GRG36: Novel EPSP Synthase Gene Conferring Herbicide Resistance

Abstract

Compositions and methods for conferring herbicide resistance to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molecules encoding herbicide resistance or tolerance polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. The nucleotide sequences of the invention can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, the present invention provides for isolated nucleic acid molecules comprising the nucleotide sequence set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22, the herbicide resistance nucleotide sequence deposited in a bacterial host as Accession Nos. NRRL B-30932, B-30933, B-30934, B-30945, B-30946, B-30947, or B-30948, as well as variants and fragments thereof.

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. patent application Ser. No. 11/769,327, filed Jun. 27, 2007, which claims the benefit of U.S. Provisional Application Ser. Nos. 60/816,676, filed Jun. 27, 2006; 60/819,122, filed Jul. 7, 2006; and, 60/819,119, filed Jul. 7, 2006, the contents of which are herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "APA046US01DSEQLIST.txt", created on Apr. 26, 2011, and having a size of 150 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] This invention provides novel genes encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase that provide herbicide resistance. These genes are useful in plant biology, crop breeding, and plant cell culture.

BACKGROUND OF THE INVENTION

[0004] N-phosphonomethylglycine, commonly referred to as glyphosate, is an important agronomic chemical. Glyphosate inhibits the enzyme that converts phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid. Inhibition of this enzyme (5-enolpyruvylshikimate-3-phosphate synthase; referred to herein as "EPSP synthase") kills plant cells by shutting down the shikimate pathway, thereby inhibiting aromatic acid biosynthesis.

[0005] Since glyphosate-class herbicides inhibit aromatic amino acid biosynthesis, they not only kill plant cells, but are also toxic to bacterial cells. Glyphosate inhibits many bacterial EPSP synthases, and thus is toxic to these bacteria. However, certain bacterial EPSP synthases have a high tolerance to glyphosate.

[0006] Plant cells resistant to glyphosate toxicity can be produced by transforming plant cells to express glyphosate-resistant bacterial EPSP synthases. Notably, the bacterial gene from Agrobacterium tumefaciens strain CP4 has been used to confer herbicide resistance on plant cells following expression in plants. A mutated EPSP synthase from Salmonella typhimurium strain CT7 confers glyphosate resistance in bacterial cells, and confers glyphosate resistance on plant cells (U.S. Pat. Nos. 4,535,060; 4,769,061; and 5,094,945).

[0007] U.S. Pat. No. 6,040,497 reports mutant maize EPSP synthase enzymes having substitutions of threonine to isoleucine at position 102 and proline to serine at position 106 (the "TIPS" mutation). Such alterations confer glyphosate resistance upon the maize enzyme. A mutated EPSP synthase from Salmonella typhimurium strain CT7 confers glyphosate resistance in bacterial cells, and is reported to confer glyphosate resistance upon plant cells (U.S. Pat. Nos. 4,535,060; 4,769,061; and 5,094,945). He et al. ((2001) Biochim et Biophysica Acta 1568:1-6) have developed EPSP synthases with increased glyphosate tolerance by mutagenesis and recombination between the E. coli and Salmonella typhimurium EPSP synthase genes, and suggest that mutations at position 42 (T42M) and position 230 (Q230K) are likely responsible for the observed resistance.

[0008] Subsequent work (He et al. (2003) Biosci. Biotech. Biochem. 67:1405-1409) shows that the T42M mutation (threonine to methionine) is sufficient to improve tolerance of both the E. coli and Salmonella typhimurium enzymes. These enzymes contain amino acid substitutions in their active sites that prevent the binding of glyphosate without affecting binding by PEP or S3P. Mutations that occur in the hinge region between the two globular domains of EPSP synthase have been shown to alter the binding affinity of glyphosate but not PEP (He et al., 2003, supra). Therefore, such enzymes have high catalytic activity, even in the presence of glyphosate.

[0009] Due to the many advantages herbicide resistance plants provide, methods for identifying herbicide resistance genes with glyphosate resistance activity are desirable.

SUMMARY OF INVENTION

[0010] Compositions and methods for conferring herbicide resistance or tolerance to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molecules encoding herbicide resistance or tolerance polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include antibodies to the herbicide resistance or tolerance polypeptides. As noted the nucleotide sequences of the invention can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In addition, methods are provided for producing the polypeptides encoded by the synthetic nucleotides of the invention.

[0011] In particular, isolated nucleic acid molecules and variants thereof encoding herbicide resistance- or tolerance polypeptides are provided. Additionally, amino acid sequences and variants thereof encoded by the polynucleotides that confer herbicide resistance or tolerance are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising the nucleotide sequence set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22, the herbicide resistance nucleotide sequence deposited in a bacterial host as Accession Nos. NRRL B-30932, B-30933, B-30934, B-30945, B-30946, B-30947, or B-30948, as well as variants and fragments thereof. Nucleotide sequences that are complementary to a nucleotide sequence of the invention, or that hybridize to a sequence of the invention or a complement of a sequence of the invention are also encompassed.

DESCRIPTION OF FIGURES

[0012] FIG. 1 shows an alignment of the amino acid sequence of GRG33 (SEQ ID NO:2) and GRG35 (SEQ ID NO:5) with EPSP synthase sequences from Streptomyces cooelicolor (SEQ ID NO:24), Streptomyces avermitilis (SEQ ID NO:25), Zea mays (SEQ ID NO:38), and E. coli (SEQ ID NO:37). The alignment shows the most highly conserved amino acid residues highlighted in black and highly conserved amino acid residues highlighted in gray.

[0013] FIG. 2 shows an alignment of the amino acid sequence of GRG36 (SEQ ID NO:8) with EPSP synthase sequences from Bacillus halodurans (SEQ ID NO:26), Bacillus claussi (SEQ ID NO:27), Zea mays (SEQ ID NO:38), and E. coli (SEQ ID NO:37). The alignment shows the most highly conserved amino acid residues highlighted in black and highly conserved amino acid residues highlighted in gray.

[0014] FIG. 3 shows an alignment of GRG38 (SEQ ID NO:16) and GRG50 (SEQ ID NO:22) with other EPSP synthase enzymes, including GRG8 (SEQ ID NO:29), GRG12 (SEQ ID NO:30), GRG15 (SEQ ID NO:31), GRG5 (SEQ ID NO:32), GRG6 (SEQ ID NO:33), GRG7 (SEQ ID NO:34), GRG9 (SEQ ID NO:35), GRG1 (SEQ ID NO:41), the EPSP synthase described in International Patent Application No. WO2005014820 (SEQ ID NO:36), and EPSP synthase enzymes from E. coli (SEQ ID NO:37), Zea mays (SEQ ID NO:38), Agrobacterium tumefaciens (SEQ ID NO:39), and Bacillus subtilis (SEQ ID NO:40). The alignment shows the most highly conserved amino acid residues highlighted in black, and highly conserved amino acid residues highlighted in gray.

DETAILED DESCRIPTION

[0015] The present invention is drawn to compositions and methods for regulating herbicide resistance in organisms, particularly in plants or plant cells. The methods involve transforming organisms with a nucleotide sequence encoding a glyphosate resistance gene of the invention. In particular, a nucleotide sequence of the invention is useful for preparing plants that show increased tolerance to the herbicide glyphosate. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided.

[0016] Compositions include nucleic acids and proteins relating to herbicide tolerance in microorganisms and plants as well as transformed bacteria, plants, plant tissues and seeds. More particularly, nucleotide sequences of the glyphosate resistance genes (grg33, syngrg33, grg35, syngrg35, grg36, syngrg36, grg37, syngrg37, grg38, syngrg38, grg39, syngrg39, grg50, syngrg50) and the amino acid sequences of the proteins encoded thereby are disclosed. The sequences find use in the construction of expression vectors for subsequent transformation into plants of interest, as probes for the isolation of other glyphosate resistance genes, as selectable markers, and the like. Thus, by "glyphosate resistance gene of the invention" is intended the nucleotide sequence set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, and fragments and variants thereof that encode a glyphosate resistance or tolerance polypeptide. Likewise, a "glyphosate resistance polypeptide of the invention" is a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22, and fragments and variants thereof that confer glyphosate resistance or tolerance to a host cell.

[0017] Plasmids containing the herbicide resistance nucleotide sequences of the invention were deposited in the permanent collection of the Agricultural Research Service Culture Collection, Northern Regional Research Laboratory (NRRL), 1815 North University Street, Peoria, Ill. 61604, United States of America, on Jun. 9, 2006, and assigned Accession Nos. NRRL B-30932 (for grg33), NRRL B-30933 (for grg35), and NRRL B-30934 (for grg36); and on Jun. 26, 2006, and assigned Accession Nos. NRRL B-30945 (for grg37), NRRL B-30946 (for grg38), NRRL B-30947 (for grg39), and NRRL B-30948 (for grg50). This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Access to these deposits will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicants will make available to the public, pursuant to 37 C.F.R. .sctn.1.808, sample(s) of the deposit with the ATCC. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. .sctn.112.

[0018] By "glyphosate" is intended any herbicidal form of N-phosphonomethylglycine (including any salt thereof) and other forms that result in the production of the glyphosate anion in planta. An "herbicide resistance protein" or a protein resulting from expression of an "herbicide resistance-encoding nucleic acid molecule" includes proteins that confer upon a cell the ability to tolerate a higher concentration of an herbicide than cells that do not express the protein, or to tolerate a certain concentration of an herbicide for a longer period of time than cells that do not express the protein. A "glyphosate resistance protein" includes a protein that confers upon a cell the ability to tolerate a higher concentration of glyphosate than cells that do not express the protein, or to tolerate a certain concentration of glyphosate for a longer period of time than cells that do not express the protein. By "tolerate" or "tolerance" is intended either to survive, or to carry out essential cellular functions such as protein synthesis and respiration in a manner that is not readily discernable from untreated cells.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

[0019] One aspect of the invention pertains to isolated or recombinant nucleic acid molecules comprising nucleotide sequences encoding herbicide resistance proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify herbicide resistance-encoding nucleic acids. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA, recombinant DNA, or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecules can be single-stranded or double-stranded, but preferably are double-stranded DNA.

[0020] Nucleotide sequences encoding the proteins of the present invention include the sequences set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, the herbicide resistance nucleotide sequence deposited in a bacterial host as Accession Nos. NRRL B-30932, B-30933, B-30934, B-30945, B-30946, B-30947, or B-30948, and variants, fragments, and complements thereof. By "complement" is intended a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex. In some embodiments, the complement hybridizes across the full length of the sequence of the invention. In another embodiment, the complement hybridizes across at least about 50% of the sequence of the invention, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of a sequence of the invention. The corresponding amino acid sequences for the herbicide resistance proteins encoded by these nucleotide sequences are set forth in SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22. The invention also encompasses nucleic acid molecules comprising nucleotide sequences encoding partial-length herbicide resistance proteins, and complements thereof.

[0021] In some embodiments, the polynucleotides of the present invention encode polypeptides that are Class III EPSP synthase enzymes. For the purposes of the present invention, a "Class III EPSP synthase enzyme" is an herbicide tolerant or herbicide resistant polypeptide containing one or more of the amino acid sequence domains described in U.S. patent application Ser. No. 11/400,598, which is herein incorporated by reference in its entirety.

[0022] An "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the invention, "isolated" when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the isolated glyphosate resistance-encoding nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flanks the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. An herbicide resistance protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-herbicide resistance protein (also referred to herein as a "contaminating protein").

[0023] Nucleic acid molecules that are fragments of these herbicide resistance-encoding nucleotide sequences are also encompassed by the present invention. By "fragment" is intended a portion of a nucleotide sequence encoding an herbicide resistance protein. A fragment of a nucleotide sequence may encode a biologically active portion of an herbicide resistance protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of an herbicide resistance nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 contiguous nucleotides, or up to the number of nucleotides present in a full-length herbicide resistance-encoding nucleotide sequence disclosed herein (for example, 1329 nucleotides for SEQ ID NO:1; 1353 nucleotides for SEQ ID NO:4; 1344 nucleotides for SEQ ID NO:7, etc) depending upon the intended use. By "contiguous" nucleotides is intended nucleotide residues that are immediately adjacent to one another.

[0024] Fragments of the nucleotide sequences of the present invention generally will encode protein fragments that retain the biological activity of the full-length glyphosate resistance protein; i.e., herbicide-resistance activity. By "retains herbicide resistance activity" is intended that the fragment will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the herbicide resistance activity of the full-length glyphosate resistance protein disclosed herein as SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.

[0025] A fragment of an herbicide resistance-encoding nucleotide sequence that encodes a biologically active portion of a protein of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400 contiguous amino acids, or up to the total number of amino acids present in a full-length herbicide resistance protein of the invention (for example, 442 amino acids for SEQ ID NO:2; 450 for SEQ ID NO:5; 447 amino acids for SEQ ID NO:8, etc).

[0026] Preferred herbicide resistance proteins of the present invention are encoded by a nucleotide sequence sufficiently identical to the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23. The term "sufficiently identical" is intended an amino acid or nucleotide sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0027] To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions).times.100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

[0028] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to glyphosate-resistant nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to herbicide resistance protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. See www.ncbi.nlm.nih.gov. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOC.TM.. GENEDOC.TM. (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package (available from Accelrys, Inc., 9865 Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0029] Unless otherwise stated, GAP Version 10, which uses the algorithm of Needleman and Wunsch (1970) supra, will be used to determine sequence identity or similarity using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0030] The invention also encompasses variant nucleic acid molecules. "Variants" of the herbicide resistance-encoding nucleotide sequences include those sequences that encode an herbicide resistance protein disclosed herein but that differ conservatively because of the degeneracy of the genetic code, as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the herbicide resistance proteins disclosed in the present invention as discussed below. Variant proteins encompassed by the present invention are biologically active, that is they retain the desired biological activity of the native protein, that is, herbicide resistance activity. By "retains herbicide resistance activity" is intended that the variant will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the herbicide resistance activity of the native protein. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.

[0031] The skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded herbicide resistance protein, without altering the biological activity of the protein. Thus, variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.

[0032] For example, conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of an herbicide resistance protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in the alignment of FIG. 1, 2, or 3. Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in the alignment of FIG. 1, 2, or 3.

[0033] Lys-22, Arg-124, Asp-313, Arg-344, Arg-386, and Lys-411, are conserved residues of the EPSP synthase from E. coli (Schonbrunn et al. (2001) Proc. Natl. Acad. Sci. USA 98:1376-1380). Conserved residues important for EPSP synthase activity also include Arg-100, Asp-242, and Asp-384 (Selvapandiyan et al. (1995) FEBS Letters 374:253-256). Arg-27 binds to S3P (Shuttleworth et al. (1999) Biochemistry 38:296-302).

[0034] Alternatively, variant nucleotide sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability to confer herbicide resistance activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.

[0035] Using methods such as PCR, hybridization, and the like, corresponding herbicide resistance sequences can be identified, such sequences having substantial identity to the sequences of the invention. See, for example, Sambrook J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).

[0036] In a hybridization method, all or part of the herbicide resistance nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as .sup.32P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known herbicide resistance-encoding nucleotide sequences disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleotide sequences or encoded amino acid sequences can additionally be used. The probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, at least about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300 consecutive nucleotides of an herbicide resistance-encoding nucleotide sequence of the invention or a fragment or variant thereof. Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), both of which are herein incorporated by reference.

[0037] For example, an entire herbicide resistance sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding herbicide resistance sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and are at least about 10 nucleotides in length, or at least about 20 nucleotides in length. Such probes may be used to amplify corresponding herbicide resistance sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0038] Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

[0039] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. Unless otherwise specified, hybridization conditions are under high stringency.

[0040] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with .gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

[0041] Herbicide resistance proteins are also encompassed within the present invention. By "herbicide resistance protein" is intended a protein having the amino acid sequence set forth in SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22. Fragments, biologically active portions, and variants thereof are also provided, and may be used to practice the methods of the present invention.

[0042] "Fragments" or "biologically active portions" include polypeptide fragments comprising a portion of an amino acid sequence encoding an herbicide resistance protein as set forth SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22, and that retains herbicide resistance activity. A biologically active portion of an herbicide resistance protein can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for herbicide resistance activity. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety. As used here, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22. The invention encompasses other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0043] By "variants" is intended proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, 80%, 85%, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2, 5, 8, 11, 14, 16, 19, or 22. Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, or a complement thereof, under stringent conditions. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining herbicide resistance activity. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.

[0044] Bacterial genes, such as the grg and syngrg genes of this invention, quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. Furthermore, it is not often determined a priori which of these codons are used naturally in the bacterium. Thus, it is understood that use of one of the alternate methionine codons may lead to generation of variants of grg and syngrg that confer herbicide resistance. These herbicide resistance proteins are encompassed in the present invention and may be used in the methods of the present invention.

[0045] Antibodies to the polypeptides of the present invention, or to variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

[0046] It is recognized that the DNA sequences of the grg or syngrg genes of the invention may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by the grg or syngrg sequences disclosed herein. This protein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 13, 15, 17, 18, 20, 21, or 23, including up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100 or more amino acid substitutions, deletions or insertions.

[0047] Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the GRG proteins disclosed herein can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect function of the protein. Such variants will possess the desired herbicide resistance activity. However, it is understood that the ability of the GRG proteins disclosed herein to confer herbicide resistance may be improved by one use of such techniques upon the compositions of this invention. For example, one may express the grg or syngrg sequences disclosed herein in host cells that exhibit high rates of base misincorporation during DNA replication, such as XL-1 Red (Stratagene, La Jolla, Calif.). After propagation in such strains, one can isolate the DNA of the invention (for example by preparing plasmid DNA, or by amplifying by PCR and cloning the resulting PCR fragment into a vector), culture the grg mutations in a non-mutagenic strain, and identify mutated genes with improved resistance to an herbicide such as glyphosate, for example by growing cells in increasing concentrations of glyphosate and testing for clones that confer ability to tolerate increased concentrations of glyphosate.

[0048] Alternatively, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression of a protein of interest, (2) introduce a binding domain, enzymatic activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art, or, (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.

[0049] Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different herbicide resistance protein coding regions can be used to create a new herbicide resistance protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the herbicide resistance gene of the invention and other known herbicide resistance genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased glyphosate resistance activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Transformation of Bacterial or Plant Cells

[0050] Provided herein are novel isolated genes that confer resistance to an herbicide. Also provided are amino acid sequences of the GRG proteins of the invention. The protein resulting from translation of this gene allows cells to function in the presence of concentrations of an herbicide that are otherwise toxic to cells including plant cells and bacterial cells. In one aspect of the invention, the grg or syngrg genes are useful as markers to assess transformation of bacterial or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny of the same are well known in the art.

[0051] By engineering the genes of the invention to be expressed from a promoter known to stimulate transcription in the organism to be tested and properly translated to generate an intact GRG peptide, and placing the cells in an otherwise toxic concentration of herbicide, one can identify cells that have been transformed with the DNA by virtue of their resistance to herbicide. By "promoter" is intended a nucleic acid sequence that functions to direct transcription of a downstream coding sequence. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences, (also termed as "control sequences") are necessary for the expression of a DNA sequence of interest.

[0052] Transformation of bacterial cells is accomplished by one of several techniques known in the art, including but not limited to electroporation or chemical transformation (see, for example, Ausubel, ed. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Indianapolis, Ind.). Markers conferring resistance to toxic substances are useful in identifying transformed cells (having taken up and expressed the test DNA) from non-transformed cells (those not containing or not expressing the test DNA). In one aspect of the invention, the grg or syngrg genes disclosed herein are useful as markers to assess transformation of bacterial or plant cells.

[0053] Transformation of plant cells can be accomplished in similar fashion. By "plant" is intended whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues refer to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. By "stable transformation" is intended that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof.

[0054] The grg genes of the invention may be modified to obtain or enhance expression in plant cells. The herbicide resistance sequences of the invention may be provided in expression cassettes for expression in the plant of interest. "Plant expression cassette" includes DNA constructs, including recombinant DNA constructs, that are capable of resulting in the expression of a protein from an open reading frame in a plant cell. The cassette will include in the 5'-3' direction of transcription, a transcriptional initiation region (i.e., promoter, particularly a heterologous promoter) operably-linked to a DNA sequence of the invention, and/or a transcriptional and translational termination region (i.e., termination region) functional in plants. The cassette may additionally contain at least one additional gene to be cotransformed into the organism, such as a selectable marker gene. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites for insertion of the herbicide resistance sequence to be under the transcriptional regulation of the regulatory regions.

[0055] The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "native" or "homologous" to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is "foreign" or "heterologous" to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention. "Heterologous" generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like. By "operably linked" is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

[0056] Often, such constructs will also contain 5' and 3' untranslated regions. Such constructs may contain a "signal sequence" or "leader sequence" to facilitate co-translational or post-translational transport of the peptide of interest to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted. For example, the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. By "signal sequence" is intended a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. By "leader sequence" is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. It may also be preferable to engineer the plant expression cassette to contain an intron, such that mRNA processing of the intron is required for expression.

[0057] By "3' untranslated region" is intended a nucleotide sequence located downstream of a coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor are 3' untranslated regions. By "5' untranslated region" is intended a nucleotide sequence located upstream of a coding sequence.

[0058] Other upstream or downstream untranslated elements include enhancers. Enhancers are nucleotide sequences that act to increase the expression of a promoter region. Enhancers are well known in the art and include, but are not limited to, the SV40 enhancer region and the 35S enhancer element.

[0059] The termination region may be native with the transcriptional initiation region, may be native with the herbicide resistance sequence of the present invention, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0060] In one aspect of the invention, synthetic DNA sequences are designed for a given polypeptide, such as the polypeptides of the invention. Expression of the open reading frame of the synthetic DNA sequence in a cell results in production of the polypeptide of the invention. Synthetic DNA sequences can be useful to simply remove unwanted restriction endonuclease sites, to facilitate DNA cloning strategies, to alter or remove any potential codon bias, to alter or improve GC content, to remove or alter alternate reading frames, and/or to alter or remove intron/exon splice recognition sites, polyadenylation sites, Shine-Delgarno sequences, unwanted promoter elements and the like that may be present in a native DNA sequence. It is also possible that synthetic DNA sequences may be utilized to introduce other improvements to a DNA sequence, such as introduction of an intron sequence, creation of a DNA sequence that in expressed as a protein fusion to organelle targeting sequences, such as chloroplast transit peptides, apoplast/vacuolar targeting peptides, or peptide sequences that result in retention of the resulting peptide in the endoplasmic reticulum. Synthetic genes can also be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11; U.S. Pat. Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. Published Application Nos. 20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0061] In one embodiment, the nucleic acids of interest are targeted to the chloroplast for expression. In this manner, where the nucleic acid of interest is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

[0062] The nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.

[0063] Typically this "plant expression cassette" will be inserted into a "plant transformation vector." By "transformation vector" is intended a DNA molecule that is necessary for efficient transformation of a cell. Such a molecule may consist of one or more expression cassettes, and may be organized into more than one "vector" DNA molecule. For example, binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). "Vector" refers to a nucleic acid construct designed for transfer between different host cells. "Expression vector" refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell.

[0064] This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as "binary vectors." Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a "gene of interest" (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the gene of interest are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science, 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

Plant Transformation

[0065] Methods of the invention involve introducing a nucleotide construct into a plant. By "introducing" is intended to present to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant is used, only that the nucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0066] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene and in this case "glyphosate") to recover the transformed plant cells from a group of untransformed cell mass. Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent (e.g. "glyphosate"). The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grow into mature plant and produce fertile seeds (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Since the transformed material contains many cells, both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest in the genome of transgenic plant.

[0067] Generation of transgenic plants may be performed by one of several methods, including but not limited to introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, and various other non-particle direct-mediated methods (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750; Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239; Bommineni and Jauhar (1997) Maydica 42:107-120) to transfer DNA.

[0068] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0069] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

Plants

[0070] The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.

[0071] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

[0072] This invention is particularly suitable for any member of the monocot plant family including, but not limited to, maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut, and dates.

Evaluation of Plant Transformation

[0073] Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

[0074] PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.

[0075] Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, radiolabeled .sup.32P target DNA fragments to confirm the integration of the introduced gene in the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).

[0076] In Northern analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the genes disclosed herein is then tested by hybridizing the filter to a radioactive probe derived from a polynucleotide of the invention, by methods known in the art (Sambrook and Russell, 2001, supra)

[0077] Western blot and biochemical assays and the like may be carried out on the transgenic plants to determine the presence of protein encoded by the herbicide resistance gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the herbicide resistance protein.

Methods for Increasing Plant Yield

[0078] Methods for increasing plant yield are provided. The methods comprise introducing into a plant or plant cell a polynucleotide comprising a grg sequence disclosed herein. As defined herein, the "yield" of the plant refers to the quality and/or quantity of biomass produced by the plant. By "biomass" is intended any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase.

[0079] In specific methods, the plant is treated with an effective concentration of an herbicide, where the herbicide application results in enhanced plant yield. By "effective concentration" is intended the concentration which allows the increased yield in the plant. Such effective concentrations for herbicides of interest are generally known in the art. The herbicide may be applied either pre- or post emergence in accordance with usual techniques for herbicide application to fields comprising crops which have been rendered resistant to the herbicide by heterologous expression of a grg gene of the invention.

[0080] Methods for conferring herbicide resistance in a plant or plant part are also provided. In such methods, a grg polynucleotide disclosed herein is introduced into the plant, wherein expression of the polynucleotide results in glyphosate tolerance or resistance. Plants produced via this method can be treated with an effective concentration of an herbicide and display an increased tolerance to the herbicide. An "effective concentration" of an herbicide in this application is an amount sufficient to slow or stop the growth of plants or plant parts that are not naturally resistant or rendered resistant to the herbicide.

[0081] In another embodiment, methods for conferring herbicide resistance in a plant or plant part are provided, wherein the plant or plant part is grown under higher or lower than ambient environmental temperatures as described supra. Glyphosate tolerant EPSP synthase enzymes having thermal stability at higher or lower temperatures, or have temperature optima at higher or lower temperatures, are useful for conferring glyphosate tolerance in plants that are grown under such conditions.

Methods of Controlling Weeds in a Field

[0082] Methods for selectively controlling weeds in a field containing a plant are also provided. In one embodiment, the plant seeds or plants are glyphosate resistant as a result of a grg polynucleotide disclosed herein being inserted into the plant seed or plant. In specific methods, the plant is treated with an effective concentration of an herbicide, where the herbicide application results in a selective control of weeds or other untransformed plants. By "effective concentration" is intended the concentration which controls the growth or spread of weeds or other untransformed plants without significantly affecting the glyphosate-resistant plant or plant seed. Such effective concentrations for herbicides of interest are generally known in the art. The herbicide may be applied either pre- or post emergence in accordance with usual techniques for herbicide application to fields comprising plants or plant seeds which have been rendered resistant to the herbicide.

[0083] The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1

Isolation of Glyphosate Resistant EPSP Synthases

[0084] Strains capable of growth in presence of glyphosate were isolated by plating samples of soil on HEPES Mineral Salts Medium (HMSM) containing glyphosate as the sole source of phosphorus. Since HMSM contains no aromatic amino acids, a strain must be resistant to glyphosate in order to grow on this media.

[0085] Two grams of soil were suspended in approximately 10 ml of water, vortexed for 15 seconds and permitted to settle for 15 minutes. A 10 .mu.l loopful of this suspension was added to 3 ml of HMSM supplemented with 10 mM glyphosate (pH 7.0). HMSM contains (per liter): 10 g glucose, 2 g NH.sub.4SO.sub.4, 9.53 g HEPES, 1.0 ml 0.8 M MgSO.sub.4, 1.0 ml 0.1 M CaCl.sub.2, 1.0 ml Trace Elements Solution (In 100 ml of 1000.times. solution: 0.1 g FeSO.sub.4.7H.sub.2O, 0.5 mg CuSO.sub.4.5H.sub.2O, 1.0 mg H.sub.3BO.sub.3, 1.0 mg MnSO.sub.4.5H.sub.2O, 7.0 mg ZnSO.sub.4.7H.sub.2O, 1.0 mg MoO.sub.3, 4.0 g KCl). The culture was grown in a shaker incubator for four days at 28.degree. C. and then 20 .mu.l was used to inoculate 2.5 ml of fresh HMSM containing 10 mM glyphosate as the only phosphorus source. After two days, 20 .mu.l was used to inoculate another fresh 2.5 ml culture. After 5 days, 20 .mu.l was used to inoculate a fresh 2.5 ml culture. After sufficient growth, the culture was plated onto solid media by streaking a 1 .mu.l loop onto the surface of agar plate containing HMSM agar containing 100 mM glyphosate as the sole phosphorus source and stored at 28.degree. C. The culture was then replated for isolation. The strains listed in Table 1 were among the strains selected due to their ability to grow in the presence of high glyphosate concentrations.

TABLE-US-00001 TABLE 1 EPSP synthase Strain Name Strain ID Gene Name ATX21561 Unknown grg33 ATX21563 Unknown grg35 ATX21567 Unknown grg36

Example 2

Isolation of Glyphosate Resistant EPSP Synthases grg37 and grg39

[0086] Strains capable of growth in presence of glyphosate were isolated by plating samples of soil on various growth media containing glyphosate. Some strains were isolated on mineral salts media supplemented with glyphosate. Other strains were isolated under rich media in the presence of glyphosate and later tested on mineral salts media supplemented with glyphosate. Since the mineral salts media contain no aromatic amino acids, a strain must be resistant to glyphosate in order to grow on this media.

[0087] Strains ATX21800 and ATX21804 were isolated by incubation under rich conditions and supplemention with glyphosate. These strains were then tested for their ability to grow in the presence of glyphosate without aromatic amino acids. Strain ATX21804 was isolated from soil (0.01 grams) that was air dried for two days and plated onto nutrient broth agar supplemented with 100 mM glyphosate. A small amount (10 .mu.l) was then used to inoculate an eosin methylene blue agar plate containing 300 mM glyphosate. ATX21800 was isolated by incubating 0.01 grams soil with 3 ml nutrient broth supplemented with 100 mM glyphosate. After initial isolation, each strain was inoculated into Luria Bertani agar plates to confirm single colony type. These strains were then tested on Brunner minimal medium containing 100 mM glyphosate and were confirmed to grow in the presence of glyphosate without aromatic amino acids.

[0088] The strains listed in Table 2 were among the strains selected due to their ability to grow in the presence of high glyphosate concentrations.

TABLE-US-00002 TABLE 2 Strain EPSP synthase Gene Name Strain ID Name ATX21800 Unknown grg37 ATX21804 Unknown grg39

Example 3

Isolation of Glyphosate Resistant EPSP Synthases grg38 and grg50

[0089] Strains capable of growth in presence of glyphosate were isolated by plating samples of soil on various growth media containing glyphosate. Some strains were isolated on mineral salts media supplemented with glyphosate. Other strains were isolated under rich media in the presence of glyphosate and later tested on mineral salts media supplemented with glyphosate. Since the mineral salts media contain no aromatic amino acids, a strain must be resistant to glyphosate in order to grow on this media.

[0090] Strain ATX20103 was isolated by suspending approximately 2 grams of soil in 10 ml of water, vortexing for 15 seconds and permitting to settle for 15 minutes. A 10 .mu.l loopful of this suspension was added to 3 ml of Tris MSM (TMSM) supplemented with 10 mM glyphosate (pH 7.0). TMSM contains (per liter): 10 g glucose, 2 g NH.sub.4SO.sub.4, 12.12 g Tris, 1.0 ml 0.8 M MgSO.sub.4, 1.0 ml 0.1 M CaCl.sub.2, 1.0 ml Trace Elements Solution (In 100 ml of 1000.times. solution: 0.1 g FeSO.sub.4.7H.sub.2O, 0.5 mg CuSO.sub.4.5H.sub.2O, 1.0 mg H.sub.3BO.sub.3, 1.0 mg MnSO.sub.4.5H.sub.2O, 7.0 mg ZnSO.sub.4.7H.sub.2O, 1.0 mg MoO.sub.3, 4.0 g KCl). The culture was then incubated at 28.degree. C. for isolation over repeated rounds of selection and then inoculated onto Luria Bertani agar to confirm single colony type. ATX20103 was then reconfirmed to grow on TMSM in the presence of glyphosate without aromatic acids.

[0091] Strain ATX21806 was isolated by incubating under rich conditions and supplementing with glyphosate. This strain was then tested for its ability to growth in the presence of glyphosate without aromatic amino acids. Strain ATX21806 was isolated from soil (0.01 grams) that had been suspended in 10 ml water overnight. A small amount (10 .mu.l) was then used to inoculate an eosin methylene blue agar plate containing 300 mM glyphosate. After each initial isolation, the strain was inoculated into Luria Bertani agar plates to confirm single colony type. The strain was then tested on Brunner minimal medium at 100 mM glyphosate and was confirmed to grow in the presence of glyphosate without aromatic amino acids.

[0092] The strains listed in Table 3 were among the strains selected due to their ability to grow in the presence of high glyphosate concentrations.

TABLE-US-00003 TABLE 3 Strain EPSP synthase Gene Name Strain ID Name ATX21806 Unknown grg38 ATX20103 Rhizobium leguminosarum grg50

Example 4

Cloning of Glyphosate-Resistant EPSP Synthases

[0093] Genomic DNA was extracted from the strains described in Tables 1, 2, and 3, and the resulting DNA was partially digested with restriction enzyme Sau3A 1 to yield DNA fragments approximately 5 kilobases in size. These DNA molecules were size selected on agarose gels, purified, and ligated into LAMBDA ZAP.RTM. vector arms pre-digested with BamH I. The ligated arms were then packaged into phage particles, and phage titers determined as known in the art. The resulting libraries were amplified by methods known in the art to generate a library titer of between 3.times.10.sup.7 and 3.times.10.sup.8 PFU/mL. For each independent library, E. coli (XL1 Blue MRF') was then co-transfected with phage from an amplified library as well as M13 helper phage to allow mass excision of the library in the form of an infectious, circular ssDNA as known in the art (Short et al. (1988) Nucleic Acids Research 16:7583-7600). After centrifugation of the co-infected cells, the phage-containing supernatant was heated to 65-70.degree. C. for 15-20 minutes to incapacitate any residual lambda phage particles. Dilutions of the resulting ssDNA plasmid library were transfected into a fresh culture of competent E. coli XL-Blue MRF' (aroA) cells (XL1 Blue MRF'). The resulting transfected cells were plated onto M63 plates containing kanamycin, 0.1 mM IPTG and either 0 mM, 20 mM or 50 mM glyphosate.

[0094] The E. coli XL-Blue MRF'(aroA) used for the transfection expresses the F-pilus, and also contains a deletion of the aroA gene encoding the endogenous E. coli EPSP synthase enzyme. This strain is also referred to as herein as .DELTA.aroA. This .DELTA.aroA strain is unable to grow on minimal media lacking aromatic amino acids, unless complemented by a functional EPSP synthase. Since glyphosate is a potent inhibitor of typical, glyphosate-sensitive EPSP synthases, such as type I EPSP synthases, transfected clones expressing a non-glyphosate resistant EPSP synthase would be able to grown on M63 plates lacking glyphosate, but would be unable to grow on M63 containing either 20 mM or 50 mM glyphosate. In order to grow on M63 plates containing 20 mM or 50 mM glyphosate, the cells must contain a plasmid that expresses an EPSP synthase that is both (1) capable of complementing the .DELTA.aroA mutation of these cells, and (2) resistant to glyphosate. Thus, this screening method allows identification of clones containing glyphosate-resistant EPSP synthases.

[0095] Colonies growing on 20 mM or 50 mM glyphosate were picked and their plasmids analyzed by restriction digest to identify plasmids with shared restriction patterns. Individual plasmids were sequenced by methods known in the art.

[0096] Using this approach, as sometimes modified for each library as known and appreciated in the art, library clones containing EPSP synthase genes were identified for each of the strains listed in Table 4.

Example 5

DNA and Protein Sequences of EPSP Synthases

[0097] The DNA sequences of the glyphosate-resistant EPSP synthases was determined for each of the clones described above by methods well known in the art.

[0098] grg33. The DNA sequence of grg33 is provided herein as SEQ ID NO:1. The predicted translation product of grg33 (GRG33) is provided herein as SEQ ID NO:2. A synthetic sequence encoding GRG33 (syngrg33) was also designed and is provided herein as SEQ ID NO:3.

[0099] grg35. The DNA sequence of grg35 is provided herein as SEQ ID NO:4. The predicted translation product of grg35 (GRG35) is provided herein as SEQ ID NO:5. A synthetic sequence encoding GRG35 (syngrg35) was also designed and is provided herein as SEQ ID NO:6.

[0100] grg36. The DNA sequence of grg36 is provided herein as SEQ ID NO:7. The predicted translation product of grg36 (GRG36) is provided herein as SEQ ID NO:8. A synthetic sequence encoding GRG36 (syngrg36) was also designed and is provided herein as SEQ ID NO:9.

[0101] grg37. The DNA sequence of grg37 is provided herein as SEQ ID NO:10. The predicted translation product of grg37 (GRG37) is provided herein as SEQ ID NO:11. A synthetic sequence encoding GRG37 (syngrg37) was also designed and is provided herein as SEQ ID NO:12.

[0102] grg38. The DNA sequence of grg38 is provided herein as SEQ ID NO:15. The predicted translation product of grg38 (GRG38) is provided herein as SEQ ID NO:16. A synthetic sequence encoding GRG38 (syngrg38) was also designed and is provided herein as SEQ ID NO:17.

[0103] grg39. The DNA sequence of grg39 is provided herein as SEQ ID NO:18. The predicted translation product of grg39 (GRG39) is provided herein as SEQ ID NO:19. A synthetic sequence encoding GRG39 (syngrg39) was also designed and is provided herein as SEQ ID NO:20.

[0104] grg50. The DNA sequence of grg50 is provided herein as SEQ ID NO:21. The predicted translation product of grg50 (GRG50) is provided herein as SEQ ID NO:22. A synthetic sequence encoding GRG50 (syngrg50) was also designed and is provided herein as SEQ ID NO:23.

Clones containing each of the grg33, grg35, grg36, grg37, grg38, grg39, and grg50 EPSP synthase genes were deposited at NRRL on Jun. 9, 2006 or Jun. 26, 2007 and assigned deposit numbers as in Table 4.

TABLE-US-00004 TABLE 4 Clones containing glyphosate-resistant EPSP synthases Strain yielding Original Isolate NRRL EPSPS EPSPS in pBKCMV Number GRG33 ATX21561 pAX1947 B-30932 GRG35 ATX21563 pAX1948 B-30933 GRG36 ATX21567 pAX1949 B-30934 GRG37 ATX21800 pAX1963 B-30945 GRG38 ATX21806 pAX1964 B-30946 GRG39 ATX21804 pAX1965 B-30947 GRG50 ATX20103 pAX1966 B-30948

Each of the proteins GRG33, GRG35, and GRG36 showed regions of homology to EPSP synthase enzymes in the NCBI database by BLAST search. The EPSPS enzyme with the highest protein sequence identity to each GRG enzyme is listed in Table 5.

TABLE-US-00005 TABLE 5 Homology of GRG33-GRG36 to known EPSP synthases Strain with homologous EPSPS Protein enzyme % Identity GRG33 GRG35, S. coelicolor 88%, 86% GRG35 GRG33, S. coelicolor 88%, 85% GRG36 Bacillus halodurans 53%

[0105] The amino acid sequences of GRG33 and GRG35 are 88% identical. A search of public protein databases with the amino acid sequence of GRG33 shows that this protein is 86% identical over 430 amino acids to the EPSP synthase from Streptomyces coelicolor (SEQ ID NO:24 GENBANK.RTM. Accession No. NP 629359.1), and 82% identical over 430 amino acids to the EPSP synthase from Streptomyces avermitilis (SEQ ID NO:25; GENBANK.RTM. Accession No. NP824218.1).

[0106] The amino acid sequence of GRG35 similarly is 85% identical over 434 amino acids to the EPSP synthase from Streptomyces coelicolor (SEQ ID NO:24; GENBANK.RTM.Accession No. NP 629359.1), and 81% identical over 441 amino acids to the EPSP synthase from Streptomyces avermitilis (SEQ ID NO:25; GENBANK.RTM. Accession No. NP 824218.1).

TABLE-US-00006 TABLE 6 Amino acid identity of GRG33 and GRG35 with Streptomyces EPSP synthases Identity with Identity with EPSP synthase GRG33 GRG35 GRG33 -- 88% GRG35 88% -- Streptomyces coelicolor 84% 82% A3(2) Streptomyces avermitilis MA- 80% 78% 4680 E. coli 30% 29% Maize 30% 29%

[0107] A search of public protein databases with the amino acid sequence of GRG36 shows that this protein is related to the EPSP synthase from Bacillus halodurans, (64% identical over 441 amino acids, SEQ ID NO:26; GENBANK.RTM. Accession No. BAB06432.1), and to a lesser extent to the EPSP synthase from Bacillus clausii (55% identical over 439 amino acids; SEQ ID NO:27; GENBANK.RTM. Accession No. BAD63759.1)

TABLE-US-00007 TABLE 7 Amino acid identity of GRG36 with EPSP synthases Identity with EPSP synthase GRG36 Bacillus halodurans 62% Bacillus clausii 54% E. coli 31% Maize 34%

[0108] A search of public protein databases with the amino acid sequence of GRG37 shows that this protein is 81% identical to the EPSP synthase from Arthrobacter sp. FB24 (SEQ ID NO:28, GENBANK.RTM. Accession No. ZP.sub.--00413033.1)

[0109] The grg37 open reading frame has two potential start codons. The upstream ATG (predicted amino acid sequence MTASPMGASADNS (corresponding to amino acid positions 1 through 13 of SEQ ID NO:10)) contains the best ribosome binding site in correct proximity. However, a second downstream ATG may be used. This ORF yields the predicted amino acid sequence MGASADNS . . . (corresponding to amino acid positions 6 through 13 of SEQ ID NO:10)). The upstream ATG appears to have a ribosome binding site ("RBS") that is a better match to the consensus RBS sequence. However, the open reading frame originating from this upstream ATG appears to be translationally coupled to an upstream open reading frame. Translational coupling is one strategy known in the art to be employed by bacteria to ensure good initiation and can substitute for a ribosome binding site. The nucleotide sequence for the downstream start site is provided herein as SEQ ID NO:13, and the encoded amino acid sequence is provided herein as SEQ ID NO:14.

[0110] GRG39 shows 96% amino acid identity to the GRG30 EPSP synthase sequence, and is highly homologous to the GRG29 EPSP synthase sequence described in U.S. patent application Ser. No. 11/760,570 filed Jun. 8, 2007.

[0111] GRG38 shows 94% amino acid identity to the GRG12 EPSP synthase described in U.S. patent application Ser. No. 11/400,598, filed Apr. 7, 2006 (Table 8). GRG38 also contains the domains of the Class III EPSP synthases described in U.S. patent application Ser. No. 11/400,598.

[0112] GRG50 shows 95% amino acid identity to the GRG8 EPSP synthase described in U.S. patent application Ser. No. 11/315,678 filed Dec. 22, 2005 (Table 8). GRG50 also contains the domains of the Class III EPSP synthases described in U.S. application Ser. No. 11/400,598, filed Apr. 7, 2006.

TABLE-US-00008 TABLE 8 Comparison with other Class III EPSP synthases Amino acid Amino acid identity with identity with EPSPS GRG38 GRG50 GRG38 -- 65% GRG50 65% -- GRG8 65% 95% GRG12 87% 62% GRG6 67% 67% GRG9 64% 70% GRG15 64% 71% GRG5 68% 68% GRG37 67% 68% E. coli (non-Class III) 32% 34% Maize (non-Class III) 32% 31%

Example 6

Cloning of Novel Glyphosate-Resistant EPSP Synthases into an E. coli Expression Vector

[0113] The EPSP synthase genes contained in the clones of Table 4 were sub-cloned into the E. coli expression vector pRSF1b (Invitrogen). Resulting clones were confirmed by DNA sequencing, and used to induce expression of each EPSP synthase in E. coli. The expressed His-tagged protein was then purified as known in the art.

Example 7

Glyphosate Resistance of GRG33, GRG35, and GRG36 EPSP Synthases

[0114] The pRSF1b clones were plated onto M63+ plates containing antibiotic and either 0 mM or 50 mM glyphosate. Growth was scored after two days growth at 37.degree. C. Each of the three EPSP synthases was observed to confer resistance to 50 mM glyphosate in E. coli cells (Table 9).

TABLE-US-00009 TABLE 9 Glyphosate screen Clone in Growth on 50 mM EPSPS pRSF1B glyphosate Vector -- - GRG33 pAX1951 +++ GRG35 pAX1952 +++ GRG36 pAX1953 +++

Example 8

Glyphosate Resistance of GRG37 and GRG39 EPSP Synthases

[0115] Cells containing the plasmid clones shown in Table 4 were plated onto M63+ plates containing antibiotic and either 0 mM or 20 mM glyphosate. Growth was scored after two days growth at 37.degree. C. Each of the EPSP synthases was observed to confer resistance to 20 mM glyphosate in E. coli cells (Table 10).

TABLE-US-00010 TABLE 10 Glyphosate screen Growth on 20 mM EPSPS Plasmid Clone glyphosate Vector -- - GRG37 pAX1963 ++ GRG39 pAX1965 ++

Example 9

Glyphosate Resistance of GRG38 and GRG50 EPSP Synthases

[0116] Cells containing the plasmid clones shown in Table 4 were plated onto M63+ plates containing antibiotic and either 0 mM or 20 mM glyphosate. Growth was scored after two days growth at 37.degree. C. Each of the EPSP synthases was observed to confer resistance to 20 mM glyphosate in E. coli cells (Table 11).

TABLE-US-00011 TABLE 11 Glyphosate screen Growth on 20 mM EPSPS Plasmid Clone glyphosate Vector -- - GRG38 pAX1964 ++ GRG50 pAX1966 ++

Example 10

Engineering grg33, grg35, grg36, grg37, grg38, grg39, grg50, syngrg33, syngrg35, syngrg36, syngrg37, syngrg38, syngrg39, and syngrg50 for Plant Transformation

[0117] The open reading frame (ORF) for each of the grg genes is amplified by PCR from a full-length cDNA template. Hind III restriction sites are added to each end of the or F during PCR. Additionally, the nucleotide sequence ACC is added immediately 5' to the start codon of the gene to increase translational efficiency (Kozak (1987) Nucleic Acids Research 15:8125-8148; Joshi (1987) Nucleic Acids Research 15:6643-6653). The PCR product is cloned and sequenced, using techniques well known in the art, to ensure that no mutations are introduced during PCR. The plasmid containing the grg PCR product is digested with, for example, Hind III and the fragment containing the intact or F is isolated.

[0118] One may generate similar constructs that contain a chloroplast targeting sequence linked to the polynucleotide of the invention by methods known in the art.

[0119] A DNA fragment containing the EPSP synthase (and either containing or not containing a chloroplast targeting sequence) is cloned into a plasmid, for example at the Hind III site of pAX200. pAX200 is a plant expression vector containing the rice actin promoter (McElroy et al. (1991) Molec. Gen. Genet. 231:150-160), and the PinII terminator (An et al. (1989) The Plant Cell 1:115-122). The promoter--gene--terminator fragment (or the promoter-leader-gene-terminator fragment) from this intermediate plasmid is subcloned into a plasmid such as pSB11 (Japan Tobacco, Inc.) to form a final plasmid, referred to herein as, for example, pSB11GRG33. pSB11GRG33 is organized such that the DNA fragment containing, for example, the promoter--grg36--terminator construct (or the promoter-leader-grg36--terminator construct) may be excised by appropriate restriction enzymes and also used for transformation into plants, for example, by aerosol beam injection. The structure of pSB11GRG33 is verified by restriction digest and gel electrophoresis, as well as by sequencing across the various cloning junctions. The same methods can be used to generate a final plasmid for each of the grg genes described herein.

[0120] The plasmid is mobilized into Agrobacterium tumefaciens strain LBA4404 which also harbors the plasmid pSB1 (Japan Tobacco, Inc.), using triparental mating procedures well known in the art, and plating on media containing antibiotic. Plasmid pSB11GRG36 carries spectinomycin resistance but is a narrow host range plasmid and cannot replicate in Agrobacterium. Antibiotic resistant colonies arise when pSB11GRG36 integrates into the broad host range plasmid pSB1 through homologous recombination. The resulting cointegrate product is verified by Southern hybridization. The Agrobacterium strain harboring the cointegrate can be used to transform maize, for example, by the PureIntro method (Japan Tobacco).

Example 11

Transformation grg33, grg35, grg36, grg37, grg38, grg39, grg50, syngrg33, syngrg35, syngrg36, syngrg37, syngrg38, syngrg39, and syngrg50 into Plant Cells

[0121] Maize ears are best collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are preferred for use in transformation. Embryos are plated scutellum side-up on a suitable incubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of 1000.times. Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and salts other than DN62A5S are suitable and are known in the art. Embryos are incubated overnight at 25.degree. C. in the dark. However, it is not necessary per se to incubate the embryos overnight.

[0122] The resulting explants are transferred to mesh squares (30-40 per plate), transferred onto osmotic media for about 30-45 minutes, then transferred to a beaming plate (see, for example, PCT Publication No. WO/0138514 and U.S. Pat. No. 5,240,842).

[0123] DNA constructs designed to express the GRG proteins of the present invention in plant cells are accelerated into plant tissue using an aerosol beam accelerator, using conditions essentially as described in PCT Publication No. WO/0138514. After beaming, embryos are incubated for about 30 min on osmotic media, and placed onto incubation media overnight at 25.degree. C. in the dark. To avoid unduly damaging beamed explants, they are incubated for at least 24 hours prior to transfer to recovery media. Embryos are then spread onto recovery period media, for about 5 days, 25.degree. C. in the dark, then transferred to a selection media. Explants are incubated in selection media for up to eight weeks, depending on the nature and characteristics of the particular selection utilized. After the selection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of regeneration is initiated by methods known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants.

Materials

TABLE-US-00012 [0124] TABLE 12 DN62A5S Media Components Per Liter Source Chu's N6 Basal 3.98 g/L Phytotechnology Salt Mixture Labs (Prod. No. C 416) Chu's N6 1 mL/L (of 1000x Stock) Phytotechnology Vitamin Solution Labs (Prod. No. C 149) L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L Sigma L-Proline 1.4 g/L Phytotechnology Labs Casamino acids 100 mg/L Fisher Scientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/L (of 1 mg/mL Stock) Sigma D-7299)

[0125] The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite (Sigma) is added at a concentration up to 3 g/L, and the media is autoclaved. After cooling to 50.degree. C., 2 ml/L of a 5 mg/ml stock solution of silver nitrate (Phytotechnology Labs) is added.

Example 12

Transformation of grg33, grg35, grg36, grg37, grg38, grg39, grg50, syngrg33, syngrg35, syngrg36, syngrg37, syngrg38, syngrg39, and syngrg50 into Maize Plant Cells by Agrobacterium-Mediated Transformation

[0126] Ears are best collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are preferred for use in transformation. Embryos are plated scutellum side-up on a suitable incubation media, and incubated overnight at 25.degree. C. in the dark. However, it is not necessary per se to incubate the embryos overnight. Embryos are contacted with an Agrobacterium strain containing the appropriate vectors for Ti plasmid mediated transfer for about 5-10 min, and then plated onto co-cultivation media for about 3 days (25.degree. C. in the dark). After co-cultivation, explants are transferred to recovery period media for about five days (at 25.degree. C. in the dark). Explants are incubated in selection media for up to eight weeks, depending on the nature and characteristics of the particular selection utilized. After the selection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of regeneration is initiated as known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants.

[0127] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0128] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Sequence CWU 1

1

4111329DNAUnknownIsolated from soil sample 1atg acc gac agc aac ccg tcc acc gac ccg ctc acc ggc cgc tgg ccc 48Met Thr Asp Ser Asn Pro Ser Thr Asp Pro Leu Thr Gly Arg Trp Pro1 5 10 15gcc ccg tac gcg gcc ggc gcc gtc gac gcc acc gtc acc gtg ccc gga 96Ala Pro Tyr Ala Ala Gly Ala Val Asp Ala Thr Val Thr Val Pro Gly 20 25 30tcg aag tcg gtc acc aac cgc gcc ctg gtg ctc gcc gcg ctg gcc gcc 144Ser Lys Ser Val Thr Asn Arg Ala Leu Val Leu Ala Ala Leu Ala Ala 35 40 45gag ccg ggc tgg gtg cgc cgc ccg ctg cgc tcg cgc gac acc ctg ctg 192Glu Pro Gly Trp Val Arg Arg Pro Leu Arg Ser Arg Asp Thr Leu Leu 50 55 60atg gcg gag gcg ctg cgc acc ctg ggc gtg aag atc gac gag ggc gtg 240Met Ala Glu Ala Leu Arg Thr Leu Gly Val Lys Ile Asp Glu Gly Val65 70 75 80ggc ccg gac ggc acc ggc gag gcc tgg cgg atc atc ccg gcc ggg ctg 288Gly Pro Asp Gly Thr Gly Glu Ala Trp Arg Ile Ile Pro Ala Gly Leu 85 90 95cgc ggc ccg gcg acc gtc gac gtc ggc aac gcg ggc acg gtc atg cgc 336Arg Gly Pro Ala Thr Val Asp Val Gly Asn Ala Gly Thr Val Met Arg 100 105 110ttc ctg ccg ccg gtg gcg gcg ctc gcg aac ggc gcg gtg cgc ttc gac 384Phe Leu Pro Pro Val Ala Ala Leu Ala Asn Gly Ala Val Arg Phe Asp 115 120 125ggc gac ccg cgc tcc cac gag cgc ccg ctg cac ggg gtg atc gac gcg 432Gly Asp Pro Arg Ser His Glu Arg Pro Leu His Gly Val Ile Asp Ala 130 135 140ctg cgc gcg ctg ggc gcc cgg atc gac gac gac ggg cgc ggc gcg ctg 480Leu Arg Ala Leu Gly Ala Arg Ile Asp Asp Asp Gly Arg Gly Ala Leu145 150 155 160ccg atg acc gtg cac ggc gcc ggc ggc ctg gag ggc ggg gtc gtg gag 528Pro Met Thr Val His Gly Ala Gly Gly Leu Glu Gly Gly Val Val Glu 165 170 175atc gac gcc tcc tcg tcc tcc cag ttc gtc agc gcg ctg ctg ctc tcc 576Ile Asp Ala Ser Ser Ser Ser Gln Phe Val Ser Ala Leu Leu Leu Ser 180 185 190ggc gcc cgc ttc aac cag ggc gtg gag gtg cgg cac gtc ggc acc cgg 624Gly Ala Arg Phe Asn Gln Gly Val Glu Val Arg His Val Gly Thr Arg 195 200 205ctg ccc tcg ctg ccg cac atc cgg atg acg gtc gac atg ctg cgc gcg 672Leu Pro Ser Leu Pro His Ile Arg Met Thr Val Asp Met Leu Arg Ala 210 215 220gtc ggc gcc cag gtc gac gag ccg gag cac ggc ggg cgt ccc gac gtg 720Val Gly Ala Gln Val Asp Glu Pro Glu His Gly Gly Arg Pro Asp Val225 230 235 240tgg cgg gtc acc ccg tcc gcg ctg ctc ggc cgg gac ctg gtg gtg gag 768Trp Arg Val Thr Pro Ser Ala Leu Leu Gly Arg Asp Leu Val Val Glu 245 250 255ccg gac ctg tcg aac gcc cag ccg ttc ctg gcg gcg gcg ctg gtc acc 816Pro Asp Leu Ser Asn Ala Gln Pro Phe Leu Ala Ala Ala Leu Val Thr 260 265 270ggc ggc cgg gtc acc gtg ccg gac tgg ccg gcc agg acc acc cag ccc 864Gly Gly Arg Val Thr Val Pro Asp Trp Pro Ala Arg Thr Thr Gln Pro 275 280 285ggt gac gcg ctg cgg cag atc ttc acc gag atg ggt ggc tcc tgc gag 912Gly Asp Ala Leu Arg Gln Ile Phe Thr Glu Met Gly Gly Ser Cys Glu 290 295 300ctc acc gac cgg ggt ctc acc ttc acc gga acc ggc cgg atc cac ggc 960Leu Thr Asp Arg Gly Leu Thr Phe Thr Gly Thr Gly Arg Ile His Gly305 310 315 320atc gac gtc gac ctc ggc gag gtc ggc gag ctg acc ccg ggc atc gcg 1008Ile Asp Val Asp Leu Gly Glu Val Gly Glu Leu Thr Pro Gly Ile Ala 325 330 335gcg gtc gcc gcg ctc gcc gac tcc ccg tcc acc ctg cgc ggg gtg gcg 1056Ala Val Ala Ala Leu Ala Asp Ser Pro Ser Thr Leu Arg Gly Val Ala 340 345 350cac ctg cgg ctg cac gag acc gac cgg ctc gcc gcg ctc acc cgg gag 1104His Leu Arg Leu His Glu Thr Asp Arg Leu Ala Ala Leu Thr Arg Glu 355 360 365atc aac gcg ctg ggc ggc gac gtc acg gag acc gag gac ggc ctg cac 1152Ile Asn Ala Leu Gly Gly Asp Val Thr Glu Thr Glu Asp Gly Leu His 370 375 380atc cgc ccg cgc ccg ctg cac ggc ggc ctc ttc cac acg tac cac gac 1200Ile Arg Pro Arg Pro Leu His Gly Gly Leu Phe His Thr Tyr His Asp385 390 395 400cac cgg atg gcg acc gcg ggc gcg ctc atc ggc ctg gcc gtg aag ggc 1248His Arg Met Ala Thr Ala Gly Ala Leu Ile Gly Leu Ala Val Lys Gly 405 410 415gtg gag atc gag aac gtg aag acg acc gag aag acc ttg ccc gac ttc 1296Val Glu Ile Glu Asn Val Lys Thr Thr Glu Lys Thr Leu Pro Asp Phe 420 425 430ccc agg atg tgg acc gaa atg ctc gga gtc tga 1329Pro Arg Met Trp Thr Glu Met Leu Gly Val 435 4402442PRTUnknownIsolated from soil sample 2Met Thr Asp Ser Asn Pro Ser Thr Asp Pro Leu Thr Gly Arg Trp Pro1 5 10 15Ala Pro Tyr Ala Ala Gly Ala Val Asp Ala Thr Val Thr Val Pro Gly 20 25 30Ser Lys Ser Val Thr Asn Arg Ala Leu Val Leu Ala Ala Leu Ala Ala 35 40 45Glu Pro Gly Trp Val Arg Arg Pro Leu Arg Ser Arg Asp Thr Leu Leu 50 55 60Met Ala Glu Ala Leu Arg Thr Leu Gly Val Lys Ile Asp Glu Gly Val65 70 75 80Gly Pro Asp Gly Thr Gly Glu Ala Trp Arg Ile Ile Pro Ala Gly Leu 85 90 95Arg Gly Pro Ala Thr Val Asp Val Gly Asn Ala Gly Thr Val Met Arg 100 105 110Phe Leu Pro Pro Val Ala Ala Leu Ala Asn Gly Ala Val Arg Phe Asp 115 120 125Gly Asp Pro Arg Ser His Glu Arg Pro Leu His Gly Val Ile Asp Ala 130 135 140Leu Arg Ala Leu Gly Ala Arg Ile Asp Asp Asp Gly Arg Gly Ala Leu145 150 155 160Pro Met Thr Val His Gly Ala Gly Gly Leu Glu Gly Gly Val Val Glu 165 170 175Ile Asp Ala Ser Ser Ser Ser Gln Phe Val Ser Ala Leu Leu Leu Ser 180 185 190Gly Ala Arg Phe Asn Gln Gly Val Glu Val Arg His Val Gly Thr Arg 195 200 205Leu Pro Ser Leu Pro His Ile Arg Met Thr Val Asp Met Leu Arg Ala 210 215 220Val Gly Ala Gln Val Asp Glu Pro Glu His Gly Gly Arg Pro Asp Val225 230 235 240Trp Arg Val Thr Pro Ser Ala Leu Leu Gly Arg Asp Leu Val Val Glu 245 250 255Pro Asp Leu Ser Asn Ala Gln Pro Phe Leu Ala Ala Ala Leu Val Thr 260 265 270Gly Gly Arg Val Thr Val Pro Asp Trp Pro Ala Arg Thr Thr Gln Pro 275 280 285Gly Asp Ala Leu Arg Gln Ile Phe Thr Glu Met Gly Gly Ser Cys Glu 290 295 300Leu Thr Asp Arg Gly Leu Thr Phe Thr Gly Thr Gly Arg Ile His Gly305 310 315 320Ile Asp Val Asp Leu Gly Glu Val Gly Glu Leu Thr Pro Gly Ile Ala 325 330 335Ala Val Ala Ala Leu Ala Asp Ser Pro Ser Thr Leu Arg Gly Val Ala 340 345 350His Leu Arg Leu His Glu Thr Asp Arg Leu Ala Ala Leu Thr Arg Glu 355 360 365Ile Asn Ala Leu Gly Gly Asp Val Thr Glu Thr Glu Asp Gly Leu His 370 375 380Ile Arg Pro Arg Pro Leu His Gly Gly Leu Phe His Thr Tyr His Asp385 390 395 400His Arg Met Ala Thr Ala Gly Ala Leu Ile Gly Leu Ala Val Lys Gly 405 410 415Val Glu Ile Glu Asn Val Lys Thr Thr Glu Lys Thr Leu Pro Asp Phe 420 425 430Pro Arg Met Trp Thr Glu Met Leu Gly Val 435 44031326DNAArtificial Sequencesynthetic gene encoding GRG33 (syngrg33) 3atg aca gac agc aac cca agc acc gac ccg ctc acc ggc cgc tgg ccg 48gcg ccc tac gcc gcc ggc gcc gtc gac gcc acc gtc acc gtg cct gga 96agc aag agc gtc acc aac agg gcg ctg gtg ctg gcg gcg ctg gct gct 144gaa cct gga tgg gtg cgg cgg ccg ctg agg agc agg gac acc tta ttg 192atg gcg gag gcg ctg agg acg ctc ggc gtc aag att gat gaa gga gtt 240gga cct gat gga act gga gaa gca tgg agg atc atc ccc gcc ggc ctc 288cgc ggc ccg gcg acg gtg gat gtt ggc aac gcc ggc acc gtg atg agg 336ttc ctg ccg ccg gtg gcg gcg ctg gcc aac ggc gcc gtc cgc ttc gac 384ggc gac cca aga agt cat gaa agg cct cta cat ggc gtc atc gac gcg 432ctc cgc gcg ctg gga gca agg atc gac gac gac ggc cgc ggc gcg ctg 480cca atg aca gtt cat ggc gcc ggc ggc ctg gag ggc ggc gtg gtg gag 528att gat gca agc agc agc agc cag ttc gtc tcg gcg ctg ctg ctg agc 576ggc gcg cgc ttc aac caa gga gtg gag gtg cgg cat gtt gga aca agg 624ctg cca tca ttg ccg cac atc agg atg acg gtg gac atg ctg cgc gcc 672gtc ggc gct caa gtt gat gag ccg gag cat gga gga agg cca gat gtt 720tgg agg gtg acg ccg tcg gcg ctg ctg gga aga gat ctg gtg gtg gag 768cca gat ctc tca aat gct caa ccc ttc ctg gcg gcg gcg ctg gtg acc 816ggc ggc cgc gtc acc gtg cca gat tgg ccg gca agg acg acg cag ccc 864ggc gac gcg ctg cgg cag atc ttc acc gag atg gga gga tca tgt gag 912ctc acc gac cgc ggc ctc acc ttc acc ggc act gga agg att cat ggc 960att gat gtg gac ctc ggc gag gtg gga gag ctg acg ccg ggc atc gcc 1008gcc gtg gcg gcg ctc gcc gac tcg ccg tcg acg ctc cgc ggc gtg gct 1056cac ctc cgc cta cat gaa aca gat cgg ctg gcg gcg ctg aca agg gag 1104atc aac gcg ctc ggc ggc gac gtc acc gag aca gaa gat ggc ctc cac 1152atc agg ccg cgg ccg cta cat gga ggc ctc ttc cac acc tac cat gat 1200cac agg atg gcc acc gcc ggc gcg ctc atc ggc ctc gcc gtc aag ggc 1248gtg gag att gaa aat gtg aag aca aca gag aag acg ctg ccg gac ttc 1296cca agg atg tgg acg gag atg ctg ggc gtc 132641353DNAUnknownisolated from soil 4atg acc gca gct tcc ccc gcg cac ccc gat ccg tcg cag ccc gcc gtc 48Met Thr Ala Ala Ser Pro Ala His Pro Asp Pro Ser Gln Pro Ala Val1 5 10 15ccc gcc ggc ccg gac ctc tgg ccc gcc ccg tac gcg agc ggc ccc gtc 96Pro Ala Gly Pro Asp Leu Trp Pro Ala Pro Tyr Ala Ser Gly Pro Val 20 25 30gac gcg acc gtg acc gtg ccc ggc tcg aag tcg gtc acc aac cgc gcc 144Asp Ala Thr Val Thr Val Pro Gly Ser Lys Ser Val Thr Asn Arg Ala 35 40 45ctg gtg ctc gcc gcg ctc gcc gcc gag ccc ggc tgg gtg cgg cgc ccg 192Leu Val Leu Ala Ala Leu Ala Ala Glu Pro Gly Trp Val Arg Arg Pro 50 55 60ctc cgc tcc cgc gac acc ctc ctg atg gcc gag ggc ctg cgc acc ctg 240Leu Arg Ser Arg Asp Thr Leu Leu Met Ala Glu Gly Leu Arg Thr Leu65 70 75 80ggc gtg aag atc gag gag ggc gtg ggc ccg gac ggc acc ggc gag gcc 288Gly Val Lys Ile Glu Glu Gly Val Gly Pro Asp Gly Thr Gly Glu Ala 85 90 95tgg cgg atc atc ccg gcc ccg ctg cgc ggc ggc gcc acg gtc gac gtc 336Trp Arg Ile Ile Pro Ala Pro Leu Arg Gly Gly Ala Thr Val Asp Val 100 105 110ggc aac gcg ggc acg gtc atg cgc ttc ctg ccg ccg gtc gcc gcg ctc 384Gly Asn Ala Gly Thr Val Met Arg Phe Leu Pro Pro Val Ala Ala Leu 115 120 125gcc gac ggc ccg gtc cgc ttc gac ggc gac ccc cgc tcc cac gag cgc 432Ala Asp Gly Pro Val Arg Phe Asp Gly Asp Pro Arg Ser His Glu Arg 130 135 140ccg ctg cac ggg gtg atc gac gcg ctg cgc gcg ctc ggc gcc cgg atc 480Pro Leu His Gly Val Ile Asp Ala Leu Arg Ala Leu Gly Ala Arg Ile145 150 155 160gac gac gag ggc cgc ggc gcc ctc ccg atg acc gtg cac ggc gcc ggc 528Asp Asp Glu Gly Arg Gly Ala Leu Pro Met Thr Val His Gly Ala Gly 165 170 175ggc ctg gag ggc ggc acg gtc gag atc gac gcc tcc tcg tcc tcc cag 576Gly Leu Glu Gly Gly Thr Val Glu Ile Asp Ala Ser Ser Ser Ser Gln 180 185 190ttc gtc agc gcc ctg ctg ctc tcc ggc gcc cgc ttc aac cag ggc gtc 624Phe Val Ser Ala Leu Leu Leu Ser Gly Ala Arg Phe Asn Gln Gly Val 195 200 205gag gtc cgg cac gcg ggc ggc cgg ctg ccg tcg atg ccg cac atc cgg 672Glu Val Arg His Ala Gly Gly Arg Leu Pro Ser Met Pro His Ile Arg 210 215 220atg acc gtg gac atg ctc cgc gcg gtg ggc gcg aag gtc gac gag ccg 720Met Thr Val Asp Met Leu Arg Ala Val Gly Ala Lys Val Asp Glu Pro225 230 235 240gag cac ggc ggc gag ccg gac gtg tgg cgg gtg gcc ccg tcg gcg ctg 768Glu His Gly Gly Glu Pro Asp Val Trp Arg Val Ala Pro Ser Ala Leu 245 250 255cgc ggc cgc gac ctg gtc atc gag ccc gac ctg tcg aac gcc cag ccg 816Arg Gly Arg Asp Leu Val Ile Glu Pro Asp Leu Ser Asn Ala Gln Pro 260 265 270ttc ctg gcg gcc gcg ctg gtc acc ggc ggc cgg gtg acc gtc ccg gac 864Phe Leu Ala Ala Ala Leu Val Thr Gly Gly Arg Val Thr Val Pro Asp 275 280 285tgg ccg gcc cgc acc acc cag ccc ggc gac gag ctg cgc cgc atc ttc 912Trp Pro Ala Arg Thr Thr Gln Pro Gly Asp Glu Leu Arg Arg Ile Phe 290 295 300acc gag atg ggc ggc gcc tgc gag ctc acc gac gcc ggt ctc acc ttc 960Thr Glu Met Gly Gly Ala Cys Glu Leu Thr Asp Ala Gly Leu Thr Phe305 310 315 320acc ggc acc ggc cgg atc cac ggc atc gac gtg gac ctc ggc gag gtc 1008Thr Gly Thr Gly Arg Ile His Gly Ile Asp Val Asp Leu Gly Glu Val 325 330 335ggc gag ctg acc ccc ggc atc gcg gcg gtc gcc gcc ctc gcc gac tcc 1056Gly Glu Leu Thr Pro Gly Ile Ala Ala Val Ala Ala Leu Ala Asp Ser 340 345 350ccc tcc acc ctg cgc ggc gtg gcc cac ctg cgg ctc cac gag acc gac 1104Pro Ser Thr Leu Arg Gly Val Ala His Leu Arg Leu His Glu Thr Asp 355 360 365cgg ctc gcg gcg ctc acc cgg gag atc aac ggt ctg ggc ggc gac gtc 1152Arg Leu Ala Ala Leu Thr Arg Glu Ile Asn Gly Leu Gly Gly Asp Val 370 375 380acc gag acc gag gac ggc ctg cac atc cgg ccg cgc ccg ctg acc ggc 1200Thr Glu Thr Glu Asp Gly Leu His Ile Arg Pro Arg Pro Leu Thr Gly385 390 395 400ggc gtc ttc cac acg tac cac gac cac cgg atg gcg acc gcg ggc gcg 1248Gly Val Phe His Thr Tyr His Asp His Arg Met Ala Thr Ala Gly Ala 405 410 415atc atc ggc ctc gcc gtg aag ggc gtg gag atc gag gac gtg gcg acg 1296Ile Ile Gly Leu Ala Val Lys Gly Val Glu Ile Glu Asp Val Ala Thr 420 425 430acc gcc aag acc ttg ccg gac ttc ccg ggg atg tgg acc gaa atg ctc 1344Thr Ala Lys Thr Leu Pro Asp Phe Pro Gly Met Trp Thr Glu Met Leu 435 440 445gga gcc tga 1353Gly Ala 4505450PRTUnknownisolated from soil 5Met Thr Ala Ala Ser Pro Ala His Pro Asp Pro Ser Gln Pro Ala Val1 5 10 15Pro Ala Gly Pro Asp Leu Trp Pro Ala Pro Tyr Ala Ser Gly Pro Val 20 25 30Asp Ala Thr Val Thr Val Pro Gly Ser Lys Ser Val Thr Asn Arg Ala 35 40 45Leu Val Leu Ala Ala Leu Ala Ala Glu Pro Gly Trp Val Arg Arg Pro 50 55 60Leu Arg Ser Arg Asp Thr Leu Leu Met Ala Glu Gly Leu Arg Thr Leu65 70 75 80Gly Val Lys Ile Glu Glu Gly Val Gly Pro Asp Gly Thr Gly Glu Ala 85 90 95Trp Arg Ile Ile Pro Ala Pro Leu Arg Gly Gly Ala Thr Val Asp Val 100 105 110Gly Asn Ala Gly Thr Val Met Arg Phe Leu Pro Pro Val Ala Ala Leu 115 120 125Ala Asp Gly Pro Val Arg Phe Asp Gly Asp Pro Arg Ser His Glu Arg 130 135 140Pro Leu His Gly Val Ile Asp Ala Leu Arg Ala Leu Gly Ala Arg Ile145 150 155 160Asp Asp Glu Gly Arg Gly Ala Leu Pro Met Thr Val His Gly Ala Gly 165 170 175Gly Leu Glu Gly Gly Thr Val Glu Ile Asp Ala Ser Ser Ser Ser Gln 180 185 190Phe Val Ser Ala Leu Leu Leu Ser Gly Ala Arg Phe Asn Gln Gly Val 195 200 205Glu Val Arg His Ala Gly Gly Arg Leu Pro Ser Met Pro His Ile Arg 210 215 220Met Thr Val Asp Met Leu Arg Ala Val Gly Ala Lys Val Asp Glu Pro225 230 235 240Glu His Gly Gly Glu Pro Asp Val Trp Arg Val Ala Pro Ser Ala Leu 245 250 255Arg Gly Arg Asp Leu Val Ile Glu Pro Asp Leu Ser Asn Ala Gln Pro 260 265 270Phe Leu Ala Ala Ala Leu Val Thr Gly Gly Arg Val Thr Val Pro Asp

275 280 285Trp Pro Ala Arg Thr Thr Gln Pro Gly Asp Glu Leu Arg Arg Ile Phe 290 295 300Thr Glu Met Gly Gly Ala Cys Glu Leu Thr Asp Ala Gly Leu Thr Phe305 310 315 320Thr Gly Thr Gly Arg Ile His Gly Ile Asp Val Asp Leu Gly Glu Val 325 330 335Gly Glu Leu Thr Pro Gly Ile Ala Ala Val Ala Ala Leu Ala Asp Ser 340 345 350Pro Ser Thr Leu Arg Gly Val Ala His Leu Arg Leu His Glu Thr Asp 355 360 365Arg Leu Ala Ala Leu Thr Arg Glu Ile Asn Gly Leu Gly Gly Asp Val 370 375 380Thr Glu Thr Glu Asp Gly Leu His Ile Arg Pro Arg Pro Leu Thr Gly385 390 395 400Gly Val Phe His Thr Tyr His Asp His Arg Met Ala Thr Ala Gly Ala 405 410 415Ile Ile Gly Leu Ala Val Lys Gly Val Glu Ile Glu Asp Val Ala Thr 420 425 430Thr Ala Lys Thr Leu Pro Asp Phe Pro Gly Met Trp Thr Glu Met Leu 435 440 445Gly Ala 45061326DNAArtificial Sequencesynthetic gene encoding GRG35 (syngrg35) 6gttgatccaa gccagccggc ggtgccggcg gggccggacc tctggccggc gccatatgct 60tctgggccgg tggacgccac cgtcaccgtg cctggaagca agagcgtcac caacagggcg 120ctggtgctgg cggcgctggc tgctgaacct ggatgggtgc ggcggccgct gaggagcagg 180gacaccttgc tgatggcaga agggctaagg acgctcggcg tcaagatcga ggagggcgtc 240ggccctgatg gaactggaga agcatggagg atcatcccgg cgccgctccg cggcggcgcc 300accgtcgacg tcggcaacgc cggcaccgtg atgaggttcc tgccgccggt ggcggcgctc 360gccgacgggc cggtgagatt tgatggagat ccaagaagtc atgaaaggcc tctacatggc 420gtcatcgacg cgctccgcgc gctcggcgcc aggattgatg atgaaggccg cggcgcgctg 480ccaatgacag ttcatggcgc cggcggcctg gaaggaggaa cagtggagat tgatgcaagc 540agcagcagcc agttcgtctc ggcgctgctg ctgagcggcg cgcgcttcaa ccaaggagtg 600gaggtgcggc acgccggcgg ccggctgcca tcaatgcctc acatcaggat gacggtggac 660atgctgcgcg ccgtcggcgc caaggtggat gagccggagc atggaggaga acctgatgtt 720tggagggtgg cgccgtcggc gctccgcggc cgcgacctgg tgattgagcc agatctctca 780aatgctcaac ccttcctggc ggcggcgctg gtgaccggcg gccgcgtcac cgtgccagat 840tggccggcaa ggacgacgca gcccggcgac gagctaagga ggatcttcac cgagatgggc 900ggcgcctgcg agctcaccga cgccggcctc accttcaccg gcactggaag gattcatggc 960attgatgtgg acctcggcga ggtgggagag ctgacgccgg gcatcgccgc cgtggcggcg 1020ctcgccgact cgccgtcgac gctccgcggc gtggctcacc tccgcctaca tgaaacagat 1080cggctggcgg cgctgacaag ggagatcaat ggcctcggcg gcgacgtcac cgagacagaa 1140gatggactac acatcaggcc gcggccgctc accggcggcg tcttccacac ctaccatgat 1200cacaggatgg ccaccgccgg cgccatcatc ggcctcgccg tgaagggcgt ggagattgaa 1260gatgtggcca ccaccgccaa gacgctgccg gacttccctg ggatgtggac ggagatgctg 1320ggcgcc 132671344DNAUnknownisolated from soil 7atg aaa aat caa aat ttt gat gct aaa gcc cgt agc ccg tgg aca ccg 48Met Lys Asn Gln Asn Phe Asp Ala Lys Ala Arg Ser Pro Trp Thr Pro1 5 10 15tta aaa ggt gta aat aag ata agt gtt tca cct agt aaa gga aga ata 96Leu Lys Gly Val Asn Lys Ile Ser Val Ser Pro Ser Lys Gly Arg Ile 20 25 30aat gga acc gtt act att cct ggt agc aag agt tta acc aac aga gct 144Asn Gly Thr Val Thr Ile Pro Gly Ser Lys Ser Leu Thr Asn Arg Ala 35 40 45tta atc ata agt tct cta gct agt gga aag tca aaa gtg caa ggt att 192Leu Ile Ile Ser Ser Leu Ala Ser Gly Lys Ser Lys Val Gln Gly Ile 50 55 60tta aaa agc gat gat tca ttt tgg tgt tta gac tcc ttg aaa aag cta 240Leu Lys Ser Asp Asp Ser Phe Trp Cys Leu Asp Ser Leu Lys Lys Leu65 70 75 80gga gta aat gtg aaa att caa gga gat aca gct ttt atc gaa gga aac 288Gly Val Asn Val Lys Ile Gln Gly Asp Thr Ala Phe Ile Glu Gly Asn 85 90 95ggt ggt aaa tgg gaa tca ggt gat tta tat att ggt gca gca gga acg 336Gly Gly Lys Trp Glu Ser Gly Asp Leu Tyr Ile Gly Ala Ala Gly Thr 100 105 110att gca cga ttt cta cct gga gca tta gca gtt tca ggt aca ggc ata 384Ile Ala Arg Phe Leu Pro Gly Ala Leu Ala Val Ser Gly Thr Gly Ile 115 120 125tgg gag tta gaa gcc agt aaa agt atg agt aaa cga cct att tca ccc 432Trp Glu Leu Glu Ala Ser Lys Ser Met Ser Lys Arg Pro Ile Ser Pro 130 135 140tta gta gat gct tta aaa gag ctt ggg gct gaa ata aca tat cta agc 480Leu Val Asp Ala Leu Lys Glu Leu Gly Ala Glu Ile Thr Tyr Leu Ser145 150 155 160gat caa ggc tac tat ccg ttg tta gtt aaa gga aaa caa cta aat ggg 528Asp Gln Gly Tyr Tyr Pro Leu Leu Val Lys Gly Lys Gln Leu Asn Gly 165 170 175ggc gag gtt gaa ctc tca ggt aga att tct agt cag ttt ata agt ggt 576Gly Glu Val Glu Leu Ser Gly Arg Ile Ser Ser Gln Phe Ile Ser Gly 180 185 190cta ttg att gcc tcg cct tat tta aat gat cca atc aag att aat att 624Leu Leu Ile Ala Ser Pro Tyr Leu Asn Asp Pro Ile Lys Ile Asn Ile 195 200 205aaa gat cac atc gtt caa cac tca tat gta ctc tta act ttg gaa tta 672Lys Asp His Ile Val Gln His Ser Tyr Val Leu Leu Thr Leu Glu Leu 210 215 220atg aaa aag ttt ggt gca aaa gtt aaa tac gat agt agc cta aaa gaa 720Met Lys Lys Phe Gly Ala Lys Val Lys Tyr Asp Ser Ser Leu Lys Glu225 230 235 240ata gtc gtc tat cca tct aag tac act cca caa gat ata aat tta gaa 768Ile Val Val Tyr Pro Ser Lys Tyr Thr Pro Gln Asp Ile Asn Leu Glu 245 250 255gca gat gtt tcc act gca tgt tat ttt ctg gct ctt gct gca gtg acc 816Ala Asp Val Ser Thr Ala Cys Tyr Phe Leu Ala Leu Ala Ala Val Thr 260 265 270aac ggt aaa gta caa att gat aat cta act tat gaa aca aaa caa cca 864Asn Gly Lys Val Gln Ile Asp Asn Leu Thr Tyr Glu Thr Lys Gln Pro 275 280 285gat ata aaa atg gtt gat atc ctt gaa cgg atg gga tgc aaa gta aca 912Asp Ile Lys Met Val Asp Ile Leu Glu Arg Met Gly Cys Lys Val Thr 290 295 300aga ggt tct tca ttc att gaa ata gag gga gtt agt caa tta aaa ggt 960Arg Gly Ser Ser Phe Ile Glu Ile Glu Gly Val Ser Gln Leu Lys Gly305 310 315 320gga ttt gaa atc tct atg agg gaa atg tct gac caa gtg tta act cta 1008Gly Phe Glu Ile Ser Met Arg Glu Met Ser Asp Gln Val Leu Thr Leu 325 330 335gca gca att gct cca ttc gca gat gaa cca ata acc ata aaa gac gtt 1056Ala Ala Ile Ala Pro Phe Ala Asp Glu Pro Ile Thr Ile Lys Asp Val 340 345 350gaa cat ata cgc cat cac gaa tca aat cga atc agt gta cta gtt gat 1104Glu His Ile Arg His His Glu Ser Asn Arg Ile Ser Val Leu Val Asp 355 360 365tca cta tct agg tta gga att ata gta gaa gaa ttt aaa gat gga cta 1152Ser Leu Ser Arg Leu Gly Ile Ile Val Glu Glu Phe Lys Asp Gly Leu 370 375 380aaa gtg tat ccg ggt aat ccg aaa gcc act tta cta gat aca cac gat 1200Lys Val Tyr Pro Gly Asn Pro Lys Ala Thr Leu Leu Asp Thr His Asp385 390 395 400gat cat aga gtt gca atg gca tta tca ctt ata ggt tca aga gtt gaa 1248Asp His Arg Val Ala Met Ala Leu Ser Leu Ile Gly Ser Arg Val Glu 405 410 415ggt ata caa ata aat gat cca gga tgt gta tct aaa act tgt cct cag 1296Gly Ile Gln Ile Asn Asp Pro Gly Cys Val Ser Lys Thr Cys Pro Gln 420 425 430tat ttt gaa tta ttg gaa aaa cta ggt ttg aat ata att aaa cat tga 1344Tyr Phe Glu Leu Leu Glu Lys Leu Gly Leu Asn Ile Ile Lys His 435 440 4458447PRTUnknownisolated from soil 8Met Lys Asn Gln Asn Phe Asp Ala Lys Ala Arg Ser Pro Trp Thr Pro1 5 10 15Leu Lys Gly Val Asn Lys Ile Ser Val Ser Pro Ser Lys Gly Arg Ile 20 25 30Asn Gly Thr Val Thr Ile Pro Gly Ser Lys Ser Leu Thr Asn Arg Ala 35 40 45Leu Ile Ile Ser Ser Leu Ala Ser Gly Lys Ser Lys Val Gln Gly Ile 50 55 60Leu Lys Ser Asp Asp Ser Phe Trp Cys Leu Asp Ser Leu Lys Lys Leu65 70 75 80Gly Val Asn Val Lys Ile Gln Gly Asp Thr Ala Phe Ile Glu Gly Asn 85 90 95Gly Gly Lys Trp Glu Ser Gly Asp Leu Tyr Ile Gly Ala Ala Gly Thr 100 105 110Ile Ala Arg Phe Leu Pro Gly Ala Leu Ala Val Ser Gly Thr Gly Ile 115 120 125Trp Glu Leu Glu Ala Ser Lys Ser Met Ser Lys Arg Pro Ile Ser Pro 130 135 140Leu Val Asp Ala Leu Lys Glu Leu Gly Ala Glu Ile Thr Tyr Leu Ser145 150 155 160Asp Gln Gly Tyr Tyr Pro Leu Leu Val Lys Gly Lys Gln Leu Asn Gly 165 170 175Gly Glu Val Glu Leu Ser Gly Arg Ile Ser Ser Gln Phe Ile Ser Gly 180 185 190Leu Leu Ile Ala Ser Pro Tyr Leu Asn Asp Pro Ile Lys Ile Asn Ile 195 200 205Lys Asp His Ile Val Gln His Ser Tyr Val Leu Leu Thr Leu Glu Leu 210 215 220Met Lys Lys Phe Gly Ala Lys Val Lys Tyr Asp Ser Ser Leu Lys Glu225 230 235 240Ile Val Val Tyr Pro Ser Lys Tyr Thr Pro Gln Asp Ile Asn Leu Glu 245 250 255Ala Asp Val Ser Thr Ala Cys Tyr Phe Leu Ala Leu Ala Ala Val Thr 260 265 270Asn Gly Lys Val Gln Ile Asp Asn Leu Thr Tyr Glu Thr Lys Gln Pro 275 280 285Asp Ile Lys Met Val Asp Ile Leu Glu Arg Met Gly Cys Lys Val Thr 290 295 300Arg Gly Ser Ser Phe Ile Glu Ile Glu Gly Val Ser Gln Leu Lys Gly305 310 315 320Gly Phe Glu Ile Ser Met Arg Glu Met Ser Asp Gln Val Leu Thr Leu 325 330 335Ala Ala Ile Ala Pro Phe Ala Asp Glu Pro Ile Thr Ile Lys Asp Val 340 345 350Glu His Ile Arg His His Glu Ser Asn Arg Ile Ser Val Leu Val Asp 355 360 365Ser Leu Ser Arg Leu Gly Ile Ile Val Glu Glu Phe Lys Asp Gly Leu 370 375 380Lys Val Tyr Pro Gly Asn Pro Lys Ala Thr Leu Leu Asp Thr His Asp385 390 395 400Asp His Arg Val Ala Met Ala Leu Ser Leu Ile Gly Ser Arg Val Glu 405 410 415Gly Ile Gln Ile Asn Asp Pro Gly Cys Val Ser Lys Thr Cys Pro Gln 420 425 430Tyr Phe Glu Leu Leu Glu Lys Leu Gly Leu Asn Ile Ile Lys His 435 440 44591344DNAArtificial Sequencesynthetic gene encoding GRG36 (syngrg36) 9atgaagaacc agaacttcga cgccaaggca agaagcccct ggacgccgct caagggcgtc 60aacaagatct ccgtctcgcc aagcaaagga aggatcaatg gcaccgtcac catccctgga 120agcaagagct tgacaaatag agctctcatc atcagcagct tagcaagcgg caagagcaag 180gttcaaggca tcctcaagag cgacgacagc ttctggtgcc tggattcatt gaagaagctc 240ggcgtcaatg tgaagatcca aggagacacc gccttcattg aaggaaatgg aggaaaatgg 300gagagtggag atctctacat cggcgccgcc ggcaccattg caagatttct tcctggcgcg 360ctggcggtga gcggcaccgg catctgggag ctggaggcaa gcaagagcat gagcaagagg 420cccatctcac cgctggtgga tgctctcaag gagctcggcg ccgagatcac ctacctctct 480gatcaaggct actacccgct gctggtgaag ggcaagcagc tcaatggagg agaggtggag 540ctctctggaa ggatcagcag ccagttcatc agcggcctgc tgattgcttc accatatttg 600aatgatccca tcaagatcaa catcaaggac cacatcgtgc agcacagcta tgtgctgctg 660acgctggagc tgatgaagaa gttcggcgcc aaggtgaagt acgacagcag cttgaaggag 720atcgtcgtct acccaagcaa gtacacgccg caggacatca acctggaagc tgatgtttca 780acagcatgct acttcctggc gctggcggcg gtgacaaatg gcaaggtgca aattgacaac 840ctcacctacg agaccaagca gccggacatc aagatggtgg acatcctgga gaggatgggc 900tgcaaggtga caagaggaag cagcttcatc gagattgaag gagtgagcca gctgaagggc 960ggcttcgaga tctcaatgag ggagatgagc gaccaggtgc tgacgctggc ggccatcgcg 1020ccatttgctg atgagcccat caccatcaag gatgtggagc acatccgcca tcatgaaagc 1080aacaggatct ccgtgctggt ggacagcctc tcaaggctgg gcatcatcgt ggaggagttc 1140aaggatggcc tcaaggtgta ccctggaaat ccaaaggcga cgctgctgga cacccatgat 1200gatcaccgcg tggcaatggc gctgagcttg attggatcaa gggtggaagg catccagatc 1260aatgatcctg gctgcgtcag caagacctgc cctcaatatt ttgagctgct ggagaagctg 1320ggcctcaaca tcatcaagca ctag 1344101407DNAUnknownisolated from soil 10atg aca gca tca ccc atg ggc gcg tcc gct gac aac tcc ggc gct gct 48Met Thr Ala Ser Pro Met Gly Ala Ser Ala Asp Asn Ser Gly Ala Ala1 5 10 15ccg cac tgg ccc gcg ccg ttt gcg gag cag ccc gtt gac gcc acg gtc 96Pro His Trp Pro Ala Pro Phe Ala Glu Gln Pro Val Asp Ala Thr Val 20 25 30cgc gtg ccg ggc tcc aag tcg ctg acc aac agg tac ctg gtg ctc gcc 144Arg Val Pro Gly Ser Lys Ser Leu Thr Asn Arg Tyr Leu Val Leu Ala 35 40 45gcg ctg gcc gac ggg ccg tcc cgg ctc cgg gcg ccg ctg cat tcc cgc 192Ala Leu Ala Asp Gly Pro Ser Arg Leu Arg Ala Pro Leu His Ser Arg 50 55 60gac tca gcg ctg atg atc gcc gcc ctg cgc cag ctc ggc gcg gac atc 240Asp Ser Ala Leu Met Ile Ala Ala Leu Arg Gln Leu Gly Ala Asp Ile65 70 75 80cgg gag gtg ccg ggc gac ggc gcc ttc ggc ccg gac ctg gag gtg acg 288Arg Glu Val Pro Gly Asp Gly Ala Phe Gly Pro Asp Leu Glu Val Thr 85 90 95ccg att ccg gca aac gcc ggg ggt gcc gac gtc gcc atc gac tgc ggc 336Pro Ile Pro Ala Asn Ala Gly Gly Ala Asp Val Ala Ile Asp Cys Gly 100 105 110ctg gcc ggg acc gtc atg agg ttt gtt ccg ccg ctg gcg gcg ctc cgc 384Leu Ala Gly Thr Val Met Arg Phe Val Pro Pro Leu Ala Ala Leu Arg 115 120 125agc ggt acc acc gtt ttc gac ggc gat ccc cac gcc cgg gag cgc ccg 432Ser Gly Thr Thr Val Phe Asp Gly Asp Pro His Ala Arg Glu Arg Pro 130 135 140atg ggg acc atc atc gag gcg ctt gcg ggc ctt ggc gtt acc gtc agc 480Met Gly Thr Ile Ile Glu Ala Leu Ala Gly Leu Gly Val Thr Val Ser145 150 155 160ggc gag gac ggc gga acg cct gct tca ctg ccg ttc cgc gtc gaa ggc 528Gly Glu Asp Gly Gly Thr Pro Ala Ser Leu Pro Phe Arg Val Glu Gly 165 170 175acc ggg cag gtc cgc ggc ggg cac ctc gtc ata gac gcc agc gcc tcc 576Thr Gly Gln Val Arg Gly Gly His Leu Val Ile Asp Ala Ser Ala Ser 180 185 190tcc cag ttc gtc tcc gcg ctg ctg ctg gtg ggg gcc cgg ttc acg gac 624Ser Gln Phe Val Ser Ala Leu Leu Leu Val Gly Ala Arg Phe Thr Asp 195 200 205ggc ctg cat ctg gag cac tcc ggc agc acc gtg ccg agc ctg gac cac 672Gly Leu His Leu Glu His Ser Gly Ser Thr Val Pro Ser Leu Asp His 210 215 220atc aac atg acc atc gct acg ctg cgc ggc gcg ggc gtg gcg gtg gat 720Ile Asn Met Thr Ile Ala Thr Leu Arg Gly Ala Gly Val Ala Val Asp225 230 235 240gac tcc acg ccc aac cac tgg att gtg ggc ccc ggg ccg atc cgc gcc 768Asp Ser Thr Pro Asn His Trp Ile Val Gly Pro Gly Pro Ile Arg Ala 245 250 255ttc gac cag cgg atc gag cag gac ctg tcc aac gcc ggc ccg ttc ctg 816Phe Asp Gln Arg Ile Glu Gln Asp Leu Ser Asn Ala Gly Pro Phe Leu 260 265 270gcg gcc gcg ctg gca acc ggc ggc acg gtg cgg att ccg gac tgg ccg 864Ala Ala Ala Leu Ala Thr Gly Gly Thr Val Arg Ile Pro Asp Trp Pro 275 280 285gag cac acc acg cag gtg ggt gac atg tgg cgc agt atc ctc agt gac 912Glu His Thr Thr Gln Val Gly Asp Met Trp Arg Ser Ile Leu Ser Asp 290 295 300atg ggg gca acc gtc acc ctg cag gac ggc acc ctg acc gtg acc ggc 960Met Gly Ala Thr Val Thr Leu Gln Asp Gly Thr Leu Thr Val Thr Gly305 310 315 320ggc agc aaa atc aac ggc gcg gac ttc gcc gag acc agc gaa ctg gcg 1008Gly Ser Lys Ile Asn Gly Ala Asp Phe Ala Glu Thr Ser Glu Leu Ala 325 330 335ccc acc gtc gct gcg ctc tgc gcg ctg gca tcc ggt ccc tcg cgg ctg 1056Pro Thr Val Ala Ala Leu Cys Ala Leu Ala Ser Gly Pro Ser Arg Leu 340 345 350acc ggc atc gcc cac ctc cgg gga cat gag acg gac cgg ctc gcc gcg 1104Thr Gly Ile Ala His Leu Arg Gly His Glu Thr Asp Arg Leu Ala Ala 355 360 365ctc gtc acc gaa atc aac cgc ctc ggc ggc gat gcc gag gaa acc gcc 1152Leu Val Thr Glu Ile Asn Arg Leu Gly Gly Asp Ala Glu Glu Thr Ala 370 375 380gac gga ctc gtc atc cgc ccc gcg gaa ctg cac gcg ggg gtg gtg cac 1200Asp Gly Leu Val Ile Arg Pro Ala Glu Leu His Ala Gly Val Val His385 390 395 400agc tac gcg gac cac cgg atg gcc aca gcc gga gcc atc ctc ggc ctg 1248Ser Tyr Ala Asp

His Arg Met Ala Thr Ala Gly Ala Ile Leu Gly Leu 405 410 415gcg gtc agg ggc gtg gaa gtg cag gac atc gcc acc aca tcc aag acc 1296Ala Val Arg Gly Val Glu Val Gln Asp Ile Ala Thr Thr Ser Lys Thr 420 425 430atg ccg gac ttc ccc aag ctg tgg gcg gac atg ctc ggc cgg cag acc 1344Met Pro Asp Phe Pro Lys Leu Trp Ala Asp Met Leu Gly Arg Gln Thr 435 440 445gat gca gct aca aac aca gcg ggt acc aac acc gcg ggg acc gcc ggt 1392Asp Ala Ala Thr Asn Thr Ala Gly Thr Asn Thr Ala Gly Thr Ala Gly 450 455 460ggc acg cag cac tga 1407Gly Thr Gln His46511468PRTUnknownisolated from soil 11Met Thr Ala Ser Pro Met Gly Ala Ser Ala Asp Asn Ser Gly Ala Ala1 5 10 15Pro His Trp Pro Ala Pro Phe Ala Glu Gln Pro Val Asp Ala Thr Val 20 25 30Arg Val Pro Gly Ser Lys Ser Leu Thr Asn Arg Tyr Leu Val Leu Ala 35 40 45Ala Leu Ala Asp Gly Pro Ser Arg Leu Arg Ala Pro Leu His Ser Arg 50 55 60Asp Ser Ala Leu Met Ile Ala Ala Leu Arg Gln Leu Gly Ala Asp Ile65 70 75 80Arg Glu Val Pro Gly Asp Gly Ala Phe Gly Pro Asp Leu Glu Val Thr 85 90 95Pro Ile Pro Ala Asn Ala Gly Gly Ala Asp Val Ala Ile Asp Cys Gly 100 105 110Leu Ala Gly Thr Val Met Arg Phe Val Pro Pro Leu Ala Ala Leu Arg 115 120 125Ser Gly Thr Thr Val Phe Asp Gly Asp Pro His Ala Arg Glu Arg Pro 130 135 140Met Gly Thr Ile Ile Glu Ala Leu Ala Gly Leu Gly Val Thr Val Ser145 150 155 160Gly Glu Asp Gly Gly Thr Pro Ala Ser Leu Pro Phe Arg Val Glu Gly 165 170 175Thr Gly Gln Val Arg Gly Gly His Leu Val Ile Asp Ala Ser Ala Ser 180 185 190Ser Gln Phe Val Ser Ala Leu Leu Leu Val Gly Ala Arg Phe Thr Asp 195 200 205Gly Leu His Leu Glu His Ser Gly Ser Thr Val Pro Ser Leu Asp His 210 215 220Ile Asn Met Thr Ile Ala Thr Leu Arg Gly Ala Gly Val Ala Val Asp225 230 235 240Asp Ser Thr Pro Asn His Trp Ile Val Gly Pro Gly Pro Ile Arg Ala 245 250 255Phe Asp Gln Arg Ile Glu Gln Asp Leu Ser Asn Ala Gly Pro Phe Leu 260 265 270Ala Ala Ala Leu Ala Thr Gly Gly Thr Val Arg Ile Pro Asp Trp Pro 275 280 285Glu His Thr Thr Gln Val Gly Asp Met Trp Arg Ser Ile Leu Ser Asp 290 295 300Met Gly Ala Thr Val Thr Leu Gln Asp Gly Thr Leu Thr Val Thr Gly305 310 315 320Gly Ser Lys Ile Asn Gly Ala Asp Phe Ala Glu Thr Ser Glu Leu Ala 325 330 335Pro Thr Val Ala Ala Leu Cys Ala Leu Ala Ser Gly Pro Ser Arg Leu 340 345 350Thr Gly Ile Ala His Leu Arg Gly His Glu Thr Asp Arg Leu Ala Ala 355 360 365Leu Val Thr Glu Ile Asn Arg Leu Gly Gly Asp Ala Glu Glu Thr Ala 370 375 380Asp Gly Leu Val Ile Arg Pro Ala Glu Leu His Ala Gly Val Val His385 390 395 400Ser Tyr Ala Asp His Arg Met Ala Thr Ala Gly Ala Ile Leu Gly Leu 405 410 415Ala Val Arg Gly Val Glu Val Gln Asp Ile Ala Thr Thr Ser Lys Thr 420 425 430Met Pro Asp Phe Pro Lys Leu Trp Ala Asp Met Leu Gly Arg Gln Thr 435 440 445Asp Ala Ala Thr Asn Thr Ala Gly Thr Asn Thr Ala Gly Thr Ala Gly 450 455 460Gly Thr Gln His465121404DNAArtificial Sequencesynthetic gene encoding GRG37 (syngrg37) 12atgacagctt ctccaatggg cgcctccgcc gacaacagcg gcgccgcgcc gcactggccg 60gcgcccttcg ccgagcagcc ggtggacgcc accgtccgcg tgcctggaag caagagcctc 120accaacagat atttggtgct ggcggcgctg gctgatgggc catcaaggct ccgcgcgccg 180ctgcattcaa gagattcggc gctgatgatc gccgcgctcc gtcagctcgg cgccgacatc 240agggaggtgc ccggcgacgg cgccttcggc cctgatctgg aggtgacgcc catcccggcc 300aacgccggcg gcgccgacgt cgccatcgac tgcggcctcg ccggcaccgt gatgaggttc 360gtgccgccgc tggcggcgct gagatcaggc accaccgtct ttgatggaga tcctcatgca 420agagaaaggc caatgggcac catcatcgag gcgctcgccg gcctcggcgt caccgtcagc 480ggcgaggacg gcggcacgcc ggcgtcgctg cccttccgcg tggaaggaac tggtcaagtt 540cgcggcggcc acctggtgat tgatgcttca gcaagcagcc agttcgtctc ggcgctgctg 600ctggtgggag caaggttcac cgacggcctc cacctggagc acagcggcag caccgtgcca 660agcctggatc acatcaacat gaccatcgcc accctccgcg gcgccggcgt cgccgtggat 720gattcaacgc ccaaccactg gatcgtcggc cccggcccca tcagggcatt tgatcaaaga 780attgagcaag atctttcaaa tgctggaccc ttcctggcgg cggcgctggc caccggcggc 840accgtgagga ttccagattg gccggagcac accacccaag ttggtgacat gtggaggagc 900atcctctccg acatgggcgc caccgtcacc cttcaagatg gcaccttgac cgtcaccggc 960ggcagcaaga tcaacggcgc cgacttcgcc gagacatcag agctggcgcc gacggtggcg 1020gcgctctgcg cgctggcttc tgggccaagc cgcctcaccg gcattgctca cctccgcggc 1080catgaaacag atcggctggc ggcgctggtg acagagatca acaggctcgg cggcgacgcc 1140gaggagaccg ccgacggcct ggtgatcagg ccggcggagc tacatgctgg agtggtgcac 1200agctatgctg atcacaggat ggccaccgcc ggcgccatcc tcggcctcgc cgtcagagga 1260gtggaggtgc aagatatagc aacaacaagc aagaccatgc cggacttccc caagctctgg 1320gccgacatgc tgggccgcca aacagatgct gccaccaaca ccgccggcac caacaccgcc 1380ggcaccgccg gcggcaccca gcac 1404131392DNAUnknownisolated from soil 13atg ggc gcg tcc gct gac aac tcc ggc gct gct ccg cac tgg ccc gcg 48Met Gly Ala Ser Ala Asp Asn Ser Gly Ala Ala Pro His Trp Pro Ala1 5 10 15ccg ttt gcg gag cag ccc gtt gac gcc acg gtc cgc gtg ccg ggc tcc 96Pro Phe Ala Glu Gln Pro Val Asp Ala Thr Val Arg Val Pro Gly Ser 20 25 30aag tcg ctg acc aac agg tac ctg gtg ctc gcc gcg ctg gcc gac ggg 144Lys Ser Leu Thr Asn Arg Tyr Leu Val Leu Ala Ala Leu Ala Asp Gly 35 40 45ccg tcc cgg ctc cgg gcg ccg ctg cat tcc cgc gac tca gcg ctg atg 192Pro Ser Arg Leu Arg Ala Pro Leu His Ser Arg Asp Ser Ala Leu Met 50 55 60atc gcc gcc ctg cgc cag ctc ggc gcg gac atc cgg gag gtg ccg ggc 240Ile Ala Ala Leu Arg Gln Leu Gly Ala Asp Ile Arg Glu Val Pro Gly65 70 75 80gac ggc gcc ttc ggc ccg gac ctg gag gtg acg ccg att ccg gca aac 288Asp Gly Ala Phe Gly Pro Asp Leu Glu Val Thr Pro Ile Pro Ala Asn 85 90 95gcc ggg ggt gcc gac gtc gcc atc gac tgc ggc ctg gcc ggg acc gtc 336Ala Gly Gly Ala Asp Val Ala Ile Asp Cys Gly Leu Ala Gly Thr Val 100 105 110atg agg ttt gtt ccg ccg ctg gcg gcg ctc cgc agc ggt acc acc gtt 384Met Arg Phe Val Pro Pro Leu Ala Ala Leu Arg Ser Gly Thr Thr Val 115 120 125ttc gac ggc gat ccc cac gcc cgg gag cgc ccg atg ggg acc atc atc 432Phe Asp Gly Asp Pro His Ala Arg Glu Arg Pro Met Gly Thr Ile Ile 130 135 140gag gcg ctt gcg ggc ctt ggc gtt acc gtc agc ggc gag gac ggc gga 480Glu Ala Leu Ala Gly Leu Gly Val Thr Val Ser Gly Glu Asp Gly Gly145 150 155 160acg cct gct tca ctg ccg ttc cgc gtc gaa ggc acc ggg cag gtc cgc 528Thr Pro Ala Ser Leu Pro Phe Arg Val Glu Gly Thr Gly Gln Val Arg 165 170 175ggc ggg cac ctc gtc ata gac gcc agc gcc tcc tcc cag ttc gtc tcc 576Gly Gly His Leu Val Ile Asp Ala Ser Ala Ser Ser Gln Phe Val Ser 180 185 190gcg ctg ctg ctg gtg ggg gcc cgg ttc acg gac ggc ctg cat ctg gag 624Ala Leu Leu Leu Val Gly Ala Arg Phe Thr Asp Gly Leu His Leu Glu 195 200 205cac tcc ggc agc acc gtg ccg agc ctg gac cac atc aac atg acc atc 672His Ser Gly Ser Thr Val Pro Ser Leu Asp His Ile Asn Met Thr Ile 210 215 220gct acg ctg cgc ggc gcg ggc gtg gcg gtg gat gac tcc acg ccc aac 720Ala Thr Leu Arg Gly Ala Gly Val Ala Val Asp Asp Ser Thr Pro Asn225 230 235 240cac tgg att gtg ggc ccc ggg ccg atc cgc gcc ttc gac cag cgg atc 768His Trp Ile Val Gly Pro Gly Pro Ile Arg Ala Phe Asp Gln Arg Ile 245 250 255gag cag gac ctg tcc aac gcc ggc ccg ttc ctg gcg gcc gcg ctg gca 816Glu Gln Asp Leu Ser Asn Ala Gly Pro Phe Leu Ala Ala Ala Leu Ala 260 265 270acc ggc ggc acg gtg cgg att ccg gac tgg ccg gag cac acc acg cag 864Thr Gly Gly Thr Val Arg Ile Pro Asp Trp Pro Glu His Thr Thr Gln 275 280 285gtg ggt gac atg tgg cgc agt atc ctc agt gac atg ggg gca acc gtc 912Val Gly Asp Met Trp Arg Ser Ile Leu Ser Asp Met Gly Ala Thr Val 290 295 300acc ctg cag gac ggc acc ctg acc gtg acc ggc ggc agc aaa atc aac 960Thr Leu Gln Asp Gly Thr Leu Thr Val Thr Gly Gly Ser Lys Ile Asn305 310 315 320ggc gcg gac ttc gcc gag acc agc gaa ctg gcg ccc acc gtc gct gcg 1008Gly Ala Asp Phe Ala Glu Thr Ser Glu Leu Ala Pro Thr Val Ala Ala 325 330 335ctc tgc gcg ctg gca tcc ggt ccc tcg cgg ctg acc ggc atc gcc cac 1056Leu Cys Ala Leu Ala Ser Gly Pro Ser Arg Leu Thr Gly Ile Ala His 340 345 350ctc cgg gga cat gag acg gac cgg ctc gcc gcg ctc gtc acc gaa atc 1104Leu Arg Gly His Glu Thr Asp Arg Leu Ala Ala Leu Val Thr Glu Ile 355 360 365aac cgc ctc ggc ggc gat gcc gag gaa acc gcc gac gga ctc gtc atc 1152Asn Arg Leu Gly Gly Asp Ala Glu Glu Thr Ala Asp Gly Leu Val Ile 370 375 380cgc ccc gcg gaa ctg cac gcg ggg gtg gtg cac agc tac gcg gac cac 1200Arg Pro Ala Glu Leu His Ala Gly Val Val His Ser Tyr Ala Asp His385 390 395 400cgg atg gcc aca gcc gga gcc atc ctc ggc ctg gcg gtc agg ggc gtg 1248Arg Met Ala Thr Ala Gly Ala Ile Leu Gly Leu Ala Val Arg Gly Val 405 410 415gaa gtg cag gac atc gcc acc aca tcc aag acc atg ccg gac ttc ccc 1296Glu Val Gln Asp Ile Ala Thr Thr Ser Lys Thr Met Pro Asp Phe Pro 420 425 430aag ctg tgg gcg gac atg ctc ggc cgg cag acc gat gca gct aca aac 1344Lys Leu Trp Ala Asp Met Leu Gly Arg Gln Thr Asp Ala Ala Thr Asn 435 440 445aca gcg ggt acc aac acc gcg ggg acc gcc ggt ggc acg cag cac tga 1392Thr Ala Gly Thr Asn Thr Ala Gly Thr Ala Gly Gly Thr Gln His 450 455 46014463PRTUnknownisolated from soil 14Met Gly Ala Ser Ala Asp Asn Ser Gly Ala Ala Pro His Trp Pro Ala1 5 10 15Pro Phe Ala Glu Gln Pro Val Asp Ala Thr Val Arg Val Pro Gly Ser 20 25 30Lys Ser Leu Thr Asn Arg Tyr Leu Val Leu Ala Ala Leu Ala Asp Gly 35 40 45Pro Ser Arg Leu Arg Ala Pro Leu His Ser Arg Asp Ser Ala Leu Met 50 55 60Ile Ala Ala Leu Arg Gln Leu Gly Ala Asp Ile Arg Glu Val Pro Gly65 70 75 80Asp Gly Ala Phe Gly Pro Asp Leu Glu Val Thr Pro Ile Pro Ala Asn 85 90 95Ala Gly Gly Ala Asp Val Ala Ile Asp Cys Gly Leu Ala Gly Thr Val 100 105 110Met Arg Phe Val Pro Pro Leu Ala Ala Leu Arg Ser Gly Thr Thr Val 115 120 125Phe Asp Gly Asp Pro His Ala Arg Glu Arg Pro Met Gly Thr Ile Ile 130 135 140Glu Ala Leu Ala Gly Leu Gly Val Thr Val Ser Gly Glu Asp Gly Gly145 150 155 160Thr Pro Ala Ser Leu Pro Phe Arg Val Glu Gly Thr Gly Gln Val Arg 165 170 175Gly Gly His Leu Val Ile Asp Ala Ser Ala Ser Ser Gln Phe Val Ser 180 185 190Ala Leu Leu Leu Val Gly Ala Arg Phe Thr Asp Gly Leu His Leu Glu 195 200 205His Ser Gly Ser Thr Val Pro Ser Leu Asp His Ile Asn Met Thr Ile 210 215 220Ala Thr Leu Arg Gly Ala Gly Val Ala Val Asp Asp Ser Thr Pro Asn225 230 235 240His Trp Ile Val Gly Pro Gly Pro Ile Arg Ala Phe Asp Gln Arg Ile 245 250 255Glu Gln Asp Leu Ser Asn Ala Gly Pro Phe Leu Ala Ala Ala Leu Ala 260 265 270Thr Gly Gly Thr Val Arg Ile Pro Asp Trp Pro Glu His Thr Thr Gln 275 280 285Val Gly Asp Met Trp Arg Ser Ile Leu Ser Asp Met Gly Ala Thr Val 290 295 300Thr Leu Gln Asp Gly Thr Leu Thr Val Thr Gly Gly Ser Lys Ile Asn305 310 315 320Gly Ala Asp Phe Ala Glu Thr Ser Glu Leu Ala Pro Thr Val Ala Ala 325 330 335Leu Cys Ala Leu Ala Ser Gly Pro Ser Arg Leu Thr Gly Ile Ala His 340 345 350Leu Arg Gly His Glu Thr Asp Arg Leu Ala Ala Leu Val Thr Glu Ile 355 360 365Asn Arg Leu Gly Gly Asp Ala Glu Glu Thr Ala Asp Gly Leu Val Ile 370 375 380Arg Pro Ala Glu Leu His Ala Gly Val Val His Ser Tyr Ala Asp His385 390 395 400Arg Met Ala Thr Ala Gly Ala Ile Leu Gly Leu Ala Val Arg Gly Val 405 410 415Glu Val Gln Asp Ile Ala Thr Thr Ser Lys Thr Met Pro Asp Phe Pro 420 425 430Lys Leu Trp Ala Asp Met Leu Gly Arg Gln Thr Asp Ala Ala Thr Asn 435 440 445Thr Ala Gly Thr Asn Thr Ala Gly Thr Ala Gly Gly Thr Gln His 450 455 460151239DNAUnknownisolated from soil 15gtg acc gtt aca ccg ccc aat ttc ccc ctc aat ggc aag gtc gcg ccc 48Val Thr Val Thr Pro Pro Asn Phe Pro Leu Asn Gly Lys Val Ala Pro1 5 10 15ccc ggc tcc aaa tcc att acc aac cgc gcc ctg ttg ctg gcg gcc ctg 96Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala Leu Leu Leu Ala Ala Leu 20 25 30gcc aag ggc acc agc cgt ctc agc ggc gcg ctc aag agc gac gac acc 144Ala Lys Gly Thr Ser Arg Leu Ser Gly Ala Leu Lys Ser Asp Asp Thr 35 40 45cgc cat atg tcg gtg gcc ctg cgc caa atg ggc gtg acc atc gat gag 192Arg His Met Ser Val Ala Leu Arg Gln Met Gly Val Thr Ile Asp Glu 50 55 60ccg gac gac acc acc ttc gtt gtc acc ggc aac ggc aaa ctg cac ctg 240Pro Asp Asp Thr Thr Phe Val Val Thr Gly Asn Gly Lys Leu His Leu65 70 75 80ccg tcg caa ccg ctg ttc ctc ggc aat gcc ggt act gcc atg cgc ttt 288Pro Ser Gln Pro Leu Phe Leu Gly Asn Ala Gly Thr Ala Met Arg Phe 85 90 95ctc acg gct gcg gtg gcc acg gtc gaa ggc acc gtg gtg ctg acc ggc 336Leu Thr Ala Ala Val Ala Thr Val Glu Gly Thr Val Val Leu Thr Gly 100 105 110gac gac tac atg caa aaa cgc ccg atc ggc ccg ttg ctg gcg acc ctc 384Asp Asp Tyr Met Gln Lys Arg Pro Ile Gly Pro Leu Leu Ala Thr Leu 115 120 125ggc cag aac ggc atc cag gtc gac agc ccg acc ggt tgc ccg ccg gtc 432Gly Gln Asn Gly Ile Gln Val Asp Ser Pro Thr Gly Cys Pro Pro Val 130 135 140acc gtg cat ggc gtg ggc aag atc aag gcc aag cgc ttc gag atc gac 480Thr Val His Gly Val Gly Lys Ile Lys Ala Lys Arg Phe Glu Ile Asp145 150 155 160ggc ggc ctg tcc agc cag tac gtg tcg gcg ctg ctg atg ctc gcg gcc 528Gly Gly Leu Ser Ser Gln Tyr Val Ser Ala Leu Leu Met Leu Ala Ala 165 170 175tgc ggc gaa gcg ccg att gaa gtg gcg ctg acc ggc aag gac atc ggt 576Cys Gly Glu Ala Pro Ile Glu Val Ala Leu Thr Gly Lys Asp Ile Gly 180 185 190gcc cgt ggc tat gtc gac ctg acc ctg gac tgc atg cgc gcc ttc ggt 624Ala Arg Gly Tyr Val Asp Leu Thr Leu Asp Cys Met Arg Ala Phe Gly 195 200 205gcc cag gtt gaa gcc gtt gac gac acc acc tgg tgc gtg gcc ccg acc 672Ala Gln Val Glu Ala Val Asp Asp Thr Thr Trp Cys Val Ala Pro Thr 210 215 220ggc tac atc gcc cac gac tac ctg atc gaa ccc gac gcc tcc gcc gcc 720Gly Tyr Ile Ala His Asp Tyr Leu Ile Glu Pro Asp Ala Ser Ala Ala225 230 235 240act tac ctg tgg gcc gct gaa gtg ttg acc ggg ggg cgc atc gac atc 768Thr Tyr Leu Trp Ala Ala Glu Val Leu Thr Gly Gly Arg Ile Asp Ile 245 250 255ggc gtg gcc gcg cag gat ttc acc cag ccg gac gcc aag gcc cag gcc 816Gly Val Ala Ala Gln Asp Phe Thr Gln Pro Asp Ala Lys Ala Gln Ala 260 265

270gtg atc gcg cag ttc ccg cat atg caa gcc acg gtg gtc ggc tcg cag 864Val Ile Ala Gln Phe Pro His Met Gln Ala Thr Val Val Gly Ser Gln 275 280 285atg cag gac gcc atc ccg acc ctg gcg gtg ctc gcg gct ttc aac aac 912Met Gln Asp Ala Ile Pro Thr Leu Ala Val Leu Ala Ala Phe Asn Asn 290 295 300acg ccg gtg cgt ttc acc gag ttg gcc aac ctg cgg gtc aag gag tgc 960Thr Pro Val Arg Phe Thr Glu Leu Ala Asn Leu Arg Val Lys Glu Cys305 310 315 320gac cgt gtg cag gca ctg cat gac ggc ctc aat gca atc cgc ccg ggc 1008Asp Arg Val Gln Ala Leu His Asp Gly Leu Asn Ala Ile Arg Pro Gly 325 330 335ctg gcg acc atc gaa ggc gac gac ctg ctg gta gcc agc gac ccc gca 1056Leu Ala Thr Ile Glu Gly Asp Asp Leu Leu Val Ala Ser Asp Pro Ala 340 345 350ttg gcc ggc acc gcg tgc acc gcg ctg atc gac acc cac gcc gac cac 1104Leu Ala Gly Thr Ala Cys Thr Ala Leu Ile Asp Thr His Ala Asp His 355 360 365cgc atc gcc atg tgc ttt gcc ctg gcc ggg ctg aag gtt tcg ggt att 1152Arg Ile Ala Met Cys Phe Ala Leu Ala Gly Leu Lys Val Ser Gly Ile 370 375 380cgc atc cag gac ccg gat tgc gtg gca aag acc tat ccc gag tac tgg 1200Arg Ile Gln Asp Pro Asp Cys Val Ala Lys Thr Tyr Pro Glu Tyr Trp385 390 395 400aag gcc ctg ggc agt ctc ggg gtg cag ctg agc tat taa 1239Lys Ala Leu Gly Ser Leu Gly Val Gln Leu Ser Tyr 405 41016412PRTUnknownisolated from soil 16Val Thr Val Thr Pro Pro Asn Phe Pro Leu Asn Gly Lys Val Ala Pro1 5 10 15Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala Leu Leu Leu Ala Ala Leu 20 25 30Ala Lys Gly Thr Ser Arg Leu Ser Gly Ala Leu Lys Ser Asp Asp Thr 35 40 45Arg His Met Ser Val Ala Leu Arg Gln Met Gly Val Thr Ile Asp Glu 50 55 60Pro Asp Asp Thr Thr Phe Val Val Thr Gly Asn Gly Lys Leu His Leu65 70 75 80Pro Ser Gln Pro Leu Phe Leu Gly Asn Ala Gly Thr Ala Met Arg Phe 85 90 95Leu Thr Ala Ala Val Ala Thr Val Glu Gly Thr Val Val Leu Thr Gly 100 105 110Asp Asp Tyr Met Gln Lys Arg Pro Ile Gly Pro Leu Leu Ala Thr Leu 115 120 125Gly Gln Asn Gly Ile Gln Val Asp Ser Pro Thr Gly Cys Pro Pro Val 130 135 140Thr Val His Gly Val Gly Lys Ile Lys Ala Lys Arg Phe Glu Ile Asp145 150 155 160Gly Gly Leu Ser Ser Gln Tyr Val Ser Ala Leu Leu Met Leu Ala Ala 165 170 175Cys Gly Glu Ala Pro Ile Glu Val Ala Leu Thr Gly Lys Asp Ile Gly 180 185 190Ala Arg Gly Tyr Val Asp Leu Thr Leu Asp Cys Met Arg Ala Phe Gly 195 200 205Ala Gln Val Glu Ala Val Asp Asp Thr Thr Trp Cys Val Ala Pro Thr 210 215 220Gly Tyr Ile Ala His Asp Tyr Leu Ile Glu Pro Asp Ala Ser Ala Ala225 230 235 240Thr Tyr Leu Trp Ala Ala Glu Val Leu Thr Gly Gly Arg Ile Asp Ile 245 250 255Gly Val Ala Ala Gln Asp Phe Thr Gln Pro Asp Ala Lys Ala Gln Ala 260 265 270Val Ile Ala Gln Phe Pro His Met Gln Ala Thr Val Val Gly Ser Gln 275 280 285Met Gln Asp Ala Ile Pro Thr Leu Ala Val Leu Ala Ala Phe Asn Asn 290 295 300Thr Pro Val Arg Phe Thr Glu Leu Ala Asn Leu Arg Val Lys Glu Cys305 310 315 320Asp Arg Val Gln Ala Leu His Asp Gly Leu Asn Ala Ile Arg Pro Gly 325 330 335Leu Ala Thr Ile Glu Gly Asp Asp Leu Leu Val Ala Ser Asp Pro Ala 340 345 350Leu Ala Gly Thr Ala Cys Thr Ala Leu Ile Asp Thr His Ala Asp His 355 360 365Arg Ile Ala Met Cys Phe Ala Leu Ala Gly Leu Lys Val Ser Gly Ile 370 375 380Arg Ile Gln Asp Pro Asp Cys Val Ala Lys Thr Tyr Pro Glu Tyr Trp385 390 395 400Lys Ala Leu Gly Ser Leu Gly Val Gln Leu Ser Tyr 405 410171236DNAArtificial Sequencesynthetic gene encoding GRG38 (syngrg38) 17atgacggtga cgccgccaaa cttcccgctc aatggcaagg tggcgccgcc gggcagcaag 60agcatcacca acagggcgct gctgctggcg gcgctggcca agggcacctc aaggctgagc 120ggcgcgctga agagcgacga cacccgtcac atgagcgtgg cgctgcggca gatgggcgtc 180accattgatg aacctgatga caccaccttc gtcgtcaccg gcaatggcaa gctgcatctt 240ccatcacagc cgctcttcct cggcaacgcc ggcaccgcca tgaggttctt gacggcggcg 300gtggccaccg tggaaggaac agtggtgctc actggagatg actacatgca aaagaggcca 360attggacctc tactggcgac gctgggccaa aatggcatcc aggtggactc gccgacgggc 420tgcccgccgg tgacagttca tggcgtcggc aagatcaagg ccaagagatt tgagatcgac 480ggcggcctca gcagccaata tgtttcagcg ctgctgatgc tggcggcctg cggcgaggcg 540cccatcgagg tggcgctcac cggcaaggac atcggcgcgc gcggctatgt ggacctcacc 600ttggactgca tgagggcctt cggcgctcaa gtggaggcgg tggatgacac cacctggtgc 660gtggcgccga cgggctacat tgctcatgac tacctcatcg agccagatgc ctccgccgcc 720acctacctat gggcggcgga ggtgctcacc ggcggcagga tcgacatcgg cgtcgccgct 780caagatttca cccaacctga tgccaaggct caagctgtga ttgctcaatt tcctcacatg 840caagcaacag tggtgggcag ccagatgcaa gatgccatcc cgacgctggc ggtgctggcg 900gccttcaaca acacgccggt gaggttcacc gagctggcca acctacgagt gaaggaatgt 960gacagggtgc aagctcttca tgatggcctc aacgccatca ggccagggct ggccaccatt 1020gaaggagatg atctgctggt ggcttcagat ccggcgctcg ccggcaccgc ctgcacggcg 1080ctcatcgaca cccacgccga ccaccgcatc gccatgtgct tcgcgctcgc cggcctcaag 1140gtgagcggca tcaggattca agatccagat tgtgtggcca agacctaccc ggagtactgg 1200aaggcgctgg gcagcctcgg cgtgcagctg agctac 1236181296DNAUnknownisolated from soil 18atg gac gtt atc gtt aaa cca acc cca tcc ctg aac ggg gaa att gga 48Met Asp Val Ile Val Lys Pro Thr Pro Ser Leu Asn Gly Glu Ile Gly1 5 10 15gct ttg tct tcc aaa aac tac acc aca cgc tac ttg cta gct gct gcg 96Ala Leu Ser Ser Lys Asn Tyr Thr Thr Arg Tyr Leu Leu Ala Ala Ala 20 25 30ctg gca gaa ggc aca agt aca atc cat tac ccg gct cat agt gaa gat 144Leu Ala Glu Gly Thr Ser Thr Ile His Tyr Pro Ala His Ser Glu Asp 35 40 45agt gat gct atg cgc aga tgt att cgt gac ctt ggg gcg gtg ctt gaa 192Ser Asp Ala Met Arg Arg Cys Ile Arg Asp Leu Gly Ala Val Leu Glu 50 55 60gaa gat gat agc aaa atc gtt atc caa gga ttc ggc agc cat ccg cgt 240Glu Asp Asp Ser Lys Ile Val Ile Gln Gly Phe Gly Ser His Pro Arg65 70 75 80gat gtg cgt gaa tta aat gta ggc aat gcg ggt gca gtg ctg cgt ttc 288Asp Val Arg Glu Leu Asn Val Gly Asn Ala Gly Ala Val Leu Arg Phe 85 90 95ctg atg gga gta acg gca ctt tgt cca gag gtg acg ttt gta aat acg 336Leu Met Gly Val Thr Ala Leu Cys Pro Glu Val Thr Phe Val Asn Thr 100 105 110tac ccg gat tct ctt ggc aaa cgc cca cat gat gac ctg atc gat gcg 384Tyr Pro Asp Ser Leu Gly Lys Arg Pro His Asp Asp Leu Ile Asp Ala 115 120 125ctt ggt cag ctc ggt gtt gag gta cag cac gaa caa gga cgc ttg cca 432Leu Gly Gln Leu Gly Val Glu Val Gln His Glu Gln Gly Arg Leu Pro 130 135 140atc acg atc aaa ggg ggt cag gct aag ggt gga cat atc cgt gta tcc 480Ile Thr Ile Lys Gly Gly Gln Ala Lys Gly Gly His Ile Arg Val Ser145 150 155 160ggt tct gtc agc tcc cag tat ttg agc gcg ttg ctg ttt gta act cct 528Gly Ser Val Ser Ser Gln Tyr Leu Ser Ala Leu Leu Phe Val Thr Pro 165 170 175ctt ctg gcc gaa gac agc aca att gaa gta tta aac gac ttg aaa tcc 576Leu Leu Ala Glu Asp Ser Thr Ile Glu Val Leu Asn Asp Leu Lys Ser 180 185 190aaa gtg gtt att ggt cag acg ctg gaa gta ctg gaa cag gcg ggg att 624Lys Val Val Ile Gly Gln Thr Leu Glu Val Leu Glu Gln Ala Gly Ile 195 200 205gtc att cat gcg agt gat gat tac atg tcc ttc cgt gta cct ggt ggt 672Val Ile His Ala Ser Asp Asp Tyr Met Ser Phe Arg Val Pro Gly Gly 210 215 220caa gct tat aaa ccg caa aca tat acc gtt caa ggc gac tat cca gga 720Gln Ala Tyr Lys Pro Gln Thr Tyr Thr Val Gln Gly Asp Tyr Pro Gly225 230 235 240tca gca gct gtc ctc gcg gcc gcg gct gtc acc caa tca gat gtt aaa 768Ser Ala Ala Val Leu Ala Ala Ala Ala Val Thr Gln Ser Asp Val Lys 245 250 255att ttg cga ttg atg gaa cag agc aaa cag ggt gag cgt gct att gtt 816Ile Leu Arg Leu Met Glu Gln Ser Lys Gln Gly Glu Arg Ala Ile Val 260 265 270gac gtt ctg cgt atg atg gaa gtg cca ttg acg cat gag aac gat gtg 864Asp Val Leu Arg Met Met Glu Val Pro Leu Thr His Glu Asn Asp Val 275 280 285gta cac gtg caa ggt aac ggt aca ttg aaa gcc gtg gaa ttc gac gga 912Val His Val Gln Gly Asn Gly Thr Leu Lys Ala Val Glu Phe Asp Gly 290 295 300gat gcc gcg aca gac gcg gtt ttg gcc atg gta gcg gcg gca gtg ttt 960Asp Ala Ala Thr Asp Ala Val Leu Ala Met Val Ala Ala Ala Val Phe305 310 315 320gcg gaa ggc acc tca cgg ttc tat aat gta gag aac tta cgt tac aag 1008Ala Glu Gly Thr Ser Arg Phe Tyr Asn Val Glu Asn Leu Arg Tyr Lys 325 330 335gaa tgt gac cga att acg gat tat ttg aac gaa ctg cgg aag gca gga 1056Glu Cys Asp Arg Ile Thr Asp Tyr Leu Asn Glu Leu Arg Lys Ala Gly 340 345 350gcc aac gta gaa gaa cgt cag gcc gag att atc gta cat ggt cgt ccg 1104Ala Asn Val Glu Glu Arg Gln Ala Glu Ile Ile Val His Gly Arg Pro 355 360 365gaa ggc gtc gaa ggc ggc gtt gag att aac gct cac tac gat cat cgc 1152Glu Gly Val Glu Gly Gly Val Glu Ile Asn Ala His Tyr Asp His Arg 370 375 380gta att atg gca ctg acc gtt gtt ggt ttg cgt tcc aaa gaa ccg ctt 1200Val Ile Met Ala Leu Thr Val Val Gly Leu Arg Ser Lys Glu Pro Leu385 390 395 400cgt att cgg gat gca cac cat gta gcg aag tct tat cca caa tat ttc 1248Arg Ile Arg Asp Ala His His Val Ala Lys Ser Tyr Pro Gln Tyr Phe 405 410 415gat cat ttg cag gcg ctt ggc gcc tcg gtt caa tgg gta aaa gag taa 1296Asp His Leu Gln Ala Leu Gly Ala Ser Val Gln Trp Val Lys Glu 420 425 43019431PRTUnknownisolated from soil 19Met Asp Val Ile Val Lys Pro Thr Pro Ser Leu Asn Gly Glu Ile Gly1 5 10 15Ala Leu Ser Ser Lys Asn Tyr Thr Thr Arg Tyr Leu Leu Ala Ala Ala 20 25 30Leu Ala Glu Gly Thr Ser Thr Ile His Tyr Pro Ala His Ser Glu Asp 35 40 45Ser Asp Ala Met Arg Arg Cys Ile Arg Asp Leu Gly Ala Val Leu Glu 50 55 60Glu Asp Asp Ser Lys Ile Val Ile Gln Gly Phe Gly Ser His Pro Arg65 70 75 80Asp Val Arg Glu Leu Asn Val Gly Asn Ala Gly Ala Val Leu Arg Phe 85 90 95Leu Met Gly Val Thr Ala Leu Cys Pro Glu Val Thr Phe Val Asn Thr 100 105 110Tyr Pro Asp Ser Leu Gly Lys Arg Pro His Asp Asp Leu Ile Asp Ala 115 120 125Leu Gly Gln Leu Gly Val Glu Val Gln His Glu Gln Gly Arg Leu Pro 130 135 140Ile Thr Ile Lys Gly Gly Gln Ala Lys Gly Gly His Ile Arg Val Ser145 150 155 160Gly Ser Val Ser Ser Gln Tyr Leu Ser Ala Leu Leu Phe Val Thr Pro 165 170 175Leu Leu Ala Glu Asp Ser Thr Ile Glu Val Leu Asn Asp Leu Lys Ser 180 185 190Lys Val Val Ile Gly Gln Thr Leu Glu Val Leu Glu Gln Ala Gly Ile 195 200 205Val Ile His Ala Ser Asp Asp Tyr Met Ser Phe Arg Val Pro Gly Gly 210 215 220Gln Ala Tyr Lys Pro Gln Thr Tyr Thr Val Gln Gly Asp Tyr Pro Gly225 230 235 240Ser Ala Ala Val Leu Ala Ala Ala Ala Val Thr Gln Ser Asp Val Lys 245 250 255Ile Leu Arg Leu Met Glu Gln Ser Lys Gln Gly Glu Arg Ala Ile Val 260 265 270Asp Val Leu Arg Met Met Glu Val Pro Leu Thr His Glu Asn Asp Val 275 280 285Val His Val Gln Gly Asn Gly Thr Leu Lys Ala Val Glu Phe Asp Gly 290 295 300Asp Ala Ala Thr Asp Ala Val Leu Ala Met Val Ala Ala Ala Val Phe305 310 315 320Ala Glu Gly Thr Ser Arg Phe Tyr Asn Val Glu Asn Leu Arg Tyr Lys 325 330 335Glu Cys Asp Arg Ile Thr Asp Tyr Leu Asn Glu Leu Arg Lys Ala Gly 340 345 350Ala Asn Val Glu Glu Arg Gln Ala Glu Ile Ile Val His Gly Arg Pro 355 360 365Glu Gly Val Glu Gly Gly Val Glu Ile Asn Ala His Tyr Asp His Arg 370 375 380Val Ile Met Ala Leu Thr Val Val Gly Leu Arg Ser Lys Glu Pro Leu385 390 395 400Arg Ile Arg Asp Ala His His Val Ala Lys Ser Tyr Pro Gln Tyr Phe 405 410 415Asp His Leu Gln Ala Leu Gly Ala Ser Val Gln Trp Val Lys Glu 420 425 430201293DNAArtificial Sequencesynthetic gene encoding GRG39 (syngrg39) 20atggatgtca tcgtcaagcc gacgccaagc ctcaatggag agatcggcgc gctgagcagc 60aagaactaca caacaagata tttgctggcg gcggcgctgg cagaaggaac aagcaccatc 120cactaccctg ctcattcaga agattctgat gccatgagaa gatgcatcag ggacctcggc 180gccgtgctgg aagaagatga cagcaagatc gtcatccaag gcttcggctc acatccaaga 240gatgtgaggg agctcaatgt tggaaatgct ggcgccgtgc tgcgcttctt gatgggcgtg 300acggcgctct gcccggaggt gaccttcgtc aacacctacc ccgacagcct cggcaagagg 360cctcatgatg acctcatcga cgcgctgggg cagctaggag tggaggtgca gcatgagcaa 420ggaaggctgc ccatcaccat caagggcggc caagcaaaag gaggccacat cagagtttct 480ggaagcgtca gctctcagta cctctcggcg ctgctcttcg tgacgccgct gctggcagaa 540gattccacca tcgaggtgct aaatgacctc aagagcaagg tggtgatcgg ccagacgctg 600gaggtgctgg agcaagctgg catcgtcatc catgcttcag atgactacat gagcttcaga 660gttcctggag gacaagccta caagccgcag acctacaccg tccaaggaga ttatcctgga 720agcgccgccg tgctggccgc cgccgccgtc acccaaagtg atgtgaagat cctccggctg 780atggagcaat caaagcaagg agaaagggcc atcgtggatg tgctgaggat gatggaggtg 840ccgctcaccc atgaaaatga tgtggtgcat gttcaaggaa atggcacctt gaaggcggtg 900gagtttgatg gagatgctgc aacagatgct gtgctggcaa tggtggcggc ggcggtgttt 960gctgaaggaa catcaagatt ctacaatgtg gagaacttga gatacaagga atgtgacagg 1020atcaccgact acctcaacga gctgaggaag gccggcgcca atgtggagga gaggcaagct 1080gagatcattg ttcatggaag gccagaagga gtggagggcg gcgtggagat caatgctcac 1140tacgaccacc gcgtcatcat ggcgctaaca gtggtggggc tgaggagcaa ggagccgctg 1200aggatcagag atgctcatca tgtggccaag agctaccctc aatattttga tcatcttcaa 1260gctctcggcg cctccgtgca gtgggtgaag gag 1293211260DNARhizobium leguminosarumCDS(1)...(1260) 21atg atg atg ggt aga gcc aaa ctc acg att atc ccg ccg ggc aag cct 48Met Met Met Gly Arg Ala Lys Leu Thr Ile Ile Pro Pro Gly Lys Pro1 5 10 15ttg acc gga cgc gcc atg ccg ccg gga tcg aag tcg atc acc aac cgc 96Leu Thr Gly Arg Ala Met Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg 20 25 30gcg ctg ctg ctg gca ggc ctt gcc aag ggc acg agc cgg ctg acc ggc 144Ala Leu Leu Leu Ala Gly Leu Ala Lys Gly Thr Ser Arg Leu Thr Gly 35 40 45gcg ctg aaa agc gac gat acc cgc tat atg gcc gaa gcg ttg cgc gcc 192Ala Leu Lys Ser Asp Asp Thr Arg Tyr Met Ala Glu Ala Leu Arg Ala 50 55 60atg ggc gtt gcg atc gac gag ccc gac gac acc acc ttc atc gtg aca 240Met Gly Val Ala Ile Asp Glu Pro Asp Asp Thr Thr Phe Ile Val Thr65 70 75 80ggc agt ggc aag ctg cag gcg ccg gca gcc cct ctt ttc ctc ggc aat 288Gly Ser Gly Lys Leu Gln Ala Pro Ala Ala Pro Leu Phe Leu Gly Asn 85 90 95gcc ggc acg gca acg cgc ttc ctg aca gca gca gca gcg cta gtc gaa 336Ala Gly Thr Ala Thr Arg Phe Leu Thr Ala Ala Ala Ala Leu Val Glu 100 105 110ggc aag gtc gtc gtt gat ggc gat gcc cat atg cgc aaa cgg ccg atc 384Gly Lys Val Val Val Asp Gly Asp Ala His Met Arg Lys Arg Pro Ile 115 120 125ggc ccg ctg gtc gac gcg ctc cgc tcg ctc ggc gtg gac gcc tcg acg 432Gly Pro Leu Val Asp Ala Leu Arg Ser Leu Gly Val Asp Ala Ser Thr 130 135 140gaa acc ggc tgc cca ccc gta aca atc aat ggc acc ggc cgt ttc gaa 480Glu Thr Gly Cys Pro Pro Val Thr Ile Asn Gly

Thr Gly Arg Phe Glu145 150 155 160gct agc cgc gtg cag atc gac ggc ggc ctg tcc agc cag tat gtc tcg 528Ala Ser Arg Val Gln Ile Asp Gly Gly Leu Ser Ser Gln Tyr Val Ser 165 170 175gcg ctg ttg atg atg gcg gcc ggc ggc gac cgc gcc gtc gat atc gag 576Ala Leu Leu Met Met Ala Ala Gly Gly Asp Arg Ala Val Asp Ile Glu 180 185 190ctg ctc ggc gaa cat atc ggt gct ctg gga tac atc gac ctg acc gtt 624Leu Leu Gly Glu His Ile Gly Ala Leu Gly Tyr Ile Asp Leu Thr Val 195 200 205gcc gcc atg cgc gcc ttc ggt gcc aag gtc gag cgc gtg agc cca gtc 672Ala Ala Met Arg Ala Phe Gly Ala Lys Val Glu Arg Val Ser Pro Val 210 215 220gcc tgg cgt gtc gag ccc acc ggc tac cat gct gcc gat ttc ctg atc 720Ala Trp Arg Val Glu Pro Thr Gly Tyr His Ala Ala Asp Phe Leu Ile225 230 235 240gag ccg gat gcc tct gct gcg acc tat ctc tgg gct gcc gaa gtg ctt 768Glu Pro Asp Ala Ser Ala Ala Thr Tyr Leu Trp Ala Ala Glu Val Leu 245 250 255ggt ggg ggc aag atc gat ctc ggg acg ccg gcg gaa caa ttc tcg cag 816Gly Gly Gly Lys Ile Asp Leu Gly Thr Pro Ala Glu Gln Phe Ser Gln 260 265 270ccg gat gcg aaa gca tat gat ctg atc tcg aaa ttc ccg cat ctg ccc 864Pro Asp Ala Lys Ala Tyr Asp Leu Ile Ser Lys Phe Pro His Leu Pro 275 280 285gcc gtc atc gac ggt tcg cag atg cag gac gcc atc ccg acg ctt gcg 912Ala Val Ile Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Leu Ala 290 295 300gtt ctc gcc gct ttc aac gag acg ccg gtg cgc ttc gtc ggc atc gag 960Val Leu Ala Ala Phe Asn Glu Thr Pro Val Arg Phe Val Gly Ile Glu305 310 315 320aat ctc cgc gtc aag gaa tgc gat cgt atc cgc gcg ctg tcg agc ggc 1008Asn Leu Arg Val Lys Glu Cys Asp Arg Ile Arg Ala Leu Ser Ser Gly 325 330 335ctg tcg cgc gtc gtt ccg aac ctc ggc acg gaa gag ggc gac gat ctg 1056Leu Ser Arg Val Val Pro Asn Leu Gly Thr Glu Glu Gly Asp Asp Leu 340 345 350atc gtc gca tcc gat ccg agc ctc gcc ggc aag acc ctg ccc gca gag 1104Ile Val Ala Ser Asp Pro Ser Leu Ala Gly Lys Thr Leu Pro Ala Glu 355 360 365atc gac agc ttt gcc gat cat cgc att gcc atg agc ttt gcg ctt gcc 1152Ile Asp Ser Phe Ala Asp His Arg Ile Ala Met Ser Phe Ala Leu Ala 370 375 380ggc ctg aag atc ggc ggc atc acc att ctc gac ccc gat tgc gtc gcc 1200Gly Leu Lys Ile Gly Gly Ile Thr Ile Leu Asp Pro Asp Cys Val Ala385 390 395 400aag acc ttc ccg tcc tat tgg aac gtg ctg gct tcg ctt gga gtc aca 1248Lys Thr Phe Pro Ser Tyr Trp Asn Val Leu Ala Ser Leu Gly Val Thr 405 410 415tac gaa gac tga 1260Tyr Glu Asp22419PRTRhizobium leguminosarum 22Met Met Met Gly Arg Ala Lys Leu Thr Ile Ile Pro Pro Gly Lys Pro1 5 10 15Leu Thr Gly Arg Ala Met Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg 20 25 30Ala Leu Leu Leu Ala Gly Leu Ala Lys Gly Thr Ser Arg Leu Thr Gly 35 40 45Ala Leu Lys Ser Asp Asp Thr Arg Tyr Met Ala Glu Ala Leu Arg Ala 50 55 60Met Gly Val Ala Ile Asp Glu Pro Asp Asp Thr Thr Phe Ile Val Thr65 70 75 80Gly Ser Gly Lys Leu Gln Ala Pro Ala Ala Pro Leu Phe Leu Gly Asn 85 90 95Ala Gly Thr Ala Thr Arg Phe Leu Thr Ala Ala Ala Ala Leu Val Glu 100 105 110Gly Lys Val Val Val Asp Gly Asp Ala His Met Arg Lys Arg Pro Ile 115 120 125Gly Pro Leu Val Asp Ala Leu Arg Ser Leu Gly Val Asp Ala Ser Thr 130 135 140Glu Thr Gly Cys Pro Pro Val Thr Ile Asn Gly Thr Gly Arg Phe Glu145 150 155 160Ala Ser Arg Val Gln Ile Asp Gly Gly Leu Ser Ser Gln Tyr Val Ser 165 170 175Ala Leu Leu Met Met Ala Ala Gly Gly Asp Arg Ala Val Asp Ile Glu 180 185 190Leu Leu Gly Glu His Ile Gly Ala Leu Gly Tyr Ile Asp Leu Thr Val 195 200 205Ala Ala Met Arg Ala Phe Gly Ala Lys Val Glu Arg Val Ser Pro Val 210 215 220Ala Trp Arg Val Glu Pro Thr Gly Tyr His Ala Ala Asp Phe Leu Ile225 230 235 240Glu Pro Asp Ala Ser Ala Ala Thr Tyr Leu Trp Ala Ala Glu Val Leu 245 250 255Gly Gly Gly Lys Ile Asp Leu Gly Thr Pro Ala Glu Gln Phe Ser Gln 260 265 270Pro Asp Ala Lys Ala Tyr Asp Leu Ile Ser Lys Phe Pro His Leu Pro 275 280 285Ala Val Ile Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Leu Ala 290 295 300Val Leu Ala Ala Phe Asn Glu Thr Pro Val Arg Phe Val Gly Ile Glu305 310 315 320Asn Leu Arg Val Lys Glu Cys Asp Arg Ile Arg Ala Leu Ser Ser Gly 325 330 335Leu Ser Arg Val Val Pro Asn Leu Gly Thr Glu Glu Gly Asp Asp Leu 340 345 350Ile Val Ala Ser Asp Pro Ser Leu Ala Gly Lys Thr Leu Pro Ala Glu 355 360 365Ile Asp Ser Phe Ala Asp His Arg Ile Ala Met Ser Phe Ala Leu Ala 370 375 380Gly Leu Lys Ile Gly Gly Ile Thr Ile Leu Asp Pro Asp Cys Val Ala385 390 395 400Lys Thr Phe Pro Ser Tyr Trp Asn Val Leu Ala Ser Leu Gly Val Thr 405 410 415Tyr Glu Asp231257DNAArtificial Sequencesynthetic gene encoding GRG50 (syngrg50) 23atgatgatgg gccgcgccaa gctcaccatc atcccgccgg gcaagccgct caccggccgc 60gccatgccgc cgggcagcaa gagcatcacc aacagggcgc tgctgctggc cggcctcgcc 120aagggcacct caaggctcac cggcgcgctg aagagcgacg acacccgcta catggcggag 180gcgctgagag caatgggcgt cgccatcgac gagccagatg acaccacctt catcgtcacc 240ggctcaggca agctgcaagc gccggcggcg ccgctcttcc tcggcaacgc cggcacggca 300acaagattct tgacggcggc ggcggcgctg gtggaaggca aggtggtggt ggatggtgat 360gctcacatga ggaagaggcc aattgggccg ctggtggacg cgctgaggag cctcggcgtg 420gatgcttcaa cagaaactgg ctgcccgccg gtgaccatca atggaactgg aagatttgaa 480gcatcaagag ttcagatcga cggcggcctc agctctcaat atgtctcggc gctgctgatg 540atggccgccg gcggcgaccg cgccgtggac attgagctgc tgggagagca catcggcgcg 600ctgggctaca tcgacctcac cgtcgccgcc atgagggcct tcggcgccaa ggtggagagg 660gtgtcaccag tggcatggag ggtggagcca actggctacc atgctgctga cttcctcatc 720gagccagatg cttctgctgc cacctacctc tgggcggcgg aggtgctggg cggcggcaag 780atagatcttg ggacgccggc ggagcagttc agccaacctg atgccaaggc ctacgacctc 840atctcaaaat ttcctcatct tcccgccgtc atcgacggca gccagatgca agatgccatc 900ccgacgctgg cggtgctggc ggccttcaat gagacgccgg tgaggttcgt cggcatcgag 960aacctccgcg tcaaggaatg tgacaggatc agggcgctga gcagcggcct ctcaagggtg 1020gtgcccaacc tcggcacaga agaaggagat gacctcatcg tggcttcaga tccaagcctc 1080gccggcaaga cgctgccggc ggagatcgac agctttgctg atcaccgcat cgccatgagc 1140ttcgcgctcg ccggcctcaa gatcggcggc atcaccatcc ttgatccaga ttgtgtggcc 1200aagaccttcc caagctactg gaatgtgctg gcaagcctcg gcgtcaccta tgaagat 125724438PRTStreptomyces coelicolor A3(2) 24Met Thr Val Asn Pro Thr His Thr Ala Leu Trp Pro Ala Pro His Ala1 5 10 15Ser Gly Ala Val Asp Ala Thr Val His Val Pro Gly Ser Lys Ser Val 20 25 30Thr Asn Arg Ala Leu Val Leu Ala Ala Leu Ala Ser Glu Pro Gly Trp 35 40 45Leu Arg Arg Pro Leu Arg Ser Arg Asp Thr Leu Leu Met Ala Glu Ala 50 55 60Leu Arg Thr Leu Gly Val Glu Ile Glu Glu Gly Val Gly Pro Glu Gly65 70 75 80Thr Gly Glu Phe Trp Arg Val Ile Pro Ala Gly Leu Arg Gly Pro Ala 85 90 95Thr Val Asp Val Gly Asn Ala Gly Thr Val Met Arg Phe Leu Pro Pro 100 105 110Val Ala Thr Leu Ala Asp Gly Ala Val Arg Phe Asp Gly Asp Pro Arg 115 120 125Ser Tyr Glu Arg Pro Leu His Gly Val Ile Asp Ala Leu Arg Val Leu 130 135 140Gly Ala Arg Ile Asp Asp Asp Gly Arg Gly Ala Leu Pro Leu Thr Val145 150 155 160His Gly Gly Gly Ala Leu Glu Gly Gly Pro Val Glu Ile Asp Ala Ser 165 170 175Ser Ser Ser Gln Phe Val Ser Ala Leu Leu Leu Ser Gly Pro Arg Phe 180 185 190Asn Gln Gly Val Glu Val Arg His Thr Gly Ser Ala Leu Pro Ser Met 195 200 205Pro His Ile Arg Met Thr Val Asp Met Leu Arg Ala Val Gly Ala Gln 210 215 220Val Asp Thr Pro Glu Ser Gly Gly Glu Pro Asn Val Trp Arg Val Thr225 230 235 240Pro Gly Ala Leu Leu Gly Arg Asp Leu Thr Val Glu Pro Asp Leu Ser 245 250 255Asn Ala Gln Pro Phe Leu Ala Ala Ala Leu Val Thr Gly Gly Lys Val 260 265 270Val Ile Pro Asp Trp Pro Ser Arg Thr Thr Gln Pro Gly Asp Arg Leu 275 280 285Arg Glu Ile Phe Thr Asp Met Gly Gly Ser Cys Glu Leu Thr Asp Phe 290 295 300Gly Leu Val Phe Thr Gly Ser Gly Ala Ile His Gly Ile Asp Val Asp305 310 315 320Leu Ser Glu Val Gly Glu Leu Thr Pro Gly Ile Ala Ala Val Ala Ala 325 330 335Leu Ala Asp Ser Pro Ser Thr Leu Arg Gly Val Ala His Leu Arg Leu 340 345 350His Glu Thr Asp Arg Leu Ala Ala Leu Thr Lys Glu Ile Asn Glu Leu 355 360 365Gly Gly Asp Val Thr Glu Thr Ala Asp Gly Leu His Ile Arg Pro Arg 370 375 380Arg Leu His Gly Gly Val Phe His Thr Tyr Asp Asp His Arg Met Ala385 390 395 400Thr Ala Gly Ala Val Leu Gly Leu Ala Val Glu Gly Val Gln Ile Glu 405 410 415Asn Val Ala Thr Thr Ala Lys Thr Leu Pro Asp Phe Pro Asp Leu Trp 420 425 430Thr Gly Met Leu Gly Ala 43525446PRTStreptomyces avermitilis MA-4680 25Met Thr Val Asn Pro Ala His Thr Ala Leu Trp Pro Ala Pro His Ala1 5 10 15Ser Gly Ala Val Asp Ala Thr Val His Val Pro Gly Ser Lys Ser Val 20 25 30Thr Asn Arg Ala Leu Val Leu Ala Ala Leu Ala Ser Glu Pro Gly Trp 35 40 45Leu Arg Arg Pro Leu Arg Ser Arg Asp Thr Leu Leu Met Ala Ala Ala 50 55 60Leu Arg Glu Met Gly Val Gly Ile Glu Glu Thr Val Ser Ser Ser Ser65 70 75 80Ser Val Gly Gly Gly Ser Asp Gly Ser Gly Glu Ala Trp Arg Val Ile 85 90 95Pro Ala Ala Leu His Gly Pro Ala Thr Val Asp Val Gly Asn Ala Gly 100 105 110Thr Val Met Arg Phe Leu Pro Pro Val Ala Ala Leu Ala Asp Gly Pro 115 120 125Ile Arg Phe Asp Gly Asp Pro Arg Ser Tyr Glu Arg Pro Leu Asn Gly 130 135 140Val Ile Asp Ala Leu Arg Ala Leu Gly Ala Arg Ile Asp Asp Asp Gly145 150 155 160Arg Gly Ala Leu Pro Leu Thr Val His Gly Gly Gly Ala Leu Asp Gly 165 170 175Gly Pro Val Ala Ile Asp Ala Ser Ser Ser Ser Gln Phe Val Ser Ala 180 185 190Leu Leu Leu Ser Gly Pro Arg Phe Asn Gln Gly Val Glu Val Arg His 195 200 205Thr Gly Ser Thr Leu Pro Ser Met Pro His Ile Arg Met Thr Val Asp 210 215 220Met Leu Arg Ala Val Gly Ala Gln Val Asp Thr Pro Glu Ser Gly Gly225 230 235 240Glu Ala Asn Val Trp Arg Val Thr Pro Gly Ala Leu Leu Gly Arg Asp 245 250 255Leu Thr Val Glu Pro Asp Leu Ser Asn Ala Gln Pro Phe Leu Ala Ala 260 265 270Ala Leu Val Thr Gly Gly Lys Val Val Ile Pro Asp Trp Pro Glu Arg 275 280 285Thr Thr Gln Pro Gly Asp Lys Leu Arg Glu Ile Phe Thr Glu Met Gly 290 295 300Gly Ser Cys Glu Leu Thr Glu Gln Gly Leu Glu Phe Thr Gly Ser Gly305 310 315 320Ala Val His Gly Ile Asp Val Asp Leu Ser Glu Val Gly Glu Leu Thr 325 330 335Pro Gly Ile Ala Ala Val Ala Ala Leu Ala Asp Ser Pro Ser Thr Leu 340 345 350Arg Gly Val Ala His Leu Arg Leu His Glu Thr Asp Arg Leu Ala Ala 355 360 365Leu Thr Lys Glu Ile Asn Glu Leu Gly Gly Asp Val Thr Glu Thr Ala 370 375 380Asp Gly Leu Ser Ile Arg Pro Arg Arg Leu His Gly Gly Ile Phe His385 390 395 400Thr Tyr Asp Asp His Arg Met Ala Thr Ala Gly Ala Ile Ile Gly Leu 405 410 415Ala Val Asp Gly Val Gln Ile Glu Asn Val Ala Thr Thr Ala Lys Thr 420 425 430Leu Pro Asp Phe Pro Asp Leu Trp Thr Gly Met Leu Gly Asn 435 440 44526447PRTBacillus halodurans C-125 26Met Thr Arg Phe Asp Glu Asn Ala Arg Ser Pro Trp Thr Pro Leu His1 5 10 15Asp Val Lys Thr Val Glu Leu Phe Pro Leu Asn Gln Arg Leu Asp Gly 20 25 30Ser Ile Thr Leu Pro Gly Ser Lys Ser Leu Thr Asn Arg Ala Leu Ile 35 40 45Ile Ser Ala Leu Ala Asn Ser Asp Ser Met Leu Thr Gly Met Leu Lys 50 55 60Ser Asp Asp Thr Tyr Trp Cys Ile Gln Ala Leu Lys Arg Leu Gly Val65 70 75 80Gln Ile Asn Val Gln Gly Glu Thr Thr Ser Ile Arg Gly Ile Gly Gly 85 90 95Gln Trp Lys Ser Ser Ser Leu Tyr Ile Gly Ala Ala Gly Thr Leu Ala 100 105 110Arg Phe Leu Leu Gly Ala Leu Ala Ile Ser Arg Ser Gly Asn Trp Glu 115 120 125Ile Glu Ala Ser Gln Ser Met Ser Lys Arg Pro Ile Glu Pro Leu Val 130 135 140Gly Val Leu Arg Glu Leu Gly Ala Thr Ile His Tyr Leu Arg Arg Glu145 150 155 160Gly Phe Tyr Pro Leu Ser Ile His Gly Asn Gly Leu Ala Gly Gly Thr 165 170 175Val Arg Leu Ser Gly Gln Met Ser Ser Gln Tyr Ile Ser Gly Leu Leu 180 185 190Ile Ala Ala Pro Tyr Ala Asp Thr Pro Val Thr Ile Thr Val Gln Gly 195 200 205Ser Ile Val Gln His Ala Tyr Val Phe Leu Thr Leu His Leu Met Lys 210 215 220Ser Phe Gly Ala Gln Val Glu Tyr Asp Gln Gln Leu Gln Thr Ile Val225 230 235 240Val His Pro Thr Pro Tyr Thr Cys Gln Asp Ile Asp Leu Glu Ala Asp 245 250 255Ala Ser Thr Ala Cys Tyr Phe Leu Ala Leu Ala Ala Leu Thr Lys Gly 260 265 270Arg Ile Arg Leu Asn Asn Leu Thr Ala Ser Thr Thr Gln Pro Asp Leu 275 280 285His Met Leu Thr Val Phe Glu Lys Met Gly Cys Thr Val Thr Arg Gly 290 295 300Ser Ser Phe Ile Glu Leu Glu Gly Val Ser Gln Leu Lys Gly Gly Phe305 310 315 320Gln Ile Ser Met Asn Glu Met Ser Asp Gln Ala Leu Thr Leu Ala Ala 325 330 335Ile Ala Pro Phe Ala Asp Gly Pro Ile Thr Ile Thr Asp Val Glu His 340 345 350Ile Arg Tyr His Glu Ser Asp Arg Ile Ala Val Ile Cys Glu Ala Leu 355 360 365Thr Arg Leu Gly Ile Gln Val Asp Glu Phe Glu Asp Gly Leu Thr Val 370 375 380Tyr Pro Gly Thr Pro Lys Pro Thr Leu His Pro Leu Ser Thr Tyr Asp385 390 395 400Asp His Arg Val Ala Met Ser Leu Ser Leu Ile Gly Thr Lys Val Lys 405 410 415Gly Leu Arg Leu Asn Asp Pro Gly Cys Val Ala Lys Thr Cys Pro Ser 420 425 430Tyr Phe Gln Leu Leu Glu Gln Leu Gly Ile Gln Val His Tyr Gln 435 440 44527446PRTBacillus clausii KSM-K16 27Met Val Gln Phe Asp Ser Gln Ala Arg Ser Pro Trp Thr Pro Leu Ala1 5 10 15Gly Val Glu Arg Leu Arg Leu Thr Pro Ser Gln Lys Arg Ile Asn Ala 20 25 30Thr Leu Glu Val Pro Gly Ser Lys Ser Ala Thr Asn Arg Ala Leu Leu 35

40 45Leu Ala Ala Val Ala Ser Gly Thr Ser Thr Leu Arg Asn Ala Leu Lys 50 55 60Ser Asp Asp Thr Tyr Trp Cys Ile Glu Ala Leu Lys Lys Thr Gly Val65 70 75 80Glu Ile Ala Val Asp Gly Ser Asn Val Thr Val Tyr Gly Arg Gly Gly 85 90 95Val Phe His Ser Gly Ser Leu Tyr Ile Gly Ser Ala Gly Thr Ala Gly 100 105 110Arg Phe Leu Pro Gly Met Leu Ala Ala Ala Thr Gly Asn Trp His Val 115 120 125Glu Ala Ser His Ser Met Asn Lys Arg Pro Ile Ala Pro Leu Val Lys 130 135 140Thr Leu Gln Ala Leu Gly Ala Asn Ile Gln Tyr Gly Ser Arg Arg Gly145 150 155 160His Tyr Pro Leu Ser Ile Ser Gly Glu Gly Leu Asn Gly Gly Lys Val 165 170 175Asn Met Ser Gly Gln Leu Ser Ser Gln Phe Ile Ser Gly Cys Leu Leu 180 185 190Ala Ala Pro Leu Ala Lys Asn Pro Val Ser Ile Thr Val Lys Asp Gly 195 200 205Ile Val Gln Gln Ala Tyr Val Arg Ile Thr Ile Asp Leu Met Ala Ala 210 215 220Phe Gly Val Glu Val Lys Ala Ala Pro Asp Trp Ser Leu Leu Glu Val225 230 235 240Asn Pro Ser Pro Tyr Val Ala Asn Asp Ile Ala Ile Glu Ala Asp Ala 245 250 255Ser Thr Ala Cys Tyr Phe Leu Ala Leu Ala Ala Ile Thr Ala Gly Lys 260 265 270Ile Arg Ile Arg His Phe Ser Thr Lys Thr Ser Gln Pro Asp Ile Leu 275 280 285Phe Val Ser Ile Leu Lys Arg Met Gly Cys Asn Phe Glu Ile Gly Pro 290 295 300Ser Phe Val Glu Gly Glu Gly Pro Thr Arg Leu Arg Gly Gly Phe Thr305 310 315 320Val Asn Met Asn Glu Leu Ser Asp Gln Ala Leu Thr Leu Ala Ala Ile 325 330 335Ser Pro Phe Ala Asp Gly Pro Ile Ala Ile Glu Gly Val Gly His Ile 340 345 350Arg His His Glu Cys Asp Arg Ile Arg Ala Ile Cys Thr Glu Leu Ser 355 360 365Arg Leu Gly Ile Arg Val Glu Glu Arg His Asp Gly Leu Thr Val Tyr 370 375 380Pro Gly Gln Pro Lys Pro Thr Val Val Asn Thr Tyr Asp Asp His Arg385 390 395 400Met Ala Met Ala Leu Ala Leu Ile Gly Ala Lys Val Asp Gly Ile Glu 405 410 415Leu Asp Asp Pro Gly Cys Val Ala Lys Thr Cys Pro Ser Tyr Phe Ser 420 425 430Met Leu Ala Gln Thr Gly Ile Gly Val Lys Ala Val Ser Pro 435 440 44528460PRTArthrobacter sp. FB24 28Met Thr Gly Thr Ala Pro Thr Glu Ser Ala Thr Ser Gly Pro Val Ala1 5 10 15Asp Val Pro His Trp Pro Ala Pro Phe Ala Glu Ala Pro Val Asp Ala 20 25 30Thr Val Thr Val Pro Gly Ser Lys Ser Leu Thr Asn Arg Tyr Leu Val 35 40 45Leu Ala Ala Leu Ala Asp Gly Pro Ser Arg Leu Arg Ala Pro Leu His 50 55 60Ser Arg Asp Ser Ala Leu Met Ile Glu Ala Leu Arg Gln Leu Gly Ala65 70 75 80Gly Ile Arg Glu Val His Ser Asp Gly Ala Phe Gly Pro Asp Leu Glu 85 90 95Val Thr Pro Leu Arg Ala Asp Ala Ala Ala Thr Asp Ala Ala Ile Asp 100 105 110Cys Gly Leu Ala Gly Thr Val Met Arg Phe Val Pro Pro Val Ala Ala 115 120 125Leu Arg Asn Gly Ala Thr Val Phe Asp Gly Asp Pro His Ala Arg Lys 130 135 140Arg Pro Met Gly Thr Ile Ile Glu Ala Leu Ala Ala Leu Gly Val Asp145 150 155 160Val Arg Ala Ala Asp Gly Thr Pro Pro Ser Ala Leu Pro Phe Thr Val 165 170 175Ala Gly Ser Gly His Val Arg Gly Gly His Leu Val Ile Asp Ala Ser 180 185 190Ala Ser Ser Gln Phe Val Ser Ala Leu Leu Leu Val Gly Ala Arg Phe 195 200 205Thr Glu Gly Leu His Leu Glu His Val Gly Lys Pro Val Pro Ser Leu 210 215 220Asp His Ile Asn Met Thr Val Ala Val Leu Arg Glu Val Gly Val Ser225 230 235 240Val Asp Asp Ser Val Pro Asn His Trp Val Val Ala Pro Gly Arg Ile 245 250 255Arg Ala Phe Asp Arg Arg Ile Glu Gln Asp Leu Ser Asn Ala Gly Pro 260 265 270Phe Leu Ala Ala Ala Leu Ala Thr Arg Gly Thr Val Arg Ile Pro Asn 275 280 285Trp Pro Ser Pro Thr Thr Gln Val Gly Asp Leu Trp Arg Ser Ile Leu 290 295 300Thr Ala Met Gly Ala Thr Val Thr Leu Asp Asn Gly Thr Leu Thr Val305 310 315 320Thr Gly Gly Pro Glu Ile Thr Gly Ala Asp Phe Ala Asp Thr Ser Glu 325 330 335Leu Ala Pro Thr Val Ala Ala Leu Cys Ala Leu Ala Thr Gly Pro Ser 340 345 350Arg Leu Thr Gly Ile Ala His Leu Arg Gly His Glu Thr Asp Arg Leu 355 360 365Ala Ala Leu Val Thr Glu Ile Asn Arg Leu Gly Gly Asp Ala Glu Glu 370 375 380Thr Ser Asp Gly Leu Val Ile Arg Pro Ala Lys Leu His Gly Gly Val385 390 395 400Val His Ser Tyr Ala Asp His Arg Met Ala Thr Ala Gly Ala Ile Leu 405 410 415Gly Leu Ala Val Pro Gly Val Glu Val Glu Asp Ile Gly Thr Thr Ser 420 425 430Lys Thr Met Pro Asp Phe Pro Gln Leu Trp Glu Ser Met Leu Thr Gln 435 440 445Gln Pro Gly Arg Gln Thr Glu Gln Ala Arg Gly Ala 450 455 46029419PRTBrevundomonas vesicularis 29Met Met Met Gly Arg Ala Lys Leu Thr Ile Ile Pro Pro Gly Lys Pro1 5 10 15Leu Thr Gly Arg Ala Met Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg 20 25 30Ala Leu Leu Leu Ala Gly Leu Ala Lys Gly Thr Ser Arg Leu Thr Gly 35 40 45Ala Leu Lys Ser Asp Asp Thr Arg Tyr Met Ala Glu Ala Leu Arg Ala 50 55 60Met Gly Val Thr Ile Asp Glu Pro Asp Asp Thr Thr Phe Ile Val Lys65 70 75 80Gly Ser Gly Lys Leu Gln Pro Pro Ala Ala Pro Leu Phe Leu Gly Asn 85 90 95Ala Gly Thr Ala Thr Arg Phe Leu Thr Ala Ala Ala Ala Leu Val Asp 100 105 110Gly Lys Val Ile Val Asp Gly Asp Ala His Met Arg Lys Arg Pro Ile 115 120 125Gly Pro Leu Val Asp Ala Leu Arg Ser Leu Gly Ile Asp Ala Ser Ala 130 135 140Glu Thr Gly Cys Pro Pro Val Thr Ile Asn Gly Thr Gly Arg Phe Glu145 150 155 160Ala Ser Arg Val Gln Ile Asp Gly Gly Leu Ser Ser Gln Tyr Val Ser 165 170 175Ala Leu Leu Met Met Ala Ala Gly Gly Asp Arg Ala Val Asp Val Glu 180 185 190Leu Leu Gly Glu His Ile Gly Ala Leu Gly Tyr Ile Asp Leu Thr Val 195 200 205Ala Ala Met Arg Ala Phe Gly Ala Lys Val Glu Arg Val Ser Pro Val 210 215 220Ala Trp Arg Val Glu Pro Thr Gly Tyr His Ala Ala Asp Phe Val Ile225 230 235 240Glu Pro Asp Ala Ser Ala Ala Thr Tyr Leu Trp Ala Ala Glu Val Leu 245 250 255Ser Gly Gly Lys Ile Asp Leu Gly Thr Pro Ala Glu Gln Phe Ser Gln 260 265 270Pro Asp Ala Lys Ala Tyr Asp Leu Ile Ser Lys Phe Pro His Leu Pro 275 280 285Ala Val Ile Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Leu Ala 290 295 300Val Leu Ala Ala Phe Asn Glu Met Pro Val Arg Phe Val Gly Ile Glu305 310 315 320Asn Leu Arg Val Lys Glu Cys Asp Arg Ile Arg Ala Leu Ser Ser Gly 325 330 335Leu Ser Arg Ile Val Pro Asn Leu Gly Thr Glu Glu Gly Asp Asp Leu 340 345 350Ile Ile Ala Ser Asp Pro Ser Leu Ala Gly Lys Ile Leu Thr Ala Glu 355 360 365Ile Asp Ser Phe Ala Asp His Arg Ile Ala Met Ser Phe Ala Leu Ala 370 375 380Gly Leu Lys Ile Gly Gly Ile Thr Ile Leu Asp Pro Asp Cys Val Ala385 390 395 400Lys Thr Phe Pro Ser Tyr Trp Asn Val Leu Ser Ser Leu Gly Val Ala 405 410 415Tyr Glu Asp30444PRTOchrobactrum/Brucella strain C58 30Met Ala Cys Leu Pro Asp Asp Ser Gly Pro His Val Gly His Ser Thr1 5 10 15Pro Pro Cys Leu Asp Gln Glu Pro Cys Thr Leu Ser Ser Gln Lys Thr 20 25 30Val Thr Val Thr Pro Pro Asn Phe Pro Leu Thr Gly Lys Val Ala Pro 35 40 45Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala Leu Leu Leu Ala Ala Leu 50 55 60Ala Lys Gly Thr Ser Arg Leu Ser Gly Ala Leu Lys Ser Asp Asp Thr65 70 75 80Arg His Met Ser Val Ala Leu Arg Gln Met Gly Val Thr Ile Asp Glu 85 90 95Pro Asp Asp Thr Thr Phe Val Val Thr Ser Gln Gly Ser Leu Gln Leu 100 105 110Pro Ala Gln Pro Leu Phe Leu Gly Asn Ala Gly Thr Ala Met Arg Phe 115 120 125Leu Thr Ala Ala Val Ala Thr Val Gln Gly Thr Val Val Leu Asp Gly 130 135 140Asp Glu Tyr Met Gln Lys Arg Pro Ile Gly Pro Leu Leu Ala Thr Leu145 150 155 160Gly Gln Asn Gly Ile Gln Val Asp Ser Pro Thr Gly Cys Pro Pro Val 165 170 175Thr Val His Gly Ala Gly Lys Val Gln Ala Arg Arg Phe Glu Ile Asp 180 185 190Gly Gly Leu Ser Ser Gln Tyr Val Ser Ala Leu Leu Met Leu Ala Ala 195 200 205Cys Gly Glu Ala Pro Ile Glu Val Ala Leu Thr Gly Lys Asp Ile Gly 210 215 220Ala Arg Gly Tyr Val Asp Leu Thr Leu Asp Cys Met Arg Ala Phe Gly225 230 235 240Ala Gln Val Asp Ile Val Asp Asp Thr Thr Trp Arg Val Ala Pro Thr 245 250 255Gly Tyr Thr Ala His Asp Tyr Leu Ile Glu Pro Asp Ala Ser Ala Ala 260 265 270Thr Tyr Leu Trp Ala Ala Glu Val Leu Thr Gly Gly Arg Ile Asp Ile 275 280 285Gly Val Ala Ala Gln Asp Phe Thr Gln Pro Asp Ala Lys Ala Gln Ala 290 295 300Val Ile Ala Gln Phe Pro Asn Met Gln Ala Thr Val Val Gly Ser Gln305 310 315 320Met Gln Asp Ala Ile Pro Thr Leu Ala Val Leu Ala Ala Phe Asn Asn 325 330 335Thr Pro Val Arg Phe Thr Glu Leu Ala Asn Leu Arg Val Lys Glu Cys 340 345 350Asp Arg Val Gln Ala Leu His Asp Gly Leu Asn Glu Ile Arg Pro Gly 355 360 365Leu Ala Thr Ile Glu Gly Asp Asp Leu Leu Val Ala Ser Asp Pro Ala 370 375 380Leu Ala Gly Thr Ala Cys Thr Ala Leu Ile Asp Thr His Ala Asp His385 390 395 400Arg Ile Ala Met Cys Phe Ala Leu Ala Gly Leu Lys Val Ser Gly Ile 405 410 415Arg Ile Gln Asp Pro Asp Cys Val Ala Lys Thr Tyr Pro Asp Tyr Trp 420 425 430Lys Ala Leu Ala Ser Leu Gly Val His Leu Ser Tyr 435 44031425PRTAgrobacterium tumefaciens strain C58 31Met Ile Glu Leu Thr Ile Thr Pro Pro Gly His Pro Leu Ser Gly Lys1 5 10 15Val Glu Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala Leu Leu Leu 20 25 30Ala Gly Leu Ala Lys Gly Lys Ser Arg Leu Thr Gly Ala Leu Lys Ser 35 40 45Asp Asp Thr Leu Tyr Met Ala Glu Ala Leu Arg Glu Met Gly Val Lys 50 55 60Val Thr Glu Pro Asp Ala Thr Thr Phe Val Val Glu Ser Ser Gly Gly65 70 75 80Leu His Gln Pro Glu Lys Pro Leu Phe Leu Gly Asn Ala Gly Thr Ala 85 90 95Thr Arg Phe Leu Thr Ala Ala Ala Ala Leu Val Asp Gly Ala Val Ile 100 105 110Ile Asp Gly Asp Glu His Met Arg Lys Arg Pro Ile Met Pro Leu Val 115 120 125Glu Ala Leu Arg Ser Leu Gly Val Glu Ala Glu Ala Pro Thr Gly Cys 130 135 140Pro Pro Val Thr Val Cys Gly Lys Gly Thr Gly Phe Pro Lys Gly Ser145 150 155 160Val Thr Ile Asp Ala Asn Leu Ser Ser Gln Tyr Val Ser Ala Leu Leu 165 170 175Met Ala Ala Ala Cys Gly Asp Lys Pro Val Asp Ile Ile Leu Lys Gly 180 185 190Glu Glu Ile Gly Ala Lys Gly Tyr Ile Asp Leu Thr Thr Ser Ala Met 195 200 205Glu Ala Phe Gly Ala Lys Val Glu Arg Val Ser Asn Ala Ile Trp Arg 210 215 220Val His Pro Thr Gly Tyr Thr Ala Thr Asp Phe His Ile Glu Pro Asp225 230 235 240Ala Ser Ala Ala Thr Tyr Leu Trp Gly Ala Glu Leu Leu Thr Gly Gly 245 250 255Ala Ile Asp Ile Gly Thr Pro Ala Asp Lys Phe Thr Gln Pro Asp Ala 260 265 270Lys Ala His Glu Val Met Ala Gln Phe Pro His Leu Pro Ala Glu Ile 275 280 285Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Ile Ala Val Leu Ala 290 295 300Ala Phe Asn Glu Thr Pro Val Arg Phe Val Gly Ile Ala Asn Leu Arg305 310 315 320Val Lys Glu Cys Asp Arg Ile Arg Ala Val Ser Leu Gly Leu Asn Glu 325 330 335Ile Arg Asp Gly Leu Ala His Glu Glu Gly Asp Asp Leu Ile Val His 340 345 350Ser Asp Pro Ser Leu Ala Gly Gln Thr Val Asn Ala Ser Ile Asp Thr 355 360 365Phe Ala Asp His Arg Ile Ala Met Ser Phe Ala Leu Ala Ala Leu Lys 370 375 380Ile Gly Gly Ile Ala Ile Gln Asn Pro Ala Cys Val Gly Lys Thr Tyr385 390 395 400Pro Gly Tyr Trp Lys Ala Leu Ala Ser Leu Gly Val Glu Tyr Ser Glu 405 410 415Lys Glu Thr Ala Ala Glu Pro Gln His 420 42532418PRTPseudomonas syringae strain 1448a 32Met Arg Pro Gln Ala Thr Leu Thr Val Leu Pro Val Glu Arg Pro Leu1 5 10 15Val Gly Arg Val Ser Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala 20 25 30Leu Leu Leu Ala Gly Leu Ala Lys Gly Thr Ser Arg Leu Thr Gly Ala 35 40 45Leu Lys Ser Asp Asp Thr Arg Val Met Ser Glu Ala Leu Arg Leu Met 50 55 60Gly Val Gln Val Asp Glu Pro Asp Asp Ser Thr Phe Val Val Thr Ser65 70 75 80Ser Gly His Trp Gln Ala Pro Gln Gln Ala Leu Phe Leu Gly Asn Ala 85 90 95Gly Thr Ala Thr Arg Phe Leu Thr Ala Ala Leu Ala Asn Phe Glu Gly 100 105 110Asp Phe Val Val Asp Gly Asp Glu Tyr Met Arg Lys Arg Pro Ile Gly 115 120 125Pro Leu Val Asp Ala Leu Gln Arg Met Gly Val Glu Val Ser Ala Pro 130 135 140Ser Gly Cys Pro Pro Val Ala Ile Lys Gly Lys Gly Gly Leu Glu Ala145 150 155 160Gly Arg Ile Glu Ile Asp Gly Asn Leu Ser Ser Gln Tyr Val Ser Ala 165 170 175Leu Leu Met Ala Gly Ala Cys Gly Lys Gly Pro Val Glu Val Ala Leu 180 185 190Thr Gly Ser Glu Ile Gly Ala Arg Gly Tyr Val Asp Leu Thr Leu Ala 195 200 205Ala Met Gln Ala Phe Gly Ala Glu Val Gln Ala Ile Gly Glu Thr Ala 210 215 220Trp Lys Val Ser Ala Thr Gly Tyr Arg Ala Thr Asp Phe His Ile Glu225 230 235 240Pro Asp Ala Ser Ala Ala Thr Tyr Leu Trp Ala Ala Gln Ala Leu Thr 245 250 255Glu Gly Asp Ile Asp Leu Gly Val Ala Ser Asp Ala Phe Thr Gln Pro 260 265 270Asp Ala Leu Ala Ser Gln Ile Ile Ala Ser Phe Pro Asn Met Pro Ala 275 280 285Val Ile Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Leu Ala Val 290

295 300Leu Ala Ala Phe Asn Arg Gln Pro Val Arg Phe Val Gly Ile Ala Asn305 310 315 320Leu Arg Val Lys Glu Cys Asp Arg Ile Ser Ala Leu Ser His Gly Leu 325 330 335Cys Ala Ile Ala Pro Gly Leu Ala Val Glu Glu Gly Asp Asp Leu Leu 340 345 350Val His Ala Asn Pro Ala Leu Ala Gly Thr Thr Val Asp Ala Leu Ile 355 360 365Asp Thr His Ser Asp His Arg Ile Ala Met Cys Phe Ala Leu Ala Gly 370 375 380Leu Lys Ile Ala Gly Ile Arg Ile Leu Asp Pro Asp Cys Val Gly Lys385 390 395 400Thr Tyr Pro Gly Tyr Trp Asp Ala Leu Ala Ser Leu Gly Val Arg Val 405 410 415Gln Arg 33418PRTPseudomonas syringae strain DC3000 33Met Arg Pro Gln Ala Thr Leu Thr Val Met Pro Val Glu Arg Pro Leu1 5 10 15Val Gly Arg Val Ser Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala 20 25 30Leu Leu Leu Ala Gly Leu Ala Lys Gly Thr Ser Arg Leu Thr Gly Ala 35 40 45Leu Lys Ser Asp Asp Thr Arg Val Met Ser Glu Ala Leu Arg Leu Met 50 55 60Gly Val Gln Val Asp Glu Pro Asp Asp Ser Thr Phe Val Val Thr Ser65 70 75 80Ser Gly His Trp Gln Ala Pro Gln Gln Ala Leu Phe Leu Gly Asn Ala 85 90 95Gly Thr Ala Thr Arg Phe Leu Thr Ala Ala Leu Ala Asn Phe Glu Gly 100 105 110Asp Phe Val Val Asp Gly Asp Glu Tyr Met Arg Lys Arg Pro Ile Gly 115 120 125Pro Leu Val Asp Ala Leu Gln Arg Met Gly Val Glu Ile Ser Ala Pro 130 135 140Ser Gly Cys Pro Pro Val Ala Ile Lys Gly Lys Gly Gly Leu Gln Ala145 150 155 160Gly Arg Ile Glu Ile Asp Gly Asn Leu Ser Ser Gln Tyr Val Ser Ala 165 170 175Leu Leu Met Ala Gly Ala Cys Gly Lys Gly Ser Leu Glu Val Ala Leu 180 185 190Thr Gly Ser Glu Ile Gly Ala Arg Gly Tyr Val Asp Leu Thr Leu Ala 195 200 205Ala Met Gln Ala Phe Gly Ala Glu Val Gln Ala Ile Gly Asp Ala Ala 210 215 220Trp Lys Val Ser Ala Thr Gly Tyr His Ala Thr Asp Phe His Ile Glu225 230 235 240Pro Asp Ala Ser Ala Ala Thr Tyr Leu Trp Ala Ala Gln Ala Leu Thr 245 250 255Glu Gly Asn Ile Asp Leu Gly Val Ala Ser Asp Ala Phe Thr Gln Pro 260 265 270Asp Ala Leu Ala Ser Gln Ile Ile Asp Ser Phe Pro Asn Met Pro Ala 275 280 285Val Ile Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Leu Ala Val 290 295 300Leu Ala Ala Phe Asn Arg Gln Pro Val Arg Phe Val Gly Ile Ala Asn305 310 315 320Leu Arg Val Lys Glu Cys Asp Arg Ile Ser Ala Leu Cys Asp Gly Leu 325 330 335Cys Ala Ile Ala Pro Gly Leu Ala Val Glu Glu Gly Asp Asp Leu Ile 340 345 350Val His Ala Asn Pro Ala Leu Ala Gly Thr Thr Val Asn Ala Leu Ile 355 360 365Asp Thr His Ser Asp His Arg Ile Ala Met Cys Phe Ala Leu Ala Gly 370 375 380Leu Lys Ile Lys Gly Ile His Ile Gln Asp Pro Asp Cys Val Ala Lys385 390 395 400Thr Tyr Pro Gly Tyr Trp Asp Ala Leu Ala Ser Leu Gly Val Ser Val 405 410 415Gln Arg34418PRTPseudomonas syringae strain B728 34Met Arg Pro Gln Ala Thr Leu Thr Val Leu Pro Val Glu Arg Pro Leu1 5 10 15Val Gly Arg Val Ser Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala 20 25 30Leu Leu Leu Ala Gly Leu Ala Lys Gly Thr Ser Arg Leu Thr Gly Ala 35 40 45Leu Lys Ser Asp Asp Thr Arg Val Met Ser Glu Ala Leu Arg Leu Met 50 55 60Gly Val Gln Val Asp Glu Pro Asp Asp Ser Thr Phe Val Val Thr Ser65 70 75 80Ser Gly His Trp Gln Ala Pro Gln Gln Ala Leu Phe Leu Gly Asn Ala 85 90 95Gly Thr Ala Thr Arg Phe Leu Thr Ala Ala Leu Ala Asn Phe Glu Gly 100 105 110Asp Phe Val Val Asp Gly Asp Glu Tyr Met Arg Lys Arg Pro Ile Gly 115 120 125Pro Leu Val Asp Ala Leu Gln Arg Met Gly Val Glu Val Ser Ala Pro 130 135 140Ser Gly Cys Pro Pro Val Ala Ile Lys Gly Lys Gly Gly Leu Glu Ala145 150 155 160Gly Arg Ile Glu Ile Asp Gly Asn Leu Ser Ser Gln Tyr Val Ser Ala 165 170 175Leu Leu Met Ala Gly Ala Cys Gly Lys Gly Pro Val Glu Val Ala Leu 180 185 190Thr Gly Ser Glu Ile Gly Ala Arg Gly Tyr Leu Asp Leu Thr Leu Ala 195 200 205Ala Met Arg Ala Phe Gly Ala Glu Val Gln Ala Ile Gly Asp Ala Ala 210 215 220Trp Lys Val Ser Ala Thr Gly Tyr Arg Ala Thr Asp Phe His Ile Glu225 230 235 240Pro Asp Ala Ser Ala Ala Thr Tyr Leu Trp Ala Ala Gln Ala Leu Thr 245 250 255Glu Gly Ala Ile Asp Leu Gly Val Ala Ser Asn Ala Phe Thr Gln Pro 260 265 270Asp Ala Leu Ala Ser Gln Ile Ile Ala Ser Phe Pro Asn Met Pro Ala 275 280 285Val Ile Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Leu Ala Val 290 295 300Leu Ala Ala Phe Asn Arg Gln Pro Val Arg Phe Val Gly Ile Ala Asn305 310 315 320Leu Arg Val Lys Glu Cys Asp Arg Ile Ser Ala Leu Ser Asn Gly Leu 325 330 335Cys Ala Ile Ala Pro Gly Leu Ala Val Glu Glu Gly Asp Asp Leu Ile 340 345 350Val Thr Ala Asn Pro Thr Leu Ala Gly Thr Thr Val Asp Ala Leu Ile 355 360 365Asp Thr His Ser Asp His Arg Ile Ala Met Cys Phe Ala Leu Ala Gly 370 375 380Leu Lys Ile Ala Gly Ile Arg Ile Leu Asp Pro Asp Cys Val Ala Lys385 390 395 400Thr Tyr Pro Gly Tyr Trp Asp Ala Leu Ala Ser Leu Gly Val Ser Val 405 410 415Gln Arg35425PRTAgrobacterium tumefaciens strain C58 35Met Ile Glu Leu Thr Ile Thr Pro Pro Gly His Pro Leu Ser Gly Lys1 5 10 15Val Glu Pro Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala Leu Leu Leu 20 25 30Ala Gly Leu Ala Lys Gly Lys Ser His Leu Ser Gly Ala Leu Lys Ser 35 40 45Asp Asp Thr Leu Tyr Met Ala Glu Ala Leu Arg Glu Met Gly Val Lys 50 55 60Val Thr Glu Pro Asp Ala Thr Thr Phe Val Val Glu Gly Thr Gly Val65 70 75 80Leu Gln Gln Pro Glu Lys Pro Leu Phe Leu Gly Asn Ala Gly Thr Ala 85 90 95Thr Arg Phe Leu Thr Ala Ala Gly Ala Leu Val Asp Gly Ala Val Ile 100 105 110Ile Asp Gly Asp Glu His Met Arg Lys Arg Pro Ile Leu Pro Leu Val 115 120 125Gln Ala Leu Arg Ala Leu Gly Val Glu Ala Asp Ala Pro Thr Gly Cys 130 135 140Pro Pro Val Thr Val Arg Gly Lys Gly Met Gly Phe Pro Lys Gly Ser145 150 155 160Val Thr Ile Asp Ala Asn Leu Ser Ser Gln Tyr Val Ser Ala Leu Leu 165 170 175Met Ala Ala Ala Cys Gly Asp Lys Pro Val Asp Ile Ile Leu Lys Gly 180 185 190Glu Glu Ile Gly Ala Lys Gly Tyr Ile Asp Leu Thr Thr Ser Ala Met 195 200 205Glu Ala Phe Gly Ala Lys Val Glu Arg Val Ser Asn Ala Ile Trp Arg 210 215 220Val His Pro Thr Gly Tyr Thr Ala Thr Asp Phe His Ile Glu Pro Asp225 230 235 240Ala Ser Ala Ala Thr Tyr Leu Trp Gly Ala Glu Leu Leu Thr Gly Gly 245 250 255Ala Ile Asp Ile Gly Thr Pro Ala Asp Lys Phe Thr Gln Pro Asp Ala 260 265 270Lys Ala Tyr Glu Val Met Ala Gln Phe Pro His Leu Pro Ala Glu Ile 275 280 285Asp Gly Ser Gln Met Gln Asp Ala Ile Pro Thr Ile Ala Val Ile Ala 290 295 300Ala Phe Asn Glu Thr Pro Val Arg Phe Val Gly Ile Ala Asn Leu Arg305 310 315 320Val Lys Glu Cys Asp Arg Ile Arg Ala Val Ser Leu Gly Leu Asn Glu 325 330 335Ile Arg Glu Gly Leu Ala His Glu Glu Gly Asp Asp Leu Ile Val His 340 345 350Ala Asp Pro Ser Leu Ala Gly Gln Thr Val Asp Ala Ser Ile Asp Thr 355 360 365Phe Ala Asp His Arg Ile Ala Met Ser Phe Ala Leu Ala Ala Leu Lys 370 375 380Ile Gly Gly Ile Ala Ile Gln Asn Pro Ala Cys Val Ala Lys Thr Tyr385 390 395 400Pro Gly Tyr Trp Lys Ala Leu Ala Ser Leu Gly Val Asp Tyr Thr Glu 405 410 415Lys Glu Ser Ala Ala Glu Pro Gln His 420 42536444PRTUnknownIsolated from soil sample 36Met Ala Cys Leu Pro Asp Asp Ser Gly Pro His Val Gly His Ser Thr1 5 10 15Pro Pro Arg Leu Asp Gln Glu Pro Cys Thr Leu Ser Ser Gln Lys Thr 20 25 30Val Thr Val Thr Pro Pro Asn Phe Pro Leu Thr Gly Lys Val Ala Pro 35 40 45Pro Gly Ser Lys Ser Ile Thr Asn Arg Ala Leu Leu Leu Ala Ala Leu 50 55 60Ala Lys Gly Thr Ser Arg Leu Ser Gly Ala Leu Lys Ser Asp Asp Thr65 70 75 80Arg His Met Ser Val Ala Leu Arg Gln Met Gly Val Thr Ile Asp Glu 85 90 95Pro Asp Asp Thr Thr Phe Val Val Thr Ser Gln Gly Ser Leu Gln Leu 100 105 110Pro Ala Gln Pro Leu Phe Leu Gly Asn Ala Gly Thr Ala Met Arg Phe 115 120 125Leu Thr Ala Ala Val Ala Thr Val Gln Gly Thr Val Val Leu Asp Gly 130 135 140Asp Glu Tyr Met Gln Lys Arg Pro Ile Gly Pro Leu Leu Ala Thr Leu145 150 155 160Gly Gln Asn Gly Ile Gln Val Asp Ser Pro Thr Gly Cys Pro Pro Val 165 170 175Thr Val His Gly Met Gly Lys Val Gln Ala Lys Arg Phe Glu Ile Asp 180 185 190Gly Gly Leu Ser Ser Gln Tyr Val Ser Ala Leu Leu Met Leu Ala Ala 195 200 205Cys Gly Glu Ala Pro Ile Glu Val Ala Leu Thr Gly Lys Asp Ile Gly 210 215 220Ala Arg Gly Tyr Val Asp Leu Thr Leu Asp Cys Met Arg Ala Phe Gly225 230 235 240Ala Gln Val Asp Ala Val Asp Asp Thr Thr Trp Arg Val Ala Pro Thr 245 250 255Gly Tyr Thr Ala His Asp Tyr Leu Ile Glu Pro Asp Ala Ser Ala Ala 260 265 270Thr Tyr Leu Trp Ala Ala Glu Val Leu Thr Gly Gly Arg Ile Asp Ile 275 280 285Gly Val Ala Ala Gln Asp Phe Thr Gln Pro Asp Ala Lys Ala Gln Ala 290 295 300Val Ile Ala Gln Phe Pro Asn Met Gln Ala Thr Val Val Gly Ser Gln305 310 315 320Met Gln Asp Ala Ile Pro Thr Leu Ala Val Leu Ala Ala Phe Asn Asn 325 330 335Thr Pro Val Arg Phe Thr Glu Leu Ala Asn Leu Arg Val Lys Glu Cys 340 345 350Asp Arg Val Gln Ala Leu His Asp Gly Leu Asn Glu Ile Arg Pro Gly 355 360 365Leu Ala Thr Ile Glu Gly Asp Asp Leu Leu Val Ala Ser Asp Pro Ala 370 375 380Leu Ala Gly Thr Ala Cys Thr Ala Leu Ile Asp Thr His Ala Asp His385 390 395 400Arg Ile Ala Met Cys Phe Ala Leu Ala Gly Leu Lys Val Ser Gly Ile 405 410 415Arg Ile Gln Asp Pro Asp Cys Val Ala Lys Thr Tyr Pro Asp Tyr Trp 420 425 430Lys Ala Trp Pro Ser Leu Gly Val His Leu Asn Asp 435 44037427PRTEscherichia coli 37Met Glu Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp Gly Thr Ile1 5 10 15Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu Ala 20 25 30Ala Leu Ala His Gly Lys Thr Val Leu Thr Asn Leu Leu Asp Ser Asp 35 40 45Asp Val Arg His Met Leu Asn Ala Leu Thr Ala Leu Gly Val Ser Tyr 50 55 60Thr Leu Ser Ala Asp Arg Thr Arg Cys Glu Ile Ile Gly Asn Gly Gly65 70 75 80Pro Leu His Ala Glu Gly Ala Leu Glu Leu Phe Leu Gly Asn Ala Gly 85 90 95Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Ser Asn Asp 100 105 110Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His 115 120 125Leu Val Asp Ala Leu Arg Leu Gly Gly Ala Lys Ile Thr Tyr Leu Glu 130 135 140Gln Glu Asn Tyr Pro Pro Leu Arg Leu Gln Gly Gly Phe Thr Gly Gly145 150 155 160Asn Val Asp Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 165 170 175Leu Met Thr Ala Pro Leu Ala Pro Glu Asp Thr Val Ile Arg Ile Lys 180 185 190Gly Asp Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu Asn Leu Met 195 200 205Lys Thr Phe Gly Val Glu Ile Glu Asn Gln His Tyr Gln Gln Phe Val 210 215 220Val Lys Gly Gly Gln Ser Tyr Gln Ser Pro Gly Thr Tyr Leu Val Glu225 230 235 240Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Ala Ala Ile Lys 245 250 255Gly Gly Thr Val Lys Val Thr Gly Ile Gly Arg Asn Ser Met Gln Gly 260 265 270Asp Ile Arg Phe Ala Asp Val Leu Glu Lys Met Gly Ala Thr Ile Cys 275 280 285Trp Gly Asp Asp Tyr Ile Ser Cys Thr Arg Gly Glu Leu Asn Ala Ile 290 295 300Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr305 310 315 320Ala Ala Leu Phe Ala Lys Gly Thr Thr Thr Leu Arg Asn Ile Tyr Asn 325 330 335Trp Arg Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu 340 345 350Arg Lys Val Gly Ala Glu Val Glu Glu Gly His Asp Tyr Ile Arg Ile 355 360 365Thr Pro Pro Glu Lys Leu Asn Phe Ala Glu Ile Ala Thr Tyr Asn Asp 370 375 380His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro385 390 395 400Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 405 410 415Phe Glu Gln Leu Ala Arg Ile Ser Gln Ala Ala 420 42538444PRTZea mays 38Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser Gly1 5 10 15Thr Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu 20 25 30Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn 35 40 45Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu Arg Thr Leu Gly Leu 50 55 60Ser Val Glu Ala Asp Lys Ala Ala Lys Arg Ala Val Val Val Gly Cys65 70 75 80Gly Gly Lys Phe Pro Val Glu Asp Ala Lys Glu Glu Val Gln Leu Phe 85 90 95Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 100 105 110Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg Met 115 120 125Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu Gly 130 135 140Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val Arg Val145 150 155 160Asn Gly Ile Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 165 170 175Ile Ser Ser Gln Tyr Leu Ser Ala Leu Leu Met Ala Ala Pro Leu Ala 180 185 190Leu Gly Asp Val Glu Ile

Glu Ile Ile Asp Lys Leu Ile Ser Ile Pro 195 200 205Tyr Val Glu Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys Ala 210 215 220Glu His Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln Lys225 230 235 240Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala 245 250 255Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr Val 260 265 270Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 275 280 285Val Leu Glu Met Met Gly Ala Lys Val Thr Trp Thr Glu Thr Ser Val 290 295 300Thr Val Thr Gly Pro Pro Arg Glu Pro Phe Gly Arg Lys His Leu Lys305 310 315 320Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 325 330 335Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp Val 340 345 350Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg Thr 355 360 365Glu Leu Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Pro Asp Tyr Cys 370 375 380Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala Ile Asp Thr Tyr385 390 395 400Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Glu 405 410 415Val Pro Val Thr Ile Arg Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro 420 425 430Asp Tyr Phe Asp Val Leu Ser Thr Phe Val Lys Asn 435 44039455PRTAgrobacterium sp. CP4 39Met Ser His Gly Ala Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser1 5 10 15Gly Leu Ser Gly Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 20 25 30Arg Ser Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 35 40 45Gly Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 50 55 60Ala Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp65 70 75 80Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu Asp Phe 85 90 95Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly Leu Val Gly Val 100 105 110Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp Ala Ser Leu Thr Lys Arg 115 120 125Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val 130 135 140Lys Ser Glu Asp Gly Asp Arg Leu Pro Val Thr Leu Arg Gly Pro Lys145 150 155 160Thr Pro Thr Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 165 170 175Lys Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 180 185 190Val Ile Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 195 200 205Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly Val Arg 210 215 220Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly Gln Val Ile Asp225 230 235 240Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu 245 250 255Leu Val Pro Gly Ser Asp Val Thr Ile Leu Asn Val Leu Met Asn Pro 260 265 270Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 275 280 285Glu Val Ile Asn Pro Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 290 295 300Arg Val Arg Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg305 310 315 320Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala Ala 325 330 335Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu Leu Arg Val 340 345 350Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn Gly Leu Lys Leu Asn 355 360 365Gly Val Asp Cys Asp Glu Gly Glu Thr Ser Leu Val Val Arg Gly Arg 370 375 380Pro Asp Gly Lys Gly Leu Gly Asn Ala Ser Gly Ala Ala Val Ala Thr385 390 395 400His Leu Asp His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 405 410 415Ser Glu Asn Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 420 425 430Phe Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu 435 440 445Leu Ser Asp Thr Lys Ala Ala 450 45540428PRTBacillus subtilis 40Met Lys Arg Asp Lys Val Gln Thr Leu His Gly Glu Ile His Ile Pro1 5 10 15Gly Asp Lys Ser Ile Ser His Arg Ser Val Met Phe Gly Ala Leu Ala 20 25 30Ala Gly Thr Thr Thr Val Lys Asn Phe Leu Pro Gly Ala Asp Cys Leu 35 40 45Ser Thr Ile Asp Cys Phe Arg Lys Met Gly Val His Ile Glu Gln Ser 50 55 60Ser Ser Asp Val Val Ile His Gly Lys Gly Ile Asp Ala Leu Lys Glu65 70 75 80Pro Glu Ser Leu Leu Asp Val Gly Asn Ser Gly Thr Thr Ile Arg Leu 85 90 95Met Leu Gly Ile Leu Ala Gly Arg Pro Phe Tyr Ser Ala Val Ala Gly 100 105 110Asp Glu Ser Ile Ala Lys Arg Pro Met Lys Arg Val Thr Glu Pro Leu 115 120 125Lys Lys Met Gly Ala Lys Ile Asp Gly Arg Ala Gly Gly Glu Phe Thr 130 135 140Pro Leu Ser Val Ser Gly Ala Ser Leu Lys Gly Ile Asp Tyr Val Ser145 150 155 160Pro Val Ala Ser Ala Gln Ile Lys Ser Ala Val Leu Leu Ala Gly Leu 165 170 175Gln Ala Glu Gly Thr Thr Thr Val Thr Glu Pro His Lys Ser Arg Asp 180 185 190His Thr Glu Arg Met Leu Ser Ala Phe Gly Val Lys Leu Ser Glu Asp 195 200 205Gln Thr Ser Val Ser Ile Ala Gly Gly Gln Lys Leu Thr Ala Ala Asp 210 215 220Ile Phe Val Pro Gly Asp Ile Ser Ser Ala Ala Phe Phe Leu Ala Ala225 230 235 240Gly Ala Met Val Pro Asn Ser Arg Ile Val Leu Lys Asn Val Gly Leu 245 250 255Asn Pro Thr Arg Thr Gly Ile Ile Asp Val Leu Gln Asn Met Gly Ala 260 265 270Lys Leu Glu Ile Lys Pro Ser Ala Asp Ser Gly Ala Glu Pro Tyr Gly 275 280 285Asp Leu Ile Ile Glu Thr Ser Ser Leu Lys Ala Val Glu Ile Gly Gly 290 295 300Asp Ile Ile Pro Arg Leu Ile Asp Glu Ile Pro Ile Ile Ala Leu Leu305 310 315 320Ala Thr Gln Ala Glu Gly Thr Thr Val Ile Lys Asp Ala Ala Glu Leu 325 330 335Lys Val Lys Glu Thr Asn Arg Ile Asp Thr Val Val Ser Glu Leu Arg 340 345 350Lys Leu Gly Ala Glu Ile Glu Pro Thr Ala Asp Gly Met Lys Val Tyr 355 360 365Gly Lys Gln Thr Leu Lys Gly Gly Ala Ala Val Ser Ser His Gly Asp 370 375 380His Arg Ile Gly Met Met Leu Gly Ile Ala Ser Cys Ile Thr Glu Glu385 390 395 400Pro Ile Glu Ile Glu His Thr Asp Ala Ile His Val Ser Tyr Pro Thr 405 410 415Phe Phe Glu His Leu Asn Lys Leu Ser Lys Lys Ser 420 42541431PRTEnterobacteriaceae 41Met Lys Val Thr Ile Gln Pro Gly Asp Leu Thr Gly Ile Leu Gln Ser1 5 10 15Pro Ala Ser Lys Ser Ser Met Gln Arg Ala Cys Ala Ala Ala Leu Val 20 25 30Ala Lys Gly Ile Ser Glu Ile Ile Asn Pro Gly His Ser Asn Asp Asp 35 40 45Lys Ala Ala Arg Asp Ile Val Ser Arg Leu Gly Ala Arg Leu Glu Asp 50 55 60Gln Pro Asp Gly Ser Leu Gln Ile Thr Ser Glu Gly Val Lys Pro Val65 70 75 80Ala Pro Phe Ile Asp Cys Gly Glu Ser Gly Leu Ser Ile Arg Met Phe 85 90 95Thr Pro Ile Val Ala Leu Ser Lys Glu Glu Val Thr Ile Lys Gly Ser 100 105 110Gly Ser Leu Val Thr Arg Pro Met Asp Phe Phe Asp Glu Ile Leu Pro 115 120 125His Leu Gly Val Lys Val Lys Ser Asn Gln Gly Lys Leu Pro Leu Val 130 135 140Ile Gln Gly Pro Leu Lys Pro Ala Asp Val Thr Val Asp Gly Ser Leu145 150 155 160Ser Ser Gln Phe Leu Thr Gly Leu Leu Leu Ala Tyr Ala Ala Ala Asp 165 170 175Ala Ser Asp Val Ala Ile Lys Val Thr Asn Leu Lys Ser Arg Pro Tyr 180 185 190Ile Asp Leu Thr Leu Asp Val Met Lys Arg Phe Gly Leu Lys Thr Pro 195 200 205Glu Asn Arg Asn Tyr Glu Glu Phe Tyr Phe Lys Ala Gly Asn Val Tyr 210 215 220Asp Glu Thr Lys Met Gln Arg Tyr Thr Val Glu Gly Asp Trp Ser Gly225 230 235 240Gly Ala Phe Leu Leu Val Ala Gly Ala Ile Ala Gly Pro Ile Thr Val 245 250 255Arg Gly Leu Asp Ile Ala Ser Thr Gln Ala Asp Lys Ala Ile Val Gln 260 265 270Ala Leu Met Ser Ala Asn Ala Gly Ile Ala Ile Asp Ala Lys Glu Ile 275 280 285Lys Leu His Pro Ala Asp Leu Asn Ala Phe Glu Phe Asp Ala Thr Asp 290 295 300Cys Pro Asp Leu Phe Pro Pro Leu Val Ala Leu Ala Ser Tyr Cys Lys305 310 315 320Gly Glu Thr Lys Ile Lys Gly Val Ser Arg Leu Ala His Lys Glu Ser 325 330 335Asp Arg Gly Leu Thr Leu Gln Asp Glu Phe Gly Lys Met Gly Val Glu 340 345 350Ile His Leu Glu Gly Asp Leu Met Arg Val Ile Gly Gly Lys Gly Val 355 360 365Lys Gly Ala Glu Val Ser Ser Arg His Asp His Arg Ile Ala Met Ala 370 375 380Cys Ala Val Ala Ala Leu Lys Ala Val Gly Glu Thr Thr Ile Glu His385 390 395 400Ala Glu Ala Val Asn Lys Ser Tyr Pro Asp Phe Tyr Ser Asp Leu Lys 405 410 415Gln Leu Gly Gly Val Val Ser Leu Asn His Gln Phe Asn Phe Ser 420 425 430

Claims

1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:8; b) a polypeptide encoded by the nucleotide sequence of SEQ ID NO:7 or 9; c) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:8, wherein said polypeptide has herbicide resistance activity; d) a polypeptide that is encoded by the herbicide resistance nucleotide sequence of the DNA insert of the plasmid deposited as Accession No. NRRL B-30934.

2. The polypeptide of claim 11 further comprising a heterologous amino acid sequence.