 75 80 Asp Phe Phe Lys Ile Asp Gly Asn Val Ile Glu Ala Glu Cys Gly Ala 85 90 95 Met Leu Ser Gln Val Ala Arg Lys Ala Cys Glu Ala Gly Leu Lys Gly 100 105 110 Phe Glu Phe Ala Val Gly Ile Pro Gly Thr Val Gly Gly Ala Val Tyr 115 120 125 Met Asn Ala Gly Ala Tyr Asp Gly Glu Ile Lys Asp Val Phe Glu Trp 130 135 140 Ala Glu Val Leu Asp Glu Asn Leu Asn Pro Val Glu Leu Gly Arg Ala 145 150 155 160 Asp Met Arg Phe Ser Tyr Arg His Ser Arg Leu Lys Glu Glu Lys Met 165 170 175 Val Leu Leu Arg Ala Ala Phe Cys Leu Lys Phe Ala Asp Lys Glu Asp 180 185 190 Ile Ser Pro Leu Gln Lys Ala Asn Glu Phe Ser Lys Arg Arg Arg Glu 195 200 205 Lys Gln Pro Leu Ser Tyr Pro Ser Ala Gly Ser Val Phe Lys Arg Pro 210 215 220 Pro Asn Asn Tyr Ala Gly Lys Leu Ile Glu Asp Ala Gly Leu Lys Gly 225 230 235 240 Tyr Arg Ile Gly Asp Ala Cys Ile Ser Glu Lys His Ala Gly Phe Ile 245 250 255 Ile Asn Leu Gly Asp Ala Lys Ala Glu Asp Val Arg Lys Leu Ile Tyr 260 265 270 Leu Ala Gln Lys Thr Val Tyr Glu Lys Phe Gly Ile Leu Leu Glu Pro 275 280 285 Glu Ile Gln Phe Ile Gly Glu Phe Glu Thr Pro Leu Phe Val Pro Glu 290 295 300 Asn Val Gln Asn Arg Arg 305 310 19971DNACaldicellulosiruptor hydrothermalisCDS(15)..(965) 19aggagataaa acat atg tat ttg aaa aac tct ggt att gaa ttc ttg aag 50 Met Tyr Leu Lys Asn Ser Gly Ile Glu Phe Leu Lys 1 5 10 gac cac ccg ctg aaa gat ctg acc acc ttt aag atc ggc ggt aag gca 98Asp His Pro Leu Lys Asp Leu Thr Thr Phe Lys Ile Gly Gly Lys Ala 15 20 25 cgt tac atc atc ttt ccg aaa tcc acg gaa gaa ctg gtg aag gtg ctg 146Arg Tyr Ile Ile Phe Pro Lys Ser Thr Glu Glu Leu Val Lys Val Leu 30 35 40 acc ctg gcg aaa gac aag gcg atc aac cat gtc gtt gtt ggc aac tgc 194Thr Leu Ala Lys Asp Lys Ala Ile Asn His Val Val Val Gly Asn Cys 45 50 55 60 agc aat atc ctg gtg agc gat aag ggc ttt gac ggt acc att att gca 242Ser Asn Ile Leu Val Ser Asp Lys Gly Phe Asp Gly Thr Ile Ile Ala 65 70 75 acg gtg aaa att gat ttc ttc aaa att gac ggc aac gtt atc gag gcc 290Thr Val Lys Ile Asp Phe Phe Lys Ile Asp Gly Asn Val Ile Glu Ala 80 85 90 gaa tgc ggt gcg atg ctg agc cag gtc gcc cgt aaa gcg tgt gaa gcg 338Glu Cys Gly Ala Met Leu Ser Gln Val Ala Arg Lys Ala Cys Glu Ala 95 100 105 ggc ctg aag ggt ttt gag ttc gcg gtt ggt att ccg ggt act gtc ggc 386Gly Leu Lys Gly Phe Glu Phe Ala Val Gly Ile Pro Gly Thr Val Gly 110 115 120 ggt gcg gtt tac atg aat gca ggc gcg tat gac ggt gag att aaa gat 434Gly Ala Val Tyr Met Asn Ala Gly Ala Tyr Asp Gly Glu Ile Lys Asp 125 130 135 140 gtg ttt gaa tgg gca gag gtt ctg gat gag aat ttg aac ccg gtt gag 482Val Phe Glu Trp Ala Glu Val Leu Asp Glu Asn Leu Asn Pro Val Glu 145 150 155 ctg ggt cgt gcg gat atg cgc ttc agc tat cgc cac agc cgc ctg aaa 530Leu Gly Arg Ala Asp Met Arg Phe Ser Tyr Arg His Ser Arg Leu Lys 160 165 170 gaa gaa aag atg gtc ctg ctg cgt gcc gcc ttt tgt ttg aaa ttc gca 578Glu Glu Lys Met Val Leu Leu Arg Ala Ala Phe Cys Leu Lys Phe Ala 175 180 185 gac aaa gag gac atc tcc cca ctg caa aag gcg aat gag ttt agc aag 626Asp Lys Glu Asp Ile Ser Pro Leu Gln Lys Ala Asn Glu Phe Ser Lys 190 195 200 cgt cgc cgt gaa aaa cag ccg ctg agc tac ccg tct gca ggc agc gtt 674Arg Arg Arg Glu Lys Gln Pro Leu Ser Tyr Pro Ser Ala Gly Ser Val 205 210 215 220 ttc aag cgt ccg ccg aat aac tat gcg ggt aaa ctg atc gag gac gct 722Phe Lys Arg Pro Pro Asn Asn Tyr Ala Gly Lys Leu Ile Glu Asp Ala 225 230 235 ggt ctg aaa ggt tac cgc atc ggt gat gct tgc att agc gag aag cat 770Gly Leu Lys Gly Tyr Arg Ile Gly Asp Ala Cys Ile Ser Glu Lys His 240 245 250 gct ggc ttc atc att aat ctg ggt gac gca aag gcc gag gat gtc cgt 818Ala Gly Phe Ile Ile Asn Leu Gly Asp Ala Lys Ala Glu Asp Val Arg 255 260 265 aag ctg att tac ctg gcg caa aag acg gtg tac gag aaa ttt ggt atc 866Lys Leu Ile Tyr Leu Ala Gln Lys Thr Val Tyr Glu Lys Phe Gly Ile 270 275 280 ttg ctg gaa ccg gag att cag ttc atc ggc gag ttt gaa acc ccg ctg 914Leu Leu Glu Pro Glu Ile Gln Phe Ile Gly Glu Phe Glu Thr Pro Leu 285 290 295 300 ttc gtg cct gag aac gtc caa aat cgt cgt cat cac cac cat cac cac 962Phe Val Pro Glu Asn Val Gln Asn Arg Arg His His His His His His 305 310 315 taa ctcgag 97120316PRTCaldicellulosiruptor hydrothermalis 20Met Tyr Leu Lys Asn Ser Gly Ile Glu Phe Leu Lys Asp His Pro Leu 1 5 10 15 Lys Asp Leu Thr Thr Phe Lys Ile Gly Gly Lys Ala Arg Tyr Ile Ile 20 25 30 Phe Pro Lys Ser Thr Glu Glu Leu Val Lys Val Leu Thr Leu Ala Lys 35 40 45 Asp Lys Ala Ile Asn His Val Val Val Gly Asn Cys Ser Asn Ile Leu 50 55 60 Val Ser Asp Lys Gly Phe Asp Gly Thr Ile Ile Ala Thr Val Lys Ile 65 70 75 80 Asp Phe Phe Lys Ile Asp Gly Asn Val Ile Glu Ala Glu Cys Gly Ala 85 90 95 Met Leu Ser Gln Val Ala Arg Lys Ala Cys Glu Ala Gly Leu Lys Gly 100 105 110 Phe Glu Phe Ala Val Gly Ile Pro Gly Thr Val Gly Gly Ala Val Tyr 115 120 125 Met Asn Ala Gly Ala Tyr Asp Gly Glu Ile Lys Asp Val Phe Glu Trp 130 135 140 Ala Glu Val Leu Asp Glu Asn Leu Asn Pro Val Glu Leu Gly Arg Ala 145 150 155 160 Asp Met Arg Phe Ser Tyr Arg His Ser Arg Leu Lys Glu Glu Lys Met 165 170 175 Val Leu Leu Arg Ala Ala Phe Cys Leu Lys Phe Ala Asp Lys Glu Asp 180 185 190 Ile Ser Pro Leu Gln Lys Ala Asn Glu Phe Ser Lys Arg Arg Arg Glu 195 200 205 Lys Gln Pro Leu Ser Tyr Pro Ser Ala Gly Ser Val Phe Lys Arg Pro 210 215 220 Pro Asn Asn Tyr Ala Gly Lys Leu Ile Glu Asp Ala Gly Leu Lys Gly 225 230 235 240 Tyr Arg Ile Gly Asp Ala Cys Ile Ser Glu Lys His Ala Gly Phe Ile 245 250 255 Ile Asn Leu Gly Asp Ala Lys Ala Glu Asp Val Arg Lys Leu Ile Tyr 260 265 270 Leu Ala Gln Lys Thr Val Tyr Glu Lys Phe Gly Ile Leu Leu Glu Pro 275 280 285 Glu Ile Gln Phe Ile Gly Glu Phe Glu Thr Pro Leu Phe Val Pro Glu 290 295 300 Asn Val Gln Asn Arg Arg His His His His His His 305 310 315 21894DNADictyoglomus turgidumCDS(1)..(894) 21atg cta att tgg aat att ctc aat aaa tat aat ttt aaa tcc aaa atc 48Met Leu Ile Trp Asn Ile Leu Asn Lys Tyr Asn Phe Lys Ser Lys Ile 1 5 10 15 tat aaa gat gtg aat tta tct cat tat acc tct ttt aag att ggg gga 96Tyr Lys Asp Val Asn Leu Ser His Tyr Thr Ser Phe Lys Ile Gly Gly 20 25 30 aaa gta gac tta ttc att att cca tat tct tgg gaa gaa ctt atc tta 144Lys Val Asp Leu Phe Ile Ile Pro Tyr Ser Trp Glu Glu Leu Ile Leu 35 40 45 att ctt caa att ctt aaa gag aat aat atc cca aca aaa gta atg gga 192Ile Leu Gln Ile Leu Lys Glu Asn Asn Ile Pro Thr Lys Val Met Gly 50 55 60 caa ggt act aat att ctt gca cct gat gaa ggg att aaa gga gca gta 240Gln Gly Thr Asn Ile Leu Ala Pro Asp Glu Gly Ile Lys Gly Ala Val 65 70 75 80 att aga ttg aat cag aac ctg ggg aaa atc aat ttt gta gat aat agc 288Ile Arg Leu Asn Gln Asn Leu Gly Lys Ile Asn Phe Val Asp Asn Ser 85 90 95 cat ctg gaa gta gaa tcg gga tgc tta atc tct aaa tta atc tct ttt 336His Leu Glu Val Glu Ser Gly Cys Leu Ile Ser Lys Leu Ile Ser Phe 100 105 110 atg gtt gaa aaa aat atg ggt ggg ctt gaa ttt atg atg ggt att cct 384Met Val Glu Lys Asn Met Gly Gly Leu Glu Phe Met Met Gly Ile Pro 115 120 125 gga act ata gga gga gcg gtg atg ggc aat gct gga gcc ttt aga aaa 432Gly Thr Ile Gly Gly Ala Val Met Gly Asn Ala Gly Ala Phe Arg Lys 130 135 140 gct ata ggt gat ttt gta gag gga gtc tat gtt tta aat gaa cat ttt 480Ala Ile Gly Asp Phe Val Glu Gly Val Tyr Val Leu Asn Glu His Phe 145 150 155 160 gag gaa atg ttt ttg ggc aaa aaa gaa ctt aat ttt aat tat aga agc 528Glu Glu Met Phe Leu Gly Lys Lys Glu Leu Asn Phe Asn Tyr Arg Ser 165 170 175 tca aac att cca aaa gac tgg att att aaa aaa gtg ctg tta aga tta 576Ser Asn Ile Pro Lys Asp Trp Ile Ile Lys Lys Val Leu Leu Arg Leu 180 185 190 gag gaa aaa cct aag gaa gaa tct tta aaa gag att aag ttt ttt ata 624Glu Glu Lys Pro Lys Glu Glu Ser Leu Lys Glu Ile Lys Phe Phe Ile 195 200 205

aag gaa aga agt aaa aag ctt cct aaa tat ccg tct gct ggg agt gta 672Lys Glu Arg Ser Lys Lys Leu Pro Lys Tyr Pro Ser Ala Gly Ser Val 210 215 220 ttt aaa aat cct aag gaa gga cct gca gga tat ttt att gat aac tta 720Phe Lys Asn Pro Lys Glu Gly Pro Ala Gly Tyr Phe Ile Asp Asn Leu 225 230 235 240 ggt ttt aga ggc ttt agg att gga gat gct atg gtt tct tat gag cat 768Gly Phe Arg Gly Phe Arg Ile Gly Asp Ala Met Val Ser Tyr Glu His 245 250 255 gca aac act ata ata aat gtt ggc agg gca aga agt aag gat gtt tta 816Ala Asn Thr Ile Ile Asn Val Gly Arg Ala Arg Ser Lys Asp Val Leu 260 265 270 gag att ata aat att ata aaa gac aaa gtg aag gag gcc tat ggt ata 864Glu Ile Ile Asn Ile Ile Lys Asp Lys Val Lys Glu Ala Tyr Gly Ile 275 280 285 gat ttg gag cca gag att att att tgg taa 894Asp Leu Glu Pro Glu Ile Ile Ile Trp 290 295 22297PRTDictyoglomus turgidum 22Met Leu Ile Trp Asn Ile Leu Asn Lys Tyr Asn Phe Lys Ser Lys Ile 1 5 10 15 Tyr Lys Asp Val Asn Leu Ser His Tyr Thr Ser Phe Lys Ile Gly Gly 20 25 30 Lys Val Asp Leu Phe Ile Ile Pro Tyr Ser Trp Glu Glu Leu Ile Leu 35 40 45 Ile Leu Gln Ile Leu Lys Glu Asn Asn Ile Pro Thr Lys Val Met Gly 50 55 60 Gln Gly Thr Asn Ile Leu Ala Pro Asp Glu Gly Ile Lys Gly Ala Val 65 70 75 80 Ile Arg Leu Asn Gln Asn Leu Gly Lys Ile Asn Phe Val Asp Asn Ser 85 90 95 His Leu Glu Val Glu Ser Gly Cys Leu Ile Ser Lys Leu Ile Ser Phe 100 105 110 Met Val Glu Lys Asn Met Gly Gly Leu Glu Phe Met Met Gly Ile Pro 115 120 125 Gly Thr Ile Gly Gly Ala Val Met Gly Asn Ala Gly Ala Phe Arg Lys 130 135 140 Ala Ile Gly Asp Phe Val Glu Gly Val Tyr Val Leu Asn Glu His Phe 145 150 155 160 Glu Glu Met Phe Leu Gly Lys Lys Glu Leu Asn Phe Asn Tyr Arg Ser 165 170 175 Ser Asn Ile Pro Lys Asp Trp Ile Ile Lys Lys Val Leu Leu Arg Leu 180 185 190 Glu Glu Lys Pro Lys Glu Glu Ser Leu Lys Glu Ile Lys Phe Phe Ile 195 200 205 Lys Glu Arg Ser Lys Lys Leu Pro Lys Tyr Pro Ser Ala Gly Ser Val 210 215 220 Phe Lys Asn Pro Lys Glu Gly Pro Ala Gly Tyr Phe Ile Asp Asn Leu 225 230 235 240 Gly Phe Arg Gly Phe Arg Ile Gly Asp Ala Met Val Ser Tyr Glu His 245 250 255 Ala Asn Thr Ile Ile Asn Val Gly Arg Ala Arg Ser Lys Asp Val Leu 260 265 270 Glu Ile Ile Asn Ile Ile Lys Asp Lys Val Lys Glu Ala Tyr Gly Ile 275 280 285 Asp Leu Glu Pro Glu Ile Ile Ile Trp 290 295 23932DNADictyoglomus turgidumCDS(15)..(926) 23aggagataaa acat atg ttg atc tgg aat att ctg aac aag tac aat ttc 50 Met Leu Ile Trp Asn Ile Leu Asn Lys Tyr Asn Phe 1 5 10 aaa agc aaa atc tat aaa gat gtg aac ctg agc cat tac acg agc ttc 98Lys Ser Lys Ile Tyr Lys Asp Val Asn Leu Ser His Tyr Thr Ser Phe 15 20 25 aaa att ggc ggt aaa gtt gac ctg ttt atc att ccg tat tct tgg gaa 146Lys Ile Gly Gly Lys Val Asp Leu Phe Ile Ile Pro Tyr Ser Trp Glu 30 35 40 gaa ctg atc ctg att ctg cag atc ttg aaa gag aac aat att ccg acc 194Glu Leu Ile Leu Ile Leu Gln Ile Leu Lys Glu Asn Asn Ile Pro Thr 45 50 55 60 aag gtg atg ggc cag ggc acg aat atc ctg gca ccg gat gaa ggc atc 242Lys Val Met Gly Gln Gly Thr Asn Ile Leu Ala Pro Asp Glu Gly Ile 65 70 75 aag ggt gcc gtt atc cgt ctg aac caa aat ctg ggc aaa atc aat ttt 290Lys Gly Ala Val Ile Arg Leu Asn Gln Asn Leu Gly Lys Ile Asn Phe 80 85 90 gtg gac aac tcg cac ttg gaa gtc gaa tcc ggt tgc ctg atc tct aaa 338Val Asp Asn Ser His Leu Glu Val Glu Ser Gly Cys Leu Ile Ser Lys 95 100 105 ctg att agc ttc atg gtt gaa aag aac atg ggt ggc ctg gag ttt atg 386Leu Ile Ser Phe Met Val Glu Lys Asn Met Gly Gly Leu Glu Phe Met 110 115 120 atg ggc att ccg ggt acc atc ggt ggt gcg gtg atg ggt aac gct ggt 434Met Gly Ile Pro Gly Thr Ile Gly Gly Ala Val Met Gly Asn Ala Gly 125 130 135 140 gcg ttt cgc aag gct att ggt gac ttt gtc gag ggt gtt tac gtg ttg 482Ala Phe Arg Lys Ala Ile Gly Asp Phe Val Glu Gly Val Tyr Val Leu 145 150 155 aac gag cac ttc gaa gag atg ttc ttg ggc aag aaa gag ctg aac ttc 530Asn Glu His Phe Glu Glu Met Phe Leu Gly Lys Lys Glu Leu Asn Phe 160 165 170 aat tat cgt agc agc aat att cca aag gat tgg atc atc aag aag gtg 578Asn Tyr Arg Ser Ser Asn Ile Pro Lys Asp Trp Ile Ile Lys Lys Val 175 180 185 ctg ctg cgc ctg gaa gag aaa ccg aaa gaa gag agc ctg aaa gag att 626Leu Leu Arg Leu Glu Glu Lys Pro Lys Glu Glu Ser Leu Lys Glu Ile 190 195 200 aag ttt ttc att aaa gag cgc agc aag aaa ctg ccg aag tat ccg agc 674Lys Phe Phe Ile Lys Glu Arg Ser Lys Lys Leu Pro Lys Tyr Pro Ser 205 210 215 220 gcc ggt agc gtt ttc aag aat ccg aaa gag ggt cct gcg ggc tac ttt 722Ala Gly Ser Val Phe Lys Asn Pro Lys Glu Gly Pro Ala Gly Tyr Phe 225 230 235 att gac aat ctg ggt ttt cgt ggt ttc cgt att ggt gat gcg atg gtc 770Ile Asp Asn Leu Gly Phe Arg Gly Phe Arg Ile Gly Asp Ala Met Val 240 245 250 agc tac gag cac gca aac acc atc atc aac gtc ggc cgt gca cgt tcc 818Ser Tyr Glu His Ala Asn Thr Ile Ile Asn Val Gly Arg Ala Arg Ser 255 260 265 aag gac gtt ctg gag att atc aac atc att aag gat aag gtc aaa gag 866Lys Asp Val Leu Glu Ile Ile Asn Ile Ile Lys Asp Lys Val Lys Glu 270 275 280 gcg tac ggc att gac ctg gaa ccg gaa atc atc att tgg cac cac cat 914Ala Tyr Gly Ile Asp Leu Glu Pro Glu Ile Ile Ile Trp His His His 285 290 295 300 cac cat cat taa ctcgag 932His His His 24303PRTDictyoglomus turgidum 24Met Leu Ile Trp Asn Ile Leu Asn Lys Tyr Asn Phe Lys Ser Lys Ile 1 5 10 15 Tyr Lys Asp Val Asn Leu Ser His Tyr Thr Ser Phe Lys Ile Gly Gly 20 25 30 Lys Val Asp Leu Phe Ile Ile Pro Tyr Ser Trp Glu Glu Leu Ile Leu 35 40 45 Ile Leu Gln Ile Leu Lys Glu Asn Asn Ile Pro Thr Lys Val Met Gly 50 55 60 Gln Gly Thr Asn Ile Leu Ala Pro Asp Glu Gly Ile Lys Gly Ala Val 65 70 75 80 Ile Arg Leu Asn Gln Asn Leu Gly Lys Ile Asn Phe Val Asp Asn Ser 85 90 95 His Leu Glu Val Glu Ser Gly Cys Leu Ile Ser Lys Leu Ile Ser Phe 100 105 110 Met Val Glu Lys Asn Met Gly Gly Leu Glu Phe Met Met Gly Ile Pro 115 120 125 Gly Thr Ile Gly Gly Ala Val Met Gly Asn Ala Gly Ala Phe Arg Lys 130 135 140 Ala Ile Gly Asp Phe Val Glu Gly Val Tyr Val Leu Asn Glu His Phe 145 150 155 160 Glu Glu Met Phe Leu Gly Lys Lys Glu Leu Asn Phe Asn Tyr Arg Ser 165 170 175 Ser Asn Ile Pro Lys Asp Trp Ile Ile Lys Lys Val Leu Leu Arg Leu 180 185 190 Glu Glu Lys Pro Lys Glu Glu Ser Leu Lys Glu Ile Lys Phe Phe Ile 195 200 205 Lys Glu Arg Ser Lys Lys Leu Pro Lys Tyr Pro Ser Ala Gly Ser Val 210 215 220 Phe Lys Asn Pro Lys Glu Gly Pro Ala Gly Tyr Phe Ile Asp Asn Leu 225 230 235 240 Gly Phe Arg Gly Phe Arg Ile Gly Asp Ala Met Val Ser Tyr Glu His 245 250 255 Ala Asn Thr Ile Ile Asn Val Gly Arg Ala Arg Ser Lys Asp Val Leu 260 265 270 Glu Ile Ile Asn Ile Ile Lys Asp Lys Val Lys Glu Ala Tyr Gly Ile 275 280 285 Asp Leu Glu Pro Glu Ile Ile Ile Trp His His His His His His 290 295 300 25927DNAGeobacillus strain Y4.1MC1CDS(1)..(927) 25atg atg tac gaa aat aat gtg att tat caa gaa tta gtg cgg att tgt 48Met Met Tyr Glu Asn Asn Val Ile Tyr Gln Glu Leu Val Arg Ile Cys 1 5 10 15 ggg gaa aaa aac gtg ctg cgg gac gaa ccg ttg aaa tat cat acg tta 96Gly Glu Lys Asn Val Leu Arg Asp Glu Pro Leu Lys Tyr His Thr Leu 20 25 30 gtg aaa att ggc ggc aag gcg gat ttc ctc gtc tgg ccg gaa aca tat 144Val Lys Ile Gly Gly Lys Ala Asp Phe Leu Val Trp Pro Glu Thr Tyr 35 40 45 gag cag gtg gtc gaa gtc att cga tta aag gaa aag cat cac ctc cct 192Glu Gln Val Val Glu Val Ile Arg Leu Lys Glu Lys His His Leu Pro 50 55 60 ttc acc ttg ctt ggc aac ggt tcg aac gtc atc gtg cgt gac ggc ggc 240Phe Thr Leu Leu Gly Asn Gly Ser Asn Val Ile Val Arg Asp Gly Gly 65 70 75 80 att cgc ggc att gtt gtg cag cta aag cat cta aca gaa atc aaa gtg 288Ile Arg Gly Ile Val Val Gln Leu Lys His Leu Thr Glu Ile Lys Val 85 90 95 gaa gga gaa aaa att atc gcg caa agc ggc gct gat att aaa gca gtt 336Glu Gly Glu Lys Ile Ile Ala Gln Ser Gly Ala Asp Ile Lys Ala Val 100 105 110 tct cgg gtt gca ctg gaa cac agc ttg acg ggg ctg gag ttc gcg tgc 384Ser Arg Val Ala Leu Glu His Ser Leu Thr Gly Leu Glu Phe Ala Cys 115 120 125 ggc att ccc ggc tct gtc ggg ggc gcg att atg atg aat gcg ggt gct 432Gly Ile Pro Gly Ser Val Gly Gly Ala Ile Met Met Asn Ala Gly Ala 130 135 140 tat gat ggg gaa ata aaa gac gtg att gac cat gtt aag gtc gtt aca 480Tyr Asp Gly Glu Ile Lys Asp Val Ile Asp His Val Lys Val Val Thr 145 150 155 160 caa acg gga gaa cag aaa att ttg cgc aaa gac gat ttg cag ctt ggt 528Gln Thr Gly Glu Gln Lys Ile Leu Arg Lys Asp Asp Leu Gln Leu Gly 165 170 175 tat cgc acg agc atc atc agc aag acg aac gac att gtg ctg gag gcg 576Tyr Arg Thr Ser Ile Ile Ser Lys Thr Asn Asp Ile Val Leu Glu Ala 180 185 190 gtg ttc cag ctt aaa aaa gga gat ccg caa aaa att aaa gaa aaa atg 624Val Phe Gln Leu Lys Lys Gly Asp Pro Gln Lys Ile Lys Glu Lys Met 195 200 205 gac gat ctc acc ttc cgg cgt gaa tcc aaa cag ccg ctt gaa tat cct 672Asp Asp Leu Thr Phe Arg Arg Glu Ser Lys Gln Pro Leu Glu Tyr Pro 210 215 220 tct gtc ggc agc gtg ttt aaa cgc cct cct gga tat ttt gcg ggc aag 720Ser Val Gly Ser Val Phe Lys Arg Pro Pro Gly Tyr Phe Ala Gly Lys 225 230 235 240 ctc att caa gac agc ggc ctg caa gga aaa gga gtc gga ggt gcg gaa 768Leu Ile Gln Asp Ser Gly Leu Gln Gly Lys Gly Val Gly Gly Ala Glu 245 250 255 gta tcg aca aag cac gca ggt ttt att att aat aaa aac aac gcc acc 816Val Ser Thr Lys His Ala Gly Phe Ile Ile Asn Lys Asn Asn Ala Thr 260 265 270 gcc tct gac tat att gcg acg atc gaa ctg gtg cgg aaa acg gtt aaa 864Ala Ser Asp Tyr Ile Ala Thr Ile Glu Leu Val Arg Lys Thr Val Lys 275 280 285 gaa aaa ttc ggc gtc gat ctg gaa tta gaa gtg aaa att atc ggg gag 912Glu Lys Phe Gly Val Asp Leu Glu Leu Glu Val Lys Ile Ile Gly Glu 290 295 300 gat atc aaa cag taa 927Asp Ile Lys Gln 305 26308PRTGeobacillus strain Y4.1MC1 26Met Met Tyr Glu Asn Asn Val Ile Tyr Gln Glu Leu Val Arg Ile Cys 1 5 10 15 Gly Glu Lys Asn Val Leu Arg Asp Glu Pro Leu Lys Tyr His Thr Leu 20 25 30 Val Lys Ile Gly Gly Lys Ala Asp Phe Leu Val Trp Pro Glu Thr Tyr 35 40 45 Glu Gln Val Val Glu Val Ile Arg Leu Lys Glu Lys His His Leu Pro 50 55 60 Phe Thr Leu Leu Gly Asn Gly Ser Asn Val Ile Val Arg Asp Gly Gly 65 70 75 80 Ile Arg Gly Ile Val Val Gln Leu Lys His Leu Thr Glu Ile Lys Val 85 90 95 Glu Gly Glu Lys Ile Ile Ala Gln Ser Gly Ala Asp Ile Lys Ala Val 100 105 110 Ser Arg Val Ala Leu Glu His Ser Leu Thr Gly Leu Glu Phe Ala Cys 115 120 125 Gly Ile Pro Gly Ser Val Gly Gly Ala Ile Met Met Asn Ala Gly Ala 130 135 140 Tyr Asp Gly Glu Ile Lys Asp Val Ile Asp His Val Lys Val Val Thr 145 150 155 160 Gln Thr Gly Glu Gln Lys Ile Leu Arg Lys Asp Asp Leu Gln Leu Gly 165 170 175 Tyr Arg Thr Ser Ile Ile Ser Lys Thr Asn Asp Ile Val Leu Glu Ala 180 185 190 Val Phe Gln Leu Lys Lys Gly Asp Pro Gln Lys Ile Lys Glu Lys Met 195 200 205 Asp Asp Leu Thr Phe Arg Arg Glu Ser Lys Gln Pro Leu Glu Tyr Pro 210 215 220 Ser Val Gly Ser Val Phe Lys Arg Pro Pro Gly Tyr Phe Ala Gly Lys 225 230 235 240 Leu Ile Gln Asp Ser Gly Leu Gln Gly Lys Gly Val Gly Gly Ala Glu 245 250 255 Val Ser Thr Lys His Ala Gly Phe Ile Ile Asn Lys Asn Asn Ala Thr 260 265 270 Ala Ser Asp Tyr Ile Ala Thr Ile Glu Leu Val Arg Lys Thr Val Lys 275 280 285 Glu Lys Phe Gly Val Asp Leu Glu Leu Glu Val Lys Ile Ile Gly Glu 290 295 300 Asp Ile Lys Gln 305 27965DNAGeobacillus strain Y4.1MC1CDS(15)..(959) 27aggagataaa acat atg atg tac gaa aac aat gta atc tat caa gag ctg 50 Met Met Tyr Glu Asn Asn Val Ile Tyr Gln Glu Leu 1 5 10 gtg cgt att tgt ggt gag aaa aac gtc ctg cgc gat gaa cct ctg aaa 98Val Arg Ile Cys Gly Glu Lys Asn Val Leu Arg Asp Glu Pro Leu Lys 15 20 25 tat cac acg ttg gtg aag att ggt ggc aag gcg gat ttc ctg gtt tgg 146Tyr His Thr Leu Val Lys Ile Gly Gly Lys Ala Asp Phe Leu Val Trp 30 35 40 ccg gaa acc tac gaa cag gtg gtc gag gtg atc cgt ttg aaa gag aag 194Pro Glu Thr Tyr Glu Gln Val Val Glu Val Ile Arg Leu Lys Glu Lys 45 50 55 60 cac cac ttg ccg ttc acg ttg

ctg ggt aat ggt agc aat gtc att gtt 242His His Leu Pro Phe Thr Leu Leu Gly Asn Gly Ser Asn Val Ile Val 65 70 75 cgc gac ggt ggt atc cgt ggc atc gtg gtg caa ctg aag cat ctg act 290Arg Asp Gly Gly Ile Arg Gly Ile Val Val Gln Leu Lys His Leu Thr 80 85 90 gag att aaa gtt gaa ggc gag aag att att gcg cag tct ggc gcc gac 338Glu Ile Lys Val Glu Gly Glu Lys Ile Ile Ala Gln Ser Gly Ala Asp 95 100 105 atc aaa gcc gtc agc cgt gtg gcg ctg gag cat tct ctg acc ggc ctg 386Ile Lys Ala Val Ser Arg Val Ala Leu Glu His Ser Leu Thr Gly Leu 110 115 120 gaa ttt gca tgc ggt att ccg ggc agc gtt ggt ggt gct att atg atg 434Glu Phe Ala Cys Gly Ile Pro Gly Ser Val Gly Gly Ala Ile Met Met 125 130 135 140 aac gct ggc gcg tac gat ggt gaa atc aaa gat gtc att gat cac gtt 482Asn Ala Gly Ala Tyr Asp Gly Glu Ile Lys Asp Val Ile Asp His Val 145 150 155 aaa gtt gtg acc cag acc ggt gag cag aag atc ctg cgt aaa gac gat 530Lys Val Val Thr Gln Thr Gly Glu Gln Lys Ile Leu Arg Lys Asp Asp 160 165 170 ctg cag ctg ggt tac cgt acc agc att atc agc aaa acg aat gat atc 578Leu Gln Leu Gly Tyr Arg Thr Ser Ile Ile Ser Lys Thr Asn Asp Ile 175 180 185 gtg ctg gaa gcg gtt ttt cag ttg aag aag ggt gac ccg caa aag atc 626Val Leu Glu Ala Val Phe Gln Leu Lys Lys Gly Asp Pro Gln Lys Ile 190 195 200 aaa gag aag atg gac gac ctg acc ttt cgt cgc gag tcc aaa caa ccg 674Lys Glu Lys Met Asp Asp Leu Thr Phe Arg Arg Glu Ser Lys Gln Pro 205 210 215 220 ctg gag tat ccg agc gtt ggc agc gtc ttt aag cgc cca ccg ggc tac 722Leu Glu Tyr Pro Ser Val Gly Ser Val Phe Lys Arg Pro Pro Gly Tyr 225 230 235 ttc gcg ggc aag ctg atc cag gac agc ggt ctg caa ggt aaa ggt gtc 770Phe Ala Gly Lys Leu Ile Gln Asp Ser Gly Leu Gln Gly Lys Gly Val 240 245 250 ggc ggt gca gag gtg agc acc aaa cat gca ggc ttc atc att aac aaa 818Gly Gly Ala Glu Val Ser Thr Lys His Ala Gly Phe Ile Ile Asn Lys 255 260 265 aac aat gcc acg gcg tcc gac tat atc gca acc att gaa ctg gtt cgt 866Asn Asn Ala Thr Ala Ser Asp Tyr Ile Ala Thr Ile Glu Leu Val Arg 270 275 280 aaa acg gtt aaa gaa aag ttc ggt gtc gat ctg gag ctg gaa gtt aag 914Lys Thr Val Lys Glu Lys Phe Gly Val Asp Leu Glu Leu Glu Val Lys 285 290 295 300 att atc ggt gag gac att aag caa cac cac cac cat cac cat taa 959Ile Ile Gly Glu Asp Ile Lys Gln His His His His His His 305 310 ctcgag 96528314PRTGeobacillus strain Y4.1MC1 28Met Met Tyr Glu Asn Asn Val Ile Tyr Gln Glu Leu Val Arg Ile Cys 1 5 10 15 Gly Glu Lys Asn Val Leu Arg Asp Glu Pro Leu Lys Tyr His Thr Leu 20 25 30 Val Lys Ile Gly Gly Lys Ala Asp Phe Leu Val Trp Pro Glu Thr Tyr 35 40 45 Glu Gln Val Val Glu Val Ile Arg Leu Lys Glu Lys His His Leu Pro 50 55 60 Phe Thr Leu Leu Gly Asn Gly Ser Asn Val Ile Val Arg Asp Gly Gly 65 70 75 80 Ile Arg Gly Ile Val Val Gln Leu Lys His Leu Thr Glu Ile Lys Val 85 90 95 Glu Gly Glu Lys Ile Ile Ala Gln Ser Gly Ala Asp Ile Lys Ala Val 100 105 110 Ser Arg Val Ala Leu Glu His Ser Leu Thr Gly Leu Glu Phe Ala Cys 115 120 125 Gly Ile Pro Gly Ser Val Gly Gly Ala Ile Met Met Asn Ala Gly Ala 130 135 140 Tyr Asp Gly Glu Ile Lys Asp Val Ile Asp His Val Lys Val Val Thr 145 150 155 160 Gln Thr Gly Glu Gln Lys Ile Leu Arg Lys Asp Asp Leu Gln Leu Gly 165 170 175 Tyr Arg Thr Ser Ile Ile Ser Lys Thr Asn Asp Ile Val Leu Glu Ala 180 185 190 Val Phe Gln Leu Lys Lys Gly Asp Pro Gln Lys Ile Lys Glu Lys Met 195 200 205 Asp Asp Leu Thr Phe Arg Arg Glu Ser Lys Gln Pro Leu Glu Tyr Pro 210 215 220 Ser Val Gly Ser Val Phe Lys Arg Pro Pro Gly Tyr Phe Ala Gly Lys 225 230 235 240 Leu Ile Gln Asp Ser Gly Leu Gln Gly Lys Gly Val Gly Gly Ala Glu 245 250 255 Val Ser Thr Lys His Ala Gly Phe Ile Ile Asn Lys Asn Asn Ala Thr 260 265 270 Ala Ser Asp Tyr Ile Ala Thr Ile Glu Leu Val Arg Lys Thr Val Lys 275 280 285 Glu Lys Phe Gly Val Asp Leu Glu Leu Glu Val Lys Ile Ile Gly Glu 290 295 300 Asp Ile Lys Gln His His His His His His 305 310 291026DNARhodothermus marinus strain SG0.5JP17-172CDS(1)..(1026) 29atg gca aac gag acc tct gtg att cct ccg tcg gta tat gaa gca tta 48Met Ala Asn Glu Thr Ser Val Ile Pro Pro Ser Val Tyr Glu Ala Leu 1 5 10 15 gtg gcc cgg ttg ggg cct cgg cgc gtg cag cga aac gtg ccg ctg gcg 96Val Ala Arg Leu Gly Pro Arg Arg Val Gln Arg Asn Val Pro Leu Ala 20 25 30 ccg tac acg acg ttc aaa atc ggc ggg ccg gcg gat ctg ttc ttc gag 144Pro Tyr Thr Thr Phe Lys Ile Gly Gly Pro Ala Asp Leu Phe Phe Glu 35 40 45 gcg cac acg cgc gac gag ctg gcc gaa gcg gtg cag atc gcc cgc acg 192Ala His Thr Arg Asp Glu Leu Ala Glu Ala Val Gln Ile Ala Arg Thr 50 55 60 ctg agc atc ccc tac ttt gtg ctc ggc ctg ggc gcc aac atc cta gta 240Leu Ser Ile Pro Tyr Phe Val Leu Gly Leu Gly Ala Asn Ile Leu Val 65 70 75 80 ggc gat cgg ggc ttt cgg gga ctg gtc att cgc aac agg gcc cgc gcc 288Gly Asp Arg Gly Phe Arg Gly Leu Val Ile Arg Asn Arg Ala Arg Ala 85 90 95 tgc cgc ctg ctg ccc ggt cac cgg ctc tgg gcc gaa agc ggg gcg gtt 336Cys Arg Leu Leu Pro Gly His Arg Leu Trp Ala Glu Ser Gly Ala Val 100 105 110 gtc tat cct gac ctt att gaa acg gct gtc gag gcc ggc ctt tct ggc 384Val Tyr Pro Asp Leu Ile Glu Thr Ala Val Glu Ala Gly Leu Ser Gly 115 120 125 ctg gaa cat tac gtg ggc atc ccg tcc acc gta ggc ggc gcg ctc tgg 432Leu Glu His Tyr Val Gly Ile Pro Ser Thr Val Gly Gly Ala Leu Trp 130 135 140 cag aac ctg cat ttt ctc tcg ccg cca ccc aag cgc gag cgt acc gtc 480Gln Asn Leu His Phe Leu Ser Pro Pro Pro Lys Arg Glu Arg Thr Val 145 150 155 160 ttt atc gaa gag gtg ctg gcc gaa gcc gaa atc ctg acg gcc gaa ggc 528Phe Ile Glu Glu Val Leu Ala Glu Ala Glu Ile Leu Thr Ala Glu Gly 165 170 175 aaa cgc cgc ctc gtt ggc cct gac tac ttc cgc ttc ggc tac gac tat 576Lys Arg Arg Leu Val Gly Pro Asp Tyr Phe Arg Phe Gly Tyr Asp Tyr 180 185 190 tcg atc ctg cat gaa cgc gac gac att gtg ctg gcc gct acg ttc cag 624Ser Ile Leu His Glu Arg Asp Asp Ile Val Leu Ala Ala Thr Phe Gln 195 200 205 cta agc ccg ggc gat cca gca cgc atg cgc gaa gtc atg gcc gcc aat 672Leu Ser Pro Gly Asp Pro Ala Arg Met Arg Glu Val Met Ala Ala Asn 210 215 220 ctc gcc tgg cga cgt gaa cgc cat cca cca ctt gag acc gaa cct agt 720Leu Ala Trp Arg Arg Glu Arg His Pro Pro Leu Glu Thr Glu Pro Ser 225 230 235 240 gct ggc tcc atc ttc aaa aag atc gac ggc att ggc gct ggc cgg ctg 768Ala Gly Ser Ile Phe Lys Lys Ile Asp Gly Ile Gly Ala Gly Arg Leu 245 250 255 atc gac cag tgc ggc ctg aaa ggc acg cgc atc ggc gat gcc gag gtt 816Ile Asp Gln Cys Gly Leu Lys Gly Thr Arg Ile Gly Asp Ala Glu Val 260 265 270 tca ccg cgc cat gcc aat atc ata gtg aac cgg gga aag gca acc gct 864Ser Pro Arg His Ala Asn Ile Ile Val Asn Arg Gly Lys Ala Thr Ala 275 280 285 gcg caa gtg cgc gcg ctc atc gcc tac gtg cag cag gtg gtc gaa gcc 912Ala Gln Val Arg Ala Leu Ile Ala Tyr Val Gln Gln Val Val Glu Ala 290 295 300 cgc acc ggc tat cac ctg gaa cct gaa atc cgt ttt gtc ggc gaa ttt 960Arg Thr Gly Tyr His Leu Glu Pro Glu Ile Arg Phe Val Gly Glu Phe 305 310 315 320 gat ccc cct gct gaa aac gaa ata ctt ctt ccg aac cat gcc gca atc 1008Asp Pro Pro Ala Glu Asn Glu Ile Leu Leu Pro Asn His Ala Ala Ile 325 330 335 cgc cat cca gac cgt tga 1026Arg His Pro Asp Arg 340 30341PRTRhodothermus marinus strain SG0.5JP17-172 30Met Ala Asn Glu Thr Ser Val Ile Pro Pro Ser Val Tyr Glu Ala Leu 1 5 10 15 Val Ala Arg Leu Gly Pro Arg Arg Val Gln Arg Asn Val Pro Leu Ala 20 25 30 Pro Tyr Thr Thr Phe Lys Ile Gly Gly Pro Ala Asp Leu Phe Phe Glu 35 40 45 Ala His Thr Arg Asp Glu Leu Ala Glu Ala Val Gln Ile Ala Arg Thr 50 55 60 Leu Ser Ile Pro Tyr Phe Val Leu Gly Leu Gly Ala Asn Ile Leu Val 65 70 75 80 Gly Asp Arg Gly Phe Arg Gly Leu Val Ile Arg Asn Arg Ala Arg Ala 85 90 95 Cys Arg Leu Leu Pro Gly His Arg Leu Trp Ala Glu Ser Gly Ala Val 100 105 110 Val Tyr Pro Asp Leu Ile Glu Thr Ala Val Glu Ala Gly Leu Ser Gly 115 120 125 Leu Glu His Tyr Val Gly Ile Pro Ser Thr Val Gly Gly Ala Leu Trp 130 135 140 Gln Asn Leu His Phe Leu Ser Pro Pro Pro Lys Arg Glu Arg Thr Val 145 150 155 160 Phe Ile Glu Glu Val Leu Ala Glu Ala Glu Ile Leu Thr Ala Glu Gly 165 170 175 Lys Arg Arg Leu Val Gly Pro Asp Tyr Phe Arg Phe Gly Tyr Asp Tyr 180 185 190 Ser Ile Leu His Glu Arg Asp Asp Ile Val Leu Ala Ala Thr Phe Gln 195 200 205 Leu Ser Pro Gly Asp Pro Ala Arg Met Arg Glu Val Met Ala Ala Asn 210 215 220 Leu Ala Trp Arg Arg Glu Arg His Pro Pro Leu Glu Thr Glu Pro Ser 225 230 235 240 Ala Gly Ser Ile Phe Lys Lys Ile Asp Gly Ile Gly Ala Gly Arg Leu 245 250 255 Ile Asp Gln Cys Gly Leu Lys Gly Thr Arg Ile Gly Asp Ala Glu Val 260 265 270 Ser Pro Arg His Ala Asn Ile Ile Val Asn Arg Gly Lys Ala Thr Ala 275 280 285 Ala Gln Val Arg Ala Leu Ile Ala Tyr Val Gln Gln Val Val Glu Ala 290 295 300 Arg Thr Gly Tyr His Leu Glu Pro Glu Ile Arg Phe Val Gly Glu Phe 305 310 315 320 Asp Pro Pro Ala Glu Asn Glu Ile Leu Leu Pro Asn His Ala Ala Ile 325 330 335 Arg His Pro Asp Arg 340 311064DNARhodothermus marinus strain SG0.5JP17-172CDS(15)..(1058) 31aggagataaa acat atg gca aat gaa acg agc gta atc cca ccg tct gtg 50 Met Ala Asn Glu Thr Ser Val Ile Pro Pro Ser Val 1 5 10 tat gaa gcc ctg gtc gcc cgt ctg ggt ccg cgt cgt gtt caa cgt aat 98Tyr Glu Ala Leu Val Ala Arg Leu Gly Pro Arg Arg Val Gln Arg Asn 15 20 25 gtt ccg ttg gca ccg tac acg acc ttc aaa att ggc ggt ccg gcg gac 146Val Pro Leu Ala Pro Tyr Thr Thr Phe Lys Ile Gly Gly Pro Ala Asp 30 35 40 ctg ttt ttc gag gcg cat acc cgt gat gag ttg gcg gaa gct gtc caa 194Leu Phe Phe Glu Ala His Thr Arg Asp Glu Leu Ala Glu Ala Val Gln 45 50 55 60 att gcc cgc acc ctg tcc atc ccg tat ttt gtc ctg ggt ctg ggc gct 242Ile Ala Arg Thr Leu Ser Ile Pro Tyr Phe Val Leu Gly Leu Gly Ala 65 70 75 aac att ctg gtt ggc gat cgt ggt ttc cgc ggt ctg gtg att cgt aac 290Asn Ile Leu Val Gly Asp Arg Gly Phe Arg Gly Leu Val Ile Arg Asn 80 85 90 cgc gca cgt gcc tgt cgt ttg ctg cca ggc cac cgc ctg tgg gca gag 338Arg Ala Arg Ala Cys Arg Leu Leu Pro Gly His Arg Leu Trp Ala Glu 95 100 105 tct ggt gcc gtg gtg tat ccg gat ctg atc gaa acg gcg gtg gaa gcg 386Ser Gly Ala Val Val Tyr Pro Asp Leu Ile Glu Thr Ala Val Glu Ala 110 115 120 ggc ctg agc ggt ctg gag cac tac gtt ggc att ccg agc acc gtc ggc 434Gly Leu Ser Gly Leu Glu His Tyr Val Gly Ile Pro Ser Thr Val Gly 125 130 135 140 ggt gct ctg tgg cag aat ctg cat ttt ctg agc cca ccg ccg aag cgc 482Gly Ala Leu Trp Gln Asn Leu His Phe Leu Ser Pro Pro Pro Lys Arg 145 150 155 gaa cgc acg gtt ttc atc gaa gag gtt ctg gca gag gca gaa att ctg 530Glu Arg Thr Val Phe Ile Glu Glu Val Leu Ala Glu Ala Glu Ile Leu 160 165 170 acg gca gag ggt aaa cgt cgc ttg gtt ggc ccg gac tac ttt cgc ttt 578Thr Ala Glu Gly Lys Arg Arg Leu Val Gly Pro Asp Tyr Phe Arg Phe 175 180 185 ggt tac gat tac agc att ctg cac gaa cgt gac gac att gtc ctg gcg 626Gly Tyr Asp Tyr Ser Ile Leu His Glu Arg Asp Asp Ile Val Leu Ala 190 195 200 gcg acc ttc cag ctg tcc ccg ggt gac cct gcg cgc atg cgt gag gtc 674Ala Thr Phe Gln Leu Ser Pro Gly Asp Pro Ala Arg Met Arg Glu Val 205 210 215 220 atg gcg gcc aac ttg gcg tgg cgt cgc gaa cgc cat ccg ccg ctg gaa 722Met Ala Ala Asn Leu Ala Trp Arg Arg Glu Arg His Pro Pro Leu Glu 225 230 235 act gag ccg agc gcg ggt agc atc ttt aag aag atc gat ggt atc ggc 770Thr Glu Pro Ser Ala Gly Ser Ile Phe Lys Lys Ile Asp Gly Ile Gly 240 245 250 gct ggt cgc ctg atc gac caa tgc ggc ctg aaa ggt acc cgt atc ggc 818Ala Gly Arg Leu Ile Asp Gln Cys Gly Leu Lys Gly Thr Arg Ile Gly 255 260 265 gac gct gag gtg agc cct cgt cac gca aac atc att gtt aac cgt ggt 866Asp Ala Glu Val Ser Pro Arg His Ala Asn Ile Ile Val Asn Arg Gly 270 275 280 aaa gca acc gcc gca cag gtg cgt gcg ctg atc gcg tac gtg cag caa 914Lys Ala Thr Ala Ala Gln Val Arg Ala Leu Ile Ala Tyr Val Gln Gln 285 290 295 300 gtg gtt gag gcg cgt acc ggt tat cac ttg gag ccg gaa atc cgt ttc 962Val Val Glu Ala Arg Thr Gly Tyr His Leu Glu Pro Glu Ile Arg Phe 305 310 315 gtc ggt gag ttc gac cca ccg gcg gag aat gag att ctg ctg ccg aat 1010Val Gly Glu Phe Asp Pro Pro Ala Glu Asn Glu Ile Leu Leu Pro Asn 320 325 330

cac gcg gcg att cgt cac ccg gat cgt cat cat cat cac cac cac taa 1058His Ala Ala Ile Arg His Pro Asp Arg His His His His His His 335 340 345 ctcgag 106432347PRTRhodothermus marinus strain SG0.5JP17-172 32Met Ala Asn Glu Thr Ser Val Ile Pro Pro Ser Val Tyr Glu Ala Leu 1 5 10 15 Val Ala Arg Leu Gly Pro Arg Arg Val Gln Arg Asn Val Pro Leu Ala 20 25 30 Pro Tyr Thr Thr Phe Lys Ile Gly Gly Pro Ala Asp Leu Phe Phe Glu 35 40 45 Ala His Thr Arg Asp Glu Leu Ala Glu Ala Val Gln Ile Ala Arg Thr 50 55 60 Leu Ser Ile Pro Tyr Phe Val Leu Gly Leu Gly Ala Asn Ile Leu Val 65 70 75 80 Gly Asp Arg Gly Phe Arg Gly Leu Val Ile Arg Asn Arg Ala Arg Ala 85 90 95 Cys Arg Leu Leu Pro Gly His Arg Leu Trp Ala Glu Ser Gly Ala Val 100 105 110 Val Tyr Pro Asp Leu Ile Glu Thr Ala Val Glu Ala Gly Leu Ser Gly 115 120 125 Leu Glu His Tyr Val Gly Ile Pro Ser Thr Val Gly Gly Ala Leu Trp 130 135 140 Gln Asn Leu His Phe Leu Ser Pro Pro Pro Lys Arg Glu Arg Thr Val 145 150 155 160 Phe Ile Glu Glu Val Leu Ala Glu Ala Glu Ile Leu Thr Ala Glu Gly 165 170 175 Lys Arg Arg Leu Val Gly Pro Asp Tyr Phe Arg Phe Gly Tyr Asp Tyr 180 185 190 Ser Ile Leu His Glu Arg Asp Asp Ile Val Leu Ala Ala Thr Phe Gln 195 200 205 Leu Ser Pro Gly Asp Pro Ala Arg Met Arg Glu Val Met Ala Ala Asn 210 215 220 Leu Ala Trp Arg Arg Glu Arg His Pro Pro Leu Glu Thr Glu Pro Ser 225 230 235 240 Ala Gly Ser Ile Phe Lys Lys Ile Asp Gly Ile Gly Ala Gly Arg Leu 245 250 255 Ile Asp Gln Cys Gly Leu Lys Gly Thr Arg Ile Gly Asp Ala Glu Val 260 265 270 Ser Pro Arg His Ala Asn Ile Ile Val Asn Arg Gly Lys Ala Thr Ala 275 280 285 Ala Gln Val Arg Ala Leu Ile Ala Tyr Val Gln Gln Val Val Glu Ala 290 295 300 Arg Thr Gly Tyr His Leu Glu Pro Glu Ile Arg Phe Val Gly Glu Phe 305 310 315 320 Asp Pro Pro Ala Glu Asn Glu Ile Leu Leu Pro Asn His Ala Ala Ile 325 330 335 Arg His Pro Asp Arg His His His His His His 340 345 33867DNASulfurihydrogenibium,yellowstonense SS-5CDS(1)..(867) 33atg att tta aaa acg att gaa cat caa gaa aat ata gac ctt agg aat 48Met Ile Leu Lys Thr Ile Glu His Gln Glu Asn Ile Asp Leu Arg Asn 1 5 10 15 ttt tgc act ata aaa gtt ggc gaa aaa gga aag att gta tat ttt cca 96Phe Cys Thr Ile Lys Val Gly Glu Lys Gly Lys Ile Val Tyr Phe Pro 20 25 30 aaa gat tac aaa gaa atc tct att tta att aaa gag tat gat aac atc 144Lys Asp Tyr Lys Glu Ile Ser Ile Leu Ile Lys Glu Tyr Asp Asn Ile 35 40 45 tat cca ctt gga att gga agc aat cta att ttt tcc gat gga gtt gtg 192Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Val Val 50 55 60 aat aaa gtt ttt gtc cat tct aag aat cta aaa aag tat gag ata gaa 240Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys Tyr Glu Ile Glu 65 70 75 80 aat caa aac gat att ttt tac atc acg gca gaa gcc ggc gta agc ttt 288Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 aaa acc ata gtt tca gta gta aaa aag tac aac ctt gaa gga ttt gaa 336Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 aac cta tcc ggc att cct gca acc gtt ggc ggg gca act gca atg aac 384Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Thr Ala Met Asn 115 120 125 gca gga gct tac gga agc gaa atc ttt gat tta ata gaa gag gtt tgg 432Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Leu Ile Glu Glu Val Trp 130 135 140 tgg ata gac aga gaa gga cgg cta aat cac tca aag aag gac gag att 480Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys Lys Asp Glu Ile 145 150 155 160 aaa tac tct tac aga tac tct caa ttt caa aac gaa ggt ttt gtt tat 528Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Glu Gly Phe Val Tyr 165 170 175 aaa gtg aag cta aaa ctt aga aaa agt gat aaa aac ata tca gag att 576Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn Ile Ser Glu Ile 180 185 190 ata aaa aac cat ctg ctt gat aga aac agt aag caa ccc ctt gat cta 624Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln Pro Leu Asp Leu 195 200 205 cca aca gcc gga tca act tac aaa aat cca gcg gga aca tat gcc ggc 672Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Ala Gly Thr Tyr Ala Gly 210 215 220 aag ctg att gaa gca gtt ggc tta aaa ggt tat aga ata aac gat ata 720Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 gga ttt tct gaa aaa cat gca aac ttt ctt gta aat tat gga aat gct 768Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Asn Ala 245 250 255 gag ttt aaa gac cta ata aaa ctc tta gaa ctt gcc gaa aga aaa ata 816Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala Glu Arg Lys Ile 260 265 270 tca gat gaa ttt agg ata aat ctt gaa aga gag gtt aaa atc att gat 864Ser Asp Glu Phe Arg Ile Asn Leu Glu Arg Glu Val Lys Ile Ile Asp 275 280 285 taa 86734288PRTSulfurihydrogenibium,yellowstonense SS-5 34Met Ile Leu Lys Thr Ile Glu His Gln Glu Asn Ile Asp Leu Arg Asn 1 5 10 15 Phe Cys Thr Ile Lys Val Gly Glu Lys Gly Lys Ile Val Tyr Phe Pro 20 25 30 Lys Asp Tyr Lys Glu Ile Ser Ile Leu Ile Lys Glu Tyr Asp Asn Ile 35 40 45 Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Val Val 50 55 60 Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys Tyr Glu Ile Glu 65 70 75 80 Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Thr Ala Met Asn 115 120 125 Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Leu Ile Glu Glu Val Trp 130 135 140 Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys Lys Asp Glu Ile 145 150 155 160 Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Glu Gly Phe Val Tyr 165 170 175 Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn Ile Ser Glu Ile 180 185 190 Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Ala Gly Thr Tyr Ala Gly 210 215 220 Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Asn Ala 245 250 255 Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala Glu Arg Lys Ile 260 265 270 Ser Asp Glu Phe Arg Ile Asn Leu Glu Arg Glu Val Lys Ile Ile Asp 275 280 285 35905DNASulfurihydrogenibium yellowstonense SS-5CDS(15)..(899) 35aggagataaa acat atg att ctg aaa acc att gaa cac caa gag aac att 50 Met Ile Leu Lys Thr Ile Glu His Gln Glu Asn Ile 1 5 10 gat ttg cgt aat ttc tgt act atc aaa gtc ggt gaa aag ggc aag atc 98Asp Leu Arg Asn Phe Cys Thr Ile Lys Val Gly Glu Lys Gly Lys Ile 15 20 25 gtt tac ttt ccg aaa gac tac aaa gaa atc agc atc ctg att aaa gag 146Val Tyr Phe Pro Lys Asp Tyr Lys Glu Ile Ser Ile Leu Ile Lys Glu 30 35 40 tac gac aac att tat ccg ctg ggc att ggc tcg aat ttg att ttc agc 194Tyr Asp Asn Ile Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser 45 50 55 60 gat ggc gtt gtc aac aag gtt ttc gtt cac agc aaa aat ctg aag aag 242Asp Gly Val Val Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys 65 70 75 tat gaa att gag aat caa aat gac atc ttc tat atc acc gca gag gcc 290Tyr Glu Ile Glu Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala 80 85 90 ggt gtt agc ttc aag acg atc gtt agc gtg gtc aaa aag tac aat ctg 338Gly Val Ser Phe Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu 95 100 105 gaa ggt ttt gaa aac ttg agc ggt att ccg gct acg gtg ggt ggt gcg 386Glu Gly Phe Glu Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala 110 115 120 acc gcc atg aat gca ggc gcg tac ggt tct gaa atc ttt gac ctg att 434Thr Ala Met Asn Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Leu Ile 125 130 135 140 gaa gaa gtg tgg tgg atc gat cgc gag ggt cgc ctg aac cac tct aaa 482Glu Glu Val Trp Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys 145 150 155 aag gac gag atc aaa tac agc tac cgt tac agc cag ttc cag aac gaa 530Lys Asp Glu Ile Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Glu 160 165 170 ggc ttt gtg tat aag gtc aag ctg aaa ctg cgc aaa agc gac aag aac 578Gly Phe Val Tyr Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn 175 180 185 att tcc gag atc atc aaa aat cat ctg ctg gac cgt aac agc aag cag 626Ile Ser Glu Ile Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln 190 195 200 ccg ctg gat ctg cca acc gcg ggt agc acg tat aag aac ccg gct ggt 674Pro Leu Asp Leu Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Ala Gly 205 210 215 220 acc tat gcg ggc aaa ctg atc gag gcc gtg ggc ctg aag ggt tat cgt 722Thr Tyr Ala Gly Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg 225 230 235 atc aat gac att ggt ttc tcc gag aaa cat gcg aat ttt ctg gtc aac 770Ile Asn Asp Ile Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn 240 245 250 tac ggt aac gcg gag ttc aaa gat ctg att aag ctg ctg gag ctg gca 818Tyr Gly Asn Ala Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala 255 260 265 gag cgt aag atc agc gat gag ttt cgt att aac ttg gaa cgc gag gtg 866Glu Arg Lys Ile Ser Asp Glu Phe Arg Ile Asn Leu Glu Arg Glu Val 270 275 280 aaa atc att gat cac cat cat cac cac cac taa ctcgag 905Lys Ile Ile Asp His His His His His His 285 290 36294PRTSulfurihydrogenibium yellowstonense SS-5 36Met Ile Leu Lys Thr Ile Glu His Gln Glu Asn Ile Asp Leu Arg Asn 1 5 10 15 Phe Cys Thr Ile Lys Val Gly Glu Lys Gly Lys Ile Val Tyr Phe Pro 20 25 30 Lys Asp Tyr Lys Glu Ile Ser Ile Leu Ile Lys Glu Tyr Asp Asn Ile 35 40 45 Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Val Val 50 55 60 Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys Tyr Glu Ile Glu 65 70 75 80 Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Thr Ala Met Asn 115 120 125 Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Leu Ile Glu Glu Val Trp 130 135 140 Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys Lys Asp Glu Ile 145 150 155 160 Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Glu Gly Phe Val Tyr 165 170 175 Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn Ile Ser Glu Ile 180 185 190 Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Ala Gly Thr Tyr Ala Gly 210 215 220 Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Asn Ala 245 250 255 Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala Glu Arg Lys Ile 260 265 270 Ser Asp Glu Phe Arg Ile Asn Leu Glu Arg Glu Val Lys Ile Ile Asp 275 280 285 His His His His His His 290 37888DNAThermodesulfovibrio yellowstonii strain ATCC 51303CDS(1)..(888) 37atg aaa aat att gaa att ttc tta aaa gaa aat agg ata cct tat aaa 48Met Lys Asn Ile Glu Ile Phe Leu Lys Glu Asn Arg Ile Pro Tyr Lys 1 5 10 15 aaa tat gag tct ttg gct aaa tat aca act ctt aga att gga gga tat 96Lys Tyr Glu Ser Leu Ala Lys Tyr Thr Thr Leu Arg Ile Gly Gly Tyr 20 25 30 gca gac ttt gtt gta ttt cca gat gaa gac tct gtt tta aaa ttg ctt 144Ala Asp Phe Val Val Phe Pro Asp Glu Asp Ser Val Leu Lys Leu Leu 35 40 45 gaa att ata aga aat gaa gga aca gca tac tat gtt ata gga gga ggc 192Glu Ile Ile Arg Asn Glu Gly Thr Ala Tyr Tyr Val Ile Gly Gly Gly 50 55 60 agt aat tta gtt gtg cat gat gaa gga ttt aag gga gtg ata atc aat 240Ser Asn Leu Val Val His Asp Glu Gly Phe Lys Gly Val Ile Ile Asn 65 70 75 80 aca aag caa atg aag aga ata aat ctt gaa gga ttt acc ata agg act 288Thr Lys Gln Met Lys Arg Ile Asn Leu Glu Gly Phe Thr Ile Arg Thr 85 90 95 tca gct ggc gta atg ttg cca aga ctt ctt gct ttt gtt tta aaa ata 336Ser Ala Gly Val Met Leu Pro Arg Leu Leu Ala Phe Val Leu Lys Ile 100 105 110 aag ctt tca gga ata gaa ggt ctt att ggt ata cca gga aca gtt gga 384Lys Leu Ser Gly Ile Glu Gly Leu Ile Gly Ile Pro Gly Thr Val Gly 115 120 125 ggt gca ata aaa ggc aat gca ggc tct ttt gga tat gaa att agt gat 432Gly Ala Ile Lys Gly Asn Ala Gly Ser Phe Gly Tyr Glu Ile Ser Asp 130 135 140 tgc ctt gca gaa gtt gaa att ata acc gat aaa ctg gaa act aaa att 480Cys Leu Ala Glu Val Glu Ile Ile Thr Asp Lys Leu Glu Thr Lys Ile

145 150 155 160 cta aaa aaa caa gat att act ttt caa tat cgt agc tca aat ttg gtt 528Leu Lys Lys Gln Asp Ile Thr Phe Gln Tyr Arg Ser Ser Asn Leu Val 165 170 175 gaa aca tgg ctt ata aaa tct gca acc ttt agt tta aag gaa gat gat 576Glu Thr Trp Leu Ile Lys Ser Ala Thr Phe Ser Leu Lys Glu Asp Asp 180 185 190 gga gag gct ttt aac agg atg aaa cag ttt ctc caa aga aaa aaa cag 624Gly Glu Ala Phe Asn Arg Met Lys Gln Phe Leu Gln Arg Lys Lys Gln 195 200 205 act caa cct tta aga gag tat tct gca gga tgc gta ttt aaa aat cca 672Thr Gln Pro Leu Arg Glu Tyr Ser Ala Gly Cys Val Phe Lys Asn Pro 210 215 220 gaa gga caa tca gca ggt tat tta ata gaa aaa gca gga cta aaa gga 720Glu Gly Gln Ser Ala Gly Tyr Leu Ile Glu Lys Ala Gly Leu Lys Gly 225 230 235 240 ttc aga gtt gga gat ata ctt ata agt cat ctc cat gct aac tat ttc 768Phe Arg Val Gly Asp Ile Leu Ile Ser His Leu His Ala Asn Tyr Phe 245 250 255 att aat gtt ggt aaa ggt aag gca aat gat ttt tta aag tta atg gat 816Ile Asn Val Gly Lys Gly Lys Ala Asn Asp Phe Leu Lys Leu Met Asp 260 265 270 att gtt aag gaa aag gta ttc aag ctt ttt tca ata gaa ttg gtg cct 864Ile Val Lys Glu Lys Val Phe Lys Leu Phe Ser Ile Glu Leu Val Pro 275 280 285 gag ata aaa att ttg gag gct taa 888Glu Ile Lys Ile Leu Glu Ala 290 295 38295PRTThermodesulfovibrio yellowstonii strain ATCC 51303 38Met Lys Asn Ile Glu Ile Phe Leu Lys Glu Asn Arg Ile Pro Tyr Lys 1 5 10 15 Lys Tyr Glu Ser Leu Ala Lys Tyr Thr Thr Leu Arg Ile Gly Gly Tyr 20 25 30 Ala Asp Phe Val Val Phe Pro Asp Glu Asp Ser Val Leu Lys Leu Leu 35 40 45 Glu Ile Ile Arg Asn Glu Gly Thr Ala Tyr Tyr Val Ile Gly Gly Gly 50 55 60 Ser Asn Leu Val Val His Asp Glu Gly Phe Lys Gly Val Ile Ile Asn 65 70 75 80 Thr Lys Gln Met Lys Arg Ile Asn Leu Glu Gly Phe Thr Ile Arg Thr 85 90 95 Ser Ala Gly Val Met Leu Pro Arg Leu Leu Ala Phe Val Leu Lys Ile 100 105 110 Lys Leu Ser Gly Ile Glu Gly Leu Ile Gly Ile Pro Gly Thr Val Gly 115 120 125 Gly Ala Ile Lys Gly Asn Ala Gly Ser Phe Gly Tyr Glu Ile Ser Asp 130 135 140 Cys Leu Ala Glu Val Glu Ile Ile Thr Asp Lys Leu Glu Thr Lys Ile 145 150 155 160 Leu Lys Lys Gln Asp Ile Thr Phe Gln Tyr Arg Ser Ser Asn Leu Val 165 170 175 Glu Thr Trp Leu Ile Lys Ser Ala Thr Phe Ser Leu Lys Glu Asp Asp 180 185 190 Gly Glu Ala Phe Asn Arg Met Lys Gln Phe Leu Gln Arg Lys Lys Gln 195 200 205 Thr Gln Pro Leu Arg Glu Tyr Ser Ala Gly Cys Val Phe Lys Asn Pro 210 215 220 Glu Gly Gln Ser Ala Gly Tyr Leu Ile Glu Lys Ala Gly Leu Lys Gly 225 230 235 240 Phe Arg Val Gly Asp Ile Leu Ile Ser His Leu His Ala Asn Tyr Phe 245 250 255 Ile Asn Val Gly Lys Gly Lys Ala Asn Asp Phe Leu Lys Leu Met Asp 260 265 270 Ile Val Lys Glu Lys Val Phe Lys Leu Phe Ser Ile Glu Leu Val Pro 275 280 285 Glu Ile Lys Ile Leu Glu Ala 290 295 39926DNAThermodesulfovibrio yellowstonii strain ATCC 51303CDS(15)..(920) 39aggagataaa acat atg aag aat att gag att ttc ctg aaa gaa aac cgc 50 Met Lys Asn Ile Glu Ile Phe Leu Lys Glu Asn Arg 1 5 10 atc ccg tac aag aaa tat gag agc ctg gcg aaa tac acg acc ctg cgt 98Ile Pro Tyr Lys Lys Tyr Glu Ser Leu Ala Lys Tyr Thr Thr Leu Arg 15 20 25 att ggt ggc tat gca gac ttt gtt gta ttt cca gac gag gac tcg gtg 146Ile Gly Gly Tyr Ala Asp Phe Val Val Phe Pro Asp Glu Asp Ser Val 30 35 40 ttg aag ctg ttg gag atc atc cgc aat gaa ggt acc gcg tac tat gtg 194Leu Lys Leu Leu Glu Ile Ile Arg Asn Glu Gly Thr Ala Tyr Tyr Val 45 50 55 60 atc ggt ggc ggt tct aat ttg gtg gtt cac gac gag ggc ttc aaa ggc 242Ile Gly Gly Gly Ser Asn Leu Val Val His Asp Glu Gly Phe Lys Gly 65 70 75 gtt atc att aac acc aag caa atg aag cgt att aac ctg gaa ggt ttc 290Val Ile Ile Asn Thr Lys Gln Met Lys Arg Ile Asn Leu Glu Gly Phe 80 85 90 acc atc cgt acc tct gca ggc gtc atg ctg ccg cgt ctg ctg gct ttt 338Thr Ile Arg Thr Ser Ala Gly Val Met Leu Pro Arg Leu Leu Ala Phe 95 100 105 gtt ctg aag att aag ctg agc ggt att gag ggt ctg att ggc att ccg 386Val Leu Lys Ile Lys Leu Ser Gly Ile Glu Gly Leu Ile Gly Ile Pro 110 115 120 ggt acg gtg ggt ggc gcg atc aaa ggt aac gca ggc agc ttc ggt tat 434Gly Thr Val Gly Gly Ala Ile Lys Gly Asn Ala Gly Ser Phe Gly Tyr 125 130 135 140 gag atc agc gat tgc ctg gcg gaa gtt gaa atc atc act gat aag ttg 482Glu Ile Ser Asp Cys Leu Ala Glu Val Glu Ile Ile Thr Asp Lys Leu 145 150 155 gaa acc aaa att ctg aaa aag cag gac att acc ttc caa tac cgt tcc 530Glu Thr Lys Ile Leu Lys Lys Gln Asp Ile Thr Phe Gln Tyr Arg Ser 160 165 170 agc aat ttg gtc gaa acc tgg ctg att aag agc gca acg ttc agc ctg 578Ser Asn Leu Val Glu Thr Trp Leu Ile Lys Ser Ala Thr Phe Ser Leu 175 180 185 aaa gag gat gac ggt gag gcc ttc aat cgc atg aaa caa ttt ctg cag 626Lys Glu Asp Asp Gly Glu Ala Phe Asn Arg Met Lys Gln Phe Leu Gln 190 195 200 cgc aag aag caa acg cag ccg ctg cgt gaa tat agc gct ggt tgt gtg 674Arg Lys Lys Gln Thr Gln Pro Leu Arg Glu Tyr Ser Ala Gly Cys Val 205 210 215 220 ttc aaa aat ccg gaa ggc cag agc gcg ggt tac ctg att gag aaa gcc 722Phe Lys Asn Pro Glu Gly Gln Ser Ala Gly Tyr Leu Ile Glu Lys Ala 225 230 235 ggc ctg aag ggt ttc cgt gtc ggt gat atc ttg att agc cat ctg cat 770Gly Leu Lys Gly Phe Arg Val Gly Asp Ile Leu Ile Ser His Leu His 240 245 250 gcg aac tac ttt atc aac gtg ggt aag ggc aaa gcg aac gat ttt ctg 818Ala Asn Tyr Phe Ile Asn Val Gly Lys Gly Lys Ala Asn Asp Phe Leu 255 260 265 aaa ctg atg gat atc gtt aaa gag aaa gtc ttt aaa ctg ttt tcc atc 866Lys Leu Met Asp Ile Val Lys Glu Lys Val Phe Lys Leu Phe Ser Ile 270 275 280 gag ctg gtc ccg gag att aag atc ctg gaa gcc cac cac cat cac cac 914Glu Leu Val Pro Glu Ile Lys Ile Leu Glu Ala His His His His His 285 290 295 300 cac taa ctcgag 926His 40301PRTThermodesulfovibrio yellowstonii strain ATCC 51303 40Met Lys Asn Ile Glu Ile Phe Leu Lys Glu Asn Arg Ile Pro Tyr Lys 1 5 10 15 Lys Tyr Glu Ser Leu Ala Lys Tyr Thr Thr Leu Arg Ile Gly Gly Tyr 20 25 30 Ala Asp Phe Val Val Phe Pro Asp Glu Asp Ser Val Leu Lys Leu Leu 35 40 45 Glu Ile Ile Arg Asn Glu Gly Thr Ala Tyr Tyr Val Ile Gly Gly Gly 50 55 60 Ser Asn Leu Val Val His Asp Glu Gly Phe Lys Gly Val Ile Ile Asn 65 70 75 80 Thr Lys Gln Met Lys Arg Ile Asn Leu Glu Gly Phe Thr Ile Arg Thr 85 90 95 Ser Ala Gly Val Met Leu Pro Arg Leu Leu Ala Phe Val Leu Lys Ile 100 105 110 Lys Leu Ser Gly Ile Glu Gly Leu Ile Gly Ile Pro Gly Thr Val Gly 115 120 125 Gly Ala Ile Lys Gly Asn Ala Gly Ser Phe Gly Tyr Glu Ile Ser Asp 130 135 140 Cys Leu Ala Glu Val Glu Ile Ile Thr Asp Lys Leu Glu Thr Lys Ile 145 150 155 160 Leu Lys Lys Gln Asp Ile Thr Phe Gln Tyr Arg Ser Ser Asn Leu Val 165 170 175 Glu Thr Trp Leu Ile Lys Ser Ala Thr Phe Ser Leu Lys Glu Asp Asp 180 185 190 Gly Glu Ala Phe Asn Arg Met Lys Gln Phe Leu Gln Arg Lys Lys Gln 195 200 205 Thr Gln Pro Leu Arg Glu Tyr Ser Ala Gly Cys Val Phe Lys Asn Pro 210 215 220 Glu Gly Gln Ser Ala Gly Tyr Leu Ile Glu Lys Ala Gly Leu Lys Gly 225 230 235 240 Phe Arg Val Gly Asp Ile Leu Ile Ser His Leu His Ala Asn Tyr Phe 245 250 255 Ile Asn Val Gly Lys Gly Lys Ala Asn Asp Phe Leu Lys Leu Met Asp 260 265 270 Ile Val Lys Glu Lys Val Phe Lys Leu Phe Ser Ile Glu Leu Val Pro 275 280 285 Glu Ile Lys Ile Leu Glu Ala His His His His His His 290 295 300 41855DNAThermotoga maritima strain ATCC 43589CDS(1)..(855) 41atg ttc gaa aaa ctt tcc tgt cac acc agc ata aaa atc ggt gga aga 48Met Phe Glu Lys Leu Ser Cys His Thr Ser Ile Lys Ile Gly Gly Arg 1 5 10 15 gtg aaa tat ctt gtt ctt cca aac gat gtt ttt tct ctg gaa cga gcc 96Val Lys Tyr Leu Val Leu Pro Asn Asp Val Phe Ser Leu Glu Arg Ala 20 25 30 atc act gtt ctg aag gat ctt ccg ttt caa ata atg gga ctt ggc acg 144Ile Thr Val Leu Lys Asp Leu Pro Phe Gln Ile Met Gly Leu Gly Thr 35 40 45 aac ctt ctg gtt caa gat gaa gat ctg gat atc gca gtg ttg aaa aca 192Asn Leu Leu Val Gln Asp Glu Asp Leu Asp Ile Ala Val Leu Lys Thr 50 55 60 gaa agg ttg aat caa att gaa ata aag gga gaa aaa gta ctg gta gaa 240Glu Arg Leu Asn Gln Ile Glu Ile Lys Gly Glu Lys Val Leu Val Glu 65 70 75 80 agt gga act ccc ctg aag aga ctc tgt cta ttt ttg atg gaa gcg gaa 288Ser Gly Thr Pro Leu Lys Arg Leu Cys Leu Phe Leu Met Glu Ala Glu 85 90 95 ctc ggg gga ctg gag ttc gca tac ggg ata ccg ggg agc gtg ggg gga 336Leu Gly Gly Leu Glu Phe Ala Tyr Gly Ile Pro Gly Ser Val Gly Gly 100 105 110 gcc atc tat atg aac gcg gga gcg tac gga gga gag att gga gag ttc 384Ala Ile Tyr Met Asn Ala Gly Ala Tyr Gly Gly Glu Ile Gly Glu Phe 115 120 125 gtc gaa gcg gtc gag gtt cta aga gat gga gaa aag acc tgg ctt tcg 432Val Glu Ala Val Glu Val Leu Arg Asp Gly Glu Lys Thr Trp Leu Ser 130 135 140 agg aac gag att ttt ttc ggc tac aga gac agt acg ttt aaa agg gag 480Arg Asn Glu Ile Phe Phe Gly Tyr Arg Asp Ser Thr Phe Lys Arg Glu 145 150 155 160 aaa ttg atc ata aca cgt gtg atg atg agt ttc aaa aaa gaa aag aag 528Lys Leu Ile Ile Thr Arg Val Met Met Ser Phe Lys Lys Glu Lys Lys 165 170 175 gaa acc ata aaa gcg aag atg gac gat tac atg agg agg cgt ctg gaa 576Glu Thr Ile Lys Ala Lys Met Asp Asp Tyr Met Arg Arg Arg Leu Glu 180 185 190 aaa caa ccc ctt gac ctt ccg agt gcg ggt agt gtt ttc aaa agg cca 624Lys Gln Pro Leu Asp Leu Pro Ser Ala Gly Ser Val Phe Lys Arg Pro 195 200 205 aga gag gat ttc tac gtg gga aag gcc ata gaa tcc ctg ggt ctg aaa 672Arg Glu Asp Phe Tyr Val Gly Lys Ala Ile Glu Ser Leu Gly Leu Lys 210 215 220 ggt tac aga att gga ggg gct cag ata tcg gaa aaa cat gcg gga ttc 720Gly Tyr Arg Ile Gly Gly Ala Gln Ile Ser Glu Lys His Ala Gly Phe 225 230 235 240 att gtg aac gca gga agt gct act ttt gat gac gtg atg aaa ctc att 768Ile Val Asn Ala Gly Ser Ala Thr Phe Asp Asp Val Met Lys Leu Ile 245 250 255 gat ttt gtg aga aaa aag gtg aag gag aaa tac ggt gtg gag ctg gaa 816Asp Phe Val Arg Lys Lys Val Lys Glu Lys Tyr Gly Val Glu Leu Glu 260 265 270 acg gag gtt gaa atc tgg tgg aat gga aga cgg tgg tga 855Thr Glu Val Glu Ile Trp Trp Asn Gly Arg Arg Trp 275 280 42284PRTThermotoga maritima strain ATCC 43589 42Met Phe Glu Lys Leu Ser Cys His Thr Ser Ile Lys Ile Gly Gly Arg 1 5 10 15 Val Lys Tyr Leu Val Leu Pro Asn Asp Val Phe Ser Leu Glu Arg Ala 20 25 30 Ile Thr Val Leu Lys Asp Leu Pro Phe Gln Ile Met Gly Leu Gly Thr 35 40 45 Asn Leu Leu Val Gln Asp Glu Asp Leu Asp Ile Ala Val Leu Lys Thr 50 55 60 Glu Arg Leu Asn Gln Ile Glu Ile Lys Gly Glu Lys Val Leu Val Glu 65 70 75 80 Ser Gly Thr Pro Leu Lys Arg Leu Cys Leu Phe Leu Met Glu Ala Glu 85 90 95 Leu Gly Gly Leu Glu Phe Ala Tyr Gly Ile Pro Gly Ser Val Gly Gly 100 105 110 Ala Ile Tyr Met Asn Ala Gly Ala Tyr Gly Gly Glu Ile Gly Glu Phe 115 120 125 Val Glu Ala Val Glu Val Leu Arg Asp Gly Glu Lys Thr Trp Leu Ser 130 135 140 Arg Asn Glu Ile Phe Phe Gly Tyr Arg Asp Ser Thr Phe Lys Arg Glu 145 150 155 160 Lys Leu Ile Ile Thr Arg Val Met Met Ser Phe Lys Lys Glu Lys Lys 165 170 175 Glu Thr Ile Lys Ala Lys Met Asp Asp Tyr Met Arg Arg Arg Leu Glu 180 185 190 Lys Gln Pro Leu Asp Leu Pro Ser Ala Gly Ser Val Phe Lys Arg Pro 195 200 205 Arg Glu Asp Phe Tyr Val Gly Lys Ala Ile Glu Ser Leu Gly Leu Lys 210 215 220 Gly Tyr Arg Ile Gly Gly Ala Gln Ile Ser Glu Lys His Ala Gly Phe 225 230 235 240 Ile Val Asn Ala Gly Ser Ala Thr Phe Asp Asp Val Met Lys Leu Ile 245 250 255 Asp Phe Val Arg Lys Lys Val Lys Glu Lys Tyr Gly Val Glu Leu Glu 260 265 270 Thr Glu Val Glu Ile Trp Trp Asn Gly Arg Arg Trp 275 280 43893DNAThermotoga maritima strain ATCC 43589CDS(15)..(887) 43aggagataaa acat atg ttc gaa aag ctc tcg tgt cat acc agc atc aaa 50 Met Phe Glu Lys Leu Ser Cys His Thr Ser Ile Lys 1 5 10 atc ggc ggt cgt gta aag tat ttg gtg ttg ccg aac gac gtg ttt agc 98Ile Gly Gly Arg Val Lys Tyr Leu Val Leu Pro Asn Asp Val Phe Ser 15 20 25 ctg gaa cgt gca att acc gtc ctg aaa gat ctg ccg ttc cag atc atg 146Leu Glu Arg Ala Ile Thr Val Leu Lys Asp Leu Pro Phe Gln Ile Met 30 35 40 ggc ctg ggt acg aat ctg ctg gtt cag gac gag gat ctg

gat att gca 194Gly Leu Gly Thr Asn Leu Leu Val Gln Asp Glu Asp Leu Asp Ile Ala 45 50 55 60 gtt ctg aaa acc gaa cgc ctg aac caa atc gag att aaa ggc gaa aag 242Val Leu Lys Thr Glu Arg Leu Asn Gln Ile Glu Ile Lys Gly Glu Lys 65 70 75 gtg ttg gtg gaa agc ggc acg ccg ctg aag cgt ctg tgc ctg ttc ctg 290Val Leu Val Glu Ser Gly Thr Pro Leu Lys Arg Leu Cys Leu Phe Leu 80 85 90 atg gaa gcc gag ctg ggt ggt ttg gag ttt gct tac ggc att ccg ggc 338Met Glu Ala Glu Leu Gly Gly Leu Glu Phe Ala Tyr Gly Ile Pro Gly 95 100 105 agc gtg ggt ggt gcg atc tac atg aac gct ggt gcg tac ggt ggt gaa 386Ser Val Gly Gly Ala Ile Tyr Met Asn Ala Gly Ala Tyr Gly Gly Glu 110 115 120 atc ggc gag ttt gtc gag gcg gtc gaa gtt ctg cgc gac ggt gag aaa 434Ile Gly Glu Phe Val Glu Ala Val Glu Val Leu Arg Asp Gly Glu Lys 125 130 135 140 acc tgg ctg tct cgt aat gag att ttc ttc ggt tac cgt gac agc acg 482Thr Trp Leu Ser Arg Asn Glu Ile Phe Phe Gly Tyr Arg Asp Ser Thr 145 150 155 ttc aaa cgt gag aag ctg atc atc acg cgt gtc atg atg agc ttt aag 530Phe Lys Arg Glu Lys Leu Ile Ile Thr Arg Val Met Met Ser Phe Lys 160 165 170 aaa gag aag aaa gaa acc att aag gcg aag atg gat gac tat atg cgt 578Lys Glu Lys Lys Glu Thr Ile Lys Ala Lys Met Asp Asp Tyr Met Arg 175 180 185 cgt cgt ctg gag aaa cag ccg ctg gat ctg ccg agc gca ggc agc gtt 626Arg Arg Leu Glu Lys Gln Pro Leu Asp Leu Pro Ser Ala Gly Ser Val 190 195 200 ttc aag cgt cca cgc gag gac ttt tac gtc ggt aag gcg att gag tcc 674Phe Lys Arg Pro Arg Glu Asp Phe Tyr Val Gly Lys Ala Ile Glu Ser 205 210 215 220 ctg ggc ctg aaa ggc tat cgc att ggt ggt gca caa atc agc gag aaa 722Leu Gly Leu Lys Gly Tyr Arg Ile Gly Gly Ala Gln Ile Ser Glu Lys 225 230 235 cat gcg ggt ttc atc gtg aat gcc ggc tcc gcc acc ttt gac gac gtt 770His Ala Gly Phe Ile Val Asn Ala Gly Ser Ala Thr Phe Asp Asp Val 240 245 250 atg aaa ttg att gat ttt gtt cgc aaa aag gtt aaa gag aag tat ggt 818Met Lys Leu Ile Asp Phe Val Arg Lys Lys Val Lys Glu Lys Tyr Gly 255 260 265 gtc gag ctg gaa act gaa gtg gaa att tgg tgg aac ggt cgc cgc tgg 866Val Glu Leu Glu Thr Glu Val Glu Ile Trp Trp Asn Gly Arg Arg Trp 270 275 280 cac cac cac cat cac cac taa ctcgag 893His His His His His His 285 290 44290PRTThermotoga maritima strain ATCC 43589 44Met Phe Glu Lys Leu Ser Cys His Thr Ser Ile Lys Ile Gly Gly Arg 1 5 10 15 Val Lys Tyr Leu Val Leu Pro Asn Asp Val Phe Ser Leu Glu Arg Ala 20 25 30 Ile Thr Val Leu Lys Asp Leu Pro Phe Gln Ile Met Gly Leu Gly Thr 35 40 45 Asn Leu Leu Val Gln Asp Glu Asp Leu Asp Ile Ala Val Leu Lys Thr 50 55 60 Glu Arg Leu Asn Gln Ile Glu Ile Lys Gly Glu Lys Val Leu Val Glu 65 70 75 80 Ser Gly Thr Pro Leu Lys Arg Leu Cys Leu Phe Leu Met Glu Ala Glu 85 90 95 Leu Gly Gly Leu Glu Phe Ala Tyr Gly Ile Pro Gly Ser Val Gly Gly 100 105 110 Ala Ile Tyr Met Asn Ala Gly Ala Tyr Gly Gly Glu Ile Gly Glu Phe 115 120 125 Val Glu Ala Val Glu Val Leu Arg Asp Gly Glu Lys Thr Trp Leu Ser 130 135 140 Arg Asn Glu Ile Phe Phe Gly Tyr Arg Asp Ser Thr Phe Lys Arg Glu 145 150 155 160 Lys Leu Ile Ile Thr Arg Val Met Met Ser Phe Lys Lys Glu Lys Lys 165 170 175 Glu Thr Ile Lys Ala Lys Met Asp Asp Tyr Met Arg Arg Arg Leu Glu 180 185 190 Lys Gln Pro Leu Asp Leu Pro Ser Ala Gly Ser Val Phe Lys Arg Pro 195 200 205 Arg Glu Asp Phe Tyr Val Gly Lys Ala Ile Glu Ser Leu Gly Leu Lys 210 215 220 Gly Tyr Arg Ile Gly Gly Ala Gln Ile Ser Glu Lys His Ala Gly Phe 225 230 235 240 Ile Val Asn Ala Gly Ser Ala Thr Phe Asp Asp Val Met Lys Leu Ile 245 250 255 Asp Phe Val Arg Lys Lys Val Lys Glu Lys Tyr Gly Val Glu Leu Glu 260 265 270 Thr Glu Val Glu Ile Trp Trp Asn Gly Arg Arg Trp His His His His 275 280 285 His His 290 45867DNASulfurihydrogenibium sp. strain YO3AOP1CDS(1)..(867) 45atg att tta aaa acg att gaa tat caa gaa aat ata gac ctt agg aat 48Met Ile Leu Lys Thr Ile Glu Tyr Gln Glu Asn Ile Asp Leu Arg Asn 1 5 10 15 ttt tgc act ata aaa gtt ggc gga aaa gga aag att gta tat ttt cca 96Phe Cys Thr Ile Lys Val Gly Gly Lys Gly Lys Ile Val Tyr Phe Pro 20 25 30 aaa gat cac aaa gaa atc tct att tta att aaa gag tat gat aac atc 144Lys Asp His Lys Glu Ile Ser Ile Leu Ile Lys Glu Tyr Asp Asn Ile 35 40 45 tat cca ctt gga att gga agc aat cta att ttt tcc gat ggg att gtg 192Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Ile Val 50 55 60 aat aaa gtt ttt gtc cat tct aag aat cta aaa aaa tat gaa ata gaa 240Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys Tyr Glu Ile Glu 65 70 75 80 aat caa aac gat att ttt tac atc acg gcg gaa gcc ggc gta agc ttt 288Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 aaa acc ata gtt tca gta gta aaa aaa tac aac ctt gaa gga ttt gaa 336Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 aac cta tcc ggc att cct gca acc gtt ggt ggg gca act gca atg aac 384Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Thr Ala Met Asn 115 120 125 gca gga gct tat gga agc gat atc ttt gat tta ata gaa gag gtt tgg 432Ala Gly Ala Tyr Gly Ser Asp Ile Phe Asp Leu Ile Glu Glu Val Trp 130 135 140 tgg ata gac aga gaa gga cgg cta aat cat tca aaa aaa gaa gag att 480Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys Lys Glu Glu Ile 145 150 155 160 aaa tac tct tac aga tac tcc caa ttt caa aac gga ggt ttt gtt tat 528Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Gly Gly Phe Val Tyr 165 170 175 aaa gtg aag cta aag ctt aga aaa agt gat aaa aac ata tca gag att 576Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn Ile Ser Glu Ile 180 185 190 ata aaa aat cat ctg ctt gat aga aac agt aag caa ccg cta gat tta 624Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln Pro Leu Asp Leu 195 200 205 cca aca gcc ggg tca act tac aaa aat cca ccg gga aca tat gcc ggc 672Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gly Thr Tyr Ala Gly 210 215 220 aaa ctg att gaa gca gtt ggc tta aaa ggt tat aga ata aac gat ata 720Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 gga ttt tct gaa aaa cat gca aac ttt ctt gta aac tat gga aat gct 768Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Asn Ala 245 250 255 gag ttt aaa gac cta ata aaa ctc ttg gaa ctt gcc gaa aaa aaa gta 816Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala Glu Lys Lys Val 260 265 270 tta gac gaa ttt aag ata aat ctt gaa aga gag gtt aaa atc att gac 864Leu Asp Glu Phe Lys Ile Asn Leu Glu Arg Glu Val Lys Ile Ile Asp 275 280 285 taa 86746288PRTSulfurihydrogenibium sp. strain YO3AOP1 46Met Ile Leu Lys Thr Ile Glu Tyr Gln Glu Asn Ile Asp Leu Arg Asn 1 5 10 15 Phe Cys Thr Ile Lys Val Gly Gly Lys Gly Lys Ile Val Tyr Phe Pro 20 25 30 Lys Asp His Lys Glu Ile Ser Ile Leu Ile Lys Glu Tyr Asp Asn Ile 35 40 45 Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Ile Val 50 55 60 Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys Tyr Glu Ile Glu 65 70 75 80 Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Thr Ala Met Asn 115 120 125 Ala Gly Ala Tyr Gly Ser Asp Ile Phe Asp Leu Ile Glu Glu Val Trp 130 135 140 Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys Lys Glu Glu Ile 145 150 155 160 Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Gly Gly Phe Val Tyr 165 170 175 Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn Ile Ser Glu Ile 180 185 190 Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gly Thr Tyr Ala Gly 210 215 220 Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Asn Ala 245 250 255 Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala Glu Lys Lys Val 260 265 270 Leu Asp Glu Phe Lys Ile Asn Leu Glu Arg Glu Val Lys Ile Ile Asp 275 280 285 47905DNASulfurihydrogenibium sp. strain YO3AOP1CDS(15)..(899) 47aggaggtaaa acat atg att ttg aaa act att gaa tac cag gaa aac att 50 Met Ile Leu Lys Thr Ile Glu Tyr Gln Glu Asn Ile 1 5 10 gat ttg cgt aac ttt tgt act atc aag gtg ggc ggt aag ggc aag atc 98Asp Leu Arg Asn Phe Cys Thr Ile Lys Val Gly Gly Lys Gly Lys Ile 15 20 25 gtg tac ttt cca aaa gat cac aaa gaa att agc att ctg att aaa gag 146Val Tyr Phe Pro Lys Asp His Lys Glu Ile Ser Ile Leu Ile Lys Glu 30 35 40 tat gac aat atc tat ccg ctg ggt att ggt tcg aat ctg atc ttt agc 194Tyr Asp Asn Ile Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser 45 50 55 60 gat ggt atc gtt aac aag gtt ttc gtg cat tcc aag aac ctg aaa aag 242Asp Gly Ile Val Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys 65 70 75 tat gaa att gag aac caa aat gac atc ttc tac atc acc gcg gaa gcc 290Tyr Glu Ile Glu Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala 80 85 90 ggc gtc agc ttc aaa acc att gtc agc gtt gtc aaa aag tat aat ctg 338Gly Val Ser Phe Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu 95 100 105 gaa ggt ttc gag aat ttg agc ggt att ccg gca acg gtt ggt ggc gcg 386Glu Gly Phe Glu Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala 110 115 120 acc gct atg aat gcc ggt gcg tac ggc tcc gac att ttt gat ctg atc 434Thr Ala Met Asn Ala Gly Ala Tyr Gly Ser Asp Ile Phe Asp Leu Ile 125 130 135 140 gaa gaa gtt tgg tgg att gac cgt gag ggt cgc ctg aat cac agc aag 482Glu Glu Val Trp Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys 145 150 155 aaa gaa gaa atc aag tac tct tac cgc tat tct cag ttt caa aac ggt 530Lys Glu Glu Ile Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Gly 160 165 170 ggc ttt gtg tat aag gtc aaa ctg aaa ctg cgt aaa agc gac aag aac 578Gly Phe Val Tyr Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn 175 180 185 atc agc gag att atc aaa aac cac ctg ttg gat cgt aat agc aag cag 626Ile Ser Glu Ile Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln 190 195 200 ccg ctg gat ctg ccg acc gcc ggt agc acc tac aaa aac ccg cct ggc 674Pro Leu Asp Leu Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gly 205 210 215 220 acg tac gcg ggt aag ctg atc gag gcg gtg ggc ttg aaa ggt tat cgt 722Thr Tyr Ala Gly Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg 225 230 235 atc aac gac atc ggc ttc agc gag aag cat gcg aac ttc ctg gtc aac 770Ile Asn Asp Ile Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn 240 245 250 tac ggt aat gca gag ttc aaa gac ctg atc aaa ctg ctg gag ctg gca 818Tyr Gly Asn Ala Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala 255 260 265 gag aag aaa gtt ctg gac gag ttc aag atc aat ctg gag cgc gag gtg 866Glu Lys Lys Val Leu Asp Glu Phe Lys Ile Asn Leu Glu Arg Glu Val 270 275 280 aag att att gat cac cat cac cac cat cac taa ctcgag 905Lys Ile Ile Asp His His His His His His 285 290 48294PRTSulfurihydrogenibium sp. strain YO3AOP1 48Met Ile Leu Lys Thr Ile Glu Tyr Gln Glu Asn Ile Asp Leu Arg Asn 1 5 10 15 Phe Cys Thr Ile Lys Val Gly Gly Lys Gly Lys Ile Val Tyr Phe Pro 20 25 30 Lys Asp His Lys Glu Ile Ser Ile Leu Ile Lys Glu Tyr Asp Asn Ile 35 40 45 Tyr Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Ile Val 50 55 60 Asn Lys Val Phe Val His Ser Lys Asn Leu Lys Lys Tyr Glu Ile Glu 65 70 75 80 Asn Gln Asn Asp Ile Phe Tyr Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Ile Val Ser Val Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Thr Ala Met Asn 115 120 125 Ala Gly Ala Tyr Gly Ser Asp Ile Phe Asp Leu Ile Glu Glu Val Trp 130 135 140 Trp Ile Asp Arg Glu Gly Arg Leu Asn His Ser Lys Lys Glu Glu Ile 145 150 155 160 Lys Tyr Ser Tyr Arg Tyr Ser Gln Phe Gln Asn Gly Gly Phe Val Tyr 165 170 175 Lys Val Lys Leu Lys Leu Arg Lys Ser Asp Lys Asn Ile Ser Glu Ile 180 185 190 Ile Lys Asn His Leu Leu Asp Arg Asn Ser Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gly Thr Tyr Ala Gly 210 215 220 Lys Leu Ile Glu Ala Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225

230 235 240 Gly Phe Ser Glu Lys His Ala Asn Phe Leu Val Asn Tyr Gly Asn Ala 245 250 255 Glu Phe Lys Asp Leu Ile Lys Leu Leu Glu Leu Ala Glu Lys Lys Val 260 265 270 Leu Asp Glu Phe Lys Ile Asn Leu Glu Arg Glu Val Lys Ile Ile Asp 275 280 285 His His His His His His 290 49867DNASulfurihydrogenibium azorenseCDS(1)..(867) 49atg gat aaa ata gaa cat cta aaa aac ttt agt ctt aaa gat ttt tgc 48Met Asp Lys Ile Glu His Leu Lys Asn Phe Ser Leu Lys Asp Phe Cys 1 5 10 15 act ata aaa ata ggt gga gta gga aaa gtt gtt ttt ttt cct aaa aat 96Thr Ile Lys Ile Gly Gly Val Gly Lys Val Val Phe Phe Pro Lys Asn 20 25 30 gtt gaa gaa atc tca ttt cta ata aga gag tat gga aaa gaa aat atc 144Val Glu Glu Ile Ser Phe Leu Ile Arg Glu Tyr Gly Lys Glu Asn Ile 35 40 45 ttt ccc ctt gga ata gga agt aat tta att ttt tca gat ggg ttt ata 192Phe Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Phe Ile 50 55 60 gat aaa gtc ttt atc cac tct aaa aac tta aaa aaa tgc gag ata acc 240Asp Lys Val Phe Ile His Ser Lys Asn Leu Lys Lys Cys Glu Ile Thr 65 70 75 80 caa gaa aac gat ata ttt tac ttg acc tta gaa gca ggg gtt agt ttt 288Gln Glu Asn Asp Ile Phe Tyr Leu Thr Leu Glu Ala Gly Val Ser Phe 85 90 95 aaa act att aac aat ata gta aaa aag tac aat ctt gaa gga ttt gaa 336Lys Thr Ile Asn Asn Ile Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 aat ctc tct gga att cct gcc acg gtt ggt gga gct gtt gcg atg aac 384Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn 115 120 125 gca ggc gcc tat ggg tca gag att ttt gat att tta gaa gaa gtt tgg 432Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Ile Leu Glu Glu Val Trp 130 135 140 tgg ata gat aaa gac ggt aat tta ata cat tca aaa aag cag gat ata 480Trp Ile Asp Lys Asp Gly Asn Leu Ile His Ser Lys Lys Gln Asp Ile 145 150 155 160 aag cac tat tac agg tac tcc cag ttt caa gaa gaa gga ttt gtt tac 528Lys His Tyr Tyr Arg Tyr Ser Gln Phe Gln Glu Glu Gly Phe Val Tyr 165 170 175 aaa gta aaa ata aag tta aaa aaa agt aat aaa gat att tcc agc atc 576Lys Val Lys Ile Lys Leu Lys Lys Ser Asn Lys Asp Ile Ser Ser Ile 180 185 190 atc aaa cag cat ctt tta gat aga aac aaa aaa caa ccc ctt gac tta 624Ile Lys Gln His Leu Leu Asp Arg Asn Lys Lys Gln Pro Leu Asp Leu 195 200 205 cca aca gca ggc tct aca tac aaa aat cca cca caa acc tat gca gga 672Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gln Thr Tyr Ala Gly 210 215 220 aaa ctt ata gag cag gta gga cta aaa ggt tat aga ata aat gat ata 720Lys Leu Ile Glu Gln Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 ggt ttt tcc tca aaa cac gca aat ttt tta gtt aac tac aaa gat gca 768Gly Phe Ser Ser Lys His Ala Asn Phe Leu Val Asn Tyr Lys Asp Ala 245 250 255 aga ttt aaa gac tta ata aat ctc ctt gaa ctt gcc gaa aag aaa gta 816Arg Phe Lys Asp Leu Ile Asn Leu Leu Glu Leu Ala Glu Lys Lys Val 260 265 270 tac gaa aaa ttt gga ata caa ctt gaa aga gag gta aag ata gtt gag 864Tyr Glu Lys Phe Gly Ile Gln Leu Glu Arg Glu Val Lys Ile Val Glu 275 280 285 taa 86750288PRTSulfurihydrogenibium azorense 50Met Asp Lys Ile Glu His Leu Lys Asn Phe Ser Leu Lys Asp Phe Cys 1 5 10 15 Thr Ile Lys Ile Gly Gly Val Gly Lys Val Val Phe Phe Pro Lys Asn 20 25 30 Val Glu Glu Ile Ser Phe Leu Ile Arg Glu Tyr Gly Lys Glu Asn Ile 35 40 45 Phe Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Phe Ile 50 55 60 Asp Lys Val Phe Ile His Ser Lys Asn Leu Lys Lys Cys Glu Ile Thr 65 70 75 80 Gln Glu Asn Asp Ile Phe Tyr Leu Thr Leu Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Ile Asn Asn Ile Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn 115 120 125 Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Ile Leu Glu Glu Val Trp 130 135 140 Trp Ile Asp Lys Asp Gly Asn Leu Ile His Ser Lys Lys Gln Asp Ile 145 150 155 160 Lys His Tyr Tyr Arg Tyr Ser Gln Phe Gln Glu Glu Gly Phe Val Tyr 165 170 175 Lys Val Lys Ile Lys Leu Lys Lys Ser Asn Lys Asp Ile Ser Ser Ile 180 185 190 Ile Lys Gln His Leu Leu Asp Arg Asn Lys Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gln Thr Tyr Ala Gly 210 215 220 Lys Leu Ile Glu Gln Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 Gly Phe Ser Ser Lys His Ala Asn Phe Leu Val Asn Tyr Lys Asp Ala 245 250 255 Arg Phe Lys Asp Leu Ile Asn Leu Leu Glu Leu Ala Glu Lys Lys Val 260 265 270 Tyr Glu Lys Phe Gly Ile Gln Leu Glu Arg Glu Val Lys Ile Val Glu 275 280 285 51905DNASulfurihydrogenibium azorenseCDS(15)..(899) 51aggaggtaaa acat atg gat aag att gaa cat ttg aag aac ttt agc ttg 50 Met Asp Lys Ile Glu His Leu Lys Asn Phe Ser Leu 1 5 10 aaa gat ttt tgc acc att aag att ggc ggt gtc ggc aaa gtt gtg ttc 98Lys Asp Phe Cys Thr Ile Lys Ile Gly Gly Val Gly Lys Val Val Phe 15 20 25 ttt ccg aag aac gtg gaa gag att agc ttc ctg atc cgt gag tat ggt 146Phe Pro Lys Asn Val Glu Glu Ile Ser Phe Leu Ile Arg Glu Tyr Gly 30 35 40 aaa gag aat atc ttc ccg ctg ggt att ggt agc aat ctg atc ttt tcc 194Lys Glu Asn Ile Phe Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser 45 50 55 60 gac ggc ttc atc gat aag gtg ttc att cac agc aag aac ctg aag aaa 242Asp Gly Phe Ile Asp Lys Val Phe Ile His Ser Lys Asn Leu Lys Lys 65 70 75 tgt gag att acc caa gaa aac gac atc ttc tac ctg acc ctg gaa gcc 290Cys Glu Ile Thr Gln Glu Asn Asp Ile Phe Tyr Leu Thr Leu Glu Ala 80 85 90 ggt gtt agc ttc aaa acg atc aat aac att gtg aag aaa tac aat ctg 338Gly Val Ser Phe Lys Thr Ile Asn Asn Ile Val Lys Lys Tyr Asn Leu 95 100 105 gaa ggt ttc gag aac ctg tcc ggc atc ccg gcg acg gtt ggc ggt gca 386Glu Gly Phe Glu Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala 110 115 120 gtt gcg atg aat gct ggc gcg tac ggt agc gag att ttc gac att ctg 434Val Ala Met Asn Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Ile Leu 125 130 135 140 gaa gag gtc tgg tgg att gac aaa gat ggc aac ctg att cat agc aag 482Glu Glu Val Trp Trp Ile Asp Lys Asp Gly Asn Leu Ile His Ser Lys 145 150 155 aag cag gac atc aag cat tac tac cgc tat agc cag ttt cag gaa gag 530Lys Gln Asp Ile Lys His Tyr Tyr Arg Tyr Ser Gln Phe Gln Glu Glu 160 165 170 ggc ttt gtt tac aaa gtg aaa atc aaa ctg aag aaa agc aac aaa gat 578Gly Phe Val Tyr Lys Val Lys Ile Lys Leu Lys Lys Ser Asn Lys Asp 175 180 185 att tcg tct att atc aaa cag cat ttg ctg gac cgt aac aag aaa caa 626Ile Ser Ser Ile Ile Lys Gln His Leu Leu Asp Arg Asn Lys Lys Gln 190 195 200 ccg ctg gat ttg ccg acc gcg ggt agc act tac aag aat cca ccg caa 674Pro Leu Asp Leu Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gln 205 210 215 220 acg tac gca ggc aag ctg atc gaa cag gtc ggt ctg aaa ggt tat cgt 722Thr Tyr Ala Gly Lys Leu Ile Glu Gln Val Gly Leu Lys Gly Tyr Arg 225 230 235 atc aat gac att ggt ttt agc tct aag cac gcc aat ttt ctg gtt aac 770Ile Asn Asp Ile Gly Phe Ser Ser Lys His Ala Asn Phe Leu Val Asn 240 245 250 tat aaa gat gcg cgc ttc aaa gac ctg atc aat ctg ctg gag ctg gca 818Tyr Lys Asp Ala Arg Phe Lys Asp Leu Ile Asn Leu Leu Glu Leu Ala 255 260 265 gag aag aaa gtc tat gag aag ttt ggt atc caa ctg gaa cgt gaa gtc 866Glu Lys Lys Val Tyr Glu Lys Phe Gly Ile Gln Leu Glu Arg Glu Val 270 275 280 aaa atc gtg gag cac cac cac cat cac cac taa ctcgag 905Lys Ile Val Glu His His His His His His 285 290 52294PRTSulfurihydrogenibium azorense 52Met Asp Lys Ile Glu His Leu Lys Asn Phe Ser Leu Lys Asp Phe Cys 1 5 10 15 Thr Ile Lys Ile Gly Gly Val Gly Lys Val Val Phe Phe Pro Lys Asn 20 25 30 Val Glu Glu Ile Ser Phe Leu Ile Arg Glu Tyr Gly Lys Glu Asn Ile 35 40 45 Phe Pro Leu Gly Ile Gly Ser Asn Leu Ile Phe Ser Asp Gly Phe Ile 50 55 60 Asp Lys Val Phe Ile His Ser Lys Asn Leu Lys Lys Cys Glu Ile Thr 65 70 75 80 Gln Glu Asn Asp Ile Phe Tyr Leu Thr Leu Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Ile Asn Asn Ile Val Lys Lys Tyr Asn Leu Glu Gly Phe Glu 100 105 110 Asn Leu Ser Gly Ile Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn 115 120 125 Ala Gly Ala Tyr Gly Ser Glu Ile Phe Asp Ile Leu Glu Glu Val Trp 130 135 140 Trp Ile Asp Lys Asp Gly Asn Leu Ile His Ser Lys Lys Gln Asp Ile 145 150 155 160 Lys His Tyr Tyr Arg Tyr Ser Gln Phe Gln Glu Glu Gly Phe Val Tyr 165 170 175 Lys Val Lys Ile Lys Leu Lys Lys Ser Asn Lys Asp Ile Ser Ser Ile 180 185 190 Ile Lys Gln His Leu Leu Asp Arg Asn Lys Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ala Gly Ser Thr Tyr Lys Asn Pro Pro Gln Thr Tyr Ala Gly 210 215 220 Lys Leu Ile Glu Gln Val Gly Leu Lys Gly Tyr Arg Ile Asn Asp Ile 225 230 235 240 Gly Phe Ser Ser Lys His Ala Asn Phe Leu Val Asn Tyr Lys Asp Ala 245 250 255 Arg Phe Lys Asp Leu Ile Asn Leu Leu Glu Leu Ala Glu Lys Lys Val 260 265 270 Tyr Glu Lys Phe Gly Ile Gln Leu Glu Arg Glu Val Lys Ile Val Glu 275 280 285 His His His His His His 290 53867DNAPersephonella marinaCDS(1)..(867) 53atg ata gat tat gaa gag aat gtt gac ctg tcg aag ctg tgt act ata 48Met Ile Asp Tyr Glu Glu Asn Val Asp Leu Ser Lys Leu Cys Thr Ile 1 5 10 15 aga atc ggt gga aca gct aaa agg gtt tac ttt cca aaa agt gtt gaa 96Arg Ile Gly Gly Thr Ala Lys Arg Val Tyr Phe Pro Lys Ser Val Glu 20 25 30 gat att ata cag ctt ttg aag ata tca cag gac agc gga aaa aag ata 144Asp Ile Ile Gln Leu Leu Lys Ile Ser Gln Asp Ser Gly Lys Lys Ile 35 40 45 att ccc ctt ggt gtg ggg agt aac aca gtt ttc agg gac ggc att ctt 192Ile Pro Leu Gly Val Gly Ser Asn Thr Val Phe Arg Asp Gly Ile Leu 50 55 60 gat cat ctg ttt gta tca aca tca aaa ctc aaa agg tat gag ata gaa 240Asp His Leu Phe Val Ser Thr Ser Lys Leu Lys Arg Tyr Glu Ile Glu 65 70 75 80 aga tct gaa gat cac gcc gta ata aca gct gaa gcg ggt gtc agt ttt 288Arg Ser Glu Asp His Ala Val Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 aaa aca ctg gtg agt ttg gtt aaa aga tac aat ctt gaa ggg ttt gag 336Lys Thr Leu Val Ser Leu Val Lys Arg Tyr Asn Leu Glu Gly Phe Glu 100 105 110 aat cta tcg gga ata cca gct tcg gta ggt ggt gct gtc gct atg aat 384Asn Leu Ser Gly Ile Pro Ala Ser Val Gly Gly Ala Val Ala Met Asn 115 120 125 gct gga gca ttt gga tct gag ata ttt gat att gtt gaa cag gtt gaa 432Ala Gly Ala Phe Gly Ser Glu Ile Phe Asp Ile Val Glu Gln Val Glu 130 135 140 tgg ata gac agt gag gga aaa ctt act gtc tca tct aaa gat gag ata 480Trp Ile Asp Ser Glu Gly Lys Leu Thr Val Ser Ser Lys Asp Glu Ile 145 150 155 160 gat tac ggg tac aga tac acc cag ttt cag aaa gag ggc ttt ata tac 528Asp Tyr Gly Tyr Arg Tyr Thr Gln Phe Gln Lys Glu Gly Phe Ile Tyr 165 170 175 aga gta aag ata aag cta aga aaa tca aaa agg aat ata cca cag atc 576Arg Val Lys Ile Lys Leu Arg Lys Ser Lys Arg Asn Ile Pro Gln Ile 180 185 190 ata aag gaa cat ctt aag gag aga aat ata aaa cag ccc ctt gat ctt 624Ile Lys Glu His Leu Lys Glu Arg Asn Ile Lys Gln Pro Leu Asp Leu 195 200 205 cca aca tca gga tcc acc ttt aaa aat cct gac ggt ata tct gca ggt 672Pro Thr Ser Gly Ser Thr Phe Lys Asn Pro Asp Gly Ile Ser Ala Gly 210 215 220 tat cta ctt gat aaa gct gga ctt aaa gga ttc aga gtg ggg gat gtt 720Tyr Leu Leu Asp Lys Ala Gly Leu Lys Gly Phe Arg Val Gly Asp Val 225 230 235 240 ggt ttt tca gaa aag cat gca aac ttt aca gta aat tac ggt cat gga 768Gly Phe Ser Glu Lys His Ala Asn Phe Thr Val Asn Tyr Gly His Gly 245 250 255 agt tac gat cag tta aag aaa ctg ctg gaa act gcc gaa aag tta gtg 816Ser Tyr Asp Gln Leu Lys Lys Leu Leu Glu Thr Ala Glu Lys Leu Val 260 265 270 gga gaa tat ttt gga ata aaa ctt gag aag gaa atc agg att gtt gaa 864Gly Glu Tyr Phe Gly Ile Lys Leu Glu Lys Glu Ile Arg Ile Val Glu 275 280 285 taa 86754288PRTPersephonella marina 54Met Ile Asp Tyr Glu Glu Asn Val Asp Leu Ser Lys Leu Cys Thr Ile 1 5 10 15 Arg Ile Gly Gly Thr Ala Lys Arg Val Tyr Phe Pro Lys Ser Val Glu 20 25 30 Asp Ile Ile Gln Leu Leu Lys Ile Ser Gln Asp Ser Gly Lys Lys Ile 35 40 45 Ile Pro Leu Gly Val Gly Ser Asn Thr Val Phe Arg Asp Gly Ile Leu 50 55 60 Asp His Leu Phe Val Ser Thr Ser Lys Leu Lys Arg Tyr Glu Ile Glu 65 70 75 80 Arg Ser Glu Asp His Ala Val Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Leu Val Ser Leu Val Lys Arg Tyr Asn Leu Glu Gly Phe Glu 100

105 110 Asn Leu Ser Gly Ile Pro Ala Ser Val Gly Gly Ala Val Ala Met Asn 115 120 125 Ala Gly Ala Phe Gly Ser Glu Ile Phe Asp Ile Val Glu Gln Val Glu 130 135 140 Trp Ile Asp Ser Glu Gly Lys Leu Thr Val Ser Ser Lys Asp Glu Ile 145 150 155 160 Asp Tyr Gly Tyr Arg Tyr Thr Gln Phe Gln Lys Glu Gly Phe Ile Tyr 165 170 175 Arg Val Lys Ile Lys Leu Arg Lys Ser Lys Arg Asn Ile Pro Gln Ile 180 185 190 Ile Lys Glu His Leu Lys Glu Arg Asn Ile Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ser Gly Ser Thr Phe Lys Asn Pro Asp Gly Ile Ser Ala Gly 210 215 220 Tyr Leu Leu Asp Lys Ala Gly Leu Lys Gly Phe Arg Val Gly Asp Val 225 230 235 240 Gly Phe Ser Glu Lys His Ala Asn Phe Thr Val Asn Tyr Gly His Gly 245 250 255 Ser Tyr Asp Gln Leu Lys Lys Leu Leu Glu Thr Ala Glu Lys Leu Val 260 265 270 Gly Glu Tyr Phe Gly Ile Lys Leu Glu Lys Glu Ile Arg Ile Val Glu 275 280 285 55905DNAPersephonella marinaCDS(15)..(899) 55aggaggtaaa acat atg att gat tac gaa gaa aac gtt gac ttg agc aaa 50 Met Ile Asp Tyr Glu Glu Asn Val Asp Leu Ser Lys 1 5 10 ctc tgt acg att cgc att ggt ggc acc gcg aaa cgc gtg tac ttt ccg 98Leu Cys Thr Ile Arg Ile Gly Gly Thr Ala Lys Arg Val Tyr Phe Pro 15 20 25 aag tct gtg gaa gat atc att caa ctg ctg aag atc agc cag gac agc 146Lys Ser Val Glu Asp Ile Ile Gln Leu Leu Lys Ile Ser Gln Asp Ser 30 35 40 ggt aag aag att atc ccg ctg ggt gtc ggt agc aat acc gtg ttc cgt 194Gly Lys Lys Ile Ile Pro Leu Gly Val Gly Ser Asn Thr Val Phe Arg 45 50 55 60 gac ggt atc ctg gat cac ctg ttc gtc agc acg agc aaa ctg aag cgc 242Asp Gly Ile Leu Asp His Leu Phe Val Ser Thr Ser Lys Leu Lys Arg 65 70 75 tat gag atc gag cgt agc gag gat cac gct gtc att act gca gag gcg 290Tyr Glu Ile Glu Arg Ser Glu Asp His Ala Val Ile Thr Ala Glu Ala 80 85 90 ggt gtc agc ttc aag acg ctg gtg tcc ctg gtt aaa cgc tac aat ctg 338Gly Val Ser Phe Lys Thr Leu Val Ser Leu Val Lys Arg Tyr Asn Leu 95 100 105 gaa ggt ttc gag aat ttg tcc ggt atc ccg gcc agc gta ggt ggt gcc 386Glu Gly Phe Glu Asn Leu Ser Gly Ile Pro Ala Ser Val Gly Gly Ala 110 115 120 gtt gcg atg aat gct ggc gcg ttt ggc agc gaa atc ttt gat atc gtg 434Val Ala Met Asn Ala Gly Ala Phe Gly Ser Glu Ile Phe Asp Ile Val 125 130 135 140 gaa cag gtt gaa tgg att gac agc gag ggc aaa ctg acc gtt agc agc 482Glu Gln Val Glu Trp Ile Asp Ser Glu Gly Lys Leu Thr Val Ser Ser 145 150 155 aag gac gag atc gat tac ggc tac cgt tac acg caa ttc cag aaa gag 530Lys Asp Glu Ile Asp Tyr Gly Tyr Arg Tyr Thr Gln Phe Gln Lys Glu 160 165 170 ggc ttc att tat cgt gtg aag atc aag ctg cgt aaa agc aaa cgt aac 578Gly Phe Ile Tyr Arg Val Lys Ile Lys Leu Arg Lys Ser Lys Arg Asn 175 180 185 atc cca caa atc att aaa gag cac ctg aaa gag cgt aac atc aaa caa 626Ile Pro Gln Ile Ile Lys Glu His Leu Lys Glu Arg Asn Ile Lys Gln 190 195 200 ccg ctg gat ttg ccg acc agc ggt tcg acc ttt aag aat ccg gac ggt 674Pro Leu Asp Leu Pro Thr Ser Gly Ser Thr Phe Lys Asn Pro Asp Gly 205 210 215 220 att tcc gcg ggt tat ctg ctg gac aaa gcg ggt ctg aaa ggc ttt cgc 722Ile Ser Ala Gly Tyr Leu Leu Asp Lys Ala Gly Leu Lys Gly Phe Arg 225 230 235 gtc ggt gac gtc ggc ttc tct gaa aag cat gca aac ttt acc gtt aac 770Val Gly Asp Val Gly Phe Ser Glu Lys His Ala Asn Phe Thr Val Asn 240 245 250 tac ggc cat ggc agc tat gac cag ctg aag aag ctg ttg gaa acc gca 818Tyr Gly His Gly Ser Tyr Asp Gln Leu Lys Lys Leu Leu Glu Thr Ala 255 260 265 gag aaa ctg gtg ggt gag tat ttc ggc att aag ctg gaa aaa gag att 866Glu Lys Leu Val Gly Glu Tyr Phe Gly Ile Lys Leu Glu Lys Glu Ile 270 275 280 cgt att gtt gag cac cac cat cac cac cat taa ctcgag 905Arg Ile Val Glu His His His His His His 285 290 56294PRTPersephonella marina 56Met Ile Asp Tyr Glu Glu Asn Val Asp Leu Ser Lys Leu Cys Thr Ile 1 5 10 15 Arg Ile Gly Gly Thr Ala Lys Arg Val Tyr Phe Pro Lys Ser Val Glu 20 25 30 Asp Ile Ile Gln Leu Leu Lys Ile Ser Gln Asp Ser Gly Lys Lys Ile 35 40 45 Ile Pro Leu Gly Val Gly Ser Asn Thr Val Phe Arg Asp Gly Ile Leu 50 55 60 Asp His Leu Phe Val Ser Thr Ser Lys Leu Lys Arg Tyr Glu Ile Glu 65 70 75 80 Arg Ser Glu Asp His Ala Val Ile Thr Ala Glu Ala Gly Val Ser Phe 85 90 95 Lys Thr Leu Val Ser Leu Val Lys Arg Tyr Asn Leu Glu Gly Phe Glu 100 105 110 Asn Leu Ser Gly Ile Pro Ala Ser Val Gly Gly Ala Val Ala Met Asn 115 120 125 Ala Gly Ala Phe Gly Ser Glu Ile Phe Asp Ile Val Glu Gln Val Glu 130 135 140 Trp Ile Asp Ser Glu Gly Lys Leu Thr Val Ser Ser Lys Asp Glu Ile 145 150 155 160 Asp Tyr Gly Tyr Arg Tyr Thr Gln Phe Gln Lys Glu Gly Phe Ile Tyr 165 170 175 Arg Val Lys Ile Lys Leu Arg Lys Ser Lys Arg Asn Ile Pro Gln Ile 180 185 190 Ile Lys Glu His Leu Lys Glu Arg Asn Ile Lys Gln Pro Leu Asp Leu 195 200 205 Pro Thr Ser Gly Ser Thr Phe Lys Asn Pro Asp Gly Ile Ser Ala Gly 210 215 220 Tyr Leu Leu Asp Lys Ala Gly Leu Lys Gly Phe Arg Val Gly Asp Val 225 230 235 240 Gly Phe Ser Glu Lys His Ala Asn Phe Thr Val Asn Tyr Gly His Gly 245 250 255 Ser Tyr Asp Gln Leu Lys Lys Leu Leu Glu Thr Ala Glu Lys Leu Val 260 265 270 Gly Glu Tyr Phe Gly Ile Lys Leu Glu Lys Glu Ile Arg Ile Val Glu 275 280 285 His His His His His His 290 57849DNAHydrogenobaculum sp. (strain Y04AAS1)CDS(1)..(849) 57atg ata ata aac aaa aat gcg gac tta aaa gac ttt aca acg ata aaa 48Met Ile Ile Asn Lys Asn Ala Asp Leu Lys Asp Phe Thr Thr Ile Lys 1 5 10 15 gta ggc ggt ata ggt tca tat atg ttt ttt cca gaa aac gaa aag gag 96Val Gly Gly Ile Gly Ser Tyr Met Phe Phe Pro Glu Asn Glu Lys Glu 20 25 30 ttc cta aat ata tat aaa aag cac aaa aac gat aaa ctc tac ata ctt 144Phe Leu Asn Ile Tyr Lys Lys His Lys Asn Asp Lys Leu Tyr Ile Leu 35 40 45 ggg aaa ggt tca aac acc ata ttt ggg gat ttc aac gga ata tta ata 192Gly Lys Gly Ser Asn Thr Ile Phe Gly Asp Phe Asn Gly Ile Leu Ile 50 55 60 aat aca aaa cat ttt tac gat att aaa atc tca gaa aca aaa gaa ggt 240Asn Thr Lys His Phe Tyr Asp Ile Lys Ile Ser Glu Thr Lys Glu Gly 65 70 75 80 att ttg gta aaa gcc tct gca gga gta cct ttg aaa gac tta ata aaa 288Ile Leu Val Lys Ala Ser Ala Gly Val Pro Leu Lys Asp Leu Ile Lys 85 90 95 cta tct att gaa aac aac ata gaa gag ttt tac aag ttg ata ggt ttt 336Leu Ser Ile Glu Asn Asn Ile Glu Glu Phe Tyr Lys Leu Ile Gly Phe 100 105 110 cca gca agc act ggc ggg gca ata gct atg aat gca gga gct tat ggt 384Pro Ala Ser Thr Gly Gly Ala Ile Ala Met Asn Ala Gly Ala Tyr Gly 115 120 125 gtg gaa acc ttt gat ttt ata aaa ggt gta tgg tgt ata gat gac gac 432Val Glu Thr Phe Asp Phe Ile Lys Gly Val Trp Cys Ile Asp Asp Asp 130 135 140 gaa ata gtt tac aaa cca aaa gaa gag ata ttt tat tcg tat aga aaa 480Glu Ile Val Tyr Lys Pro Lys Glu Glu Ile Phe Tyr Ser Tyr Arg Lys 145 150 155 160 acc gag ttt gaa aat aag cca gtg ttg tac gga gag ttt tta ttt aaa 528Thr Glu Phe Glu Asn Lys Pro Val Leu Tyr Gly Glu Phe Leu Phe Lys 165 170 175 aaa agt cat caa gat ata aaa cct tta gca caa aat ata aat caa aaa 576Lys Ser His Gln Asp Ile Lys Pro Leu Ala Gln Asn Ile Asn Gln Lys 180 185 190 aga ata gaa gcc cag cca ctt aac atg ccc act tct ggt tct aca ttt 624Arg Ile Glu Ala Gln Pro Leu Asn Met Pro Thr Ser Gly Ser Thr Phe 195 200 205 aaa aat cca aaa gat cac ttt gcc ggt aag ctt tta gaa acg gtt ggt 672Lys Asn Pro Lys Asp His Phe Ala Gly Lys Leu Leu Glu Thr Val Gly 210 215 220 tta aaa gga tat aga ata aaa gac ata ggt ttt tca gaa agg cat gca 720Leu Lys Gly Tyr Arg Ile Lys Asp Ile Gly Phe Ser Glu Arg His Ala 225 230 235 240 aac ttt tta ata aat tat aaa aac gca tct ttt caa aat gta att gac 768Asn Phe Leu Ile Asn Tyr Lys Asn Ala Ser Phe Gln Asn Val Ile Asp 245 250 255 ata tta aac ata gca aaa gag caa gtt tac aaa gct ttt aat ata ata 816Ile Leu Asn Ile Ala Lys Glu Gln Val Tyr Lys Ala Phe Asn Ile Ile 260 265 270 tta gaa gag gaa ata aaa ctg ata tgc gca taa 849Leu Glu Glu Glu Ile Lys Leu Ile Cys Ala 275 280 58282PRTHydrogenobaculum sp. (strain Y04AAS1) 58Met Ile Ile Asn Lys Asn Ala Asp Leu Lys Asp Phe Thr Thr Ile Lys 1 5 10 15 Val Gly Gly Ile Gly Ser Tyr Met Phe Phe Pro Glu Asn Glu Lys Glu 20 25 30 Phe Leu Asn Ile Tyr Lys Lys His Lys Asn Asp Lys Leu Tyr Ile Leu 35 40 45 Gly Lys Gly Ser Asn Thr Ile Phe Gly Asp Phe Asn Gly Ile Leu Ile 50 55 60 Asn Thr Lys His Phe Tyr Asp Ile Lys Ile Ser Glu Thr Lys Glu Gly 65 70 75 80 Ile Leu Val Lys Ala Ser Ala Gly Val Pro Leu Lys Asp Leu Ile Lys 85 90 95 Leu Ser Ile Glu Asn Asn Ile Glu Glu Phe Tyr Lys Leu Ile Gly Phe 100 105 110 Pro Ala Ser Thr Gly Gly Ala Ile Ala Met Asn Ala Gly Ala Tyr Gly 115 120 125 Val Glu Thr Phe Asp Phe Ile Lys Gly Val Trp Cys Ile Asp Asp Asp 130 135 140 Glu Ile Val Tyr Lys Pro Lys Glu Glu Ile Phe Tyr Ser Tyr Arg Lys 145 150 155 160 Thr Glu Phe Glu Asn Lys Pro Val Leu Tyr Gly Glu Phe Leu Phe Lys 165 170 175 Lys Ser His Gln Asp Ile Lys Pro Leu Ala Gln Asn Ile Asn Gln Lys 180 185 190 Arg Ile Glu Ala Gln Pro Leu Asn Met Pro Thr Ser Gly Ser Thr Phe 195 200 205 Lys Asn Pro Lys Asp His Phe Ala Gly Lys Leu Leu Glu Thr Val Gly 210 215 220 Leu Lys Gly Tyr Arg Ile Lys Asp Ile Gly Phe Ser Glu Arg His Ala 225 230 235 240 Asn Phe Leu Ile Asn Tyr Lys Asn Ala Ser Phe Gln Asn Val Ile Asp 245 250 255 Ile Leu Asn Ile Ala Lys Glu Gln Val Tyr Lys Ala Phe Asn Ile Ile 260 265 270 Leu Glu Glu Glu Ile Lys Leu Ile Cys Ala 275 280 59887DNAHydrogenobaculum sp. (strain Y04AAS1)CDS(15)..(881) 59aggaggtaaa acat atg atc att aac aaa aac gcg gac ttg aaa gat ttt 50 Met Ile Ile Asn Lys Asn Ala Asp Leu Lys Asp Phe 1 5 10 acg acg att aag gta ggc ggc att ggc tcc tat atg ttc ttt ccg gag 98Thr Thr Ile Lys Val Gly Gly Ile Gly Ser Tyr Met Phe Phe Pro Glu 15 20 25 aat gag aaa gag ttc ctg aac atc tac aag aag cac aag aac gac aag 146Asn Glu Lys Glu Phe Leu Asn Ile Tyr Lys Lys His Lys Asn Asp Lys 30 35 40 ctg tac att ctg ggc aaa ggt tcc aac acc att ttt ggt gat ttc aac 194Leu Tyr Ile Leu Gly Lys Gly Ser Asn Thr Ile Phe Gly Asp Phe Asn 45 50 55 60 ggt att ctg att aac acc aag cac ttc tac gat atc aag atc agc gag 242Gly Ile Leu Ile Asn Thr Lys His Phe Tyr Asp Ile Lys Ile Ser Glu 65 70 75 act aaa gaa ggt att ctg gtg aag gcg agc gca ggc gtc cct ctg aaa 290Thr Lys Glu Gly Ile Leu Val Lys Ala Ser Ala Gly Val Pro Leu Lys 80 85 90 gac ctg att aag ctg agc atc gag aac aat atc gaa gaa ttc tat aaa 338Asp Leu Ile Lys Leu Ser Ile Glu Asn Asn Ile Glu Glu Phe Tyr Lys 95 100 105 ctg att ggt ttt ccg gcc agc acg ggt ggt gcg att gca atg aat gcc 386Leu Ile Gly Phe Pro Ala Ser Thr Gly Gly Ala Ile Ala Met Asn Ala 110 115 120 ggt gcc tac ggc gtt gaa acc ttc gac ttc atc aaa ggt gtc tgg tgc 434Gly Ala Tyr Gly Val Glu Thr Phe Asp Phe Ile Lys Gly Val Trp Cys 125 130 135 140 att gac gac gat gag atc gtc tat aaa ccg aaa gaa gaa atc ttc tac 482Ile Asp Asp Asp Glu Ile Val Tyr Lys Pro Lys Glu Glu Ile Phe Tyr 145 150 155 agc tat cgt aag acc gag ttt gag aat aag ccg gtt ctg tat ggc gag 530Ser Tyr Arg Lys Thr Glu Phe Glu Asn Lys Pro Val Leu Tyr Gly Glu 160 165 170 ttc ctg ttc aaa aag agc cat cag gac atc aaa cca ctg gca caa aac 578Phe Leu Phe Lys Lys Ser His Gln Asp Ile Lys Pro Leu Ala Gln Asn 175 180 185 atc aat cag aag cgt att gaa gca caa ccg ttg aat atg ccg acc agc 626Ile Asn Gln Lys Arg Ile Glu Ala Gln Pro Leu Asn Met Pro Thr Ser 190 195 200 ggt tct acc ttc aag aat ccg aag gat cat ttt gct ggt aag ctg ctg 674Gly Ser Thr Phe Lys Asn Pro Lys Asp His Phe Ala Gly Lys Leu Leu 205 210 215 220 gaa acg gtg ggt ctg aaa ggt tac cgc att aaa gat att ggc ttt tcg 722Glu Thr Val Gly Leu Lys Gly Tyr Arg Ile Lys Asp Ile Gly Phe Ser 225 230 235 gag cgt cac gcg aac ttt ctg atc aat tac aaa aat gcg agc ttc cag 770Glu Arg His Ala Asn Phe Leu Ile Asn Tyr Lys Asn Ala Ser Phe Gln 240 245 250 aat gtt att gac atc ttg aac atc gcg aaa gag caa gtg tac aag gcg 818Asn Val Ile Asp Ile Leu Asn Ile Ala Lys Glu Gln Val Tyr Lys Ala 255 260 265 ttt aac atc atc ttg gaa gaa gag att aaa ctg atc tgt gct cat cac 866Phe Asn Ile Ile Leu Glu Glu Glu Ile Lys Leu Ile Cys Ala His His 270 275 280 cac cac cat cac taa ctcgag

887His His His His 285 60288PRTHydrogenobaculum sp. (strain Y04AAS1) 60Met Ile Ile Asn Lys Asn Ala Asp Leu Lys Asp Phe Thr Thr Ile Lys 1 5 10 15 Val Gly Gly Ile Gly Ser Tyr Met Phe Phe Pro Glu Asn Glu Lys Glu 20 25 30 Phe Leu Asn Ile Tyr Lys Lys His Lys Asn Asp Lys Leu Tyr Ile Leu 35 40 45 Gly Lys Gly Ser Asn Thr Ile Phe Gly Asp Phe Asn Gly Ile Leu Ile 50 55 60 Asn Thr Lys His Phe Tyr Asp Ile Lys Ile Ser Glu Thr Lys Glu Gly 65 70 75 80 Ile Leu Val Lys Ala Ser Ala Gly Val Pro Leu Lys Asp Leu Ile Lys 85 90 95 Leu Ser Ile Glu Asn Asn Ile Glu Glu Phe Tyr Lys Leu Ile Gly Phe 100 105 110 Pro Ala Ser Thr Gly Gly Ala Ile Ala Met Asn Ala Gly Ala Tyr Gly 115 120 125 Val Glu Thr Phe Asp Phe Ile Lys Gly Val Trp Cys Ile Asp Asp Asp 130 135 140 Glu Ile Val Tyr Lys Pro Lys Glu Glu Ile Phe Tyr Ser Tyr Arg Lys 145 150 155 160 Thr Glu Phe Glu Asn Lys Pro Val Leu Tyr Gly Glu Phe Leu Phe Lys 165 170 175 Lys Ser His Gln Asp Ile Lys Pro Leu Ala Gln Asn Ile Asn Gln Lys 180 185 190 Arg Ile Glu Ala Gln Pro Leu Asn Met Pro Thr Ser Gly Ser Thr Phe 195 200 205 Lys Asn Pro Lys Asp His Phe Ala Gly Lys Leu Leu Glu Thr Val Gly 210 215 220 Leu Lys Gly Tyr Arg Ile Lys Asp Ile Gly Phe Ser Glu Arg His Ala 225 230 235 240 Asn Phe Leu Ile Asn Tyr Lys Asn Ala Ser Phe Gln Asn Val Ile Asp 245 250 255 Ile Leu Asn Ile Ala Lys Glu Gln Val Tyr Lys Ala Phe Asn Ile Ile 260 265 270 Leu Glu Glu Glu Ile Lys Leu Ile Cys Ala His His His His His His 275 280 285 61864DNAHydrogenivirga sp. 128-5R1-1CDS(1)..(864) 61atg aaa ata tat gaa aat gta gat tta aaa aat ttt tca act ata aaa 48Met Lys Ile Tyr Glu Asn Val Asp Leu Lys Asn Phe Ser Thr Ile Lys 1 5 10 15 atc gga gga aaa gct aaa aag tta tat ttt cct gaa agt ctc aac gac 96Ile Gly Gly Lys Ala Lys Lys Leu Tyr Phe Pro Glu Ser Leu Asn Asp 20 25 30 ata aaa ttc cta att aaa aaa tca aaa gac gaa gat aaa aag tta gtt 144Ile Lys Phe Leu Ile Lys Lys Ser Lys Asp Glu Asp Lys Lys Leu Val 35 40 45 ttt att gga gtt gga agt aat aca att ttt aaa gat ggg acg tta gat 192Phe Ile Gly Val Gly Ser Asn Thr Ile Phe Lys Asp Gly Thr Leu Asp 50 55 60 tac ata ttt att tca aca aaa ttt cta aaa aac ata gag ata aaa gaa 240Tyr Ile Phe Ile Ser Thr Lys Phe Leu Lys Asn Ile Glu Ile Lys Glu 65 70 75 80 gaa aaa gac ctt ttt tat tta aat tta gaa gca gga gta agt ttt aaa 288Glu Lys Asp Leu Phe Tyr Leu Asn Leu Glu Ala Gly Val Ser Phe Lys 85 90 95 gaa att ata aat ctg gta aaa aaa ttt aat tta gaa ggt ttt gaa aat 336Glu Ile Ile Asn Leu Val Lys Lys Phe Asn Leu Glu Gly Phe Glu Asn 100 105 110 ctt tct gga ata cct gca tct tta ggt gga gct gtt gca atg aat gct 384Leu Ser Gly Ile Pro Ala Ser Leu Gly Gly Ala Val Ala Met Asn Ala 115 120 125 gga gcc ttt gga aat gaa ata ttt gat att ata gaa gat gta ctc tgg 432Gly Ala Phe Gly Asn Glu Ile Phe Asp Ile Ile Glu Asp Val Leu Trp 130 135 140 ata gat ttt gat aca aat gag cat ctt tca aaa aaa aat gaa ata aag 480Ile Asp Phe Asp Thr Asn Glu His Leu Ser Lys Lys Asn Glu Ile Lys 145 150 155 160 tac tct tac agg aca acc cag ttc caa aaa gaa ggt ttt atc tat aaa 528Tyr Ser Tyr Arg Thr Thr Gln Phe Gln Lys Glu Gly Phe Ile Tyr Lys 165 170 175 gct act ata aaa ctg aaa aaa agt aaa aaa gat ata gca aaa ata ata 576Ala Thr Ile Lys Leu Lys Lys Ser Lys Lys Asp Ile Ala Lys Ile Ile 180 185 190 aaa aac cat tta ata gaa aga aat aaa aaa cag cct tta aat tta cca 624Lys Asn His Leu Ile Glu Arg Asn Lys Lys Gln Pro Leu Asn Leu Pro 195 200 205 aca tca ggc tca aca tat aaa aat cct cca aac aat ttt gct gga aaa 672Thr Ser Gly Ser Thr Tyr Lys Asn Pro Pro Asn Asn Phe Ala Gly Lys 210 215 220 att ctg gaa gaa ata ggt tat aaa ggg aaa agg ata gga gac ata ggt 720Ile Leu Glu Glu Ile Gly Tyr Lys Gly Lys Arg Ile Gly Asp Ile Gly 225 230 235 240 ttt tca gat aaa cat gca aat ttt tta gta aat tac tca aat gca aca 768Phe Ser Asp Lys His Ala Asn Phe Leu Val Asn Tyr Ser Asn Ala Thr 245 250 255 ttc aaa gat tta atg aat tta tta gaa tct gcc gaa agg aaa gtt gaa 816Phe Lys Asp Leu Met Asn Leu Leu Glu Ser Ala Glu Arg Lys Val Glu 260 265 270 agg gtt ttt aat ata aaa ttt gaa agg gaa ata aga ata gtt gag taa 864Arg Val Phe Asn Ile Lys Phe Glu Arg Glu Ile Arg Ile Val Glu 275 280 285 62287PRTHydrogenivirga sp. 128-5R1-1 62Met Lys Ile Tyr Glu Asn Val Asp Leu Lys Asn Phe Ser Thr Ile Lys 1 5 10 15 Ile Gly Gly Lys Ala Lys Lys Leu Tyr Phe Pro Glu Ser Leu Asn Asp 20 25 30 Ile Lys Phe Leu Ile Lys Lys Ser Lys Asp Glu Asp Lys Lys Leu Val 35 40 45 Phe Ile Gly Val Gly Ser Asn Thr Ile Phe Lys Asp Gly Thr Leu Asp 50 55 60 Tyr Ile Phe Ile Ser Thr Lys Phe Leu Lys Asn Ile Glu Ile Lys Glu 65 70 75 80 Glu Lys Asp Leu Phe Tyr Leu Asn Leu Glu Ala Gly Val Ser Phe Lys 85 90 95 Glu Ile Ile Asn Leu Val Lys Lys Phe Asn Leu Glu Gly Phe Glu Asn 100 105 110 Leu Ser Gly Ile Pro Ala Ser Leu Gly Gly Ala Val Ala Met Asn Ala 115 120 125 Gly Ala Phe Gly Asn Glu Ile Phe Asp Ile Ile Glu Asp Val Leu Trp 130 135 140 Ile Asp Phe Asp Thr Asn Glu His Leu Ser Lys Lys Asn Glu Ile Lys 145 150 155 160 Tyr Ser Tyr Arg Thr Thr Gln Phe Gln Lys Glu Gly Phe Ile Tyr Lys 165 170 175 Ala Thr Ile Lys Leu Lys Lys Ser Lys Lys Asp Ile Ala Lys Ile Ile 180 185 190 Lys Asn His Leu Ile Glu Arg Asn Lys Lys Gln Pro Leu Asn Leu Pro 195 200 205 Thr Ser Gly Ser Thr Tyr Lys Asn Pro Pro Asn Asn Phe Ala Gly Lys 210 215 220 Ile Leu Glu Glu Ile Gly Tyr Lys Gly Lys Arg Ile Gly Asp Ile Gly 225 230 235 240 Phe Ser Asp Lys His Ala Asn Phe Leu Val Asn Tyr Ser Asn Ala Thr 245 250 255 Phe Lys Asp Leu Met Asn Leu Leu Glu Ser Ala Glu Arg Lys Val Glu 260 265 270 Arg Val Phe Asn Ile Lys Phe Glu Arg Glu Ile Arg Ile Val Glu 275 280 285 63902DNAHydrogenivirga sp. 128-5R1-1CDS(15)..(896) 63aggaggtaaa acat atg aag att tat gaa aat gta gac ttg aaa aac ttt 50 Met Lys Ile Tyr Glu Asn Val Asp Leu Lys Asn Phe 1 5 10 agc acg att aag att ggc ggt aaa gcg aag aaa ctg tat ttt cca gag 98Ser Thr Ile Lys Ile Gly Gly Lys Ala Lys Lys Leu Tyr Phe Pro Glu 15 20 25 agc ctg aac gac atc aag ttt ctg att aag aag agc aaa gac gag gac 146Ser Leu Asn Asp Ile Lys Phe Leu Ile Lys Lys Ser Lys Asp Glu Asp 30 35 40 aaa aag ctg gtg ttt att ggt gtg ggt tct aac acg atc ttc aaa gac 194Lys Lys Leu Val Phe Ile Gly Val Gly Ser Asn Thr Ile Phe Lys Asp 45 50 55 60 ggt acg ttg gat tac atc ttc att agc acg aaa ttt ctg aaa aac atc 242Gly Thr Leu Asp Tyr Ile Phe Ile Ser Thr Lys Phe Leu Lys Asn Ile 65 70 75 gag atc aaa gaa gaa aaa gat ctg ttt tac ttg aat ctg gaa gcc ggt 290Glu Ile Lys Glu Glu Lys Asp Leu Phe Tyr Leu Asn Leu Glu Ala Gly 80 85 90 gtg agc ttc aaa gag atc att aac ctg gtc aag aag ttt aac ctg gaa 338Val Ser Phe Lys Glu Ile Ile Asn Leu Val Lys Lys Phe Asn Leu Glu 95 100 105 ggt ttc gag aac ctg tcg ggc att ccg gca agc ctg ggt ggc gcc gtt 386Gly Phe Glu Asn Leu Ser Gly Ile Pro Ala Ser Leu Gly Gly Ala Val 110 115 120 gca atg aat gct ggc gcg ttt ggt aat gag att ttt gat atc atc gag 434Ala Met Asn Ala Gly Ala Phe Gly Asn Glu Ile Phe Asp Ile Ile Glu 125 130 135 140 gat gtc ctg tgg att gat ttc gat acc aat gag cac ctg tcc aag aaa 482Asp Val Leu Trp Ile Asp Phe Asp Thr Asn Glu His Leu Ser Lys Lys 145 150 155 aat gag atc aag tac tcc tat cgt acc acc cag ttt caa aaa gag ggt 530Asn Glu Ile Lys Tyr Ser Tyr Arg Thr Thr Gln Phe Gln Lys Glu Gly 160 165 170 ttc att tac aag gcg act atc aaa ctg aag aaa agc aag aag gac att 578Phe Ile Tyr Lys Ala Thr Ile Lys Leu Lys Lys Ser Lys Lys Asp Ile 175 180 185 gca aaa atc atc aag aac cat ctg att gaa cgt aat aag aaa cag ccg 626Ala Lys Ile Ile Lys Asn His Leu Ile Glu Arg Asn Lys Lys Gln Pro 190 195 200 ctg aat ctg ccg acc agc ggt agc acc tac aaa aat ccg ccg aac aac 674Leu Asn Leu Pro Thr Ser Gly Ser Thr Tyr Lys Asn Pro Pro Asn Asn 205 210 215 220 ttc gcg ggc aaa atc ctg gaa gag atc ggc tat aag ggt aaa cgc atc 722Phe Ala Gly Lys Ile Leu Glu Glu Ile Gly Tyr Lys Gly Lys Arg Ile 225 230 235 ggt gac att ggc ttc agc gac aaa cat gcg aat ttc ctg gtt aac tac 770Gly Asp Ile Gly Phe Ser Asp Lys His Ala Asn Phe Leu Val Asn Tyr 240 245 250 agc aac gct acc ttc aaa gat ctg atg aac ctg ttg gaa tct gcg gaa 818Ser Asn Ala Thr Phe Lys Asp Leu Met Asn Leu Leu Glu Ser Ala Glu 255 260 265 cgt aag gtc gaa cgc gtt ttc aat atc aag ttc gag cgc gag att cgt 866Arg Lys Val Glu Arg Val Phe Asn Ile Lys Phe Glu Arg Glu Ile Arg 270 275 280 atc gtt gag cac cat cac cac cac cac taa ctcgag 902Ile Val Glu His His His His His His 285 290 64293PRTHydrogenivirga sp. 128-5R1-1 64Met Lys Ile Tyr Glu Asn Val Asp Leu Lys Asn Phe Ser Thr Ile Lys 1 5 10 15 Ile Gly Gly Lys Ala Lys Lys Leu Tyr Phe Pro Glu Ser Leu Asn Asp 20 25 30 Ile Lys Phe Leu Ile Lys Lys Ser Lys Asp Glu Asp Lys Lys Leu Val 35 40 45 Phe Ile Gly Val Gly Ser Asn Thr Ile Phe Lys Asp Gly Thr Leu Asp 50 55 60 Tyr Ile Phe Ile Ser Thr Lys Phe Leu Lys Asn Ile Glu Ile Lys Glu 65 70 75 80 Glu Lys Asp Leu Phe Tyr Leu Asn Leu Glu Ala Gly Val Ser Phe Lys 85 90 95 Glu Ile Ile Asn Leu Val Lys Lys Phe Asn Leu Glu Gly Phe Glu Asn 100 105 110 Leu Ser Gly Ile Pro Ala Ser Leu Gly Gly Ala Val Ala Met Asn Ala 115 120 125 Gly Ala Phe Gly Asn Glu Ile Phe Asp Ile Ile Glu Asp Val Leu Trp 130 135 140 Ile Asp Phe Asp Thr Asn Glu His Leu Ser Lys Lys Asn Glu Ile Lys 145 150 155 160 Tyr Ser Tyr Arg Thr Thr Gln Phe Gln Lys Glu Gly Phe Ile Tyr Lys 165 170 175 Ala Thr Ile Lys Leu Lys Lys Ser Lys Lys Asp Ile Ala Lys Ile Ile 180 185 190 Lys Asn His Leu Ile Glu Arg Asn Lys Lys Gln Pro Leu Asn Leu Pro 195 200 205 Thr Ser Gly Ser Thr Tyr Lys Asn Pro Pro Asn Asn Phe Ala Gly Lys 210 215 220 Ile Leu Glu Glu Ile Gly Tyr Lys Gly Lys Arg Ile Gly Asp Ile Gly 225 230 235 240 Phe Ser Asp Lys His Ala Asn Phe Leu Val Asn Tyr Ser Asn Ala Thr 245 250 255 Phe Lys Asp Leu Met Asn Leu Leu Glu Ser Ala Glu Arg Lys Val Glu 260 265 270 Arg Val Phe Asn Ile Lys Phe Glu Arg Glu Ile Arg Ile Val Glu His 275 280 285 His His His His His 290 65885DNAHydrogenobacter thermophilusCDS(1)..(885) 65atg aag tta gag aaa aac gtg cta ctt gct cca tat acc acc ata agg 48Met Lys Leu Glu Lys Asn Val Leu Leu Ala Pro Tyr Thr Thr Ile Arg 1 5 10 15 ata ggg ggt act gcg cgt ttt atg tgt ttt ccc tct gac ttt gca gag 96Ile Gly Gly Thr Ala Arg Phe Met Cys Phe Pro Ser Asp Phe Ala Glu 20 25 30 ctt tct aaa gcc ata agg tgg gca aag gag gag gga ctg cct gta ttc 144Leu Ser Lys Ala Ile Arg Trp Ala Lys Glu Glu Gly Leu Pro Val Phe 35 40 45 ttg ctg ggt agg ggt gct aac aca att ttt ggt gat tac tat ggg ctt 192Leu Leu Gly Arg Gly Ala Asn Thr Ile Phe Gly Asp Tyr Tyr Gly Leu 50 55 60 gtg ata aac acc tca aga cta aac ggt atg aag att ttc cat gca ggg 240Val Ile Asn Thr Ser Arg Leu Asn Gly Met Lys Ile Phe His Ala Gly 65 70 75 80 gaa aag gtt ctg ctg gaa gcc cag tgt ggg gtg agg ctc tcg cag gtg 288Glu Lys Val Leu Leu Glu Ala Gln Cys Gly Val Arg Leu Ser Gln Val 85 90 95 gtt aaa ctg gct ctt gag cta aac ctg gaa ggc ata tac aag ctt gcc 336Val Lys Leu Ala Leu Glu Leu Asn Leu Glu Gly Ile Tyr Lys Leu Ala 100 105 110 ggg ttt ccc gca acg gtg gga ggt gct gtg gct atg aat gca gga gct 384Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn Ala Gly Ala 115 120 125 ttt ggc acg gaa ata tcg cat tac ctt aag agc ctt ctt gtc atg gac 432Phe Gly Thr Glu Ile Ser His Tyr Leu Lys Ser Leu Leu Val Met Asp 130 135 140 tgg gaa ggc aat gtg gag aag atc agc gcg gag gat gta aag ttt gat 480Trp Glu Gly Asn Val Glu Lys Ile Ser Ala Glu Asp Val Lys Phe Asp 145 150 155 160 tac aga agc tct cct ttt ccg gat atg ggc ata gtt ctt atg gct gag 528Tyr Arg Ser Ser Pro Phe Pro Asp Met Gly Ile Val Leu Met Ala Glu 165 170 175 ctg gag cta aag aga gca gag ctt gat gtt agg cac gag cag aat ctc 576Leu Glu Leu Lys Arg Ala Glu Leu Asp Val Arg His Glu Gln Asn Leu 180 185 190 ata aag gag agg aga agg cgc aca cag ccc ata aac atg ccc aca tcc 624Ile Lys Glu Arg Arg Arg Arg Thr Gln Pro Ile Asn Met Pro Thr Ser 195 200 205 ggc tct acc ttt aag aat ccg cct ggg cag tat gcg ggc aaa ctt ctt 672Gly Ser Thr Phe Lys Asn Pro Pro Gly Gln Tyr Ala Gly Lys Leu Leu

210 215 220 gag atg gtg ggt atg aag gga tac aga gta gga gat gtg gct ttt tcc 720Glu Met Val Gly Met Lys Gly Tyr Arg Val Gly Asp Val Ala Phe Ser 225 230 235 240 cac ctt cat gct aac ttt ctt gta aac tta ggt gat ggc aga tac gaa 768His Leu His Ala Asn Phe Leu Val Asn Leu Gly Asp Gly Arg Tyr Glu 245 250 255 gat gcg ctt aaa ata ctc ttg gaa gca aaa agg aga gtt tac gag gag 816Asp Ala Leu Lys Ile Leu Leu Glu Ala Lys Arg Arg Val Tyr Glu Glu 260 265 270 ttt ggt ata tat ctt gag gag gag gta aaa gtc gtt gag agt tgt agt 864Phe Gly Ile Tyr Leu Glu Glu Glu Val Lys Val Val Glu Ser Cys Ser 275 280 285 act cat ggg ggg cag gtc tga 885Thr His Gly Gly Gln Val 290 66294PRTHydrogenobacter thermophilus 66Met Lys Leu Glu Lys Asn Val Leu Leu Ala Pro Tyr Thr Thr Ile Arg 1 5 10 15 Ile Gly Gly Thr Ala Arg Phe Met Cys Phe Pro Ser Asp Phe Ala Glu 20 25 30 Leu Ser Lys Ala Ile Arg Trp Ala Lys Glu Glu Gly Leu Pro Val Phe 35 40 45 Leu Leu Gly Arg Gly Ala Asn Thr Ile Phe Gly Asp Tyr Tyr Gly Leu 50 55 60 Val Ile Asn Thr Ser Arg Leu Asn Gly Met Lys Ile Phe His Ala Gly 65 70 75 80 Glu Lys Val Leu Leu Glu Ala Gln Cys Gly Val Arg Leu Ser Gln Val 85 90 95 Val Lys Leu Ala Leu Glu Leu Asn Leu Glu Gly Ile Tyr Lys Leu Ala 100 105 110 Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn Ala Gly Ala 115 120 125 Phe Gly Thr Glu Ile Ser His Tyr Leu Lys Ser Leu Leu Val Met Asp 130 135 140 Trp Glu Gly Asn Val Glu Lys Ile Ser Ala Glu Asp Val Lys Phe Asp 145 150 155 160 Tyr Arg Ser Ser Pro Phe Pro Asp Met Gly Ile Val Leu Met Ala Glu 165 170 175 Leu Glu Leu Lys Arg Ala Glu Leu Asp Val Arg His Glu Gln Asn Leu 180 185 190 Ile Lys Glu Arg Arg Arg Arg Thr Gln Pro Ile Asn Met Pro Thr Ser 195 200 205 Gly Ser Thr Phe Lys Asn Pro Pro Gly Gln Tyr Ala Gly Lys Leu Leu 210 215 220 Glu Met Val Gly Met Lys Gly Tyr Arg Val Gly Asp Val Ala Phe Ser 225 230 235 240 His Leu His Ala Asn Phe Leu Val Asn Leu Gly Asp Gly Arg Tyr Glu 245 250 255 Asp Ala Leu Lys Ile Leu Leu Glu Ala Lys Arg Arg Val Tyr Glu Glu 260 265 270 Phe Gly Ile Tyr Leu Glu Glu Glu Val Lys Val Val Glu Ser Cys Ser 275 280 285 Thr His Gly Gly Gln Val 290 67923DNAHydrogenobacter thermophilusCDS(15)..(917) 67aggaggtaaa acat atg aag ttg gag aaa aac gta ttg ttg gca cca tat 50 Met Lys Leu Glu Lys Asn Val Leu Leu Ala Pro Tyr 1 5 10 act acg att cgc atc ggt ggc acg gct cgc ttt atg tgt ttc ccg agc 98Thr Thr Ile Arg Ile Gly Gly Thr Ala Arg Phe Met Cys Phe Pro Ser 15 20 25 gat ttc gca gaa ctg tcg aaa gcg atc cgt tgg gcg aaa gaa gag ggt 146Asp Phe Ala Glu Leu Ser Lys Ala Ile Arg Trp Ala Lys Glu Glu Gly 30 35 40 ctg ccg gtt ttc ttg ctg ggt cgt ggt gcg aac acg att ttc ggt gat 194Leu Pro Val Phe Leu Leu Gly Arg Gly Ala Asn Thr Ile Phe Gly Asp 45 50 55 60 tac tat ggc ctg gtc atc aac acc agc cgt ctg aac ggc atg aag atc 242Tyr Tyr Gly Leu Val Ile Asn Thr Ser Arg Leu Asn Gly Met Lys Ile 65 70 75 ttt cac gca ggc gag aag gtc ctg ctg gaa gcc caa tgc ggc gtt cgc 290Phe His Ala Gly Glu Lys Val Leu Leu Glu Ala Gln Cys Gly Val Arg 80 85 90 ctg tct caa gtg gtt aaa ctg gcg ctg gaa ctg aat ctg gaa ggt att 338Leu Ser Gln Val Val Lys Leu Ala Leu Glu Leu Asn Leu Glu Gly Ile 95 100 105 tac aaa ctg gct ggc ttc ccg gcc acc gtt ggt ggt gcg gtg gca atg 386Tyr Lys Leu Ala Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met 110 115 120 aat gct ggt gcg ttt ggt acc gag atc agc cat tac ctg aag agc ctg 434Asn Ala Gly Ala Phe Gly Thr Glu Ile Ser His Tyr Leu Lys Ser Leu 125 130 135 140 ctg gtt atg gat tgg gaa ggt aat gtg gag aag att agc gca gag gac 482Leu Val Met Asp Trp Glu Gly Asn Val Glu Lys Ile Ser Ala Glu Asp 145 150 155 gtg aaa ttt gac tac cgc tcc agc ccg ttc ccg gat atg ggc att gtc 530Val Lys Phe Asp Tyr Arg Ser Ser Pro Phe Pro Asp Met Gly Ile Val 160 165 170 ctg atg gcg gaa ctg gag ctg aag cgc gca gag ctg gac gtg cgt cat 578Leu Met Ala Glu Leu Glu Leu Lys Arg Ala Glu Leu Asp Val Arg His 175 180 185 gag cag aat ctg atc aaa gag cgc cgt cgt cgt acg cag ccg att aac 626Glu Gln Asn Leu Ile Lys Glu Arg Arg Arg Arg Thr Gln Pro Ile Asn 190 195 200 atg ccg acc agc ggc agc acc ttt aag aac cca ccg ggt cag tat gcg 674Met Pro Thr Ser Gly Ser Thr Phe Lys Asn Pro Pro Gly Gln Tyr Ala 205 210 215 220 ggc aaa ctg ctg gaa atg gtt ggc atg aaa ggt tac cgt gtt ggt gac 722Gly Lys Leu Leu Glu Met Val Gly Met Lys Gly Tyr Arg Val Gly Asp 225 230 235 gtc gcc ttc tcc cac ttg cac gcc aat ttc ctg gtc aat ctg ggt gat 770Val Ala Phe Ser His Leu His Ala Asn Phe Leu Val Asn Leu Gly Asp 240 245 250 ggt cgt tat gag gac gcg ctg aag att ctg ttg gaa gcg aaa cgt cgt 818Gly Arg Tyr Glu Asp Ala Leu Lys Ile Leu Leu Glu Ala Lys Arg Arg 255 260 265 gtg tat gaa gag ttt ggc atc tac ctg gaa gaa gag gtg aag gtc gtt 866Val Tyr Glu Glu Phe Gly Ile Tyr Leu Glu Glu Glu Val Lys Val Val 270 275 280 gag agc tgc agc acc cac ggt ggc caa gtg cat cac cac cat cac cat 914Glu Ser Cys Ser Thr His Gly Gly Gln Val His His His His His His 285 290 295 300 taa ctcgag 92368300PRTHydrogenobacter thermophilus 68Met Lys Leu Glu Lys Asn Val Leu Leu Ala Pro Tyr Thr Thr Ile Arg 1 5 10 15 Ile Gly Gly Thr Ala Arg Phe Met Cys Phe Pro Ser Asp Phe Ala Glu 20 25 30 Leu Ser Lys Ala Ile Arg Trp Ala Lys Glu Glu Gly Leu Pro Val Phe 35 40 45 Leu Leu Gly Arg Gly Ala Asn Thr Ile Phe Gly Asp Tyr Tyr Gly Leu 50 55 60 Val Ile Asn Thr Ser Arg Leu Asn Gly Met Lys Ile Phe His Ala Gly 65 70 75 80 Glu Lys Val Leu Leu Glu Ala Gln Cys Gly Val Arg Leu Ser Gln Val 85 90 95 Val Lys Leu Ala Leu Glu Leu Asn Leu Glu Gly Ile Tyr Lys Leu Ala 100 105 110 Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn Ala Gly Ala 115 120 125 Phe Gly Thr Glu Ile Ser His Tyr Leu Lys Ser Leu Leu Val Met Asp 130 135 140 Trp Glu Gly Asn Val Glu Lys Ile Ser Ala Glu Asp Val Lys Phe Asp 145 150 155 160 Tyr Arg Ser Ser Pro Phe Pro Asp Met Gly Ile Val Leu Met Ala Glu 165 170 175 Leu Glu Leu Lys Arg Ala Glu Leu Asp Val Arg His Glu Gln Asn Leu 180 185 190 Ile Lys Glu Arg Arg Arg Arg Thr Gln Pro Ile Asn Met Pro Thr Ser 195 200 205 Gly Ser Thr Phe Lys Asn Pro Pro Gly Gln Tyr Ala Gly Lys Leu Leu 210 215 220 Glu Met Val Gly Met Lys Gly Tyr Arg Val Gly Asp Val Ala Phe Ser 225 230 235 240 His Leu His Ala Asn Phe Leu Val Asn Leu Gly Asp Gly Arg Tyr Glu 245 250 255 Asp Ala Leu Lys Ile Leu Leu Glu Ala Lys Arg Arg Val Tyr Glu Glu 260 265 270 Phe Gly Ile Tyr Leu Glu Glu Glu Val Lys Val Val Glu Ser Cys Ser 275 280 285 Thr His Gly Gly Gln Val His His His His His His 290 295 300 69888DNAThermocrinus albusCDS(1)..(888) 69atg cag aag gaa gag aaa gtg agc ctc tca ccc ttt aca aca ctg aag 48Met Gln Lys Glu Glu Lys Val Ser Leu Ser Pro Phe Thr Thr Leu Lys 1 5 10 15 ata gga ggc gtg gct gac ctc ttc tgt agt ccg cag agg gaa gag gaa 96Ile Gly Gly Val Ala Asp Leu Phe Cys Ser Pro Gln Arg Glu Glu Glu 20 25 30 cta agg cag tgt ata cag atg gcc aag gtg aaa gac gtt cct ata ctg 144Leu Arg Gln Cys Ile Gln Met Ala Lys Val Lys Asp Val Pro Ile Leu 35 40 45 gtt atg gga agg gga gct aac ttg ctg gta ggc gac gta gag ggt ctc 192Val Met Gly Arg Gly Ala Asn Leu Leu Val Gly Asp Val Glu Gly Leu 50 55 60 gtg gtg agt acc aga tat atg aga ggt atg tgg gta agg gaa gag aag 240Val Val Ser Thr Arg Tyr Met Arg Gly Met Trp Val Arg Glu Glu Lys 65 70 75 80 gat ggt ttg aag gtt aaa gtt atg gca gga gaa ccc ctg aag acc ctc 288Asp Gly Leu Lys Val Lys Val Met Ala Gly Glu Pro Leu Lys Thr Leu 85 90 95 atc cag ctg gcc ctt aag gaa aac ttg gaa ggt cta tac cgg tta gcg 336Ile Gln Leu Ala Leu Lys Glu Asn Leu Glu Gly Leu Tyr Arg Leu Ala 100 105 110 ggt ttc ccg gcc acc gtg ggt ggt gcg gta gct atg aac gcg gga gcc 384Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn Ala Gly Ala 115 120 125 ttt ggc tac gaa ata tct cag cac ctt acc cat gtg gcc ttt ctg gac 432Phe Gly Tyr Glu Ile Ser Gln His Leu Thr His Val Ala Phe Leu Asp 130 135 140 tgg gac ggt cgg ctg cac cgt gtt cct gca aag gag ata aac ttc tcg 480Trp Asp Gly Arg Leu His Arg Val Pro Ala Lys Glu Ile Asn Phe Ser 145 150 155 160 tac cgc cac tca cct ttt ccc agg tgg gga ata gtg gtg tgg gca gag 528Tyr Arg His Ser Pro Phe Pro Arg Trp Gly Ile Val Val Trp Ala Glu 165 170 175 ttc ctc ttt cca aga tcc gaa aag cca gtt tat gaa gag tat ctc cag 576Phe Leu Phe Pro Arg Ser Glu Lys Pro Val Tyr Glu Glu Tyr Leu Gln 180 185 190 ata aga gag agg agg aag aag acc caa ccc atc cac caa ccc acc tgt 624Ile Arg Glu Arg Arg Lys Lys Thr Gln Pro Ile His Gln Pro Thr Cys 195 200 205 gga tcc acc ttc aaa aac cca cct gga gat tac gct ggt cga ctc atc 672Gly Ser Thr Phe Lys Asn Pro Pro Gly Asp Tyr Ala Gly Arg Leu Ile 210 215 220 cag ctg gtg ggt ttg aaa ggc tac cga ttg ggc aga gtt gct ttt tca 720Gln Leu Val Gly Leu Lys Gly Tyr Arg Leu Gly Arg Val Ala Phe Ser 225 230 235 240 gag ata cac gcc aac ttc atc atc aac tta gga ggc gcc acc ttc caa 768Glu Ile His Ala Asn Phe Ile Ile Asn Leu Gly Gly Ala Thr Phe Gln 245 250 255 gaa gcc aca gag ctt atc cag ata gcc aaa gat aaa gtt tac aga gag 816Glu Ala Thr Glu Leu Ile Gln Ile Ala Lys Asp Lys Val Tyr Arg Glu 260 265 270 tta ggt ata acc ttg gaa gag gag gtg aga atc gtt gaa ggt cgt cgt 864Leu Gly Ile Thr Leu Glu Glu Glu Val Arg Ile Val Glu Gly Arg Arg 275 280 285 tct gat ggg tgg aag atc cta tga 888Ser Asp Gly Trp Lys Ile Leu 290 295 70295PRTThermocrinus albus 70Met Gln Lys Glu Glu Lys Val Ser Leu Ser Pro Phe Thr Thr Leu Lys 1 5 10 15 Ile Gly Gly Val Ala Asp Leu Phe Cys Ser Pro Gln Arg Glu Glu Glu 20 25 30 Leu Arg Gln Cys Ile Gln Met Ala Lys Val Lys Asp Val Pro Ile Leu 35 40 45 Val Met Gly Arg Gly Ala Asn Leu Leu Val Gly Asp Val Glu Gly Leu 50 55 60 Val Val Ser Thr Arg Tyr Met Arg Gly Met Trp Val Arg Glu Glu Lys 65 70 75 80 Asp Gly Leu Lys Val Lys Val Met Ala Gly Glu Pro Leu Lys Thr Leu 85 90 95 Ile Gln Leu Ala Leu Lys Glu Asn Leu Glu Gly Leu Tyr Arg Leu Ala 100 105 110 Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn Ala Gly Ala 115 120 125 Phe Gly Tyr Glu Ile Ser Gln His Leu Thr His Val Ala Phe Leu Asp 130 135 140 Trp Asp Gly Arg Leu His Arg Val Pro Ala Lys Glu Ile Asn Phe Ser 145 150 155 160 Tyr Arg His Ser Pro Phe Pro Arg Trp Gly Ile Val Val Trp Ala Glu 165 170 175 Phe Leu Phe Pro Arg Ser Glu Lys Pro Val Tyr Glu Glu Tyr Leu Gln 180 185 190 Ile Arg Glu Arg Arg Lys Lys Thr Gln Pro Ile His Gln Pro Thr Cys 195 200 205 Gly Ser Thr Phe Lys Asn Pro Pro Gly Asp Tyr Ala Gly Arg Leu Ile 210 215 220 Gln Leu Val Gly Leu Lys Gly Tyr Arg Leu Gly Arg Val Ala Phe Ser 225 230 235 240 Glu Ile His Ala Asn Phe Ile Ile Asn Leu Gly Gly Ala Thr Phe Gln 245 250 255 Glu Ala Thr Glu Leu Ile Gln Ile Ala Lys Asp Lys Val Tyr Arg Glu 260 265 270 Leu Gly Ile Thr Leu Glu Glu Glu Val Arg Ile Val Glu Gly Arg Arg 275 280 285 Ser Asp Gly Trp Lys Ile Leu 290 295 71926DNAThermocrinus albusCDS(15)..(920) 71aggaggtaaa acat atg caa aaa gaa gag aaa gta tcc ctg agc cca ttt 50 Met Gln Lys Glu Glu Lys Val Ser Leu Ser Pro Phe 1 5 10 acg acc ctg aag att ggc ggt gta gcg gat ttg ttc tgc agc ccg cag 98Thr Thr Leu Lys Ile Gly Gly Val Ala Asp Leu Phe Cys Ser Pro Gln 15 20 25 cgc gaa gaa gag ttg cgc cag tgt atc caa atg gct aag gtg aaa gac 146Arg Glu Glu Glu Leu Arg Gln Cys Ile Gln Met Ala Lys Val Lys Asp 30 35 40 gtt ccg atc ctg gtt atg ggt cgt ggc gcg aac ctg ttg gtg ggt gac 194Val Pro Ile Leu Val Met Gly Arg Gly Ala Asn Leu Leu Val Gly Asp 45 50 55 60 gtt gag ggt ctg gtt gtc tcg acc cgt tac atg cgt ggt atg tgg gtg 242Val Glu Gly Leu Val Val Ser Thr Arg Tyr Met Arg Gly Met Trp Val 65 70 75 cgt gaa gag aag gac ggc ctg aag gtc aaa gtt atg gcc ggt gaa ccg 290Arg Glu Glu Lys Asp Gly Leu Lys Val Lys Val Met Ala Gly Glu Pro 80 85 90 ctg aaa acc ttg att caa ttg gca ctg aaa gaa aat ctg gaa ggt ctg 338Leu Lys Thr Leu Ile Gln Leu Ala Leu Lys Glu Asn Leu Glu Gly Leu 95 100 105

tat cgc ctg gca ggc ttt ccg gcg acc gtc ggt ggc gca gtg gca atg 386Tyr Arg Leu Ala Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met 110 115 120 aac gcg ggt gcg ttc ggc tac gag att tct caa cac ctg acc cac gtc 434Asn Ala Gly Ala Phe Gly Tyr Glu Ile Ser Gln His Leu Thr His Val 125 130 135 140 gcc ttc ctg gat tgg gac ggt cgt ctg cac cgc gtc ccg gca aaa gag 482Ala Phe Leu Asp Trp Asp Gly Arg Leu His Arg Val Pro Ala Lys Glu 145 150 155 atc aac ttc agc tac cgc cat agc ccg ttc ccg cgt tgg ggc atc gtc 530Ile Asn Phe Ser Tyr Arg His Ser Pro Phe Pro Arg Trp Gly Ile Val 160 165 170 gtt tgg gcg gag ttc ctg ttt ccg cgt agc gag aag cct gtg tat gaa 578Val Trp Ala Glu Phe Leu Phe Pro Arg Ser Glu Lys Pro Val Tyr Glu 175 180 185 gag tat ctg caa atc cgt gag cgt cgt aag aaa acc cag ccg atc cac 626Glu Tyr Leu Gln Ile Arg Glu Arg Arg Lys Lys Thr Gln Pro Ile His 190 195 200 cag ccg acg tgc ggc agc act ttt aag aat cca ccg ggc gac tac gct 674Gln Pro Thr Cys Gly Ser Thr Phe Lys Asn Pro Pro Gly Asp Tyr Ala 205 210 215 220 ggt cgc ctg atc cag ctg gtt ggt ctg aaa ggt tat cgt ttg ggt cgc 722Gly Arg Leu Ile Gln Leu Val Gly Leu Lys Gly Tyr Arg Leu Gly Arg 225 230 235 gtg gcg ttt agc gag att cat gcc aac ttt atc att aat ctg ggt ggc 770Val Ala Phe Ser Glu Ile His Ala Asn Phe Ile Ile Asn Leu Gly Gly 240 245 250 gcg acg ttc cag gaa gcc acc gag ctg att cag atc gcg aaa gat aag 818Ala Thr Phe Gln Glu Ala Thr Glu Leu Ile Gln Ile Ala Lys Asp Lys 255 260 265 gtg tac cgc gag ctg ggt att acg ctg gaa gaa gag gtg cgt att gtt 866Val Tyr Arg Glu Leu Gly Ile Thr Leu Glu Glu Glu Val Arg Ile Val 270 275 280 gag ggc cgt cgt agc gat ggt tgg aag att ctg cac cac cat cac cat 914Glu Gly Arg Arg Ser Asp Gly Trp Lys Ile Leu His His His His His 285 290 295 300 cat taa ctcgag 926His 72301PRTThermocrinus albus 72Met Gln Lys Glu Glu Lys Val Ser Leu Ser Pro Phe Thr Thr Leu Lys 1 5 10 15 Ile Gly Gly Val Ala Asp Leu Phe Cys Ser Pro Gln Arg Glu Glu Glu 20 25 30 Leu Arg Gln Cys Ile Gln Met Ala Lys Val Lys Asp Val Pro Ile Leu 35 40 45 Val Met Gly Arg Gly Ala Asn Leu Leu Val Gly Asp Val Glu Gly Leu 50 55 60 Val Val Ser Thr Arg Tyr Met Arg Gly Met Trp Val Arg Glu Glu Lys 65 70 75 80 Asp Gly Leu Lys Val Lys Val Met Ala Gly Glu Pro Leu Lys Thr Leu 85 90 95 Ile Gln Leu Ala Leu Lys Glu Asn Leu Glu Gly Leu Tyr Arg Leu Ala 100 105 110 Gly Phe Pro Ala Thr Val Gly Gly Ala Val Ala Met Asn Ala Gly Ala 115 120 125 Phe Gly Tyr Glu Ile Ser Gln His Leu Thr His Val Ala Phe Leu Asp 130 135 140 Trp Asp Gly Arg Leu His Arg Val Pro Ala Lys Glu Ile Asn Phe Ser 145 150 155 160 Tyr Arg His Ser Pro Phe Pro Arg Trp Gly Ile Val Val Trp Ala Glu 165 170 175 Phe Leu Phe Pro Arg Ser Glu Lys Pro Val Tyr Glu Glu Tyr Leu Gln 180 185 190 Ile Arg Glu Arg Arg Lys Lys Thr Gln Pro Ile His Gln Pro Thr Cys 195 200 205 Gly Ser Thr Phe Lys Asn Pro Pro Gly Asp Tyr Ala Gly Arg Leu Ile 210 215 220 Gln Leu Val Gly Leu Lys Gly Tyr Arg Leu Gly Arg Val Ala Phe Ser 225 230 235 240 Glu Ile His Ala Asn Phe Ile Ile Asn Leu Gly Gly Ala Thr Phe Gln 245 250 255 Glu Ala Thr Glu Leu Ile Gln Ile Ala Lys Asp Lys Val Tyr Arg Glu 260 265 270 Leu Gly Ile Thr Leu Glu Glu Glu Val Arg Ile Val Glu Gly Arg Arg 275 280 285 Ser Asp Gly Trp Lys Ile Leu His His His His His His 290 295 300 73840DNAThermoanaerobacter italicusCDS(1)..(840) 73atg aaa aga cac act tct ttt aaa ata ggt gga cct gca gat gtg tta 48Met Lys Arg His Thr Ser Phe Lys Ile Gly Gly Pro Ala Asp Val Leu 1 5 10 15 gtt gta cca aat aat cga aaa gaa tta ttg gaa gcc ata tct ttg ttg 96Val Val Pro Asn Asn Arg Lys Glu Leu Leu Glu Ala Ile Ser Leu Leu 20 25 30 aaa cga gaa aac ata cct ttt ttt ata cta gga aat ggt act aat cta 144Lys Arg Glu Asn Ile Pro Phe Phe Ile Leu Gly Asn Gly Thr Asn Leu 35 40 45 tta gta agt gaa aaa ggc att aga gga gtt gta ata aaa tta tcc tct 192Leu Val Ser Glu Lys Gly Ile Arg Gly Val Val Ile Lys Leu Ser Ser 50 55 60 ttg agg aat gta gtt gta gaa ggt aat agg ata att gct gaa gca gga 240Leu Arg Asn Val Val Val Glu Gly Asn Arg Ile Ile Ala Glu Ala Gly 65 70 75 80 gca cct ctt tcc tat att gcc aat gtg gca ctt gta cat gaa ctt gcg 288Ala Pro Leu Ser Tyr Ile Ala Asn Val Ala Leu Val His Glu Leu Ala 85 90 95 gga ttt gaa ttt gct agc ggg att cct ggc act tta ggt gga gca ata 336Gly Phe Glu Phe Ala Ser Gly Ile Pro Gly Thr Leu Gly Gly Ala Ile 100 105 110 gtg atg aac gca ggg gct tat ggg tct gaa atg aag gac gtg ata gaa 384Val Met Asn Ala Gly Ala Tyr Gly Ser Glu Met Lys Asp Val Ile Glu 115 120 125 aaa gta gag gtt tta gat gga gaa ggc aat ata ttg att tta tca aac 432Lys Val Glu Val Leu Asp Gly Glu Gly Asn Ile Leu Ile Leu Ser Asn 130 135 140 gaa gaa atg aat ttt tcc tat aga tac agc att att cat gaa aag gat 480Glu Glu Met Asn Phe Ser Tyr Arg Tyr Ser Ile Ile His Glu Lys Asp 145 150 155 160 tgg att gtt tta aga gca tgg ctt agt ttg gca aaa ggg aaa tac gaa 528Trp Ile Val Leu Arg Ala Trp Leu Ser Leu Ala Lys Gly Lys Tyr Glu 165 170 175 gag ata aaa agc aaa atg gag gaa cta aat gca aaa aga agg gaa aaa 576Glu Ile Lys Ser Lys Met Glu Glu Leu Asn Ala Lys Arg Arg Glu Lys 180 185 190 cag cct ttg gag tat cca agt gcc gga agt act ttt aaa agg cca cct 624Gln Pro Leu Glu Tyr Pro Ser Ala Gly Ser Thr Phe Lys Arg Pro Pro 195 200 205 gga tat tat gct ggg aaa ttg att gag gaa gca gga ctt aaa ggc tat 672Gly Tyr Tyr Ala Gly Lys Leu Ile Glu Glu Ala Gly Leu Lys Gly Tyr 210 215 220 tca att gga gga gct aaa gtt tcc gaa aag cat tcg gga ttt att ata 720Ser Ile Gly Gly Ala Lys Val Ser Glu Lys His Ser Gly Phe Ile Ile 225 230 235 240 aat act ggc aat gca act ttt tac gat gtt tta aat ttg att gag cat 768Asn Thr Gly Asn Ala Thr Phe Tyr Asp Val Leu Asn Leu Ile Glu His 245 250 255 ata caa aaa gta gta aaa gaa aag ttt gga gta gaa ctt gta cca gaa 816Ile Gln Lys Val Val Lys Glu Lys Phe Gly Val Glu Leu Val Pro Glu 260 265 270 ata aaa ata ata gga gag aaa tag 840Ile Lys Ile Ile Gly Glu Lys 275 74279PRTThermoanaerobacter italicus 74Met Lys Arg His Thr Ser Phe Lys Ile Gly Gly Pro Ala Asp Val Leu 1 5 10 15 Val Val Pro Asn Asn Arg Lys Glu Leu Leu Glu Ala Ile Ser Leu Leu 20 25 30 Lys Arg Glu Asn Ile Pro Phe Phe Ile Leu Gly Asn Gly Thr Asn Leu 35 40 45 Leu Val Ser Glu Lys Gly Ile Arg Gly Val Val Ile Lys Leu Ser Ser 50 55 60 Leu Arg Asn Val Val Val Glu Gly Asn Arg Ile Ile Ala Glu Ala Gly 65 70 75 80 Ala Pro Leu Ser Tyr Ile Ala Asn Val Ala Leu Val His Glu Leu Ala 85 90 95 Gly Phe Glu Phe Ala Ser Gly Ile Pro Gly Thr Leu Gly Gly Ala Ile 100 105 110 Val Met Asn Ala Gly Ala Tyr Gly Ser Glu Met Lys Asp Val Ile Glu 115 120 125 Lys Val Glu Val Leu Asp Gly Glu Gly Asn Ile Leu Ile Leu Ser Asn 130 135 140 Glu Glu Met Asn Phe Ser Tyr Arg Tyr Ser Ile Ile His Glu Lys Asp 145 150 155 160 Trp Ile Val Leu Arg Ala Trp Leu Ser Leu Ala Lys Gly Lys Tyr Glu 165 170 175 Glu Ile Lys Ser Lys Met Glu Glu Leu Asn Ala Lys Arg Arg Glu Lys 180 185 190 Gln Pro Leu Glu Tyr Pro Ser Ala Gly Ser Thr Phe Lys Arg Pro Pro 195 200 205 Gly Tyr Tyr Ala Gly Lys Leu Ile Glu Glu Ala Gly Leu Lys Gly Tyr 210 215 220 Ser Ile Gly Gly Ala Lys Val Ser Glu Lys His Ser Gly Phe Ile Ile 225 230 235 240 Asn Thr Gly Asn Ala Thr Phe Tyr Asp Val Leu Asn Leu Ile Glu His 245 250 255 Ile Gln Lys Val Val Lys Glu Lys Phe Gly Val Glu Leu Val Pro Glu 260 265 270 Ile Lys Ile Ile Gly Glu Lys 275 75944DNAThermoanaerobacter italicusCDS(15)..(938) 75aggaggtaaa acat atg aat gag att att gac aaa ttg aag gac att ttg 50 Met Asn Glu Ile Ile Asp Lys Leu Lys Asp Ile Leu 1 5 10 cgt gag ggt aag ctg tat ttg aac gaa ccg atg aaa cgc cat acg agc 98Arg Glu Gly Lys Leu Tyr Leu Asn Glu Pro Met Lys Arg His Thr Ser 15 20 25 ttt aag att ggc ggt ccg gcg gat gtt ctg gtg gtg ccg aat aac cgt 146Phe Lys Ile Gly Gly Pro Ala Asp Val Leu Val Val Pro Asn Asn Arg 30 35 40 aaa gaa ctg ctg gaa gca atc agc ctg ctg aaa cgt gag aac att ccg 194Lys Glu Leu Leu Glu Ala Ile Ser Leu Leu Lys Arg Glu Asn Ile Pro 45 50 55 60 ttc ttc atc ctg ggt aac ggc act aat ctg ctg gtg agc gag aag ggt 242Phe Phe Ile Leu Gly Asn Gly Thr Asn Leu Leu Val Ser Glu Lys Gly 65 70 75 atc cgt ggt gtc gtg att aag ttg agc agc ctg cgt aac gtc gtt gtc 290Ile Arg Gly Val Val Ile Lys Leu Ser Ser Leu Arg Asn Val Val Val 80 85 90 gaa ggc aat cgt atc att gcc gag gcg ggt gcg ccg ctg tcc tat atc 338Glu Gly Asn Arg Ile Ile Ala Glu Ala Gly Ala Pro Leu Ser Tyr Ile 95 100 105 gcg aac gtc gct ctg gtt cat gag ctg gca ggc ttt gag ttt gcc agc 386Ala Asn Val Ala Leu Val His Glu Leu Ala Gly Phe Glu Phe Ala Ser 110 115 120 ggc att ccg ggt acc ctg ggt ggc gca att gtt atg aac gcg ggt gca 434Gly Ile Pro Gly Thr Leu Gly Gly Ala Ile Val Met Asn Ala Gly Ala 125 130 135 140 tac ggt agc gag atg aaa gat gtg att gag aag gtg gaa gtg ctg gat 482Tyr Gly Ser Glu Met Lys Asp Val Ile Glu Lys Val Glu Val Leu Asp 145 150 155 ggc gaa ggc aac atc ctg atc ttg agc aat gaa gaa atg aat ttc tct 530Gly Glu Gly Asn Ile Leu Ile Leu Ser Asn Glu Glu Met Asn Phe Ser 160 165 170 tac cgt tac tcg att atc cac gag aag gac tgg atc gtg ctg cgc gcc 578Tyr Arg Tyr Ser Ile Ile His Glu Lys Asp Trp Ile Val Leu Arg Ala 175 180 185 tgg ctg agc ttg gcg aaa ggc aag tat gaa gag att aag tcc aaa atg 626Trp Leu Ser Leu Ala Lys Gly Lys Tyr Glu Glu Ile Lys Ser Lys Met 190 195 200 gaa gaa ctg aac gcg aaa cgc cgc gag aaa cag ccg ctg gag tat cca 674Glu Glu Leu Asn Ala Lys Arg Arg Glu Lys Gln Pro Leu Glu Tyr Pro 205 210 215 220 agc gcc ggt agc acc ttc aaa cgt ccg cct ggt tac tac gct ggc aag 722Ser Ala Gly Ser Thr Phe Lys Arg Pro Pro Gly Tyr Tyr Ala Gly Lys 225 230 235 ctg att gaa gag gcg ggt ctg aag ggc tat tcc atc ggc ggt gca aag 770Leu Ile Glu Glu Ala Gly Leu Lys Gly Tyr Ser Ile Gly Gly Ala Lys 240 245 250 gtt agc gaa aaa cac tct ggt ttc att atc aat acg ggt aac gcg acc 818Val Ser Glu Lys His Ser Gly Phe Ile Ile Asn Thr Gly Asn Ala Thr 255 260 265 ttt tac gac gtc ctg aat ctg atc gag cac atc caa aaa gtt gtt aaa 866Phe Tyr Asp Val Leu Asn Leu Ile Glu His Ile Gln Lys Val Val Lys 270 275 280 gag aag ttt ggt gtt gaa ctg gtc ccg gag atc aag atc att ggt gag 914Glu Lys Phe Gly Val Glu Leu Val Pro Glu Ile Lys Ile Ile Gly Glu 285 290 295 300 aaa cat cac cac cat cac cac taa ctcgag 944Lys His His His His His His 305 76307PRTThermoanaerobacter italicus 76Met Asn Glu Ile Ile Asp Lys Leu Lys Asp Ile Leu Arg Glu Gly Lys 1 5 10 15 Leu Tyr Leu Asn Glu Pro Met Lys Arg His Thr Ser Phe Lys Ile Gly 20 25 30 Gly Pro Ala Asp Val Leu Val Val Pro Asn Asn Arg Lys Glu Leu Leu 35 40 45 Glu Ala Ile Ser Leu Leu Lys Arg Glu Asn Ile Pro Phe Phe Ile Leu 50 55 60 Gly Asn Gly Thr Asn Leu Leu Val Ser Glu Lys Gly Ile Arg Gly Val 65 70 75 80 Val Ile Lys Leu Ser Ser Leu Arg Asn Val Val Val Glu Gly Asn Arg 85 90 95 Ile Ile Ala Glu Ala Gly Ala Pro Leu Ser Tyr Ile Ala Asn Val Ala 100 105 110 Leu Val His Glu Leu Ala Gly Phe Glu Phe Ala Ser Gly Ile Pro Gly 115 120 125 Thr Leu Gly Gly Ala Ile Val Met Asn Ala Gly Ala Tyr Gly Ser Glu 130 135 140 Met Lys Asp Val Ile Glu Lys Val Glu Val Leu Asp Gly Glu Gly Asn 145 150 155 160 Ile Leu Ile Leu Ser Asn Glu Glu Met Asn Phe Ser Tyr Arg Tyr Ser 165 170 175 Ile Ile His Glu Lys Asp Trp Ile Val Leu Arg Ala Trp Leu Ser Leu 180 185 190 Ala Lys Gly Lys Tyr Glu Glu Ile Lys Ser Lys Met Glu Glu Leu Asn 195 200 205 Ala Lys Arg Arg Glu Lys Gln Pro Leu Glu Tyr Pro Ser Ala Gly Ser 210 215 220 Thr Phe Lys Arg Pro Pro Gly Tyr Tyr Ala Gly Lys Leu Ile Glu Glu 225 230 235 240 Ala Gly Leu Lys Gly Tyr Ser Ile Gly Gly Ala Lys Val Ser Glu Lys 245 250 255 His Ser Gly Phe Ile Ile Asn Thr Gly Asn Ala Thr Phe Tyr Asp Val 260 265 270 Leu Asn Leu Ile Glu His Ile Gln Lys Val Val Lys Glu Lys Phe Gly 275 280 285 Val Glu Leu Val Pro Glu Ile Lys Ile Ile Gly Glu Lys His His His 290 295 300 His His His 305

Claims

1. A recombinant polynucleotide comprising a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

2. The recombinant polynucleotide of claim 1, wherein the nucleotide sequence further encodes at least one additional polypeptide of interest in-frame with the encoded protein.

3. The recombinant polynucleotide of claim 2, wherein the at least one additional polypeptide of interest comprises a tag.

4. The recombinant polynucleotide of claim 3, wherein the tag comprises a poly-His tag.

5. The recombinant polynucleotide of claim 1, wherein the encoded protein is thermostable.

6. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 50.degree. C. is greater than about 10% of fluorescence of the encoded protein at about 30.degree. C.

7. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 60.degree. C. is greater than about 10% of fluorescence of the encoded protein at about 30.degree. C.

8. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 70.degree. C. is greater than about 10% of fluorescence of the encoded protein at about 30.degree. C.

9. The recombinant polynucleotide of claim 1, wherein fluorescence of the encoded protein at about 75.degree. C. is greater than about 10% of fluorescence of the encoded protein at about 30.degree. C.

10. The recombinant polynucleotide of claim 1, wherein the nucleotide sequence is optimized to express the fluorescent protein in a heterologous host.

11. The recombinant polynucleotide of claim 1, wherein the nucleotide sequence encodes a fluorescent protein which is at least 95% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

12. An expression construct comprising a recombinant polynucleotide as recited in claim 1.

13. A host cell comprising an expression construct as recited in claim 12.

14. A method for making a fluorescent protein comprising cultivating the host cell of claim 13.

15. A fluorescent protein made by cultivating a host cell, wherein the host cell comprises an expression construct, the expression construct comprises a recombinant polynucleotide, and the recombinant polynucleotide comprises a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

16. A fusion protein comprising a polypeptide of interest operationally linked to a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

17. A method of increasing the aqueous solubility of a protein of interest comprising fusing or attaching the protein of interest to a protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74.

18. A method of assaying gene expression comprising: cultivating a host cell comprising an expression construct, wherein the expression construct comprises a recombinant polynucleotide and the recombinant polynucleotide comprises a nucleotide sequence encoding a fluorescent protein which is at least 90% sequence identical to a protein having a sequence selected from the group consisting of SEQ. ID. NOS: 22, 26, 34, 46, 50, 54, 66, 70, and 74; and detecting fluorescence in the cell or an extract thereof.

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