Patent application title: Methods For Producing Hyaluronan In A Recombinant Host Cell
Inventors:
Alan Sloma (Davis, CA, US)
Regine Behr (Roseville, CA, US)
William Widner (Davis, CA, US)
William Widner (Davis, CA, US)
Stephen Brown (Davis, CA, US)
Stephen Brown (Davis, CA, US)
Maria Tang (Fairfield, CA, US)
Maria Tang (Fairfield, CA, US)
David Sternberg (Davis, CA, US)
Assignees:
Novozymes Biopharma DK A/S
IPC8 Class: AC12P1926FI
USPC Class:
435 84
Class name: Micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition preparing compound containing saccharide radical preparing nitrogen-containing saccharide
Publication date: 2014-02-06
Patent application number: 20140038235
Abstract:
The present invention relates to methods for producing a hyaluronic acid,
comprising: (a) cultivating a Bacillus host cell under conditions
suitable for production of the hyaluronic acid, wherein the Bacillus host
cell comprises a nucleic acid construct comprising a hyaluronan synthase
encoding sequence operably linked to a promoter sequence foreign to the
hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic
acid from the cultivation medium. The present invention also relates to
an isolated nucleic acid sequence encoding a hyaluronan synthase operon
comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase
gene, and optionally one or more genes selected from the group consisting
of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine
pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The
present invention also relates to isolated nucleic acid sequences
encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase,
and UDP-N-acetylglucosamine pyrophosphorylaseClaims:
1. A Bacillus host cell comprising an artificial operon, wherein the
operon comprises a hyaluronan synthase encoding sequence, a UDP-glucose
6-dehydrogenase encoding sequence, and a UDP-glucose pyrophosphorylase
encoding sequence; wherein the hyaluronan synthase encoding sequence is
(i) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO:
2; or (ii) a nucleic acid sequence which hybridizes under high stringency
conditions with SEQ ID NO: 1 or its full-length complement; wherein the
UDP-glucose 6-dehydrogenase encoding sequence is (i) a nucleic acid
sequence encoding a polypeptide comprising SEQ ID NO: 12; or (ii) a
nucleic acid sequence which hybridizes under high stringency conditions
with SEQ ID NO: 11 or its full-length complement; wherein the UDP-glucose
pyrophosphorylase encoding sequence is (i) a nucleic acid sequence
encoding a polypeptide comprising SEQ ID NO: 22; or (ii) a nucleic acid
sequence which hybridizes under high stringency conditions with SEQ ID
NO: 21 or its full-length complement; and wherein high stringency
conditions are defined as prehybridization and hybridization at
42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and
denatured salmon sperm DNA, and 50% formamide, and washing three times
each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at
65.degree. C.
2. The Bacillus host cell of claim 1, wherein the hyaluronan synthase encoding sequence encodes a polypeptide comprising SEQ ID NO: 2.
3. The Bacillus host cell of claim 1, wherein the hyaluronan synthase encoding sequence is a nucleic acid sequence which hybridizes under high or very high stringency conditions with SEQ ID NO: 94 or its full-length complement; wherein high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 65.degree. C. and wherein very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 70.degree. C.
4. The Bacillus host cell of claim 1, wherein the UDP-glucose 6-dehydrogenase encoding sequence encodes a polypeptide comprising SEQ ID NO: 12.
5. The Bacillus host cell of claim 1, wherein the UDP-glucose 6-dehydrogenase encoding sequence is a nucleic acid sequence which hybridizes under high or very high stringency conditions with SEQ ID NO: 11 or its full-length complement; wherein high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 65.degree. C. and wherein very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 70.degree. C.
6. The Bacillus host cell of claim 1, wherein the UDP-glucose pyrophosphorylase encoding sequence encodes a polypeptide comprising SEQ ID NO: 22.
7. The Bacillus host cell of claim 1, wherein the UDP-glucose pyrophosphorylase encoding sequence is a nucleic acid sequence which hybridizes under high or very high stringency conditions with SEQ ID NO: 21 or its full-length complement; wherein high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 65.degree. C. and wherein very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 70.degree. C.
8. The Bacillus host cell of claim 1, wherein the artificial operon further comprises one or more additional genes encoding enzymes in the biosynthesis of a precursor sugar of the hyaluronic acid or the Bacillus host cell further comprises one or more nucleic acid constructs comprising one or more additional genes encoding enzymes in the biosynthesis of a precursor sugar of the hyaluronic acid.
9. The Bacillus host cell of claim 8, wherein the one or more additional genes encoding enzymes in the biosynthesis of a precursor sugar of the hyaluronic acid are selected from the group consisting of a UDP-N-acetylglucosamine pyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinase gene, phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyl transferase gene.
10. The Bacillus host cell of claim 9, wherein the UDP-N-acetylglucosamine pyrophosphorylase encoding sequence encodes a polypeptide comprising SEQ ID NO: 30.
11. The Bacillus host cell of claim 9, wherein the UDP-N-acetylglucosamine pyrophosphorylase encoding sequence is (a) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO: 30; or (b) a nucleic acid sequence which hybridizes under high or very high stringency conditions with SEQ ID NO: 29 or its full-length complement; wherein high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 65.degree. C. and wherein very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 70.degree. C.
12. The Bacillus host cell of claim 9, wherein the glucose-6-phosphate isomerase encoding sequence encodes a polypeptide comprising SEQ ID NO: 101.
13. The Bacillus host cell of claim 9, wherein the glucose-6-phosphate isomerase encoding sequence is (a) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO: 101; or (b) a nucleic acid sequence which hybridizes under high or very high stringency conditions with SEQ ID NO: 100 or its full-length complement; wherein high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 65.degree. C. and wherein very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 70.degree. C.
14. The Bacillus host cell of claim 9, wherein the one or more additional genes selected from the group of the UDP-N-acetylglucosamine pyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinase gene, phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyl transferase gene are under the control of the same or a different promoter(s) as the hyaluronan synthase encoding sequence.
15. The Bacillus host cell of claim 1, wherein the artificial operon further comprises an mRNA processing/stabilizing sequence located downstream of the short "consensus" amyQ promoter operably linked to the hyaluronan synthase encoding sequence, the UDP-glucose 6-dehydrogenase encoding sequence, and the UDP-glucose pyrophosphorylase encoding sequence and upstream of the hyaluronan synthase encoding sequence, the UDP-glucose 6-dehydrogenase encoding sequence, and the UDP-glucose pyrophosphorylase encoding sequence.
16. The Bacillus host cell of claim 1, wherein the artificial operon further comprises a selectable marker gene.
17. The Bacillus host cell of claim 1, wherein the artificial operon further comprises a short "consensus" amyQ promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region operably linked to the hyaluronan synthase encoding sequence, the UDP-glucose 6-dehydrogenase encoding sequence, and the UDP-glucose pyrophosphorylase encoding sequence.
18. The Bacillus host cell of claim 1, wherein the Bacillus host cell is selected from the group consisting of Bacillus agaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.
19. The Bacillus host cell of claim 1, wherein the Bacillus host cell is a Bacillus licheniformis cell or a Bacillus subtilis cell.
20. The Bacillus host cell of claim 1, wherein the Bacillus host cell is unmarked with a selectable marker.
21. The Bacillus host cell of claim 1, wherein the artificial operon is integrated into the chromosome of the Bacillus host cell.
22. A method for producing hyaluronic acid, comprising: (a) cultivating the Bacillus host cell of claim 1 in a cultivation medium under conditions suitable for production of the hyaluronic acid; and (b) recovering the hyaluronic acid from the cultivation medium.
Description:
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser. No. 13/401,663 filed Feb. 21, 2012, which is a divisional of U.S. application Ser. No. 13/084,230 filed Apr. 11, 2011, now U.S. Pat. No. 8,137,951, which is a divisional of U.S. application Ser. No. 12/891,548 filed Sep. 27, 2010, now U.S. Pat. No. 8,093,036, which is a divisional of U.S. application Ser. No. 10/326,185 filed Dec. 20, 2002, now U.S. Pat. No. 7,811,806, which claims priority from U.S. Provisional Application Ser. No. 60/342,644 filed Dec. 21, 2001. The contents of these applications are fully incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to methods for producing a hyaluronan in a recombinant host cell.
[0004] 2. Description of the Related Art
[0005] The most abundant heteropolysaccharides of the body are the glycosaminoglycans. Glycosaminoglycans are unbranched carbohydrate polymers, consisting of repeating disaccharide units (only keratan sulphate is branched in the core region of the carbohydrate). The disaccharide units generally comprise, as a first saccharide unit, one of two modified sugars --N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc). The second unit is usually an uronic acid, such as glucuronic acid (GlcUA) or iduronate.
[0006] Glycosaminoglycans are negatively charged molecules, and have an extended conformation that imparts high viscosity when in solution. Glycosaminoglycans are located primarily on the surface of cells or in the extracellular matrix. Glycosaminoglycans also have low compressibility in solution and, as a result, are ideal as a physiological lubricating fluid, e.g., joints. The rigidity of glycosaminoglycans provides structural integrity to cells and provides passageways between cells, allowing for cell migration. The glycosaminoglycans of highest physiological importance are hyaluronan, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Most glycosaminoglycans bind covalently to a proteoglycan core protein through specific oligosaccharide structures. Hyaluronan forms large aggregates with certain proteoglycans, but is an exception as free carbohydrate chains form non-covalent complexes with proteoglycans.
[0007] Numerous roles of hyaluronan in the body have been identified (see, Laurent T. C. and Fraser J. R. E., 1992, FASEB J. 6: 2397-2404; and Toole B. P., 1991, "Proteoglycans and hyaluronan in morphogenesis and differentiation." In: Cell Biology of the Extracellular Matrix, pp. 305-341, Hay E. D., ed., Plenum, N.Y.). Hyaluronan is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis. Hyaluronan is also suspected of having a role in numerous physiological functions, such as adhesion, development, cell motility, cancer, angiogenesis, and wound healing. Due to the unique physical and biological properties of hyaluronan, it is employed in eye and joint surgery and is being evaluated in other medical procedures. Products of hyaluronan have also been developed for use in orthopaedics, rheumatology, and dermatology.
[0008] Rooster combs are a significant commercial source for hyaluronan. Microorganisms are an alternative source. U.S. Pat. No. 4,801,539 discloses a fermentation method for preparing hyaluronic acid involving a strain of Streptococcus zooepidemicus with reported yields of about 3.6 g of hyaluronic acid per liter. European Patent No. EP0694616 discloses fermentation processes using an improved strain of Streptococcus zooepidemicus with reported yields of about 3.5 g of hyaluronic acid per liter.
[0009] The microorganisms used for production of hyaluronic acid by fermentation are strains of pathogenic bacteria, foremost among them being several Streptococcus spp. The group A and group C streptococci surround themselves with a nonantigenic capsule composed of hyaluronan, which is identical in composition to that found in connective tissue and joints. Pasteurella multocida, another pathogenic encapsulating bacteria, also surrounds its cells with hyaluronan.
[0010] Hyaluronan synthases have been described from vertebrates, bacterial pathogens, and algal viruses (DeAngelis, P. L., 1999, Cell. Mol. Life Sci. 56: 670-682). WO 99/23227 discloses a Group I hyaluronate synthase from Streptococcus equisimilis. WO 99/51265 and WO 00/27437 describe a Group II hyaluronate synthase from Pasturella multocida. Ferretti et al. disclose the hyaluronan synthase operon of Streptococcus pyogenes, which is composed of three genes, hasA, hasB, and hasC, that encode hyaluronate synthase, UDP glucose dehydrogenase, and UDP-glucose pyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98, 4658-4663, 2001). WO 99/51265 describes a nucleic acid segment having a coding region for a Streptococcus equisimilis hyaluronan synthase.
[0011] Bacilli are well established as host cell systems for the production of native and recombinant proteins. It is an object of the present invention to provide methods for producing a hyaluronan in a recombinant Bacillus host cell.
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium.
[0013] In preferred embodiments, the nucleic acid construct further comprises one or more genes encoding enzymes in the biosynthesis of a precursor sugar of the hyaluronic acid or the Bacillus host cell further comprises one or more second nucleic acid constructs comprising one or more genes encoding enzymes in the biosynthesis of the precursor sugar.
[0014] In another preferred embodiment, the one or more genes encoding a precursor sugar are under the control of the same or a different promoter(s) as the hyaluronan synthase encoding sequence.
[0015] The present invention also relates to Bacillus host cells comprising a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence, and to such nucleic acid constructs.
[0016] The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene or a portion thereof and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene.
[0017] The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase selected from the group consisting of: (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 41; (b) a nucleic acid sequence having at least about 75%, about 80%, about 85%, about 90%, or about 95% homology to SEQ ID NO: 40; (c) a nucleic acid sequence which hybridizes under medium or high stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 40, (ii) the cDNA sequence contained in SEQ ID NO: 40, or (iii) a complementary strand of (i) or (ii); and (d) a subsequence of (a), (b), or (c), wherein the subsequence encodes a polypeptide fragment which has UDP-glucose 6-dehydrogenase activity.
[0018] The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose pyrophosphorylase selected from the group consisting of: (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least about 90%, about 95%, or about 97% identity to SEQ ID NO: 43; (b) a nucleic acid sequence having at least about 90%, about 95%, or about 97% homology to SEQ ID NO: 42; (c) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 42, (ii) the cDNA sequence contained in SEQ ID NO: 42, or (iii) a complementary strand of (i) or (ii); and (d) a subsequence of (a), (b), or (c), wherein the subsequence encodes a polypeptide fragment which has UDP-N-acetylglucosamine pyrophosphorylase activity.
[0019] The present invention also relates to isolated nucleic acid sequences encoding a UDP-N-acetylglucosamine pyrophosphorylase selected from the group consisting of: (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 45; (b) a nucleic acid sequence having at least about 75%, about 80%, about 85%, about 90%, or about 95% homology to SEQ ID NO: 44; (c) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 44, (ii) the cDNA sequence contained in SEQ ID NO: 44, or (iii) a complementary strand of (i) or (ii); and (d) a subsequence of (a), (b), or (c), wherein the subsequence encodes a polypeptide fragment which has UDP-N-acetylglucosamine pyrophosphorylase activity.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows the chemical structure of hyaluronan.
[0021] FIG. 2 shows the biosynthetic pathway for hyaluronan synthesis.
[0022] FIG. 3 shows a restriction map of pCR2.1-sehasA.
[0023] FIG. 4 shows a restriction map of pCR2.1-tuaD.
[0024] FIG. 5 shows a restriction map of pCR2.1-gtaB.
[0025] FIG. 6 shows a restriction map of pCR2.1-gcaD.
[0026] FIG. 7 shows a restriction map of pHA1.
[0027] FIG. 8 shows a restriction map of pHA2.
[0028] FIG. 9 shows a restriction map of pHA3.
[0029] FIG. 10 shows a restriction map of pHA4.
[0030] FIG. 11 shows a restriction map of pHA5.
[0031] FIG. 12 shows a restriction map of pHA6.
[0032] FIG. 13 shows a restriction map of pHA7.
[0033] FIG. 14 shows a restriction map of pMRT106.
[0034] FIG. 15 shows a restriction map of pHA8.
[0035] FIG. 16 shows a restriction map of pHA9.
[0036] FIG. 17 shows a restriction map of pHA10.
[0037] FIG. 18 shows a restriction map of pRB157.
[0038] FIG. 19 shows a restriction map of pMRT084.
[0039] FIG. 20 shows a restriction map of pMRT086.
[0040] FIG. 21 shows a restriction map of pCJ791.
[0041] FIG. 22 shows a restriction map of pMRT032.
[0042] FIG. 23 shows a restriction map of pNNB194neo.
[0043] FIG. 24 shows a restriction map of pNNB194neo-oriT.
[0044] FIG. 25 shows a restriction map of pShV3.
[0045] FIG. 26 shows a restriction map of pShV2.1-amyEΔB.
[0046] FIG. 27 shows a restriction map of pShV3A.
[0047] FIG. 28 shows a restriction map of pMRT036.
[0048] FIG. 29 shows a restriction map of pMRT037.
[0049] FIG. 30 shows a restriction map of pMRT041.
[0050] FIG. 31 shows a restriction map of pMRT064.1.
[0051] FIG. 32 shows a restriction map of pMRT068.
[0052] FIG. 33 shows a restriction map of pMRT069.
[0053] FIG. 34 shows a restriction map of pMRT071.
[0054] FIG. 35 shows a restriction map of pMRT074.
[0055] FIG. 36 shows a restriction map of pMRT120.
[0056] FIG. 37 shows a restriction map of pMRT122.
[0057] FIG. 38 shows a restriction map of pCR2.1-pel5'.
[0058] FIG. 39 shows a restriction map of pCR2.1-pel3'.
[0059] FIG. 40 shows a restriction map of pRB161.
[0060] FIG. 41 shows a restriction map of pRB162.
[0061] FIG. 42 shows a restriction map of pRB156.
[0062] FIG. 43 shows a restriction map of pRB164.
[0063] FIG. 44 shows a summary of fermentations of various hyaluronic acid producing Bacillus subtilis strains run under fed batch at approximately 2 g sucrose/L0-hr, 37° C.
[0064] FIG. 45 shows a summary of peak hyaluronic acid weight average molecular weights (MDa) obtained from fermentations of various hyaluronic acid producing Bacillus subtilis strains run under fed batch at approximately 2 g sucrose/L0-hr, 37° C.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The present invention relates to methods for producing a hyaluronan, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronan, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronan from the cultivation medium.
[0066] The methods of the present invention represent an improvement over the production of hyaluronan from pathogenic, encapsulating bacteria. In encapsulating bacteria, a large quantity of the hyaluronan is produced in the capsule. In processing and purifying hyaluronan from such sources, it is first necessary to remove the hyaluronan from the capsule, such as by the use of a surfactant, or detergent, such as SDS. This creates a complicating step in commercial production of hyaluronan, as the surfactant must be added in order to liberate a large portion of the hyaluronan, and subsequently the surfactant must be removed prior to final purification.
[0067] The present invention allows the production of a large quantity of a hyaluronan, which is produced in a non-encapsulating host cell, as free hyaluronan. When viewed under the microscope, there is no visible capsule associated with the recombinant strains of Bacillus, whereas the pathogenic strains traditionally used in hyaluronan production comprise a capsule of hyaluronan that is at least twice the diameter of the cell itself.
[0068] Since the hyaluronan of the recombinant Bacillus cell is expressed directly to the culture medium, a simple process may be used to isolate the hyaluronan from the culture medium. First, the Bacillus cells and cellular debris are physically removed from the culture medium. The culture medium may be diluted first, if desired, to reduce the viscosity of the medium. Many methods are known to those skilled in the art for removing cells from culture medium, such as centrifugation or microfiltration. If desired, the remaining supernatant may then be filtered, such as by ultrafiltration, to concentrate and remove small molecule contaminants from the hyaluronan. Following removal of the cells and cellular debris, a simple precipitation of the hyaluronan from the medium is performed by known mechanisms. Salt, alcohol, or combinations of salt and alcohol may be used to precipitate the hyaluronan from the filtrate. Once reduced to a precipitate, the hyaluronan can be easily isolated from the solution by physical means. Alternatively, the hyaluronan may be dried or concentrated from the filtrate solution by using evaporative techniques known to the art, such as spray drying.
[0069] The methods of the present invention thus represent an improvement over existing techniques for commercially producing hyaluronan by fermentation, in not requiring the use of a surfactant in the purification of hyaluronan from cells in culture.
Hyaluronic Acid
[0070] "Hyaluronic acid" is defined herein as an unsulphated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) linked together by alternating beta-1,4 and beta-1,3 glycosidic bonds (FIG. 1). Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA. The terms hyaluronan and hyaluronic acid are used interchangeably herein.
[0071] In a preferred embodiment, the hyaluronic acid obtained by the methods of the present invention has a molecular weight of about 10,000 to about 10,000,000 Da. In a more preferred embodiment, the hyaluronic acid obtained by the methods of the present invention has a molecular weight of about 25,000 to about 5,000,000 Da. In a most preferred embodiment, the hyaluronic acid obtained by the methods of the present invention has a molecular weight of about 50,000 to about 3,000,000 Da.
[0072] The level of hyaluronic acid produced by a Bacillus host cell of the present invention may be determined according to the modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334). Moreover, the average molecular weight of the hyaluronic acid may be determined using standard methods in the art, such as those described by Ueno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, "Light Scattering University DAWN Course Manual" and "DAWN EOS Manual" Wyatt Technology Corporation, Santa Barbara, Calif.
[0073] The hyaluronic acid obtained by the methods of the present invention may be subjected to various techniques known in the art to modify the hyaluronic acid, such as crosslinking as described, for example, in U.S. Pat. Nos. 5,616,568, 5,652,347, and 5,874,417. Moreover, the molecular weight of the hyaluronic acid may be altered using techniques known in the art.
Host Cells
[0074] In the methods of the present invention, the Bacillus host cell may be any Bacillus cell suitable for recombinant production of hyaluronic acid. The Bacillus host cell may be a wild-type Bacillus cell or a mutant thereof. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus agaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. Mutant Bacillus subtilis cells particularly adapted for recombinant expression are described in WO 98/22598. Non-encapsulating Bacillus cells are particularly useful in the present invention.
[0075] In a preferred embodiment, the Bacillus host cell is a Bacillus amyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In a more preferred embodiment, the Bacillus cell is a Bacillus amyloliquefaciens cell. In another more preferred embodiment, the Bacillus cell is a Bacillus clausii cell. In another more preferred embodiment, the Bacillus cell is a Bacillus lentus cell. In another more preferred embodiment, the Bacillus cell is a Bacillus licheniformis cell. In another more preferred embodiment, the Bacillus cell is a Bacillus subtilis cell. In a most preferred embodiment, the Bacillus host cell is Bacillus subtilis A164Δ5 (see U.S. Pat. No. 5,891,701) or Bacillus subtilis 168Δ4.
[0076] Transformation of the Bacillus host cell with a nucleic acid construct of the present invention may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278).
Nucleic Acid Constructs
[0077] "Nucleic acid construct" is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence. The term "coding sequence" is defined herein as a sequence which is transcribed into mRNA and translated into an enzyme of interest when placed under the control of the below mentioned control sequences. The boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5' end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
[0078] The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are well known in the art and include, for example, isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences from such genomic DNA can be effected, e.g., by using antibody screening of expression libraries to detect cloned DNA fragments with shared structural features or the well known polymerase chain reaction (PCR). See, for example, Innis et al., 1990, PCR Protocols: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction, ligated activated transcription, and nucleic acid sequence-based amplification may be used. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a Bacillus cell where clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combinations thereof.
[0079] An isolated nucleic acid sequence encoding an enzyme may be manipulated in a variety of ways to provide for expression of the enzyme. Manipulation of the nucleic acid sequence prior to its insertion into a construct or vector may be desirable or necessary depending on the expression vector or Bacillus host cell. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art. It will be understood that the nucleic acid sequence may also be manipulated in vivo in the host cell using methods well known in the art.
[0080] A number of enzymes are involved in the biosynthesis of hyaluronic acid. These enzymes include hyaluronan synthase, UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, hexokinase, phosphoglucomutase, amidotransferase, mutase, and acetyl transferase. Hyaluronan synthase is the key enzyme in the production of hyaluronic acid.
[0081] "Hyaluronan synthase" is defined herein as a synthase that catalyzes the elongation of a hyaluronan chain by the addition of GlcUA and GlcNAc sugar precursors. The amino acid sequences of streptococcal hyaluronan synthases, vertebrate hyaluronan synthases, and the viral hyaluronan synthase are distinct from the Pasteurella hyaluronan synthase, and have been proposed for classification as Group I and Group II hyaluronan synthases, the Group I hyaluronan synthases including Streptococcal hyaluronan synthases (DeAngelis, 1999). For production of hyaluronan in Bacillus host cells, hyaluronan synthases of a eukaryotic origin, such as mammalian hyaluronan synthases, are less preferred.
[0082] The hyaluronan synthase encoding sequence may be any nucleic acid sequence capable of being expressed in a Bacillus host cell. The nucleic acid sequence may be of any origin. Preferred hyaluronan synthase genes include any of either Group I or Group II, such as the Group I hyaluronan synthase genes from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus, or the Group II hyaluronan synthase genes of Pasturella multocida.
[0083] In a preferred embodiment, the hyaluronan synthase encoding sequence is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 1, SEQ ID NO: 92, or SEQ ID NO: 102; and (c) a complementary strand of (a) or (b).
[0084] In a more preferred embodiment, the hyaluronan synthase encoding sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; or a fragment thereof having hyaluronan synthase activity.
[0085] In another preferred embodiment, the hyaluronan synthase encoding sequence is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 95; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 94; and (c) a complementary strand of (a) or (b).
[0086] In another more preferred embodiment, the hyaluronan synthase encoding sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO: 95, or a fragment thereof having hyaluronan synthase activity.
[0087] The methods of the present invention also include constructs whereby precursor sugars of hyaluronan are supplied to the host cell, either to the culture medium, or by being encoded by endogenous genes, by non-endogenous genes, or by a combination of endogenous and non-endogenous genes in the Bacillus host cell. The precursor sugar may be D-glucuronic acid or N-acetyl-glucosamine.
[0088] In the methods of the present invention, the nucleic acid construct may further comprise one or more genes encoding enzymes in the biosynthesis of a precursor sugar of a hyaluronan. Alternatively, the Bacillus host cell may further comprise one or more second nucleic acid constructs comprising one or more genes encoding enzymes in the biosynthesis of the precursor sugar. Hyaluronan production is improved by the use of constructs with a nucleic acid sequence or sequences encoding a gene or genes directing a step in the synthesis pathway of the precursor sugar of hyaluronan. By, "directing a step in the synthesis pathway of a precursor sugar of hyaluronan" is meant that the expressed protein of the gene is active in the formation of N-acetyl-glucosamine or D-glucuronic acid, or a sugar that is a precursor of either of N-acetyl-glucosamine and D-glucuronic acid (FIG. 2).
[0089] In a preferred method for supplying precursor sugars, constructs are provided for improving hyaluronan production in a host cell having a hyaluronan synthase, by culturing a host cell having a recombinant construct with a heterologous promoter region operably linked to a nucleic acid sequence encoding a gene directing a step in the synthesis pathway of a precursor sugar of hyaluronan. In a preferred method the host cell also comprises a recombinant construct having a promoter region operably linked to a hyaluronan synthase, which may use the same or a different promoter region than the nucleic acid sequence to a synthase involved in the biosynthesis of N-acetyl-glucosamine. In a further preferred embodiment, the host cell may have a recombinant construct with a promoter region operably linked to different nucleic acid sequences encoding a second gene involved in the synthesis of a precursor sugar of hyaluronan.
[0090] Thus, the present invention also relates to constructs for improving hyaluronan production by the use of constructs with a nucleic acid sequence encoding a gene directing a step in the synthesis pathway of a precursor sugar of hyaluronan. The nucleic acid sequence to the precursor sugar may be expressed from the same or a different promoter as the nucleic acid sequence encoding the hyaluronan synthase.
[0091] The genes involved in the biosynthesis of precursor sugars for the production of hyaluronic acid include a UDP-glucose 6-dehydrogenase gene, UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinase gene, phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyl transferase gene.
[0092] In a cell containing a hyaluronan synthase, any one or combination of two or more of hasB, hasC and hasD, or the homologs thereof, such as the Bacillus subtilis tuaD, gtaB, and gcaD, respectively, as well as hasE, may be expressed to increase the pools of precursor sugars available to the hyaluronan synthase. The Bacillus genome is described in Kunst, et al., Nature 390, 249-256, "The complete genome sequence of the Gram-positive bacterium Bacillus subtilis" (20 Nov. 1997). In some instances, such as where the host cell does not have a native hyaluronan synthase activity, the construct may include the hasA gene.
[0093] The nucleic acid sequence encoding the biosynthetic enzymes may be native to the host cell, while in other cases heterologous sequence may be utilized. If two or more genes are expressed they may be genes that are associated with one another in a native operon, such as the genes of the HAS operon of Streptococcus equisimilis, which comprises hasA, hasB, hasC and hasD. In other instances, the use of some combination of the precursor gene sequences may be desired, without each element of the operon included. The use of some genes native to the host cell, and others which are exogenous may also be preferred in other cases. The choice will depend on the available pools of sugars in a given host cell, the ability of the cell to accommodate overproduction without interfering with other functions of the host cell, and whether the cell regulates expression from its native genes differently than exogenous genes.
[0094] As one example, depending on the metabolic requirements and growth conditions of the cell, and the available precursor sugar pools, it may be desirable to increase the production of N-acetyl-glucosamine by expression of a nucleic acid sequence encoding UDP-N-acetylglucosamine pyrophosphorylase, such as the hasD gene, the Bacillus gcaD gene, and homologs thereof. Alternatively, the precursor sugar may be D-glucuronic acid. In one such embodiment, the nucleic acid sequence encodes UDP-glucose 6-dehydrogenase. Such nucleic acid sequences include the Bacillus tuaD gene, the hasB gene of Streptococcus, and homologs thereof. The nucleic acid sequence may also encode UDP-glucose pyrophosphorylase, such as in the Bacillus gtaB gene, the hasC gene of Streptococcus, and homologs thereof.
[0095] In the methods of the present invention, the UDP-glucose 6-dehydrogenase gene may be a hasB gene or tuaD gene; or homologs thereof.
[0096] In a preferred embodiment, the hasB gene is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 41, SEQ ID NO: 97, or SEQ ID NO: 105; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 40, SEQ ID NO: 96, or SEQ ID NO: 104; and (c) a complementary strand of (a) or (b).
[0097] In a more preferred embodiment, the hasB gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 97, or SEQ ID NO: 105; or a fragment thereof having UDP-glucose 6-dehydrogenase activity.
[0098] In another preferred embodiment, the tuaD gene is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 12; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 11; and (c) a complementary strand of (a) or (b).
[0099] In another more preferred embodiment, the tuaD gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 12, or a fragment thereof having UDP-glucose 6-dehydrogenase activity.
[0100] In the methods of the present invention, the UDP-glucose pyrophosphorylase gene may be a hasC gene or gtaB gene; or homologs thereof.
[0101] In a preferred embodiment, the hasC gene is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 43, SEQ ID NO: 99, or SEQ ID NO: 107; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 42 or SEQ ID NO: 98, or SEQ ID NO: 106; and (c) a complementary strand of (a) or (b).
[0102] In another more preferred embodiment, the hasC gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 99, or SEQ ID NO: 107; or a fragment thereof having UDP-glucose pyrophosphorylase activity.
[0103] In another preferred embodiment, the gtaB gene is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 22; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 21; and (c) a complementary strand of (a) or (b).
[0104] In another more preferred embodiment, the gtaB gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 22, or a fragment thereof having UDP-glucose pyrophosphorylase activity.
[0105] In the methods of the present invention, the UDP-N-acetylglucosamine pyrophosphorylase gene may be a hasD or gcaD gene; or homologs thereof.
[0106] In a preferred embodiment, the hasD gene is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 45; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 44; and (c) a complementary strand of (a) or (b).
[0107] In another more preferred embodiment, the hasD gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 45, or a fragment thereof having UDP-N-acetylglucosamine pyrophosphorylase activity.
[0108] In another preferred embodiment, the gcaD gene is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 30; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 29; and (c) a complementary strand of (a) or (b).
[0109] In another more preferred embodiment, the gcaD gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 30, or a fragment thereof having UDP-N-acetylglucosamine pyrophosphorylase activity.
[0110] In the methods of the present invention, the glucose-6-phosphate isomerase gene may be a hasE or homolog thereof.
[0111] In a preferred embodiment, the hasE gene is selected from the group consisting of (a) a nucleic acid sequence encoding a polypeptide with an amino acid sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 101; (b) a nucleic acid sequence which hybridizes under low, medium, or high stringency conditions with SEQ ID NO: 100; and (c) a complementary strand of (a) or (b).
[0112] In another more preferred embodiment, the hasE gene encodes a polypeptide having the amino acid sequence of SEQ ID NO: 101, or a fragment thereof having glucose-6-phosphate isomerase activity.
[0113] In the methods of the present invention, the hyaluronan synthase gene and the one or more genes encoding a precursor sugar are under the control of the same promoter. Alternatively, the one or more genes encoding a precursor sugar are under the control of the same promoter but a different promoter driving the hyaluronan synthase gene. A further alternative is that the hyaluronan synthase gene and each of the genes encoding a precursor sugar are under the control of different promoters. In a preferred embodiment, the hyaluronan synthase gene and the one or more genes encoding a precursor sugar are under the control of the same promoter.
[0114] The present invention also relates to a nucleic acid construct comprising an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. A nucleic acid sequence encoding most of the hyaluronan synthase operon of Streptococcus equisimilis is found in SEQ ID NO: 108. This sequence contains the hasB (SEQ ID NO: 40) and hasC (SEQ ID nO: 42) homologs of the Bacillus subtilis tuaD gene (SEQ ID NO: 11) and gtaB gene (SEQ ID NO: 21), respectively, as is the case for Streptococcus pyogenes, as well as a homolog of the gcaD gene (SEQ ID NO: 29), which has been designated hasD (SEQ ID NO: 44). The Bacillus subtilis gcaD encodes UDP-N-acetylglucosamine pyrophosphorylase, which is involved in the synthesis of N-acetyl-glucosamine, one of the two sugars of hyaluronan. The Streptococcus equisimilis homolog of gcaD, hasD, is arranged by Streptococcus equisimilis on the hyaluronan synthase operon. The nucleic acid sequence also contains a portion of the hasA gene (the last 1156 bp of SEQ ID NO: 1).
[0115] In some cases the host cell will have a recombinant construct with a heterologous promoter region operably linked to a nucleic acid sequence encoding a gene directing a step in the synthesis pathway of a precursor sugar of hyaluronan, which may be in concert with the expression of hyaluronan synthase from a recombinant construct. The hyaluronan synthase may be expressed from the same or a different promoter region than the nucleic acid sequence encoding an enzyme involved in the biosynthesis of the precursor. In another preferred embodiment, the host cell may have a recombinant construct with a promoter region operably linked to a different nucleic acid sequence encoding a second gene involved in the synthesis of a precursor sugar of hyaluronan.
[0116] The nucleic acid sequence encoding the enzymes involved in the biosynthesis of the precursor sugar(s) may be expressed from the same or a different promoter as the nucleic acid sequence encoding the hyaluronan synthase. In the former sense, "artificial operons" are constructed, which may mimic the operon of Streptococcus equisimilis in having each hasA, hasB, hasC and hasD, or homologs thereof, or, alternatively, may utilize less than the full complement present in the Streptococcus equisimilis operon. The artificial operons" may also comprise a glucose-6-phosphate isomerase gene (hasE) as well as one or more genes selected from the group consisting of a hexokinase gene, phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyl transferase gene. In the artificial operon, at least one of the elements is heterologous to one other of the elements, such as the promoter region being heterologous to the encoding sequences.
[0117] In a preferred embodiment, the nucleic acid construct comprises hasA, tuaD, and gtaB. In another preferred embodiment, the nucleic acid construct comprises hasA, tuaD, gtaB, and gcaD. In another preferred embodiment, the nucleic acid construct comprises hasA and tuaD. In another preferred embodiment, the nucleic acid construct comprises hasA. In another preferred embodiment, the nucleic acid construct comprises hasA, tuaD, gtaB, gcaD, and hasE. In another preferred embodiment, the nucleic acid construct comprises hasA, hasB, hasC, and hasD. In another preferred embodiment, the nucleic acid construct comprises hasA, hasB, hasC, hasD, and hasE. Based on the above preferred embodiments, the genes noted can be replaced with homologs thereof.
[0118] In the methods of the present invention, the nucleic acid constructs comprise a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence. The promoter sequence may be, for example, a single promoter or a tandem promoter.
[0119] "Promoter" is defined herein as a nucleic acid sequence involved in the binding of RNA polymerase to initiate transcription of a gene. "Tandem promoter" is defined herein as two or more promoter sequences each of which is operably linked to a coding sequence and mediates the transcription of the coding sequence into mRNA. "Operably linked" is defined herein as a configuration in which a control sequence, e.g., a promoter sequence, is appropriately placed at a position relative to a coding sequence such that the control sequence directs the production of a polypeptide encoded by the coding sequence. As noted earlier, a "coding sequence" is defined herein as a nucleic acid sequence which is transcribed into mRNA and translated into a polypeptide when placed under the control of the appropriate control sequences. The boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5' end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, genomic DNA, cDNA, semisynthetic, synthetic, and recombinant nucleic acid sequences.
[0120] In a preferred embodiment, the promoter sequences may be obtained from a bacterial source. In a more preferred embodiment, the promoter sequences may be obtained from a gram positive bacterium such as a Bacillus strain, e.g., Bacillus agaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans or Streptomyces murinus; or from a gram negative bacterium, e.g., E. coli or Pseudomonas sp.
[0121] Examples of suitable promoters for directing the transcription of a nucleic acid sequence in the methods of the present invention are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus lentus or Bacillus clausii alkaline protease gene (aprH), Bacillus licheniformis alkaline protease gene (subtilisin Carlsberg gene), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis subsp. tenebrionis CryIIIA gene (cryIIIA) or portions thereof, prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731). Other examples are the promoter of the spo1 bacterial phage promoter and the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80:21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook, Fritsch, and Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.
[0122] The promoter may also be a "consensus" promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region. The consensus promoter may be obtained from any promoter which can function in a Bacillus host cell. The construction of a "consensus" promoter may be accomplished by site-directed mutagenesis to create a promoter which conforms more perfectly to the established consensus sequences for the "-10" and "-35" regions of the vegetative "sigma A-type" promoters for Bacillus subtilis (Voskuil et al., 1995, Molecular Microbiology 17: 271-279).
[0123] In a preferred embodiment, the "consensus" promoter is obtained from a promoter obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus clausii or Bacillus lentus alkaline protease gene (aprH), Bacillus licheniformis alkaline protease gene (subtilisin Carlsberg gene), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis subsp. tenebrionis CryIIIA gene (cryIIIA) or portions thereof, or prokaryotic beta-lactamase gene spo1 bacterial phage promoter. In a more preferred embodiment, the "consensus" promoter is obtained from Bacillus amyloliquefaciens alpha-amylase gene (amyQ).
[0124] Widner, et al., U.S. Pat. Nos. 6,255,076 and 5,955,310, describe tandem promoters and constructs and methods for use in expression in Bacillus cells, including the short consensus amyQ promoter (also called scBAN). The use of the cryIIIA stabilizer sequence, and constructs using the sequence, for improved production in Bacillus are also described therein.
[0125] Each promoter sequence of the tandem promoter may be any nucleic acid sequence which shows transcriptional activity in the Bacillus cell of choice including a mutant, truncated, and hybrid promoter, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the Bacillus cell. Each promoter sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide and native or foreign to the Bacillus cell. The promoter sequences may be the same promoter sequence or different promoter sequences.
[0126] The two or more promoter sequences of the tandem promoter may simultaneously promote the transcription of the nucleic acid sequence. Alternatively, one or more of the promoter sequences of the tandem promoter may promote the transcription of the nucleic acid sequence at different stages of growth of the Bacillus cell.
[0127] In a preferred embodiment, the tandem promoter contains at least the amyQ promoter of the Bacillus amyloliquefaciens alpha-amylase gene. In another preferred embodiment, the tandem promoter contains at least a "consensus" promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region. In another preferred embodiment, the tandem promoter contains at least the amyL promoter of the Bacillus licheniformis alpha-amylase gene. In another preferred embodiment, the tandem promoter contains at least the cryIIIA promoter or portions thereof (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107).
[0128] In a more preferred embodiment, the tandem promoter contains at least the amyL promoter and the cryIIIA promoter. In another more preferred embodiment, the tandem promoter contains at least the amyQ promoter and the cryIIIA promoter. In another more preferred embodiment, the tandem promoter contains at least a "consensus" promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region and the cryIIIA promoter. In another more preferred embodiment, the tandem promoter contains at least two copies of the amyL promoter. In another more preferred embodiment, the tandem promoter contains at least two copies of the amyQ promoter. In another more preferred embodiment, the tandem promoter contains at least two copies of a "consensus" promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region. In another more preferred embodiment, the tandem promoter contains at least two copies of the cryIIIA promoter.
[0129] "An mRNA processing/stabilizing sequence" is defined herein as a sequence located downstream of one or more promoter sequences and upstream of a coding sequence to which each of the one or more promoter sequences are operably linked such that all mRNAs synthesized from each promoter sequence may be processed to generate mRNA transcripts with a stabilizer sequence at the 5' end of the transcripts. The presence of such a stabilizer sequence at the 5' end of the mRNA transcripts increases their half-life (Agaisse and Lereclus, 1994, supra, Hue et al., 1995, Journal of Bacteriology 177: 3465-3471). The mRNA processing/stabilizing sequence is complementary to the 3' extremity of a bacterial 16S ribosomal RNA. In a preferred embodiment, the mRNA processing/stabilizing sequence generates essentially single-size transcripts with a stabilizing sequence at the 5' end of the transcripts. The mRNA processing/stabilizing sequence is preferably one, which is complementary to the 3' extremity of a bacterial 16S ribosomal RNA. See, U.S. Pat. Nos. 6,255,076 and 5,955,310.
[0130] In a more preferred embodiment, the mRNA processing/stabilizing sequence is the Bacillus thuringiensis cryIIIA mRNA processing/stabilizing sequence disclosed in WO 94/25612 and Agaisse and Lereclus, 1994, supra, or portions thereof which retain the mRNA processing/stabilizing function. In another more preferred embodiment, the mRNA processing/stabilizing sequence is the Bacillus subtilis SP82 mRNA processing/stabilizing sequence disclosed in Hue et al., 1995, supra, or portions thereof which retain the mRNA processing/stabilizing function.
[0131] When the cryIIIA promoter and its mRNA processing/stabilizing sequence are employed in the methods of the present invention, a DNA fragment containing the sequence disclosed in WO 94/25612 and Agaisse and Lereclus, 1994, supra, or portions thereof which retain the promoter and mRNA processing/stabilizing functions, may be used. Furthermore, DNA fragments containing only the cryIIIA promoter or only the cryIIIA mRNA processing/stabilizing sequence may be prepared using methods well known in the art to construct various tandem promoter and mRNA processing/stabilizing sequence combinations. In this embodiment, the cryIIIA promoter and its mRNA processing/stabilizing sequence are preferably placed downstream of the other promoter sequence(s) constituting the tandem promoter and upstream of the coding sequence of the gene of interest.
[0132] The isolated nucleic acid sequence encoding the desired enzyme(s) involved in hyaluronic acid production may then be further manipulated to improve expression of the nucleic acid sequence. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.
[0133] A nucleic acid construct comprising a nucleic acid sequence encoding an enzyme may be operably linked to one or more control sequences capable of directing the expression of the coding sequence in a Bacillus cell under conditions compatible with the control sequences.
[0134] The term "control sequences" is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of a nucleic acid sequence. Each control sequence may be native or foreign to the nucleic acid sequence encoding the enzyme. In addition to promoter sequences described above, such control sequences include, but are not limited to, a leader, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding an enzyme.
[0135] The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a Bacillus cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the enzyme or the last enzyme of an operon. Any terminator which is functional in the Bacillus cell of choice may be used in the present invention.
[0136] The control sequence may also be a suitable leader sequence, a nontranslated region of a mRNA which is important for translation by the Bacillus cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the enzyme. Any leader sequence which is functional in the Bacillus cell of choice may be used in the present invention.
[0137] The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of a polypeptide which can direct the expressed polypeptide into the cell's secretory pathway. The signal peptide coding region may be native to the polypeptide or may be obtained from foreign sources. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the secreted polypeptide. The foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the polypeptide relative to the natural signal peptide coding region normally associated with the coding sequence. The signal peptide coding region may be obtained from an amylase or a protease gene from a Bacillus species. However, any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of a Bacillus cell of choice may be used in the present invention.
[0138] An effective signal peptide coding region for Bacillus cells is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis prsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.
[0139] The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE) and Bacillus subtilis neutral protease (nprT).
[0140] Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
[0141] It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.
Expression Vectors
[0142] In the methods of the present invention, a recombinant expression vector comprising a nucleic acid sequence, a promoter, and transcriptional and translational stop signals may be used for the recombinant production of an enzyme involved in hyaluronic acid production. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide or enzyme at such sites. Alternatively, the nucleic acid sequence may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.
[0143] The recombinant expression vector may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the Bacillus cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the Bacillus cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the Bacillus cell, or a transposon may be used.
[0144] The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the Bacillus host cell's genome or autonomous replication of the vector in the cell independent of the genome.
[0145] For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the Bacillus cell. The additional nucleic acid sequences enable the vector to be integrated into the Bacillus cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the Bacillus cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
[0146] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the Bacillus cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. The origin of replication may be one having a mutation to make its function temperature-sensitive in the Bacillus cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).
[0147] The vectors preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/09129, where the selectable marker is on a separate vector.
[0148] More than one copy of a nucleic acid sequence may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. A convenient method for achieving amplification of genomic DNA sequences is described in WO 94/14968.
[0149] The procedures used to ligate the elements described above to construct the recombinant expression vectors are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Production
[0150] In the methods of the present invention, the Bacillus host cells are cultivated in a nutrient medium suitable for production of the hyaluronic acid using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzymes involved in hyaluronic acid synthesis to be expressed and the hyaluronic acid to be isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). The secreted hyaluronic acid can be recovered directly from the medium.
[0151] The resulting hyaluronic acid may be isolated by methods known in the art. For example, the hyaluronic acid may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated hyaluronic acid may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
[0152] In the methods of the present invention, the Bacillus host cells produce greater than about 4 g, preferably greater than about 6 g, more preferably greater than about 8 g, even more preferably greater than about 10 g, and most preferably greater than about 12 g of hyaluronic acid per liter.
Deletions/Disruptions
[0153] Gene deletion or replacement techniques may be used for the complete removal of a selectable marker gene or other undesirable gene. In such methods, the deletion of the selectable marker gene may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the selectable marker gene. The contiguous 5' and 3' regions may be introduced into a Bacillus cell on a temperature-sensitive plasmid, e.g., pE194, in association with a second selectable marker at a permissive temperature to allow the plasmid to become established in the cell. The cell is then shifted to a non-permissive temperature to select for cells that have the plasmid integrated into the chromosome at one of the homologous flanking regions. Selection for integration of the plasmid is effected by selection for the second selectable marker. After integration, a recombination event at the second homologous flanking region is stimulated by shifting the cells to the permissive temperature for several generations without selection. The cells are plated to obtain single colonies and the colonies are examined for loss of both selectable markers (see, for example, Perego, 1993, In A. L. Sonneshein, J. A. Hoch, and R. Losick, editors, Bacillus subtilis and Other Gram-Positive Bacteria, Chapter 42, American Society of Microbiology, Washington, D.C., 1993).
[0154] A selectable marker gene may also be removed by homologous recombination by introducing into the mutant cell a nucleic acid fragment comprising 5' and 3' regions of the defective gene, but lacking the selectable marker gene, followed by selecting on the counter-selection medium. By homologous recombination, the defective gene containing the selectable marker gene is replaced with the nucleic acid fragment lacking the selectable marker gene. Other methods known in the art may also be used.
[0155] U.S. Pat. No. 5,891,701 discloses techniques for deleting several genes including spoIIAC, aprE, nprE, and amyE.
[0156] Other undesirable biological compounds may also be removed by the above described methods such as the red pigment synthesized by cypX (accession no. BG12580) and/or yvmC (accession no. BG14121).
[0157] In a preferred embodiment, the Bacillus host cell is unmarked with any heterologous or exogenous selectable markers. In another preferred embodiment, the Bacillus host cell does not produce any red pigment synthesized by cypX and yvmC.
Isolated Nucleic Acid Sequences Encoding Polypeptides Having UDP-Glucose 6-Dehydrogenase Activity, UDP-Glucose Pyrophosphorylase Activity, or UDP-N-Acetylglucosamine Pyrophosphorylase Activity
[0158] The term "UDP-glucose 6-dehydrogenase activity" is defined herein as a UDP glucose:NAD.sup.+ 6-oxidoreductase activity which catalyzes the conversion of UDP-glucose in the presence of 2NAD.sup.+ and water to UDP-glucuronate and 2NADH. For purposes of the present invention UDP-glucose 6-dehydrogenase activity is determined according to the procedure described by Jaenicke and Rudolph, 1986, Biochemistry 25: 7283-7287. One unit of UDP-glucose 6-dehydrogenase activity is defined as 1.0 μmole of UDP-glucuronate produced per minute at 25° C., pH 7.
[0159] The term "UDP-glucose pyrophosphorylase activity" is defined herein as a UTP:quadrature-D-glucose-1-phosphate uridylyltransferase activity which catalyzes the conversion of glucose-1-phosphate in the presence of UTP to diphosphate and UDP-glucose. For purposes of the present invention UDP-glucose pyrophosphorylase activity activity is determined according to the procedure described by Kamogawa et al., 1965, J. Biochem. (Tokyo) 57: 758-765 or Hansen et al., 1966, Method Enzymol. 8: 248-253. One unit of UDP-glucose pyrophosphorylase activity is defined as 1.0 μmole of UDP-glucose produced per minute at 25° C., pH 7.
[0160] The term "UDP-N-acetylglucosamine pyrophosphorylase activity" is defined herein as a UTP:N-acetyl-alpha-D-glucoamine-1-phosphate uridyltransferase activity which catalyzes the conversion of N-acetyl-alpha-D-glucosamine-1-phosphate in the presence of UTP to diphosphate and UDP-N-acetyl-alpha-D-glucoamine. For purposes of the present invention, UDP-N-acetylglucosamine pyrophosphorylase activity is determined according to the procedure described by Mangin-Lecreuix et al., 1994, J. Bacteriology 176: 5788-5795. One unit of UDP-N-acetylglucosamine pyrophosphorylase activity is defined as 1.0 μmole of UDP-N-acetyl-alpha-D-glucoamine produced per minute at 25° C., pH 7.
[0161] The term "isolated nucleic acid sequence" as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
[0162] In a first embodiment, the present invention relates to isolated nucleic acid sequences encoding polypeptides having an amino acid sequence which has a degree of identity to SEQ ID NO: 41 of at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have UDP-glucose 6-dehydrogenase activity (hereinafter "homologous polypeptides"). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from SEQ ID NO: 41.
[0163] In another first embodiment, the present invention relates to isolated nucleic acid sequences encoding polypeptides having an amino acid sequence which has a degree of identity to SEQ ID NO: 43 of at least about 90%, preferably at least about 95%, and more preferably at least about 97%, which have UDP-glucose pyrophosphorylase activity (hereinafter "homologous polypeptides"). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from SEQ ID NO: 43.
[0164] In another first embodiment, the present invention relates to isolated nucleic acid sequences encoding polypeptides having an amino acid sequence which has a degree of identity to SEQ ID NO: 45 of at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have UDP-N-acetylglucosamine pyrophosphorylase activity (hereinafter "homologous polypeptides"). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from SEQ ID NO: 45.
[0165] For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the Vector NTI AlignX software package (Informax Inc., Bethesda, Md.) with the following defaults: pairwise alignment, gap opening penalty of 10, gap extension penalty of 0.1, and score matrix: blosum62mt2.
[0166] Preferably, the nucleic acid sequences of the present invention encode polypeptides that comprise the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45; or an allelic variant thereof; or a fragment thereof that has UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity, respectively. In a more preferred embodiment, the nucleic acid sequence of the present invention encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45. In another preferred embodiment, the nucleic acid sequence of the present invention encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45; or an allelic variant thereof; or a fragment thereof, wherein the polypeptide fragment has UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity, respectively. In another preferred embodiment, the nucleic acid sequence of the present invention encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45.
[0167] The present invention also encompasses nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, which differ from SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44 which encode fragments of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, respectively, which have UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity, respectively.
[0168] A subsequence of SEQ ID NO: 40 is a nucleic acid sequence encompassed by SEQ ID NO: 40 except that one or more nucleotides from the 5' and/or 3' end have been deleted. Preferably, a subsequence contains at least 1020 nucleotides, more preferably at least 1080 nucleotides, and most preferably at least 1140 nucleotides. A fragment of SEQ ID NO: 41 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. Preferably, a fragment contains at least 340 amino acid residues, more preferably at least 360 amino acid residues, and most preferably at least 380 amino acid residues.
[0169] A subsequence of SEQ ID NO: 42 is a nucleic acid sequence encompassed by SEQ ID NO: 42 except that one or more nucleotides from the 5' and/or 3' end have been deleted. Preferably, a subsequence contains at least 765 nucleotides, more preferably at least 810 nucleotides, and most preferably at least 855 nucleotides. A fragment of SEQ ID NO: 43 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. Preferably, a fragment contains at least 255 amino acid residues, more preferably at least 270 amino acid residues, and most preferably at least 285 amino acid residues.
[0170] A subsequence of SEQ ID NO: 44 is a nucleic acid sequence encompassed by SEQ ID NO: 44 except that one or more nucleotides from the 5' and/or 3' end have been deleted. Preferably, a subsequence contains at least 1110 nucleotides, more preferably at least 1200 nucleotides, and most preferably at least 1290 nucleotides. A fragment of SEQ ID NO: 45 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. Preferably, a fragment contains at least 370 amino acid residues, more preferably at least 400 amino acid residues, and most preferably at least 430 amino acid residues.
[0171] An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. The allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
[0172] In a second embodiment, the present invention relates to isolated nucleic acid sequences which have a degree of homology to SEQ ID NO: 40 of at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%.
[0173] In another second embodiment, the present invention relates to isolated nucleic acid sequences which have a degree of homology to SEQ ID NO: 42 of at least about 90%, preferably at least about 95%, and more preferably at least about 97%.
[0174] In another second embodiment, the present invention relates to isolated nucleic acid sequences which have a degree of homology to SEQ ID NO: 44 of at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%.
[0175] For purposes of the present invention, the degree of homology between two nucleic acid sequences is determined by the Vector NTI AlignX software package (Informax Inc., Bethesda, Md.) using the following defaults: pairwise alignment, gap opening penalty of 15, gap extension penalty of 6.6, and score matrix: swgapdnamt.
[0176] In a third embodiment, the present invention relates to isolated nucleic acid sequences encoding polypeptides having UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity, which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, (ii) the cDNA sequence contained in SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, or (iii) a complementary strand of (i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). The subsequence of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the respective subsequence may encode a polypeptide fragment which has UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity.
[0177] The nucleic acid sequence of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, or subsequences thereof, as well as the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity, respectively, from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
[0178] Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, or a subsequence thereof, the carrier material is used in a Southern blot. For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, its complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.
[0179] In a preferred embodiment, the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45; or a subsequence thereof. In another preferred embodiment, the nucleic acid probe is SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. In another preferred embodiment, the nucleic acid probe is the nucleic acid sequence contained in plasmid pMRT106 which is contained in Escherichia coli NRRL B-30536, wherein the nucleic acid sequence encodes polypeptides having UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase activity.
[0180] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures.
[0181] For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 45° C. (very low stringency), more preferably at least at 50° C. (low stringency), more preferably at least at 55° C. (medium stringency), more preferably at least at 60° C. (medium-high stringency), even more preferably at least at 65° C. (high stringency), and most preferably at least at 70° C. (very high stringency).
[0182] For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at 5° C. to 10° C. below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.
[0183] For short probes which are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.
[0184] In a fourth embodiment, the present invention relates to isolated nucleic acid sequences which encode variants of the polypeptide having an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45 comprising a substitution, deletion, and/or insertion of one or more amino acids.
[0185] The amino acid sequences of the variant polypeptides may differ from the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
[0186] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.
[0187] Modification of a nucleic acid sequence of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
[0188] It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
[0189] The polypeptides encoded by the isolated nucleic acid sequences of the present invention have at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the UDP-glucose 6-dehydrogenase activity of the polypeptide of SEQ ID NO: 41, the UDP-glucose pyrophosphorylase activity of the polypeptide of SEQ ID NO: 43, or the UDP-N-acetylglucosamine pyrophosphorylase activity of the polypeptide of SEQ ID NO: 45.
[0190] The nucleic acid sequences of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted. In a preferred embodiment, the polypeptide encoded by a nucleic acid sequence of the present invention is secreted extracellularly.
[0191] The nucleic acid sequences may be obtained from a bacterial source. For example, these polypeptides may be obtained from a gram positive bacterium such as a Bacillus strain, e.g., Bacillus agaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans or Streptomyces murinus; or from a gram negative bacterium, e.g., E. coli or Pseudomonas sp.
[0192] In a preferred embodiment, the nucleic acid sequences are obtained from a Streptococcus or Pastuerella strain.
[0193] In a more preferred embodiment, the nucleic acid sequences are obtained from a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subs. zooepidemicus strain, or a Pasteurella multocida strain.
[0194] In a most preferred embodiment, the nucleic acid sequences are obtained from Streptococcus equisimilis, e.g., the nucleic acid sequence set forth in SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. In another most preferred embodiment, the nucleic acid sequence is the sequence contained in plasmid pMRT106 which is contained in Escherichia coli NRRL B-30536. In further most preferred embodiment, the nucleic acid sequence is SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.
[0195] Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
[0196] Furthermore, such nucleic acid sequences may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
[0197] The present invention also relates to mutant nucleic acid sequences comprising at least one mutation in the polypeptide coding sequence of SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, in which the mutant nucleic acid sequence encodes a polypeptide which consists of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 45, respectively.
[0198] The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from a strain of Streptococcus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.
[0199] The present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
[0200] The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals.
[0201] The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides.
[0202] The present invention also relates to methods for producing a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity comprising (a) cultivating a host cell under conditions suitable for production of the polypeptide; and (b) recovering the polypeptide.
[0203] In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
[0204] The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.
[0205] The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
[0206] The polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
[0207] The present invention further relates to the isolated polypeptides having UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activity encoded by the nucleic acid sequences described above.
[0208] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.
EXAMPLES
Primers and Oligos
[0209] All primers and oligos were purchased (MWG Biotech Inc., High Point, N.C.)
Example 1
PCR Amplification and Cloning of the Streptococcus equisimilis hasA Gene and the Bacillus subtilis tuaD, gtaB, and gcaD Genes
[0210] The Streptococcus equisimilis hyaluronan synthase gene (hasA, accession number AF023876, SEQ ID NOs: 1 [DNA sequence] and 2 [deduced amino acid sequence]) was PCR amplified from plasmid pKKseD (Weigel, 1997, Journal of Biological Chemistry 272: 32539-32546) using primers 1 and 2:
TABLE-US-00001 Primer 1: (SEQ ID NO: 3) 5'-GAGCTCTATAAAAATGAGGAGGGAACCGAATGAGAACATTAAAAAAC CT-3' Primer 2: (SEQ ID NO: 4) 5'-GTTAACGAATTCAGCTATGTAGGTACCTTATAATAATTTTTTACGTG T-3'
[0211] PCR amplifications were conducted in triplicate in 50 μl reactions composed of 1 ng of pKKseD DNA, 0.4 μM each of primers 1 and 2, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II (Applied Biosystems, Inc., Foster City, Calif.) with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase (Applied Biosystems, Inc., Foster City, Calif.). The reactions were performed in a RoboCycler 40 thermacycler (Stratagene, Inc., La Jolla, Calif.) programmed for 1 cycle at 95° C. for 9 minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and 72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes. The PCR product was visualized using a 0.8% agarose gel with 44 mM Tris Base, 44 mM boric acid, 0.5 mM EDTA buffer (0.5×TBE). The expected fragment was approximately 1200 bp.
[0212] The 1200 bp PCR fragment was cloned into pCR2.1 using the TA-TOPO Cloning Kit (Stratagene, Inc., La Jolla, Calif.) and transformed into E. coli OneShot® competent cells according to the manufacturers' instructions (Stratagene, Inc., La Jolla, Calif.). Transformants were selected at 37° C. after 16 hours of growth on 2× yeast-tryptone (YT) agar plates supplemented with 100 μg of ampicillin per ml. Plasmid DNA from these transformants was purified using a QIAGEN robot (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions and the DNA sequence of the inserts confirmed by DNA sequencing using M13 (-20) forward and M13 reverse primers (Invitrogen, Inc, Carlsbad, Calif.) and the following internal primers. The plasmid harboring the 1200 bp PCR fragment was designated pCR2.1-sehasA (FIG. 3).
TABLE-US-00002 Primer 3: 5'-GTTGACGATGGAAGTGCTGA-3' (SEQ ID NO: 5) Primer 4: 5'-ATCCGTTACAGGTAATATCC-3' (SEQ ID NO: 6) Primer 5: 5'-TCCTTTTGTAGCCCTATGGA-3' (SEQ ID NO: 7) Primer 6: 5'-TCAGCACTTCCATCGTCAAC-3' (SEQ ID NO: 8) Primer 7: 5'-GGATATTACCTGTAACGGAT-3' (SEQ ID NO: 9) Primer 8: 5'-TCCATAGGGCTACAAAAGGA-3' (SEQ ID NO: 10)
[0213] The Bacillus subtilis UDP-glucose-6-dehydrogenase gene (tuaD, accession number BG12691, SEQ ID NOs: 11 [DNA sequence] and 12 [deduced amino acid sequence]) was PCR amplified from Bacillus subtilis 168 (BGSC 1A1, Bacillus Genetic Stock Center, Columbus, Ohio) using primers 9 and 10:
TABLE-US-00003 Primer 9: (SEQ ID NO: 13) 5'-GGTACCGACACTGCGACCATTATAAA-3' Primer 10: (SEQ ID NO: 14) 5'-GTTAACGAATTCCAGCTATGTATCTAGACAGCTTCAACCAAGTAACA CT-3'
[0214] PCR amplifications were carried out in triplicate in 30 μl reactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.3 μM each of primers 9 and 10, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 programmed for 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycles each at 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5 minute; and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized in a 0.8% agarose gel using 0.5×TBE buffer. The expected fragment was approximately 1400 bp.
[0215] The 1400 bp PCR fragment was cloned into pCR2.1 using the TA-TOPO Cloning Kit and transformed into E. coli OneShot® competent cells according to the manufacturers' instructions. Plasmid DNA was purified using a QIAGEN robot according to the manufacturer's instructions and the DNA sequence of the inserts confirmed by DNA sequencing using M13 (-20) forward and M13 reverse primers and the following internal primers. The plasmid harboring the 1400 bp PCR fragment was designated pCR2.1-tuaD (FIG. 4).
TABLE-US-00004 Primer 11: 5'-AGCATCTTAACGGCTACAAA-3' (SEQ ID NO: 15) Primer 12: 5'-TGTGAGCGAGTCGGCGCAGA-3' (SEQ ID NO: 16) Primer 13: 5'-GGGCGCCCATGTAAAAGCAT-3' (SEQ ID NO: 17) Primer 14: 5'-TTTGTAGCCGTTAAGATGCT-3' (SEQ ID NO: 18) Primer 15: 5'-TCTGCGCCGACTCGCTCACA-3' (SEQ ID NO: 19) Primer 16: 5'-ATGCTTTTACATGGGCGCCC-3' (SEQ ID NO: 20)
[0216] The Bacillus subtilis UTP-glucose-1-phosphate uridylyltransferase gene (gtaB, accession number BG10402, SEQ ID NOs: 21 [DNA sequence] and 22 [deduced amino acid sequence]) was PCR amplified from Bacillus subtilis 168 using primers 17 and 18:
TABLE-US-00005 Primer 17: (SEQ ID NO: 23) 5'-TCTAGATTTTTCGATCATAAGGAAGGT-3' Primer 18: (SEQ ID NO: 24) 5'-GTTAACGAATTCCAGCTATGTAGGATCCAATGTCCAATAGCCTTTTT GT-3'
[0217] PCR amplifications were carried out in triplicate in 30 μl reactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.3 μM each of primers 17 and 18, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 programmed for 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycles each at 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5 minute; and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized in a 0.8% agarose-0.5×TBE gel. The expected fragment was approximately 900 bp.
[0218] The 900 bp PCR fragment was cloned into pCR2.1 using the TA-TOPO cloning kit and transformed into E. coli OneShot® competent cells according to the manufacturer's instructions. Plasmid DNA was purified using a QIAGEN robot according to the manufacturer's instructions and the DNA sequence of the inserts confirmed by DNA sequencing using M13 (-20) forward and M13 reverse primers and the following internal primers. The plasmid harboring the 900 bp PCR fragment was designated pCR2.1-gtaB (FIG. 5).
TABLE-US-00006 Primer 19: (SEQ ID NO: 25) 5'-AAAAAGGCTTCTAACCTGGC-3' Primer 20: (SEQ ID NO: 26) 5'-AAACCGCCTAAAGGCACAGC-3' Primer 21: (SEQ ID NO: 27) 5'-GCCAGGTTAGAAGCCTTTTT-3' Primer 22: (SEQ ID NO: 28) 5'-GCTGTGCCTTTAGGCGGTTT-3'
[0219] The Bacillus subtilis UDP-N-acetylglucosamine pyrophosphorylase gene (gcaD, accession number BG10113, SEQ ID NOs: 29 [DNA sequence] and 30 [deduced amino acid sequence]) was PCR amplified from Bacillus subtilis 168 using primers 23 and 24:
TABLE-US-00007 Primer 23: (SEQ ID NO: 31) 5'-GGATCCTTTCTATGGATAAAAGGGAT-3' Primer 24: (SEQ ID NO: 32) 5'-GTTAACAGGATTATTTTTTATGAATATTTTT-3'
[0220] PCR amplifications were carried out in triplicate in 30 μl reactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.3 μM each of primers 23 and 24, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 programmed for 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycles each at 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5 minute; and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized in a 0.8% agarose-0.5×TBE gel. The expected fragment was approximately 1500 bp.
[0221] The 1500 bp PCR fragment was cloned into pCR2.1 using the TA-TOPO cloning kit and transformed into E. coli OneShot® competent cells according to the manufacturer's instructions. Plasmid DNA was purified using a QIAGEN robot according to the manufacturer's instructions and the DNA sequence of the inserts confirmed by DNA sequencing using M13 (-20) forward and M13 reverse primers and the following internal primers. The plasmid harboring the 900 bp PCR fragment was designated pCR2.1-gcaD (FIG. 6).
TABLE-US-00008 Primer 25: (SEQ ID NO: 33) 5'-CAGAGACGATGGAACAGATG-3' Primer 26: (SEQ ID NO: 34) 5'-GGAGTTAATGATAGAGTTGC-3' Primer 27: (SEQ ID NO: 35) 5'-GAAGATCGGGAATTTTGTAG-3' Primer 28: (SEQ ID NO: 36) 5'-CATCTGTTCCATCGTCTCTG-3' Primer 29: (SEQ ID NO: 37) 5'-GCAACTCTATCATTAACTCC-3' Primer 30: (SEQ ID NO: 38) 5'-CTACAAAATTCCCGATCTTC-3'
Example 2
Construction of the hasA/tuaD/gtaB Operon
[0222] Plasmids pDG268Δneo-cryIIIAstab/Sav (U.S. Pat. No. 5,955,310) and pCR2.1-tuaD (Example 1, FIG. 4) were digested with KpnI and HpaI. The digestions were resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment (approximately 7700 bp) from pDG268Δneo-cryIIIAstab/Sav and the smaller tuaD fragment (approximately 1500 bp) from pCR2.1-tuaD were gel-purified using the QIAquick DNA Extraction kit according to the manufacturer's instructions (QIAGEN, Valencia, Calif.). The two purified fragments were ligated together with T4 DNA ligase according to the manufacturer's instructions (Roche Applied Science; Indianapolis, Ind.) and the ligation mix was transformed into E. coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.). Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml.
[0223] Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by KpnI plus HpaI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of an approximately 1500 bp KpnI/HpaI tuaD fragment and was designated pHA1 (FIG. 7).
[0224] Plasmids pHA1 and pCR2.1-gtaB (Example 1, FIG. 5) were digested with XbaI and HpaI. The digestions were resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment from pHA1 (approximately 9200 bp) and the smaller gtaB fragment (approximately 900 bp) from pCR2.1-gtaB were gel-purified from a 0.8% agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction Kit according to the manufacturer's instructions. These two purified fragments were ligated together with T4 DNA ligase and the ligation mix was used to transform E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 mg of ampicillin per ml at 37° C.
[0225] Plasmids were purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by XbaI plus HpaI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The correct plasmid was identified by the presence of an approximately 900 bp XbaI/HpaI gtaB fragment and was designated pHA2 (FIG. 8).
[0226] Plasmids pHA2 and pCR2.1-sehasA (Example 1, FIG. 3) were digested with SacI plus KpnI. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The larger vector fragment (approximately 10000 bp) from pHA2 and the smaller hasA fragment (approximately 1300 bp) from pCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction kit according to the manufacturer's instructions. The two purified fragments were ligated together with T4 DNA ligase and the ligation mix was used to transform E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C. Plasmids were purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by SacI plus KpnI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The correct plasmid was identified by the presence of an approximately 1300 bp SacI/KpnI hasA fragment and was designated pHA3 (FIG. 9).
Example 3
Construction of the hasA/tuaD/gtaB/gcaD Operon
[0227] Plasmids pHA2 (Example 2, FIG. 8) and pCR2.1-gcaD (Example 1, FIG. 6) were digested with BamHI and HpaI. The digestions were resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment (approximately 10,000 bp) from pHA2 and the smaller gcaD fragment (approximately 1,400 bp) from pCR2.1-gcaD were gel-purified from a 0.8% agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction Kit according to the manufacturer's instructions. These two purified fragments were ligated together with T4 DNA ligase and the ligation mix was used to transform E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C.
[0228] Plasmids were purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by XbaI plus HpaI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The correct plasmid was identified by the presence of an approximately 1400 bp BamHI/HpaI gcaD fragment and was designated pHA4 (FIG. 10).
[0229] Plasmids pHA4 and pCR2.1-sehasA (Example 1, FIG. 3) were digested with SacI and KpnI. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The larger vector fragment (approximately 11,000 bp) from pHA4 and the smaller hasA fragment (approximately 1,300 bp) from pCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction kit according to the manufacturer's instructions. The two purified fragments were ligated together with T4 DNA ligase and the ligation mix was used to transform E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C. Plasmids were purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by SacI plus KpnI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The correct plasmid was identified by the presence of an approximately 1,300 bp SacI/KpnI hasA fragment and was designated pHA5 (FIG. 11).
Example 4
Construction of the hasA/tuaD/gcaD Operon
[0230] Plasmids pHA1 (Example 2, FIG. 7) and pCR2.1-gcaD (Example 1, FIG. 6) were digested with BamHI and HpaI. The digestions were resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment from pHA1 (approximately 9,200 bp) and the smaller gcaD fragment (approximately 1400 bp) from pCR2.1-gcaD were gel-purified from a 0.8% agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction Kit according to the manufacturer's instructions. These two purified fragments were ligated together with T4 DNA ligase and the ligation mix was used to transform E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C.
[0231] Plasmids were purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by BamHI plus HpaI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The correct plasmid was identified by the presence of an approximately 1400 bp BamHI/HpaI gtaB fragment and was designated pHA6 (FIG. 12).
[0232] Plasmids pHA6 and pCR2.1-sehasA (Example 1, FIG. 3) were digested with SacI plus KpnI. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The larger vector fragment (approximately 10,200 bp) from pHA6 and the smaller hasA fragment (approximately 1,300 bp) from pCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction kit according to the manufacturer's instructions. The two purified fragments were ligated together with T4 DNA ligase and the ligation mix was used to transform E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml. Plasmids were purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by SacI plus KpnI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. The correct plasmid was identified by the presence of an approximately 1300 bp SacI/KpnI hasA fragment and was designated pHA7 (FIG. 13).
Example 5
Construction of Bacillus subtilis RB161
[0233] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digested with SacI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with NotI. The largest plasmid fragment of approximately 6800 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions (QIAGEN, Valencia, Calif.). The recovered vector DNA was then ligated with the DNA insert described below.
[0234] Plasmid pHA3 (Example 2, FIG. 9) was digested with SacI. The digested plasmid was then purified as described above, and finally digested with NotI. The smallest plasmid fragment of approximately 3800 bp was gel-purified as described above. The recovered vector and DNA insert were ligated using the Rapid DNA Cloning Kit (Roche Applied Science; Indianapolis, Ind.) according to the manufacturer's instructions. Prior to transformation in Bacillus subtilis, the ligation described above was linearized using ScaI to ensure double cross-over integration in the chromosome rather than single cross-over integration in the chromosome. Competent cells of Bacillus subtilis 168Δ4 were transformed with the ligation products digested with ScaI. Bacillus subtilis 168Δ4 is derived from the Bacillus subtilis type strain 168 (BGSC 1A1, Bacillus Genetic Stock Center, Columbus, Ohio) and has deletions in the spoIIAC, aprE, nprE, and amyE genes. The deletion of these four genes was performed essentially as described for Bacillus subtilis A164Δ5, which is described in detail in U.S. Pat. No. 5,891,701.
[0235] Bacillus subtilis chloramphenicol-resistant transformants were selected at 34° C. after 16 hours of growth on Tryptose blood agar base (TBAB) plates supplemented with 5 μg of chloramphenicol per ml. To screen for integration of the plasmid by double cross-over at the amyE locus, Bacillus subtilis primary transformants were patched on TBAB plates supplemented with 6 μg of neomycin per ml and on TBAB plates supplemented with 5 μg of chloramphenicol per ml. Integration of the plasmid by double cross-over at the amyE locus does not incorporate the neomycin resistance gene and therefore renders the strain neomycin sensitive. Isolates were also patched onto minimal plates to visualize whether or not these were producing hyaluronic acid. Hyaluronic acid producing isolates have a "wet" phenotype on minimal plates. Using this plate screen, chloramphenicol resistant and neomycin sensitive "wet" transformants (due to hyaluronic acid production) were isolated at 37° C.
[0236] Genomic DNA was isolated from the "wet", chloramphenicol resistant, and neomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGEN tip-20 column (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. PCR amplifications were performed on these transformants using the synthetic oligonucleotides below, which are based on the hasA, tuaD, and gtaB gene sequences, to confirm the presence and integrity of these genes in the operon of the Bacillus subtilis transformants.
[0237] The amplification reactions (25 μl) were composed of approximately 50 ng of genomic DNA of the Bacillus subtilis 168Δ4 transformants, 0.5 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II, 3 mM MgCl2, and 0.625 units of AmpliTaq Gold® DNA polymerase. The reactions were incubated in a RoboCycler 40 Temperature Cycler programmed for one cycle at 95° C. for 9 minutes; 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes; and a final cycle at 72° C. for 7 minutes.
[0238] Primers 3 and 8 were used to confirm the presence of the hasA gene, primers 3 and 16 to confirm the presence of the tuaD gene, and primers 3 and 22 to confirm the presence of the gtaB gene. The Bacillus subtilis 168Δ4 hasA/tuaD/gtaB integrant was designated Bacillus subtilis RB158.
[0239] Genomic DNA was isolated from Bacillus subtilis RB158 using a QIAGEN tip-20 column according to the manufacturer's instructions, and was used to transform competent Bacillus subtilis A164Δ5 (deleted at the spoIIAC, aprE, nprE, amyE, and srfC genes; see U.S. Pat. No. 5,891,701). Transformants were selected on TBAB plates supplemented with 5 μg of chloramphenicol per ml at 37° C. A Bacillus subtilis A164Δ5 hasA/tuaD/gtaB integrant was identified by its "wet" phenotype and designated Bacillus subtilis RB161.
Example 6
Construction of Bacillus subtilis RB163
[0240] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digested with SacI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with NotI. The largest plasmid fragment of approximately 6,800 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered vector DNA was then ligated with the DNA insert described below.
[0241] Plasmid pHA7 (Example 4, FIG. 13) was digested with SacI. The digested plasmid was then purified as described above, and finally digested with NotI. The smallest plasmid fragment of approximately 4,300 bp was gel-purified as described above. The recovered vector and DNA insert were ligated using the Rapid DNA Cloning Kit according to the manufacturer's instructions. Prior to transformation in Bacillus subtilis, the ligation described above was linearized using ScaI to ensure double cross-over integration in the chromosome rather than single cross-over integration in the chromosome. Bacillus subtilis 168Δ4 competent cells were transformed with the ligation digested with the restriction enzyme ScaI.
[0242] Bacillus subtilis chloramphenicol-resistant transformants were selected on TBAB plates supplemented with 5 μg of chloramphenicol per ml at 37° C. To screen for integration of the plasmid by double cross-over at the amyE locus, Bacillus subtilis primary transformants were patched on TBAB plates supplemented with 6 μg of neomycin per ml and on TBAB plates supplemented with 5 μg of chloramphenicol per ml to isolate chloramphenicol resistant and neomycin sensitive "wet" transformants (due to hyaluronic acid production).
[0243] Genomic DNA was isolated from the "wet", chloramphenicol resistant, and neomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGEN tip-20 column according to the manufacturer's instructions. PCR amplifications were performed on these transformants using primers 3, 8, 16, 22 and primer 30 (Example 1) to confirm the presence and integrity of these genes in the operon of the Bacillus subtilis transformants. The amplification reactions (25 μl) were composed of approximately 50 ng of genomic DNA of the Bacillus subtilis 168Δ4 transformants, 0.5 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR buffer, 3 mM MgCl2, and 0.625 units of AmpliTaq Gold® DNA polymerase. The reactions were incubated in a RoboCycler 40 Temperature Cycler programmed for one cycle at 95° C. for 9 minutes; 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes; and a final cycle at 72° C. for 7 minutes.
[0244] Primers 3 and 8 were used to confirm the presence of the hasA gene, primers 3 and 16 to confirm the presence of the tuaD gene, primers 3 and 22 to confirm the presence of the gtaB gene, and primers 3 and 30 to confirm the presence of the gcaD gene. The Bacillus subtilis 168Δ4 hasA/tuaD/gcaD integrant was designated Bacillus subtilis RB160.
[0245] Genomic DNA was isolated from Bacillus subtilis RB160 using a QIAGEN tip-20 column according to the manufacturer's instructions, and was used to transform competent Bacillus subtilis A164Δ5. Transformants were selected on TBAB plates containing 5 μg of chloramphenicol per ml, and grown at 37° C. for 16 hours. The Bacillus subtilis A164Δ5 hasA/tuaD/gcaD integrant was identified by its "wet" phenotype and designated Bacillus subtilis RB163.
Example 7
Construction of Bacillus subtilis TH-1
[0246] The hyaluronan synthase (has) operon was obtained from Streptococcus equisimilis using the following procedure. The has operon is composed of the hasA, hasB, hasC, and hasD genes. Approximately 20 μg of Streptococcus equisimilis D181 (Kumari and Weigel, 1997, Journal of Biological Chemistry 272: 32539-32546) chromosomal DNA was digested with HindIII and resolved on a 0.8% agarose-0.5×TBE gel. DNA in the 3-6 kb range was excised from the gel and purified using the QIAquick DNA Gel Extraction Kit according to the manufacturer's instructions. The recovered DNA insert was then ligated with the vector DNA described below.
[0247] Plasmid pUC18 (2 μg) was digested with HindIII and the 5' protruding ends were dephosphorylated with shrimp alkaline phosphatase according to the manufacturer's instructions (Roche Applied Science; Indianapolis, Ind.). The dephosphorylated vector and DNA insert were ligated using the Rapid DNA Cloning Kit according to the manufacturer's instructions. The ligation was used to transform E. coli XL10 Gold Kan competent cells (Stratagene, Inc., La Jolla, Calif.). Cells were plated onto Luria broth plates (100 μg/ml ampicillin) and incubated overnight at 37° C. Five plates containing approximately 500 colonies/plate were probed with oligo 952-55-1, shown below, which is a 54 bp sequence identical to the coding strand near the 3' end of the Streptococcus equisimilis D181 hasA gene (nucleotides 1098-1151 with respect to the A residue of the ATG translation start codon).
TABLE-US-00009 Primer 31: (SEQ ID NO: 39) 5'-GTGTCGGAACATTCATTACATGCTTAAGCACCCGCTGTCCTTCTTG TTATCTCC-3'
[0248] The oligonucleotide probe was DIG-labeled using the DIG Oligonucleotide 3'-end Labeling Kit according to the manufacturer's instructions (Roche Applied Science; Indianapolis, Ind.). Colony hybridization and chemiluminescent detection were performed as described in "THE DIG SYSTEM USER'S GUIDE FOR FILTER HYBRIDIZATION", Boehringer Mannheim GmbH.
[0249] Seven colonies were identified that hybridized to the probe. Plasmid DNA from one of these transformants was purified using a QIAGEN robot (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions, digested with HindIII, and resolved on a 0.8% agarose gel using 0.5×TBE buffer. The DNA insert was shown to be approximately 5 kb in size. This plasmid was designated pMRT106 (FIG. 14).
[0250] The DNA sequence of the cloned fragment was determined using the EZ::TN®<TET-1> Insertion Kit according to the manufacturer's instructions (Epicenter Technologies, Madison, Wis.). The sequencing revealed that the cloned DNA insert contained the last 1156 bp of the Streptococcus equisimilis hasA gene followed by three other genes designated hasB, hasC, and hasD; presumably all four genes are contained within a single operon and are therefore co-transcribed. The Streptococcus equisimilis hasB gene is contained in nucleotides 1411-2613 (SEQ ID NOs: 40 [DNA sequence] and 41 [deduced amino acid sequence]) of the fragment, and Streptococcus equisimilis hasC gene in nucleotides 2666-3565 (SEQ ID NOs: 42 [DNA sequence] and 43 [deduced amino acid sequence]) of the fragment, and Streptococcus equisimilis hasD gene in nucleotides 3735-5114 (SEQ ID NOs: 44 [DNA sequence] and 45 [deduced amino acid sequence]) of the fragment.
[0251] The polypeptides encoded by the Streptococcus equisimilis hasB and hasC genes show some homology to those encoded by the hasB and hasC genes, respectively, from the Streptococcus pyogenes has operon sequence (Ferretti et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98 (8), 4658-4663). The degree of identity was determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the Vector NTI AlignX software (Informax Inc., Bethesda, Md.) with the following defaults: pairwise alignment, gap opening penalty of 10, gap extension penalty of 0.1, and score matrix: blosum62mt2.
[0252] Amino acid sequence comparisons showed that the Streptococcus equisimilis HasB protein has 70% identity to the HasB protein from Streptococcus uberis (SEQ ID NO: 105); the Streptococcus equisimilis HasC protein has 91% identity to the HasC protein from Streptococcus pyogenes (SEQ ID NO: 99); and the Streptococcus equisimilis HasD protein has 73% identity to the GlmU protein (a putative UDP-N-acetylglucosamine pyrophosphorylase) of Streptococcus pyogenes (accession # Q8P286). The Streptococcus equisimilis hasD gene encodes a polypeptide that shows 50.7% identity to the UDP-N-acetyl-glucosamine pyrophosphorylase enzyme encoded by the gcaD gene of Bacillus subtilis.
[0253] Plasmid pHA5 (Example 3, FIG. 11) was digested with HpaI and BamHI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment (approximately 11,000 bp) was gel-purified using the QIAquick DNA Extraction Kit according to the manufacturer's instructions. Plasmid pMRT106 was digested with HindIII, the sticky ends were filled in with Klenow fragment, and the DNA was digested with BamHI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer and the smaller insert fragment (approximately 1000 bp, the last 2/3 of the Streptococcus equisimilis hasD gene) was gel-purified using the QIAquick DNA Extraction kit according to the manufacturer's instructions.
[0254] The two purified fragments were ligated together with T4 DNA ligase and the ligation mix was transformed into E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C.
[0255] Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by BamHI plus NotI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of an approximately 1,100 bp BamHI/NotI hasD fragment and was designated pHA8 (FIG. 15). This plasmid was digested with HindIII and ligated together with T4 DNA ligase and the ligation mix was transformed into E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml. Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by HindIII digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of a single band of approximately 9,700 bp and was designated pHA9 (FIG. 16).
[0256] Plasmid pHA9 was digested with SacI and NotI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer and the smaller fragment of approximately 2,500 bp was gel-purified using the QIAquick DNA Extraction kit according to the manufacturer's instructions. Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digested with SacI and NotI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment of approximately 6,800 bp was gel-purified using the QIAquick DNA Extraction kit according to the manufacturer's instructions. The two purified fragments were ligated together with T4 DNA ligase and the ligation mix was transformed into E. coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.). Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml.
[0257] Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by SalI plus HindIII digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of an approximately 1600 bp SalI/HindIII fragment and was designated pHA10 (FIG. 17).
[0258] Plasmid pHA10 was digested with HindIII and BamHI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment (approximately 8100 bp) was gel-purified using the QIAquick DNA Extraction kit according to the manufacturer's instructions. Plasmid pMRT106 was digested with HindIII and BamHI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer and the larger insert fragment of approximately 4,100 bp was gel-purified using the QIAquick DNA Extraction kit according to the manufacturer's instructions. The two purified fragments were ligated together with T4 DNA ligase and the ligation mix was used to transform Bacillus subtilis 168Δ4. Transformants were selected on TBAB agar plates supplemented with 5 μg of chloramphenicol per ml at 37° C. Approximately 100 transformants were patched onto TBAB supplemented with chloramphenicol (5 μg/ml) and TBAB supplemented with neomycin (10 μg/ml) to score chloramphenicol resistant, neomycin sensitive colonies; this phenotype is indicative of a double crossover into the amyE locus. A few such colonies were identified, all of which exhibited a "wet" phenotype indicating that hyaluronic acid was being produced. One colony was chosen and designated Bacillus subtilis 168Δ4::scBAN/se hasA/hasB/hasC/hasD.
[0259] Genomic DNA was isolated from Bacillus subtilis 168Δ4::scBAN/se hasA/hasB/hasC/hasD using a QIAGEN tip-20 column according to the manufacturer's instructions, and used to transform competent Bacillus subtilis A164Δ5. Transformants were selected on TBAB plates containing 5 μg of chloramphenicol per ml, and grown at 37° C. for 16 hours. The Bacillus subtilis A164Δ5 hasA/hasB/hasC/hasD integrant was identified by its "wet" phenotype and designated Bacillus subtilis TH-1.
Example 8
Construction of Bacillus subtilis RB184
[0260] The hasA gene from Streptococcus equisimilis (Example 1) and tuaD gene (a Bacillus subtilis hasB homologue) (Example 1) were cloned to be under the control of a short "consensus" amyQ (scBAN) promoter (U.S. Pat. No. 5,955,310).
[0261] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digested with SacI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with NotI. The largest plasmid fragment of approximately 6,800 bp was gel-purified from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Gel Extraction Kit according to the manufacturer's instructions. The recovered vector DNA was then ligated with the DNA insert described below.
[0262] Plasmid pHA5 (Example 3, FIG. 11) was digested with HpaI. The digested plasmid was then purified as described above, and finally digested with XbaI. The double-digested plasmid was then blunted by first inactivating XbaI at 85° C. for 30 minutes. Blunting was performed by adding 0.5 μl of 10 mM each dNTPs, 1 μl of 1 U/μl T4 DNA polymerase (Roche Applied Science; Indianapolis, Ind.) and incubating at 11° C. for 10 minutes. Finally the polymerase was inactivated by incubating the reaction at 75° C. for 10 minutes. The largest plasmid fragment of approximately 11,000 bp was then gel-purified as described above and religated using the Rapid DNA Cloning Kit according to the manufacturer's instructions. The ligation mix was transformed into E. coli SURE competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C. Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by ScaI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of an approximately 11 kb fragment and was designated pRB157 (FIG. 18).
[0263] pRB157 was digested with SacI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with NotI. The smallest plasmid fragment of approximately 2,632 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered DNA insert was then ligated with the vector DNA described above.
[0264] Prior to transformation in Bacillus subtilis, the ligation described above was linearized using ScaI to ensure double cross-over integration in the chromosome rather than single cross-over integration in the chromosome. Bacillus subtilis 168Δ4 competent cells were transformed with the ligation digested with the restriction enzyme ScaI.
[0265] Bacillus subtilis chloramphenicol-resistant transformants were selected on TBAB plates supplemented with 5 μg of chloramphenicol per ml. To screen for integration of the plasmid by double cross-over at the amyE locus, Bacillus subtilis primary transformants were patched on TBAB plates supplemented with 6 μg of neomycin per ml and on TBAB plates supplemented with 5 μg of chloramphenicol per ml to isolate chloramphenicol resistant and neomycin sensitive "wet" transformants (due to hyaluronic acid production).
[0266] Genomic DNA was isolated from the "wet", chloramphenicol resistant, and neomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGEN tip-20 column according to the manufacturer's instructions. PCR amplifications were performed on these transformants using primers 3, 8, and 16 (Example 1) to confirm the presence and integrity of hasA and tuaD in the operon of the Bacillus subtilis transformants. The amplification reactions (25 μl) were composed of approximately 50 ng of genomic DNA of the Bacillus subtilis 168Δ4 transformants, 0.5 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR buffer, 3 mM MgCl2, and 0.625 units of AmpliTaq Gold® DNA polymerase. The reactions were incubated in a RoboCycler 40 Temperature Cycler programmed for one cycle at 95° C. for 9 minutes; 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes; and a final cycle at 72° C. for 7 minutes.
[0267] Primers 3 and 8 were used to confirm the presence of the hasA gene and primers 3 and 16 to confirm the presence of the tuaD gene. A Bacillus subtilis 168Δ4 hasA/tuaD integrant was designated Bacillus subtilis RB183.
[0268] Bacillus subtilis RB183 genomic DNA was also used to transform competent Bacillus subtilis A164Δ5. Transformants were selected on TBAB plates containing 5 μg of chloramphenicol per ml, and grown at 37° C. for 16 hours. The Bacillus subtilis A164Δ5 hasA/tuaD integrant was identified by its "wet" phenotype and designated Bacillus subtilis RB184.
Example 9
Construction of Bacillus subtilis RB187
[0269] Bacillus subtilis RB161 was made competent and transformed with the cat deletion plasmid pRB115 (Widner et al., 2000, Journal of Industrial Microbiology & Biotechnology 25: 204-212). Selection for direct integration into the chromosome was performed at the non-permissive temperature of 45° C. using erythromycin (5 μg/ml) selection. At this temperature, the pE194 origin of replication is inactive. Cells are able to maintain erythromycin resistance only by integration of the plasmid into the cat gene on the bacterial chromosome. These so-called "integrants" were maintained at 45° C. to ensure growth at this temperature with selection. To allow for loss or "looping out" of the plasmid, which will result in the deletion of most of the cat gene from the chromosome, the integrants were grown in Luria-Bertani (LB) medium without selection at the permissive temperature of 34° C. for many generations. At this temperature the pE194 origin of replication is active and promotes excision of the plasmid from the genome (Molecular Biological Methods for Bacillus, edited by C. R. Harwood and S. M. Cutting, 1990, John Wiley and Sons Ltd.).
[0270] The cells were then plated on non-selective LB agar plates and colonies which contained deletions in the cat gene and loss of the pE194-based replicon were identified by the following criteria: (1) chloramphenicol sensitivity indicated the presence of the cat deletion; (2) erythromycin sensitivity indicated the absence of the erythromycin resistance gene encoded by the vector pRB115; and (3) PCR confirmed the presence of the cat deletion in the strain of interest. PCR was performed to confirm deletion of the cat gene at the amyE locus by using primers 32 and 33:
TABLE-US-00010 Primer 32: (SEQ ID NO: 46) 5'-GCGGCCGCGGTACCTGTGTTACACCTGTT-3' Primer 33: (SEQ ID NO: 47) 5'-GTCAAGCTTAATTCTCATGTTTGACAGCTTATCATCGG-3'
[0271] Chromosomal DNA from potential deletants was isolated using the REDextract-N-Amp® Plant PCR kits (Sigma Chemical Company, St. Louis, Mo.) as follows: Single Bacillus colonies were inoculated into 100 μl of Extraction Solution (Sigma Chemical Company, St. Louis, Mo.), incubated at 95° C. for 10 minutes, and then diluted with an equal volume of Dilution Solution (Sigma Chemical Company, St. Louis, Mo.). PCR was performed using 4 μl of extracted DNA in conjunction with the REDextract-N-Amp PCR Reaction Mix and the desired primers according to the manufacturer's instructions, with PCR cycling conditions described in Example 5. PCR reaction products were visualized in a 0.8% agarose-0.5×TBE gel. The verified strain was named Bacillus subtilis RB187.
Example 10
Construction of Bacillus subtilis RB192
[0272] Bacillus subtilis RB184 was made unmarked by deleting the chloramphenicol resistance gene (cat gene). This was accomplished using the method described previously in Example 9. The resultant strain was designated Bacillus subtilis RB192.
Example 11
Construction of Bacillus subtilis RB194
[0273] Bacillus subtilis RB194 was constructed by deleting the cypX region of the chromosome of Bacillus subtilis RB187 (Example 9). The cypX region includes the cypX gene which encodes a cytochrome P450-like enzyme that is involved in the synthesis of a red pigment during fermentation. In order to delete this region of the chromosome plasmid pMRT086 was constructed.
[0274] The region of the chromosome which harbors the cypX-yvmC and yvmB-yvmA operons was PCR amplified from Bacillus subtilis BRG-1 as a single fragment using primers 34 and 35. Bacillus subtilis BRG1 is essentially a chemically mutagenized isolate of an amylase-producing strain of Bacillus subtilis which is based on the Bacillus subtilis A164Δ5 genetic background that was described in Example 5. The sequence of this region is identical to the published sequence for the Bacillus subtilis 168 type strain.
TABLE-US-00011 Primer 34: (SEQ ID NO: 48) 5'-CATGGGAGAGACCTTTGG-3' Primer 35: (SEQ ID NO: 49) 5'-GTCGGTCTTCCATTTGC-3'
[0275] The amplification reactions (50 μl) were composed of 200 ng of Bacillus subtilis BRG-1 chromosomal DNA, 0.4 μM each of primers 34 and 35, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1× Expand® High Fidelity buffer (Roche Applied Science; Indianapolis, Ind.) with 1.5 mM MgCl2, and 2.6 units of Expand® High Fidelity PCR System enzyme mix (Roche Applied Science; Indianapolis, Ind.). Bacillus subtilis BRG-1 chromosomal DNA was obtained using a QIAGEN tip-20 column according to the manufacturer's instructions. Amplification reactions were performed in a RoboCycler 40 thermacycler (Stratagene, Inc, La Jolla, Calif.) programmed for 1 cycle at 95° C. for 3 minutes; 10 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and 68° C. for 4 minutes; 20 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, 68° C. for 4 minutes plus 20 seconds per cycle, followed by 1 cycle at 72° C. for 7 minutes. Reaction products were analyzed by agarose gel electrophoresis using a 0.8% agarose gel using 0.5×TBE buffer.
[0276] The resulting fragment comprising the cypX-yvmC and yvmB-yvmA operons was cloned into pCR2.1 using the TA-TOPO Cloning Kit and transformed into E. coli OneShot® cells according to the manufacturer's instructions (Invitrogen, Inc., Carlsbad, Calif.). Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml. Plasmid DNA from several transformants was isolated using QIAGEN tip-20 columns according to the manufacturer's instructions and verified by DNA sequencing with M13 (-20) forward, M13 reverse and primers 36 to 51 shown below. The resulting plasmid was designated pMRT084 (FIG. 19).
TABLE-US-00012 Primer 36: (SEQ ID NO: 50) 5'-CGACCACTGTATCTTGG-3' Primer 37: (SEQ ID NO: 51) 5'-GAGATGCCAAACAGTGC-3' Primer 38: (SEQ ID NO: 52) 5'-CATGTCCATCGTGACG-3' Primer 39: (SEQ ID NO: 53) 5'-CAGGAGCATTTGATACG-3' Primer 40: (SEQ ID NO: 54) 5'-CCTTCAGATGTGATCC-3' Primer 41: (SEQ ID NO: 55) 5'-GTGTTGACGTCAACTGC-3' Primer 42: (SEQ ID NO: 56) 5'-GTTCAGCCTTTCCTCTCG-3' Primer 43: (SEQ ID NO: 57) 5'-GCTACCTTCTTTCTTAGG-3' Primer 44: (SEQ ID NO: 58) 5'-CGTCAATATGATCTGTGC-3' Primer 45: (SEQ ID NO: 59) 5'-GGAAAGAAGGTCTGTGC-3' Primer 46: (SEQ ID NO: 60) 5'-CAGCTATCAGCTGACAG-3' Primer 47: (SEQ ID NO: 61) 5'-GCTCAGCTATGACATATTCC-3' Primer 48: (SEQ ID NO: 62) 5'-GATCGTCTTGATTACCG-3' Primer 49: (SEQ ID NO: 63) 5'-AGCTTTATCGGTGACG-3' Primer 50: (SEQ ID NO: 64) 5'-TGAGCACGATTGCAGG-3' Primer 51: (SEQ ID NO: 65) 5'-CATTGCGGAGACATTGC-3'
[0277] Plasmid pMRT084 was digested with BsgI to delete most of the cypX-yvmC and yvmB-yvmA operons, leaving about 500 bases at each end. The digested BsgI DNA was treated with T4 DNA polymerase. Plasmid pECC1 (Youngman et al., 1984, Plasmid 12: 1-9) was digested with SmaI. A fragment of approximately 5,100 bp from pMRT084 and a fragment of approximately 1,600 bp fragment from pECC1 were isolated from a 0.8% agarose-0.5×TBE gel using the QIAquick DNA Extraction Kit according to the manufacturer's instructions, ligated together, and transformed into E. coli XL1 Blue cells according to the manufacturer's instructions (Stratagene, Inc., La Jolla, Calif.). Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml. Transformants carrying the correct plasmid with most of the cypX-yvmC and yvmB-yvmA operons deleted were identified by PCR amplification using primers 52 and 53. PCR amplification was conducted in 50 μl reactions composed of 1 ng of plasmid DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized using a 0.8% agarose-0.5×TBE gel. This construct was designated pMRT086 (FIG. 20).
TABLE-US-00013 Primer 52: (SEQ ID NO: 66) 5'-TAGACAATTGGAAGAGAAAAGAGATA-3' Primer 53: (SEQ ID NO: 67) 5'-CCGTCGCTATTGTAACCAGT-3'
[0278] Plasmid pMRT086 was linearized with ScaI and transformed into Bacillus subtilis RB128 competent cells in the presence of 0.2 μg of chloramphenicol per ml. Transformants were selected on TBAB plates containing 5 μg of chloramphenicol per ml after incubation at 37° C. for 16 hours. Chromosomal DNA was prepared from several transformants using a QIAGEN tip-20 column according to the manufacturer's instructions. Chloramphenicol resistant colonies were screened by PCR for deletion of the cypX-yvmC and yvmB-yvmA operons via PCR using primers 36 and 52, 36 and 53, 37 and 52, and 37 and 53. PCR amplification was conducted in 50 μl reactions composed of 50 ng of chromosomal DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes. The PCR products were visualized using a 0.8% agarose-0.5×TBE gel. The resulting Bacillus subtilis RB128 cypX-yvmC and yvmB-yvmA deleted strain was designated Bacillus subtilis MaTa17.
[0279] Competent cells of Bacillus subtilis RB187 (Example 9) were transformed with genomic DNA from Bacillus subtilis MaTa17. Genomic DNA was obtained from this strain using a QIAGEN tip-20 column according to the manufacturer's instructions. Bacillus subtilis chloramphenicol resistant transformants were selected on TBAB plates supplemented with 5 μg of chloramphenicol per ml at 37° C. Primary transformants were streaked for single colony isolations on TBAB plates containing 5 μg of chloramphenicol per ml at 37° C. The resulting cypX-yvmC and yvmB-yvmA deleted strain was designated Bacillus subtilis RB194.
Example 12
Construction of Bacillus subtilis RB197
[0280] Bacillus subtilis RB197 is very similar to Bacillus subtilis RB194, the only difference being that RB197 contains a smaller deletion in the cypX region: only a portion of the cypX gene is deleted in this strain to generate a cypX minus phenotype. In order to accomplish this task a plasmid, pMRT122, was constructed as described below.
[0281] Plasmid pCJ791 (FIG. 21) was constructed by digestion of plasmid pSJ2739 (WO 96/23073) with EcoRI/HindIII and ligation to a fragment containing a deleted form of the wprA gene (cell wall serine protease) from Bacillus subtilis. The 5' region of wprA was amplified using primers 54 and 55 see below, and the 3' region was amplified using primers 56 and 57 shown below from chromosomal DNA obtained from Bacillus subtilis DN1885 (Diderichsen et al., 1990, Journal of Bacteriology 172: 4315-4321). PCR amplification was conducted in 50 μl reactions composed of 1 ng of Bacillus subtilis DN1885 chromosomal DNA, 0.4 μM each of primers 39 and 40, 200 μm each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes.
[0282] The 5' and 3' wprA PCR fragments were linked by digestion with BglII followed by ligation, and PCR amplification was performed on the ligation mixture fragments using primers 54 and 57. PCR amplification was conducted in 50 μl reactions composed of 1 ng of the ligated fragment, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized using a 0.8% agarose-0.5×TBE gel. The resulting PCR fragment was cloned into pSJ2739 as an EcoRI/HindIII fragment, resulting in plasmid pCJ791 (FIG. 21). Transformants were selected on TBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg of kanamycin per ml after incubation at 28° C. for 24-48 hours. Plasmid DNA from several transformants was isolated using QIAGEN tip-20 columns according to the manufacturer's instructions and verified by PCR amplification with primers 54 and 57 using the conditions above.
TABLE-US-00014 Primer 54: (SEQ ID NO: 68) 5'-GGAATTCCAAAGCTGCAGCGGCCGGCGCG-3' Primer 55: (SEQ ID NO: 69) 5'-GAAGATCTCGTATACTTGGCTTCTGCAGCTGC-3' Primer 56: (SEQ ID NO: 70) 5'-GAAGATCTGGTCAACAAGCTGGAAAGCACTC-3' Primer 57: (SEQ ID NO: 71) 5'-CCCAAGCTTCGTGACGTACAGCACCGTTCCGGC-3'
[0283] The amyL upstream sequence and 5' coding region from plasmid pDN1981 (U.S. Pat. No. 5,698,415) were fused together by SOE using the primer pairs 58/59 and 60/61 shown below. The resulting fragment was cloned into vector pCR2.1 to generate plasmid pMRT032 as follows. PCR amplifications were conducted in triplicate in 50 μl reactions composed of 1 ng of pDN1981 DNA, 0.4 μM each of appropriate primers, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 9 minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and 72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes. The PCR product was visualized in a 0.8% agarose-0.5×TBE gel. The expected fragments were approximately 530 and 466 bp, respectively. The final SOE fragment was generated using primer pair 59/60 and cloned into pCR2.1 vector using the TA-TOPO Cloning Kit. Transformants were selected on 2×YT agar plates supplemented with 100 μg/ml ampicillin after incubation at 37° C. for 16 hours. Plasmid DNA from several transformants was isolated using QIAGEN tip-20 columns according to the manufacturer's instructions and verified by DNA sequencing with M13 (-20) forward and M13 reverse primers. The plasmid harboring the amyL upstream sequence/5' coding sequence fusion fragment was designated pMRT032 (FIG. 22).
TABLE-US-00015 Primer 58: (SEQ ID NO: 72) 5'-CCTTAAGGGCCGAATATTTATACGGAGCTCCCTGAAACAACAAAAA CGGC-3' Primer 59: (SEQ ID NO: 73) 5'-GGTGTTCTCTAGAGCGGCCGCGGTTGCGGTCAGC-3' Primer 60: (SEQ ID NO: 74) 5'-GTCCTTCTTGGTACCTGGAAGCAGAGC-3' Primer 61: (SEQ ID NO: 75) 5'-GTATAAATATTCGGCCCTTAAGGCCAGTACCATTTTCCC-3'
[0284] Plasmid pNNB194 (pSK.sup.+/pE194; U.S. Pat. No. 5,958,728) was digested with NsiI and NotI, and plasmid pBEST501 (Itaya et al. 1989 Nucleic Acids Research 17: 4410) was digested with PstI and NotI. The 5,193 bp vector fragment from pNNB194 and the 1,306 bp fragment bearing the neo gene from pBEST501 were isolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions. The isolated fragments were ligated together and used to transform E. coli SURE competent cells according to the manufacturer's instructions. Ampicillin-resistant transformants were selected on 2×YT plates supplemented with 100 μg of ampicillin per ml. Plasmid DNA was isolated from one such transformant using the QIAGEN Plasmid Kit (QIAGEN Inc., Valencia, Calif.), and the plasmid was verified by digestion with NsiI and NotI. This plasmid was designated pNNB194neo (FIG. 23).
[0285] Plasmid pNNB194neo was digested with SacI/NotI and treated with T4 DNA polymerase and dNTPs to generate blunt ends using standard protocols. Plasmid pPL2419 (U.S. Pat. No. 5,958,728) was digested with Ecl136II. The 6,478 bp vector fragment from pNNB194neo and the 562 bp fragment bearing oriT from pPL2419 were isolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions. The gel-purified fragments were ligated together and used to transform E. coli SURE cells according to the manufacturer's instructions. Ampicillin-resistant transformants were selected on 2×YT plates supplemented with 100 μg of ampicillin per ml at 37° C. Plasmid DNA was isolated from one such transformant using the QIAGEN Plasmid Kit, and the plasmid was verified by digestion with NSiI, SacI, and BscI. This plasmid was designated pNNB194neo-oriT (FIG. 24).
[0286] Plasmid pNNB194neo-oriT was digested with BamHI and treated with T4 DNA polymerase and dNTPs to generate blunt ends using standard protocols. The digested plasmid was gel-purified from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions. The purified plasmid was treated with T4 DNA ligase and used to transform E. coli SURE cells according to the manufacturer's instructions. Ampicillin-resistant transformants were selected on 2×YT plates supplemented with 100 μg of ampicillin per ml at 37° C. Plasmid DNA was isolated from one such transformant using the QIAGEN Plasmid Kit, and disruption of the BamHI site was confirmed by digestion with BamHI and ScaI. The resulting plasmid was designated pShV3 (FIG. 25).
[0287] Plasmid pShV2.1-amyEΔ (U.S. Pat. No. 5,958,728) was digested with SfiI and NotI, and the 8696 bp vector fragment was gel-purified from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions. In order to insert a BamHI site between the SfiI and NotI sites of pShV2.1-amyEΔ, a synthetic linker was constructed as follows: primers 62 and 63 were annealed by mixing 50 μM of each, boiling the mixture, and allowing the mixture to cool slowly.
TABLE-US-00016 Primer 62: (SEQ ID NO: 76) 5'-GGGCCGGATCCGC-3' Primer 63: (SEQ ID NO: 77) 3'-ATTCCCGGCCTAGGCGCCGG-5'
[0288] The purified pShV2.1-amyEΔ vector and annealed oligonucleotides were ligated together and used to transform E. coli SURE competent cells according to the manufacturer's instructions. Chloramphenicol-resistant transformants were selected on LB plates supplemented with 30 μg of chloramphenicol per ml at 37° C. Plasmid DNA was isolated from one such transformant using the QIAGEN Plasmid Kit, and insertion of the BamHI site was confirmed by digestion with BamHI. This plasmid was designated pShV2.1-amyEΔB (FIG. 26).
[0289] Plasmids pShV3 and pShV2.1-amyEΔB were digested with SalI/HindIII. A 7033 bp vector fragment from pShV3 and a 1031 bp fragment bearing amyEΔ from pShV2.1-amyEΔ were gel-purified from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions. The gel-purified fragments were ligated together and used to transform E. coli SURE cells according to the manufacturer's instructions. Ampicillin-resistant transformants were selected on 2×YT plates supplemented with 100 μg of ampicillin per ml. Plasmid DNA was isolated from one such transformant using the QIAGEN Plasmid Kit, and the plasmid was verified by digestion with Sail and HindIII. This plasmid was designated pShV3A (FIG. 27).
[0290] Plasmid pMRT032 was digested with KpnI/XbaI, filled with Klenow fragment DNA polymerase in the presence of dNTPs, and a fragment of approximately 1000 bp was isolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions. This fragment was cloned into plasmid pShV3a digested with EcoRV, and transformed into E. coli XL1 Blue cells according to the manufacturer's instructions. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml after incubation at 37° C. for 16 hours. Plasmid DNA from several transformants was isolated using QIAGEN tip-20 columns according to the manufacturer's instructions and verified on a 0.8% agarose-0.5×TBE gel by restriction analysis with SacI/SphI. The resulting plasmid was designated pMRT036 (FIG. 28).
[0291] Plasmid pMRT036 was digested with SalI/HindIII, filled with Klenow fragment DNA polymerase in the presence of dNTPs, ligated and transformed into E. coli XL1 Blue cells according to the manufacturer's instructions. Transformants were selected on 2×YT-agar plates supplemented with 100 μg/ml ampicillin after incubation at 37° C. for 16 hours. Plasmid DNA from several transformants was isolated using QIAGEN tip-20 columns according to the manufacturer's instructions and verified on a 0.8% agarose-0.5×TBE gel by restriction analysis with SacI/XbaI, PstI and NdeI. The resulting plasmid was designated pMRT037 (FIG. 29).
[0292] The scBAN/cryIIIA stabilizer fragment from plasmid pDG268Δneo-cryIIIAstab/Sav (U.S. Pat. No. 5,955,310) was isolated from a 2% agarose-0.5×TBE gel as a SfiI/SacI fragment using a QIAquick DNA Purification Kit according to the manufacturer's instructions, ligated to plasmid pMRT037 digested with SfiI/SacI, and transformed into E. coli XL1 Blue cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml after incubation at 37° C. for 16 hours. Plasmid DNA from several transformants was isolated using QIAGEN tip-20 columns according to the manufacturer's instructions and verified on a 0.8% agarose-0.5×TBE gel by restriction analysis with PstI. The resulting plasmid was designated pMRT041 (FIG. 30).
[0293] Plasmids pMRT041 and pCJ791 were digested with EcoRI/HindIII. A fragment of approximately 1300 bp from pMRT041 and a fragment of approximately 4500 bp from pCJ791 were isolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions, ligated, and transformed into Bacillus subtilis 168Δ4 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at 30° C. for 24-48 hours. Plasmid DNA from several transformants was isolated using QIAGEN tip-20 columns according to the manufacturer's instructions and verified on a 0.8% agarose-0.5×TBE gel by restriction analysis with SacI and EcoRI/HindIII. The resulting plasmid was designated pMRT064.1 (FIG. 31).
[0294] The SacI site at position 2666 in plasmid pMRT064.1 was deleted by SOE using primer pairs 64 and 65, and primer pairs 66 and 67 shown below. PCR amplification was conducted in 50 μl reactions composed of 1 ng of pMRT064.1 DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized in a 0.8% agarose-0.5×TBE gel. The expected fragments were approximately 400 and 800 bp, respectively. The final fragment for cloning back into pMRT064.1 was amplified using primers 64 and 67. This fragment was cloned into pCR2.1 vector using the TA-TOPO Cloning Kit. Transformants were selected on 2×YT agar plates supplemented with 100 μg/ml ampicillin after incubation at 37° C. for 16 hours. Transformants carrying the correct plasmid were verified by DNA sequencing using M13 forward and reverse primers, and primers 65, 67, and 68. This plasmid was designated pMRT068 (FIG. 32), and was further transformed into E. coli DM1 cells (Stratagene, Inc., La Jolla, Calif.) according to the manufacturer's instructions. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml.
TABLE-US-00017 Primer 64: (SEQ ID NO: 78) 5'-GGAAATTATCGTGATCAAC-3' Primer 65: (SEQ ID NO: 79) 5'-GCACGAGCACTGATAAATATG-3' Primer 66: (SEQ ID NO: 80) 5'-CATATTTATCAGTGCTCGTGC-3' Primer 67: (SEQ ID NO: 81) 5'-TCGTAGACCTCATATGC-3' Primer 68: (SEQ ID NO: 82) 5'-GTCGTTAAACCGTGTGC-3'
[0295] The SacI sites at positions 5463 and 6025 in plasmid pMRT064.1 were deleted using PCR amplification with primers 69 and 70, and using the PCR conditions described above. The resulting fragment was cloned into pCR2.1 vector using the TA-TOPO Cloning Kit (Invitrogen, Inc., Carlsbad, Calif.). Transformants were selected on 2×YT-agar plates supplemented with 100 μg of ampicillin per ml after incubation at 37° C. for 16 hours. Transformants carrying the correct plasmid were verified by DNA sequencing using M13 forward and reverse primers. This construct was designated pMRT069 (FIG. 33).
TABLE-US-00018 Primer 69: (SEQ ID NO: 83) 5'-CTAGAGGATCCCCGGGTACCGTGCTCTGCCTTTTAGTCC-3' Primer 70: (SEQ ID NO: 84) 5'-GTACATCGAATTCGTGCTCATTATTAATCTGTTCAGC-3'
[0296] Plasmids pMRT068 and pMRT064.1 were digested with BclI/AccI. A fragment of approximately 1300 bp from pMRT068 and a fragment of approximately 3800 bp from pMRT064.1 were isolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions, ligated, and transformed into Bacillus subtilis 168Δ4 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at 30° C. for 24-48 hours. Transformants carrying the correct plasmid were identified on a 0.8% agarose-0.5×TBE gel by restriction analysis with SacI and EcoRI/AvaI. The resulting construct was designated pMRT071 (FIG. 34).
[0297] Plasmids pMRT071 and pMRT069 were digested with AvaI/EcoRI. The 578 bp fragment from pMRT069 and the 4510 bp fragment from pMRT071 were isolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions, ligated, and transformed into Bacillus subtilis 168Δ4 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at 30° C. for 24-48 hours. Transformants carrying the correct plasmid were identified on a 0.8% agarose-0.5×TBE gel by restriction analysis with SacI. The resulting construct was designated pMRT074 (FIG. 35).
[0298] Plasmid pMRT084 described in Example 11 was digested with SacII/NdeI, treated with T4 DNA polymerase, ligated, and transformed into E. coli XL1 Blue cells according to the manufacturer's instructions. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml after incubation at 37° C. for 16 hours. Transformants carrying the correct plasmid were identified on a 0.8% agarose-0.5×TBE gel by restriction analysis with DraI. The resulting plasmid was named pMRT120 (FIG. 36).
[0299] Plasmid pMRT074 was digested with HindIII, treated with Klenow fragment DNA polymerase, and digested with EcoRI. Plasmid pMRT120 was digested with EcoRI/Ecl13611. A fragment of approximately 600 bp from pMRT120 and a fragment of approximately 4300 bp from pMRT074 were isolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according to the manufacturer's instructions, ligated, and transformed into Bacillus subtilis 168Δ4 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at 30° C. for 24-48 hours. Transformants carrying the correct plasmid were identified on a 0.8% agarose-0.5×TBE gel by restriction analysis with SspI. The resulting construct was designated pMRT122 (FIG. 37).
[0300] Plasmid pMRT122 was transformed into Bacillus subtilis A164Δ5 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at 30° C. for 24-48 hours. The plasmid was introduced into the chromosome of Bacillus subtilis A164Δ5 via homologous recombination into the cypX locus by incubating a freshly streaked plate of Bacillus subtilis A164Δ5 (pMRT086) cells at 45° C. for 16 hours and selecting for healthy growing colonies. Genomic DNA was isolated from this strain using a QIAGEN tip-20 column according to the manufacturer's instructions and used to transform Bacillus subtilis RB187 (Example 9). Transformants were selected on TBAB plates supplemented with 1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at 45° C. for 16 hours. At this temperature, the pE194 replicon is unable to replicate. Cells are able to maintain erythromycin resistance only by maintaining the plasmid in the bacterial chromosome.
[0301] The plasmid was removed from the chromosome via homologous recombination resulting in the deletion of a portion of the cypX gene on the chromosome by growing the transformants in Luria-Bertani (LB) medium without selection at the permissive temperature of 34° C. for many generations. At this temperature the pE194 origin of replication is active and actually promotes the excision of the plasmid from the chromosome (Molecular Biological Methods for Bacillus, edited by C. R. Harwood and S. M. Cutting, 1990, John Wiley and Sons Ltd.).
[0302] After several generations of outgrowth the cells were plated on non-selective LB agar plates and colonies which had lost the plasmid and were now cypX-deleted and producing hyaluronic acid were identified as follows: (1) cell patches were "wet" when plated on minimal plates indicating production of hyaluronic acid, (2) erythromycin sensitivity indicated loss of the pE194-based plasmid, and (3) PCR confirmed the presence of the 800 bp cypX deletion in the strain of interest by using primers 34 and 45.
[0303] Chromosomal DNA from potential cypX deletants was isolated using the REDextract-N-Amp® Plant PCR kits as follows: Single Bacillus colonies were inoculated into 100 μl of Extraction Solution, incubated at 95° C. for 10 minutes, and then diluted with an equal volume of Dilution Solution. PCR was performed using 4 μl of extracted DNA in conjunction with the REDextract-N-Amp® PCR Reaction Mix and the desired primers according to the manufacturer's instructions, using PCR cycling conditions as described in Example 5. PCR reaction products were visualized using a 0.8% agarose-0.5×TBE gel. The verified strain was designated Bacillus subtilis RB197.
Example 13
Construction of Bacillus subtilis RB200
[0304] The cypX gene of Bacillus subtilis RB192 was deleted using the same methods described in Example 9 for Bacillus subtilis RB187. The resultant strain was designated Bacillus subtilis RB200.
Example 14
Construction of Bacillus subtilis RB202
[0305] Bacillus subtilis A164Δ5ΔcypX was constructed as follows: Plasmid pMRT122 (Example 12) was transformed into Bacillus subtilis A164Δ5 competent cells. Transformants were selected on TBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at 30° C. for 24-48 hours. The plasmid was introduced into the chromosome of Bacillus subtilis A164Δ5 via homologous recombination into the cypX locus by incubating a freshly streaked plate of Bacillus subtilis A164Δ5 (pMRT086) cells at 45° C. for 16 hours and selecting for healthy growing colonies. The plasmid was removed from the chromosome via homologous recombination resulting in the deletion of a portion of the cypX gene on the chromosome by growing the transformants in Luria-Bertani (LB) medium without selection at the permissive temperature of 34° C. for many generations. At this temperature the pE194 origin of replication is active and actually promotes the excision of the plasmid from the chromosome (Molecular Biological Methods for Bacillus, edited by C. R. Harwood and S. M. Cutting, 1990, John Wiley and Sons Ltd.). After several generations of outgrowth the cells were plated on non-selective LB agar plates and colonies which had lost the plasmid and were now cypX-deleted were identified as follows: (1) erythromycin sensitivity indicated loss of the pE194-based plasmid, and (2) PCR confirmed the presence of the 800 bp cypX deletion in the strain of interest by using primers 34 and 45 as described above. The verified strain was designated Bacillus subtilis A164quadrature5quadraturecypX.
[0306] Bacillus subtilis A164Δ5ΔcypX was made competent and transformed with Bacillus subtilis TH1 genomic DNA (Example 7) isolated using a QIAGEN tip-20 column according to the manufacturer's instructions. Transformants were selected on TBAB plates containing 5 μg of chloramphenicol per ml at 37° C. The Bacillus subtilis A164Δ5ΔcypX hasA/hasB/hasC/hasD integrant was identified by its "wet" phenotype and designated Bacillus subtilis RB201. The cat gene was deleted from Bacillus subtilis RB201 using the same method described in Example 9. The resultant strain was designated Bacillus subtilis RB202.
Example 15
Construction of Bacillus subtilis MF002 (tuaD/gtaB)
[0307] Plasmid pHA3 (Example 2, FIG. 9) was digested with Asp718. The digested plasmid was then blunted by first inactivating the restriction enzyme at 85° C. for 30 minutes. Blunting was performed by adding 0.5 μl of 10 mM each dNTPs, 1 μl of 1 U/μl T4 polymerase and incubating at 11° C. for 10 minutes. Finally the polymerase was inactivated by incubating the reaction at 75° C. for 10 minutes. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions and finally digested with NotI. The smallest plasmid fragment of approximately 2522 bp was then gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered DNA insert (tuaD/gtaB) was then ligated with the vector DNA described below.
[0308] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digested with Ecl136II. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with NotI. The largest plasmid fragment of approximately 6800 bp was gel-purified from a 0.8% agarose-0.5×TBE gel using a QIAquick DNA Gel Extraction Kit according to the manufacturer's instructions.
[0309] The recovered vector and DNA insert were ligated using the Rapid DNA Cloning Kit according to the manufacturer's instructions. Prior to transformation in Bacillus subtilis, the ligation described above was linearized using ScaI to ensure double cross-over integration in the chromosome rather than single cross-over integration in the chromosome. Bacillus subtilis 168Δ4 competent cells were transformed with the ligation digested with the restriction enzyme ScaI.
[0310] Bacillus subtilis chloramphenicol-resistant transformants were selected on TBAB plates supplemented with 5 μg of chloramphenicol per ml. To screen for integration of the plasmid by double cross-over at the amyE locus, Bacillus subtilis primary transformants were patched on TBAB plates supplemented with 6 μg of neomycin per ml and on TBAB plates supplemented with 5 μg of chloramphenicol per ml to isolate chloramphenicol resistant and neomycin sensitive transformants were isolated.
[0311] Chromosomal DNA from chloramphenicol resistant and neomycin sensitive Bacillus subtilis 168Δ4 transformants was isolated using the REDextract-N-Amp® Plant PCR kits (Sigma Chemical Company, St. Louis, Mo.) as follows: Single Bacillus colonies were inoculated into 100 μl of Extraction Solution, incubated at 95° C. for 10 minutes, and then diluted with an equal volume of Dilution Solution. PCR was performed using 4 μl of extracted DNA in conjunction with the REDextract-N-Amp PCR Reaction Mix and the desired primers according to the manufacturer's instructions, with PCR cycling conditions described in Example 5.
[0312] PCR amplifications were performed on these transformants using the synthetic oligonucleotides described below to confirm the absence/presence and integrity of the genes hasA, gtaB, and tuaD of the operon of the Bacillus subtilis transformants. Primers 3 and 8 were used to confirm the absence of the hasA gene, primer 71 and primer 15 to confirm the presence of the tuaD gene, and primers 20 and 71 to confirm the presence of the gtaB gene. PCR reaction products were visualized in a 0.8% agarose-0.5×TBE gel. The verified strain, a Bacillus subtilis 168Δ4 hasA/tuaD/gtaB integrant, was designated Bacillus subtilis RB176.
TABLE-US-00019 Primer 71: (SEQ ID NO: 85) 5'-AACTATTGCCGATGATAAGC-3' (binds upstream of tuaD)
[0313] Genomic DNA was isolated from the chloramphenicol resistant, and neomycin sensitive Bacillus subtilis RB176 transformants using a QIAGEN tip-20 column according to the manufacturer's instructions. The Bacillus subtilis RB176 genomic DNA was used to transform competent Bacillus subtilis A164Δ5. Transformants were selected on TBAB plates containing 5 μg of chloramphenicol per ml, and grown at 37° C. A Bacillus subtilis A164Δ5 tuaD/gtaB integrant was designated Bacillus subtilis RB177.
[0314] The cat gene was deleted in strain Bacillus subtilis RB177 using the method described in Example 9. The resultant strain was designated Bacillus subtilis MF002.
Example 16
Construction of the pel Integration Plasmid pRB162
[0315] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was double-digested with SacI and AatII. The largest plasmid fragment of approximately 6193 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered vector DNA was then ligated with the DNA insert described below.
[0316] The 5' and 3' fragments of a Bacillus subtilis pectate lyase gene (pel, accession number BG10840, SEQ ID NOs. 86 [DNA sequence] and 87 [deduced amino acid sequence]) was PCR amplified from Bacillus subtilis 168 (BGSC 1A1, Bacillus Genetic Stock Center, Columbus, Ohio) using primers 72 (introduces 5' SpeI restriction site) and 73 (introduces 3' SalI restriction site) for the 5' pel fragment and primers 74 (introduces 5' SacI/BamHI restriction sites) and 75 (introduces 3' NotI/AatII restriction sites) for the 3' pel fragment:
TABLE-US-00020 Primer 72: (SEQ ID NO: 88) 5'-ACTAGTAATGATGGCTGGGGCGCGTA-3' Primer 73: (SEQ ID NO: 89) 5'-GTCGACATGTTGTCGTATTGTGAGTT-3' Primer 74: (SEQ ID NO: 90) 5'-GAGCTCTACAACGCTTATGGATCCGCGGCCGCGGCGGCACACACAT CTGGAT-3' Primer 75: (SEQ ID NO: 91) 5'-GACGTCAGCCCGTTTGCAGCCGATGC-3'
[0317] PCR amplifications were carried out in triplicate in 30 μl reactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.4 μM each of primer pair 72/73 for the 5' pel fragment or primer pair 74/75 for the 3' pel fragment, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl2, and 2.5 units of AmpliTaq Gold® DNA polymerase. The reactions were performed in a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 9 minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and 72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes. The PCR products were visualized using a 0.8% agarose-0.5×TBE gel. The expected fragments were approximately 530 bp for the 5' pel fragment and 530 bp for the 3' pel fragment.
[0318] The 530 bp 5' pel and 530 bp 3' pel PCR fragments were cloned into pCR2.1 using the TA-TOPO Cloning Kit and transformed into E. coli OneShot® competent cells according to the manufacturers' instructions. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml incubated at 37° C. Plasmid DNA from these transformants was purified using a QIAGEN robot according to the manufacturer's instructions and the DNA sequence of the inserts confirmed by DNA sequencing using the primers described above (primers 72 and 73 for 5' pel and primers 74 and 75 for 3' pel). The plasmids harboring the 530 bp and the 530 bp PCR fragments were designated pCR2.1-pel 5' and pCR2.1-pel3', respectively (FIGS. 38 and 39, respectively).
[0319] Plasmid pCR2.1-pel3' was double-digested with SacI and AatII. The smallest plasmid fragment of approximately 530 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions.
[0320] The recovered vector (pDG268MCSΔneo/scBAN) and DNA insert (3' pel) were ligated using the Rapid DNA Cloning Kit according to the manufacturer's instructions. The ligation mix was transformed into E. coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.). Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C.
[0321] Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by SacI and AatII digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of an approximately 530 bp SacI/AatII 3' pel fragment and was designated pRB161 (FIG. 40).
[0322] Plasmid pRB161 was double-digested with SpeI and SalI. The largest plasmid fragment of approximately 5346 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered vector DNA was then ligated with the DNA insert described below.
[0323] Plasmid pCR2.1-pel5' was double-digested with SpeI and SalI. The smallest plasmid fragment of approximately 530 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions.
[0324] The recovered vector (pDG268MCSΔneo/scBAN/pel 3') and insert (pel 5') DNA were ligated using the Rapid DNA Cloning Kit according to the manufacturer's instructions. The ligation mix was transformed into E. coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.). Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml.
[0325] Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by SpeI and SalI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of an approximately 530 bp SpeI/SalI pel 5' fragment and was designated pRB162 (FIG. 41).
Example 17
Construction of pRB156
[0326] Plasmid pHA7 (Example 4, FIG. 13) was digested with HpaI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions and finally digested with Asp718. The double-digested plasmid was then blunted by first inactivating the restriction enzyme at 85° C. for 30 minutes. Blunting was performed by adding 0.5 μl of 10 mM each dNTPs and 1 μl of 1 U/μl of T4 polymerase and incubating at 11° C. for 10 minutes. The polymerase was then inactivated by incubating the reaction at 75° C. for 10 minutes. The largest plasmid fragment of approximately 8600 bp was then gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered DNA insert (pDG268Δneo-cryIIIAstab/sehasA) was then re-ligated using the Rapid DNA Cloning Kit according to the manufacturer's instructions.
[0327] The ligation mix was transformed into E. coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.). Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C. Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by ScaI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the presence of an approximately 8,755 bp fragment and was designated pRB156 (FIG. 42).
Example 18
Construction of Bacillus subtilis MF009
[0328] The hasA gene under control of the scBAN promoter was introduced into the pectate lyase gene (pel) locus of Bacillus subtilis MF002 to generate Bacillus subtilis MF009.
[0329] Plasmid pRB156 was digested with SacI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with NotI. The smallest plasmid fragment of approximately 1,377 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered DNA insert was then ligated with the vector DNA described below.
[0330] Plasmid pRB162 (Example 16, FIG. 41) was digested with NotI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with SacI. The largest plasmid fragment of approximately 5850 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered vector DNA was then ligated with the DNA insert described above.
[0331] The ligation mixture was transformed directly in Bacillus subtilis 168Δ4 competent cells. Bacillus subtilis chloramphenicol-resistant transformants were selected on TBAB plates supplemented with 5 μg of chloramphenicol per ml at 37° C. To screen for integration of the plasmid by double cross-over at the pel locus, Bacillus subtilis primary transformants were patched on TBAB plates supplemented with 6 μg of neomycin per ml and on TBAB plates supplemented with 5 μg of chloramphenicol per ml. Integration of the plasmid by double cross-over at the pel locus does not incorporate the neomycin resistance gene and therefore renders the strain neomycin sensitive. Using this plate screen, chloramphenicol resistant and neomycin sensitive transformants were isolated.
[0332] Genomic DNA was isolated from the chloramphenicol resistant and neomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGEN tip-20 column according to the manufacturer's instructions. This genomic DNA was used to transform competent Bacillus subtilis MF002 (Example 15). Transformants were selected on TBAB plates containing 5 μg of chloramphenicol per ml and grown at 37° C. The Bacillus subtilis A164Δ5 hasA and tuaD/gtaB integrant was identified by its "wet" phenotype and designated Bacillus subtilis MF009.
Example 19
Construction of Bacillus subtilis MF010
[0333] Plasmid pDG268MCSΔneo/BAN/Sav (U.S. Pat. No. 5,955,310) was digested with NotI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with SfiI. The smallest plasmid fragment of approximately 185 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered DNA insert was then ligated with the vector DNA described below.
[0334] Plasmid pRB162 (Example 16, FIG. 41) was digested with NotI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with SfiI. The largest plasmid fragment of approximately 5747 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered vector DNA was then ligated with the DNA insert described above.
[0335] The recovered vector and DNA insert were ligated using the Rapid DNA Cloning Kit according to the manufacturer's instructions. The ligation mix was transformed into E. coli XLI Blue competent cells. Transformants were selected on 2×YT agar plates supplemented with 100 μg of ampicillin per ml.
[0336] Plasmid DNA was purified from several transformants using a QIAGEN robot according to the manufacturer's instructions and analyzed by BamHI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correct plasmid was identified by the linearization of the plasmid which provides an approximately 7,156 bp fragment and was designated pRB164 (FIG. 43).
[0337] Plasmid pRB156 (Example 17, FIG. 42) was digested with SacI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with NotI. The smallest plasmid fragment of approximately 1377 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered DNA insert was then ligated with the vector DNA described below.
[0338] Plasmid pRB164 was digested with NotI. The digested plasmid was then purified using a QIAquick DNA Purification Kit according to the manufacturer's instructions, and finally digested with SacI. The largest plasmid fragment of approximately 5922 bp was gel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to the manufacturer's instructions. The recovered vector DNA was then ligated with the DNA insert described above.
[0339] This ligation mix was transformed directly in Bacillus subtilis 168Δ4 competent cells. Bacillus subtilis chloramphenicol-resistant transformants were selected on TBAB plates supplemented with 5 μg of chloramphenicol per ml at 37° C. To screen for integration of the plasmid by double cross-over at the amyE locus, Bacillus subtilis primary transformants were patched on TBAB plates supplemented with 6 μg of neomycin per ml and on TBAB plates supplemented with 5 μg of chloramphenicol per ml. Integration of the plasmid by double cross-over at the amyE locus does not incorporate the neomycin resistance gene and therefore renders the strain neomycin sensitive. Using this plate screen, chloramphenicol resistant and neomycin sensitive transformants were isolated.
[0340] Genomic DNA was isolated from the chloramphenicol resistant and neomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGEN tip-20 column according to the manufacturer's instructions. This genomic DNA was used to transform competent Bacillus subtilis MF002 (Example 15). Transformants were selected on minimal plates containing 5 μg of chloramphenicol per ml and grown at 37° C. for 16 hours. A Bacillus subtilis A164Δ5 BAN/hasA and scBAN/tuaD/gtaB integrant was identified by its "wet" phenotype and designated Bacillus subtilis MF010.
Example 20
Fermentations
[0341] The ability of the Bacillus subtilis strains listed in Table 1 to produce hyaluronic acid was evaluated under various growth conditions.
TABLE-US-00021 TABLE 1 B. subtilis Strain promoter/gene complement catΔ cypXΔ RB161 scBAN/hasA/tuaD/gtaB no no RB163 scBAN/hasA/tuaD/gcaD no no TH-1 scBANhasA/hasB/hasC/hasD no no RB184 scBAN/hasA/tuaD no no RB187 scBAN/hasA/tuaD/gtaB yes no RB192 scBAN/hasA/tuaD yes no RB194 scBAN/hasA/tuaD/gtaB yes yes RB197 scBAN/hasA/tuaD/gtaB yes yes RB200 scBAN/hasA/tuaD yes yes RB202 scBAN/hasA/hasb/hasC/hasD yes yes MF009 scBAN/tuaD/gtaB no no scBAN/hasA MF010 scBAN/tuaD/gtaB no no BAN/hasA
[0342] The Bacillus subtilis strains were fermented in standard small fermenters in a medium composed per liter of 6.5 g of KH2PO4, 4.5 g of Na2HPO4, 3.0 g of (NH4)2SO4, 2.0 g of Na3-citrate-2H2O, 3.0 g of MgSO4.7H2O, 6.0 ml of Mikrosoy-2, 0.15 mg of biotin (1 ml of 0.15 mg/ml ethanol), 15.0 g of sucrose, 1.0 ml of SB 2066, 2.0 ml of P2000, 0.5 g of CaCl2.2H2O. The medium was pH 6.3 to 6.4 (unadjusted) prior to autoclaving. The CaCl2.2H2O was added after autoclaving.
[0343] The seed medium used was B-3, i.e., Agar-3 without agar, or "S/S-1" medium. The Agar-3 medium was composed per liter of 4.0 g of nutrient broth, 7.5 g of hydrolyzed protein, 3.0 g of yeast extract, 1.0 g of glucose, and 2% agar. The pH was not adjusted; pH before autoclaving was approximately 6.8; after autoclaving approximately pH 7.7.
[0344] The sucrose/soy seed flask medium (S/S-1) was composed per liter of 65 g of sucrose, 35 g of soy flour, 2 g of Na3-citrate.2H2O, 4 g of KH2PO4, 5 g of Na2HPO4, and 6 ml of trace elements. The medium was adjusted pH to about 7 with NaOH; after dispensing the medium to flasks, 0.2% vegetable oil was added to suppress foaming. Trace elements was composed per liter of 100 g of citric acid-H2O, 20 g of FeSO4.7H2O, 5 g of MnSO4.H2O, 2 g of CuSO4.5H2O, and 2 g of ZnCl2.
[0345] The pH was adjusted to 6.8-7.0 with ammonia before inoculation, and controlled thereafter at pH 7.0±0.2 with ammonia and H3PO4. The temperature was maintained at 37° C. Agitation was at a maximum of 1300 RPM using two 6-bladed rushton impellers of 6 cm diameter in 3 liter tank with initial volume of 1.5 liters. The aeration had a maximum of 1.5 VVM.
[0346] For feed, a simple sucrose solution was used. Feed started at about 4 hours after inoculation, when dissolved oxygen (D.O.) was still being driven down (i.e., before sucrose depletion). The feed rate was ramped linearly from 0 to approximately 6 g sucrose/L0-hr over a 7 hour time span. A lower feed rate, ramped linearly from 0 to approximately 2 g sucrose/L0-hr, was also used in some fermentations.
[0347] Viscosity was noticeable by about 10 hours and by 24 hours viscosity was very high, causing the D.O. to bottom-out. End-point viscosity reached 3,220 cP. Cell mass development reached a near maximum (12 to 15 g/liter) by 20 hours. Cells were removed by diluting 1 part culture with 3 parts water, mixing well and centrifuging at about 30,000×g to produce a clear supernatant and cell pellet, which can be washed and dried.
[0348] Assays of hyaluronic acid concentration were performed using the ELISA method, based on a hyaluronan binding protein (protein and kits commercially available from Seikagaku America, Falmouth, Mass.).
[0349] Bacillus subtilis RB 161 and RB163 were cultured in batch and fed-batch fermentations. In the fed-batch processes, the feed rate was varied between cultures of Bacillus subtilis strains RB163 and RB161. Assays of hyaluronic acid concentrations were again performed using the ELISA method. The results are provided in Table 2.
TABLE-US-00022 TABLE 2 HA (relative yield) Strain and Growth ELISA Conditions method RB-161 0.7 ± 0.1 (hasA/tuaD/gtaB) simple batch RB-163 0.9 ± 0.1 (hasA/tuaD/gcaD) fed batch ~ 6 g sucrose/L0-hr RB161 0.9 ± 0.1 (hasA/tuaD/gtaB) fed batch ~ 6 g sucrose/L0-hr RB-163 1.0 ± 0..2 (hasA/tuaD/gcaD) fed batch ~ 2 g sucrose/L0-hr RB161 1.0 ± 0..1 (hasA/tuaD/gtaB) fed batch ~ 2 g sucrose/L0-hr
[0350] The results of the culture assays for the same strain at a fed batch rate of 2 g/L sucrose/L0-hr compared to 6 g/L sucrose/L0-hr demonstrated that a faster sucrose feed rate did not significantly improve titers.
[0351] A summary of the Bacillus strains run under same conditions (fed batch at approximately 2 g sucrose/L0-hr, 37° C.) is shown in FIG. 44. In FIG. 44, ±values indicate standard deviation of data from multiple runs under the same conditions. Data without ±values are from single runs. Hyaluronic acid concentrations were determined using the modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334).
[0352] A summary of peak hyaluronic acid weight average molecular weights (MDa) obtained from fermentation of the recombinant Bacillus subtilis strains under the same conditions (fed batch at approximately 2 g sucrose/L0-hr, 37° C.) is shown in FIG. 45. Molecular weights were determined using a GPC MALLS assay. Data was gathered from GPC MALLS assays using the following procedure. GPC-MALLS (gel permeation or size-exclusion) chromatography coupled with multi-angle laser light scattering) is widely used to characterize high molecular weight (MW) polymers. Separation of polymers is achieved by GPC, based on the differential partitioning of molecules of different MW between eluent and resin. The average molecular weight of an individual polymer is determined by MALLS based the differential scattering extent/angle of molecules of different MW. Principles of GPC-MALLS and protocols suited for hyaluronic acid are described by Ueno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, "Light Scattering University DAWN Course Manual" and "DAWN EOS Manual" Wyatt Technology Corporation, Santa Barbara, Calif.). An Agilent 1100 isocratic HPLC, a Tosoh Biosep G6000 PWxl column for the GPC, and a Wyatt Down EOS for the MALLS were used. An Agilent G1362A refractive index detector was linked downstream from the MALLS for eluate concentration determination. Various commercial hyaluronic acid products with known molecular weights served as standards.
Deposit of Biological Material
[0353] The following biological material has been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoria, Ill., 61604, and given the following accession number:
TABLE-US-00023 Accession Deposit Number Date of Deposit E. coli XL10 Gold kan (pMRT106) NRRL B-30536 Dec. 12, 2001
[0354] The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
[0355] The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.
[0356] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.
Sequence CWU
1
1
10811251DNAStreptococcus equisimilisCDS(1)..(1251) 1atg aga aca tta aaa
aac ctc ata act gtt gtg gcc ttt agt att ttt 48Met Arg Thr Leu Lys
Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe 1 5
10 15 tgg gta ctg ttg att
tac gtc aat gtt tat ctc ttt ggt gct aaa gga 96Trp Val Leu Leu Ile
Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly 20
25 30 agc ttg tca att tat
ggc ttt ttg ctg ata gct tac cta tta gtc aaa 144Ser Leu Ser Ile Tyr
Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys 35
40 45 atg tcc tta tcc ttt
ttt tac aag cca ttt aag gga agg gct ggg caa 192Met Ser Leu Ser Phe
Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln 50
55 60 tat aag gtt gca gcc
att att ccc tct tat aac gaa gat gct gag tca 240Tyr Lys Val Ala Ala
Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser 65
70 75 80 ttg cta gag acc tta
aaa agt gtt cag cag caa acc tat ccc cta gca 288Leu Leu Glu Thr Leu
Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala 85
90 95 gaa att tat gtt gtt
gac gat gga agt gct gat gag aca ggt att aag 336Glu Ile Tyr Val Val
Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys 100
105 110 cgc att gaa gac tat
gtg cgt gac act ggt gac cta tca agc aat gtc 384Arg Ile Glu Asp Tyr
Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val 115
120 125 att gtt cac cgg tca
gaa aaa aat caa gga aag cgt cat gca cag gcc 432Ile Val His Arg Ser
Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala 130
135 140 tgg gcc ttt gaa aga
tca gac gct gat gtc ttt ttg acc gtt gac tca 480Trp Ala Phe Glu Arg
Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser 145
150 155 160 gat act tat atc tac
cct gat gct tta gag gag ttg tta aaa acc ttt 528Asp Thr Tyr Ile Tyr
Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe 165
170 175 aat gac cca act gtt
ttt gct gcg acg ggt cac ctt aat gtc aga aat 576Asn Asp Pro Thr Val
Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn 180
185 190 aga caa acc aat ctc
tta aca cgc ttg aca gat att cgc tat gat aat 624Arg Gln Thr Asn Leu
Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn 195
200 205 gct ttt ggc gtt gaa
cga gct gcc caa tcc gtt aca ggt aat att ctc 672Ala Phe Gly Val Glu
Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu 210
215 220 gtt tgc tca ggc ccg
ctt agc gtt tac aga cgc gag gtg gtt gtt cct 720Val Cys Ser Gly Pro
Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro 225
230 235 240 aac ata gat aga tac
atc aac cag acc ttc ctg ggt att cct gta agt 768Asn Ile Asp Arg Tyr
Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser 245
250 255 atc ggt gat gac agg
tgc ttg acc aac tat gca act gat tta gga aag 816Ile Gly Asp Asp Arg
Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys 260
265 270 act gtt tat caa tcc
act gct aaa tgt att aca gat gtt cct gac aag 864Thr Val Tyr Gln Ser
Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys 275
280 285 atg tct act tac ttg
aag cag caa aac cgc tgg aac aag tcc ttc ttt 912Met Ser Thr Tyr Leu
Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe 290
295 300 aga gag tcc att att
tct gtt aag aaa atc atg aac aat cct ttt gta 960Arg Glu Ser Ile Ile
Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val 305
310 315 320 gcc cta tgg acc ata
ctt gag gtg tct atg ttt atg atg ctt gtt tat 1008Ala Leu Trp Thr Ile
Leu Glu Val Ser Met Phe Met Met Leu Val Tyr 325
330 335 tct gtg gtg gat ttc
ttt gta gac aat gtc aga gaa ttt gat tgg ctc 1056Ser Val Val Asp Phe
Phe Val Asp Asn Val Arg Glu Phe Asp Trp Leu 340
345 350 agg gtt ttg gcc ttt
ctg gtg att atc ttc att gtt gct ctt tgt cgt 1104Arg Val Leu Ala Phe
Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg 355
360 365 aat att cac tat atg
ctt aag cac ccg ctg tcc ttc ttg tta tct ccg 1152Asn Ile His Tyr Met
Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro 370
375 380 ttt tat ggg gta ctg
cat ttg ttt gtc cta cag ccc ttg aaa ttg tat 1200Phe Tyr Gly Val Leu
His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr 385
390 395 400 tct ctt ttt act att
aga aat gct gac tgg gga aca cgt aaa aaa tta 1248Ser Leu Phe Thr Ile
Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu 405
410 415 tta
1251Leu
2417PRTStreptococcus
equisimilis 2 Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile
Phe 1 5 10 15 Trp
Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly
20 25 30 Ser Leu Ser Ile Tyr
Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys 35
40 45 Met Ser Leu Ser Phe Phe Tyr Lys Pro
Phe Lys Gly Arg Ala Gly Gln 50 55
60 Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp
Ala Glu Ser 65 70 75
80 Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala
85 90 95 Glu Ile Tyr Val
Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys 100
105 110 Arg Ile Glu Asp Tyr Val Arg Asp Thr
Gly Asp Leu Ser Ser Asn Val 115 120
125 Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala
Gln Ala 130 135 140
Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser 145
150 155 160 Asp Thr Tyr Ile Tyr
Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe 165
170 175 Asn Asp Pro Thr Val Phe Ala Ala Thr Gly
His Leu Asn Val Arg Asn 180 185
190 Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp
Asn 195 200 205 Ala
Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu 210
215 220 Val Cys Ser Gly Pro Leu
Ser Val Tyr Arg Arg Glu Val Val Val Pro 225 230
235 240 Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu
Gly Ile Pro Val Ser 245 250
255 Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys
260 265 270 Thr Val
Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys 275
280 285 Met Ser Thr Tyr Leu Lys Gln
Gln Asn Arg Trp Asn Lys Ser Phe Phe 290 295
300 Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn
Asn Pro Phe Val 305 310 315
320 Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr
325 330 335 Ser Val Val
Asp Phe Phe Val Asp Asn Val Arg Glu Phe Asp Trp Leu 340
345 350 Arg Val Leu Ala Phe Leu Val Ile
Ile Phe Ile Val Ala Leu Cys Arg 355 360
365 Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu
Leu Ser Pro 370 375 380
Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr 385
390 395 400 Ser Leu Phe Thr
Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu 405
410 415 Leu 349DNAStreptococcus equisimilis
3gagctctata aaaatgagga gggaaccgaa tgagaacatt aaaaaacct
49448DNAStreptococcus equisimilis 4gttaacgaat tcagctatgt aggtacctta
taataatttt ttacgtgt 48520DNAStreptococcus equisimilis
5gttgacgatg gaagtgctga
20620DNAStreptococcus equisimilis 6atccgttaca ggtaatatcc
20720DNAStreptococcus equisimilis
7tccttttgta gccctatgga
20820DNAStreptococcus equisimilis 8tcagcacttc catcgtcaac
20920DNAStreptococcus equisimilis
9ggatattacc tgtaacggat
201020DNAStreptococcus equisimilis 10tccatagggc tacaaaagga
20111383DNABacillus
subtilisCDS(1)..(1383) 11gtg aaa aaa ata gct gtc att gga aca ggt tat gta
gga ctc gta tca 48Val Lys Lys Ile Ala Val Ile Gly Thr Gly Tyr Val
Gly Leu Val Ser 1 5 10
15 ggc act tgc ttt gcg gag atc ggc aat aaa gtt gtt
tgc tgt gat atc 96Gly Thr Cys Phe Ala Glu Ile Gly Asn Lys Val Val
Cys Cys Asp Ile 20 25
30 gat gaa tca aaa atc aga agc ctg aaa aat ggg gta
atc cca atc tat 144Asp Glu Ser Lys Ile Arg Ser Leu Lys Asn Gly Val
Ile Pro Ile Tyr 35 40
45 gaa cca ggg ctt gca gac tta gtt gaa aaa aat gtg
ctg gat cag cgc 192Glu Pro Gly Leu Ala Asp Leu Val Glu Lys Asn Val
Leu Asp Gln Arg 50 55 60
ctg acc ttt acg aac gat atc ccg tct gcc att cgg
gcc tca gat att 240Leu Thr Phe Thr Asn Asp Ile Pro Ser Ala Ile Arg
Ala Ser Asp Ile 65 70 75
80 att tat att gca gtc gga acg cct atg tcc aaa aca
ggt gaa gct gat 288Ile Tyr Ile Ala Val Gly Thr Pro Met Ser Lys Thr
Gly Glu Ala Asp 85 90
95 tta acg tac gtc aaa gcg gcg gcg aaa aca atc ggt
gag cat ctt aac 336Leu Thr Tyr Val Lys Ala Ala Ala Lys Thr Ile Gly
Glu His Leu Asn 100 105
110 ggc tac aaa gtg atc gta aat aaa agc aca gtc ccg
gtt gga aca ggg 384Gly Tyr Lys Val Ile Val Asn Lys Ser Thr Val Pro
Val Gly Thr Gly 115 120
125 aaa ctg gtg caa tct atc gtt caa aaa gcc tca aag
ggg aga tac tca 432Lys Leu Val Gln Ser Ile Val Gln Lys Ala Ser Lys
Gly Arg Tyr Ser 130 135 140
ttt gat gtt gta tct aac cct gaa ttc ctt cgg gaa
ggg tca gcg att 480Phe Asp Val Val Ser Asn Pro Glu Phe Leu Arg Glu
Gly Ser Ala Ile 145 150 155
160 cat gac acg atg aat atg gag cgt gcc gtg att ggt
tca aca agt cat 528His Asp Thr Met Asn Met Glu Arg Ala Val Ile Gly
Ser Thr Ser His 165 170
175 aaa gcc gct gcc atc att gag gaa ctt cat cag cca
ttc cat gct cct 576Lys Ala Ala Ala Ile Ile Glu Glu Leu His Gln Pro
Phe His Ala Pro 180 185
190 gtc att aaa aca aac cta gaa agt gca gaa atg att
aaa tac gcc gcg 624Val Ile Lys Thr Asn Leu Glu Ser Ala Glu Met Ile
Lys Tyr Ala Ala 195 200
205 aat gca ttt ctg gcg aca aag att tcc ttt atc aac
gat atc gca aac 672Asn Ala Phe Leu Ala Thr Lys Ile Ser Phe Ile Asn
Asp Ile Ala Asn 210 215 220
att tgt gag cga gtc ggc gca gac gtt tca aaa gtt
gct gat ggt gtt 720Ile Cys Glu Arg Val Gly Ala Asp Val Ser Lys Val
Ala Asp Gly Val 225 230 235
240 ggt ctt gac agc cgt atc ggc aga aag ttc ctt aaa
gct ggt att gga 768Gly Leu Asp Ser Arg Ile Gly Arg Lys Phe Leu Lys
Ala Gly Ile Gly 245 250
255 ttc ggc ggt tca tgt ttt cca aag gat aca acc gcg
ctg ctt caa atc 816Phe Gly Gly Ser Cys Phe Pro Lys Asp Thr Thr Ala
Leu Leu Gln Ile 260 265
270 gca aaa tcg gca ggc tat cca ttc aag ctc atc gaa
gct gtc att gaa 864Ala Lys Ser Ala Gly Tyr Pro Phe Lys Leu Ile Glu
Ala Val Ile Glu 275 280
285 acg aac gaa aag cag cgt gtt cat att gta gat aaa
ctt ttg act gtt 912Thr Asn Glu Lys Gln Arg Val His Ile Val Asp Lys
Leu Leu Thr Val 290 295 300
atg gga agc gtc aaa ggg aga acc att tca gtc ctg
gga tta gcc ttc 960Met Gly Ser Val Lys Gly Arg Thr Ile Ser Val Leu
Gly Leu Ala Phe 305 310 315
320 aaa ccg aat acg aac gat gtg aga tcc gct cca gcg
ctt gat att atc 1008Lys Pro Asn Thr Asn Asp Val Arg Ser Ala Pro Ala
Leu Asp Ile Ile 325 330
335 cca atg ctg cag cag ctg ggc gcc cat gta aaa gca
tac gat ccg att 1056Pro Met Leu Gln Gln Leu Gly Ala His Val Lys Ala
Tyr Asp Pro Ile 340 345
350 gct att cct gaa gct tca gcg atc ctt ggc gaa cag
gtc gag tat tac 1104Ala Ile Pro Glu Ala Ser Ala Ile Leu Gly Glu Gln
Val Glu Tyr Tyr 355 360
365 aca gat gtg tat gct gcg atg gaa gac act gat gca
tgc ctg att tta 1152Thr Asp Val Tyr Ala Ala Met Glu Asp Thr Asp Ala
Cys Leu Ile Leu 370 375 380
acg gat tgg ccg gaa gtg aaa gaa atg gag ctt gta
aaa gtg aaa acc 1200Thr Asp Trp Pro Glu Val Lys Glu Met Glu Leu Val
Lys Val Lys Thr 385 390 395
400 ctc tta aaa cag cca gtc atc att gac ggc aga aat
tta ttt tca ctt 1248Leu Leu Lys Gln Pro Val Ile Ile Asp Gly Arg Asn
Leu Phe Ser Leu 405 410
415 gaa gag atg cag gca gcc gga tac att tat cac tct
atc ggc cgt ccc 1296Glu Glu Met Gln Ala Ala Gly Tyr Ile Tyr His Ser
Ile Gly Arg Pro 420 425
430 gct gtt cgg gga acg gaa ccc tct gac aag tat ttt
ccg ggc ttg ccg 1344Ala Val Arg Gly Thr Glu Pro Ser Asp Lys Tyr Phe
Pro Gly Leu Pro 435 440
445 ctt gaa gaa ttg gct aaa gac ttg gga agc gtc aat
tta 1383Leu Glu Glu Leu Ala Lys Asp Leu Gly Ser Val Asn
Leu 450 455 460
12461PRTBacillus subtilis 12Val Lys Lys Ile Ala
Val Ile Gly Thr Gly Tyr Val Gly Leu Val Ser 1 5
10 15 Gly Thr Cys Phe Ala Glu Ile Gly Asn Lys
Val Val Cys Cys Asp Ile 20 25
30 Asp Glu Ser Lys Ile Arg Ser Leu Lys Asn Gly Val Ile Pro Ile
Tyr 35 40 45 Glu
Pro Gly Leu Ala Asp Leu Val Glu Lys Asn Val Leu Asp Gln Arg 50
55 60 Leu Thr Phe Thr Asn Asp
Ile Pro Ser Ala Ile Arg Ala Ser Asp Ile 65 70
75 80 Ile Tyr Ile Ala Val Gly Thr Pro Met Ser Lys
Thr Gly Glu Ala Asp 85 90
95 Leu Thr Tyr Val Lys Ala Ala Ala Lys Thr Ile Gly Glu His Leu Asn
100 105 110 Gly Tyr
Lys Val Ile Val Asn Lys Ser Thr Val Pro Val Gly Thr Gly 115
120 125 Lys Leu Val Gln Ser Ile Val
Gln Lys Ala Ser Lys Gly Arg Tyr Ser 130 135
140 Phe Asp Val Val Ser Asn Pro Glu Phe Leu Arg Glu
Gly Ser Ala Ile 145 150 155
160 His Asp Thr Met Asn Met Glu Arg Ala Val Ile Gly Ser Thr Ser His
165 170 175 Lys Ala Ala
Ala Ile Ile Glu Glu Leu His Gln Pro Phe His Ala Pro 180
185 190 Val Ile Lys Thr Asn Leu Glu Ser
Ala Glu Met Ile Lys Tyr Ala Ala 195 200
205 Asn Ala Phe Leu Ala Thr Lys Ile Ser Phe Ile Asn Asp
Ile Ala Asn 210 215 220
Ile Cys Glu Arg Val Gly Ala Asp Val Ser Lys Val Ala Asp Gly Val 225
230 235 240 Gly Leu Asp Ser
Arg Ile Gly Arg Lys Phe Leu Lys Ala Gly Ile Gly 245
250 255 Phe Gly Gly Ser Cys Phe Pro Lys Asp
Thr Thr Ala Leu Leu Gln Ile 260 265
270 Ala Lys Ser Ala Gly Tyr Pro Phe Lys Leu Ile Glu Ala Val
Ile Glu 275 280 285
Thr Asn Glu Lys Gln Arg Val His Ile Val Asp Lys Leu Leu Thr Val 290
295 300 Met Gly Ser Val Lys
Gly Arg Thr Ile Ser Val Leu Gly Leu Ala Phe 305 310
315 320 Lys Pro Asn Thr Asn Asp Val Arg Ser Ala
Pro Ala Leu Asp Ile Ile 325 330
335 Pro Met Leu Gln Gln Leu Gly Ala His Val Lys Ala Tyr Asp Pro
Ile 340 345 350 Ala
Ile Pro Glu Ala Ser Ala Ile Leu Gly Glu Gln Val Glu Tyr Tyr 355
360 365 Thr Asp Val Tyr Ala Ala
Met Glu Asp Thr Asp Ala Cys Leu Ile Leu 370 375
380 Thr Asp Trp Pro Glu Val Lys Glu Met Glu Leu
Val Lys Val Lys Thr 385 390 395
400 Leu Leu Lys Gln Pro Val Ile Ile Asp Gly Arg Asn Leu Phe Ser Leu
405 410 415 Glu Glu
Met Gln Ala Ala Gly Tyr Ile Tyr His Ser Ile Gly Arg Pro 420
425 430 Ala Val Arg Gly Thr Glu Pro
Ser Asp Lys Tyr Phe Pro Gly Leu Pro 435 440
445 Leu Glu Glu Leu Ala Lys Asp Leu Gly Ser Val Asn
Leu 450 455 460 1326DNABacillus
subtilis 13ggtaccgaca ctgcgaccat tataaa
261449DNABacillus subtilis 14gttaacgaat tccagctatg tatctagaca
gcttcaacca agtaacact 491520DNABacillus subtilis
15agcatcttaa cggctacaaa
201620DNABacillus subtilis 16tgtgagcgag tcggcgcaga
201720DNABacillus subtilis 17gggcgcccat
gtaaaagcat
201820DNABacillus subtilis 18tttgtagccg ttaagatgct
201920DNABacillus subtilis 19tctgcgccga
ctcgctcaca
202020DNABacillus subtilis 20atgcttttac atgggcgccc
2021876DNABacillus subtilisCDS(1)..(876) 21atg
aaa aaa gta cgt aaa gcc ata att cca gca gca ggc tta gga aca 48Met
Lys Lys Val Arg Lys Ala Ile Ile Pro Ala Ala Gly Leu Gly Thr 1
5 10 15 cgt
ttt ctt ccg gct acg aaa gca atg ccg aaa gaa atg ctt cct atc 96Arg
Phe Leu Pro Ala Thr Lys Ala Met Pro Lys Glu Met Leu Pro Ile
20 25 30 gtt
gat aaa cct acc att caa tac ata att gaa gaa gct gtt gaa gcc 144Val
Asp Lys Pro Thr Ile Gln Tyr Ile Ile Glu Glu Ala Val Glu Ala
35 40 45 ggt
att gaa gat att att atc gta aca gga aaa agc aag cgt gcg att 192Gly
Ile Glu Asp Ile Ile Ile Val Thr Gly Lys Ser Lys Arg Ala Ile
50 55 60 gag
gat cat ttt gat tac tct cct gag ctt gaa aga aac cta gaa gaa 240Glu
Asp His Phe Asp Tyr Ser Pro Glu Leu Glu Arg Asn Leu Glu Glu 65
70 75 80 aaa
gga aaa act gag ctg ctt gaa aaa gtg aaa aag gct tct aac ctg 288Lys
Gly Lys Thr Glu Leu Leu Glu Lys Val Lys Lys Ala Ser Asn Leu
85 90 95 gct
gac att cac tat atc cgc caa aaa gaa cct aaa ggt ctc gga cat 336Ala
Asp Ile His Tyr Ile Arg Gln Lys Glu Pro Lys Gly Leu Gly His
100 105 110 gct
gtc tgg tgc gca cgc aac ttt atc ggc gat gag ccg ttt gcg gta 384Ala
Val Trp Cys Ala Arg Asn Phe Ile Gly Asp Glu Pro Phe Ala Val
115 120 125 ctg
ctt ggt gac gat att gtt cag gct gaa act cca ggg ttg cgc caa 432Leu
Leu Gly Asp Asp Ile Val Gln Ala Glu Thr Pro Gly Leu Arg Gln
130 135 140 tta
atg gat gaa tat gaa aaa aca ctt tct tct att atc ggt gtt cag 480Leu
Met Asp Glu Tyr Glu Lys Thr Leu Ser Ser Ile Ile Gly Val Gln 145
150 155 160 cag
gtg ccc gaa gaa gaa aca cac cgc tac ggc att att gac ccg ctg 528Gln
Val Pro Glu Glu Glu Thr His Arg Tyr Gly Ile Ile Asp Pro Leu
165 170 175 aca
agt gaa ggc cgc cgt tat cag gtg aaa aac ttc gtt gaa aaa ccg 576Thr
Ser Glu Gly Arg Arg Tyr Gln Val Lys Asn Phe Val Glu Lys Pro
180 185 190 cct
aaa ggc aca gca cct tct aat ctt gcc atc tta ggc cgt tac gta 624Pro
Lys Gly Thr Ala Pro Ser Asn Leu Ala Ile Leu Gly Arg Tyr Val
195 200 205 ttc
acg cct gag atc ttc atg tat tta gaa gag cag cag gtt ggc gcc 672Phe
Thr Pro Glu Ile Phe Met Tyr Leu Glu Glu Gln Gln Val Gly Ala
210 215 220 ggc
gga gaa att cag ctc aca gac gcc att caa aag ctg aat gaa att 720Gly
Gly Glu Ile Gln Leu Thr Asp Ala Ile Gln Lys Leu Asn Glu Ile 225
230 235 240 caa
aga gtg ttt gct tac gat ttt gaa ggc aag cgt tat gat gtt ggt 768Gln
Arg Val Phe Ala Tyr Asp Phe Glu Gly Lys Arg Tyr Asp Val Gly
245 250 255 gaa
aag ctc ggc ttt atc aca aca act ctt gaa ttt gcg atg cag gat 816Glu
Lys Leu Gly Phe Ile Thr Thr Thr Leu Glu Phe Ala Met Gln Asp
260 265 270 aaa
gag ctt cgc gat cag ctc gtt cca ttt atg gaa ggt tta cta aac 864Lys
Glu Leu Arg Asp Gln Leu Val Pro Phe Met Glu Gly Leu Leu Asn
275 280 285 aaa
gaa gaa atc 876Lys
Glu Glu Ile
290
22292PRTBacillus subtilis 22Met Lys Lys Val Arg Lys Ala Ile Ile Pro Ala
Ala Gly Leu Gly Thr 1 5 10
15 Arg Phe Leu Pro Ala Thr Lys Ala Met Pro Lys Glu Met Leu Pro Ile
20 25 30 Val Asp
Lys Pro Thr Ile Gln Tyr Ile Ile Glu Glu Ala Val Glu Ala 35
40 45 Gly Ile Glu Asp Ile Ile Ile
Val Thr Gly Lys Ser Lys Arg Ala Ile 50 55
60 Glu Asp His Phe Asp Tyr Ser Pro Glu Leu Glu Arg
Asn Leu Glu Glu 65 70 75
80 Lys Gly Lys Thr Glu Leu Leu Glu Lys Val Lys Lys Ala Ser Asn Leu
85 90 95 Ala Asp Ile
His Tyr Ile Arg Gln Lys Glu Pro Lys Gly Leu Gly His 100
105 110 Ala Val Trp Cys Ala Arg Asn Phe
Ile Gly Asp Glu Pro Phe Ala Val 115 120
125 Leu Leu Gly Asp Asp Ile Val Gln Ala Glu Thr Pro Gly
Leu Arg Gln 130 135 140
Leu Met Asp Glu Tyr Glu Lys Thr Leu Ser Ser Ile Ile Gly Val Gln 145
150 155 160 Gln Val Pro Glu
Glu Glu Thr His Arg Tyr Gly Ile Ile Asp Pro Leu 165
170 175 Thr Ser Glu Gly Arg Arg Tyr Gln Val
Lys Asn Phe Val Glu Lys Pro 180 185
190 Pro Lys Gly Thr Ala Pro Ser Asn Leu Ala Ile Leu Gly Arg
Tyr Val 195 200 205
Phe Thr Pro Glu Ile Phe Met Tyr Leu Glu Glu Gln Gln Val Gly Ala 210
215 220 Gly Gly Glu Ile Gln
Leu Thr Asp Ala Ile Gln Lys Leu Asn Glu Ile 225 230
235 240 Gln Arg Val Phe Ala Tyr Asp Phe Glu Gly
Lys Arg Tyr Asp Val Gly 245 250
255 Glu Lys Leu Gly Phe Ile Thr Thr Thr Leu Glu Phe Ala Met Gln
Asp 260 265 270 Lys
Glu Leu Arg Asp Gln Leu Val Pro Phe Met Glu Gly Leu Leu Asn 275
280 285 Lys Glu Glu Ile 290
2327DNABacillus subtilis 23tctagatttt tcgatcataa ggaaggt
272449DNABacillus subtilis 24gttaacgaat
tccagctatg taggatccaa tgtccaatag cctttttgt
492520DNABacillus subtilis 25aaaaaggctt ctaacctggc
202620DNABacillus subtilis 26aaaccgccta
aaggcacagc
202720DNABacillus subtilis 27gccaggttag aagccttttt
202820DNABacillus subtilis 28gctgtgcctt
taggcggttt
20291368DNABacillus subtilisCDS(1)..(1368) 29atg gat aag cgg ttt gca gtt
gtt tta gcg gct gga caa gga acg aga 48Met Asp Lys Arg Phe Ala Val
Val Leu Ala Ala Gly Gln Gly Thr Arg 1 5
10 15 atg aaa tcg aag ctt tat aaa
gtc ctt cat cca gtt tgc ggt aag cct 96Met Lys Ser Lys Leu Tyr Lys
Val Leu His Pro Val Cys Gly Lys Pro 20
25 30 atg gta gag cac gtc gtg gac
gaa gcc tta aaa tta tct tta tca aag 144Met Val Glu His Val Val Asp
Glu Ala Leu Lys Leu Ser Leu Ser Lys 35
40 45 ctt gtc acg att gtc gga cat
ggt gcg gaa gaa gtg aaa aag cag ctt 192Leu Val Thr Ile Val Gly His
Gly Ala Glu Glu Val Lys Lys Gln Leu 50 55
60 ggt gat aaa agc gag tac gcg
ctt caa gca aaa cag ctt ggc act gct 240Gly Asp Lys Ser Glu Tyr Ala
Leu Gln Ala Lys Gln Leu Gly Thr Ala 65 70
75 80 cat gct gta aaa cag gca cag
cca ttt ctt gct gac gaa aaa ggc gtc 288His Ala Val Lys Gln Ala Gln
Pro Phe Leu Ala Asp Glu Lys Gly Val 85
90 95 aca att gtc att tgc gga gat
acg ccg ctt ttg aca gca gag acg atg 336Thr Ile Val Ile Cys Gly Asp
Thr Pro Leu Leu Thr Ala Glu Thr Met 100
105 110 gaa cag atg ctg aaa gaa cat
aca caa aga gaa gcg aaa gct acg att 384Glu Gln Met Leu Lys Glu His
Thr Gln Arg Glu Ala Lys Ala Thr Ile 115
120 125 tta act gcg gtt gca gaa gat
cca act gga tac ggc cgc att att cgc 432Leu Thr Ala Val Ala Glu Asp
Pro Thr Gly Tyr Gly Arg Ile Ile Arg 130 135
140 agc gaa aac gga gcg gtt caa
aaa ata gtt gag cat aag gac gcc tct 480Ser Glu Asn Gly Ala Val Gln
Lys Ile Val Glu His Lys Asp Ala Ser 145 150
155 160 gaa gaa gaa cgt ctt gta act
gag atc aac acc ggt acg tat tgt ttt 528Glu Glu Glu Arg Leu Val Thr
Glu Ile Asn Thr Gly Thr Tyr Cys Phe 165
170 175 gac aat gaa gcg cta ttt cgg
gct att gat cag gtg tct aat gat aat 576Asp Asn Glu Ala Leu Phe Arg
Ala Ile Asp Gln Val Ser Asn Asp Asn 180
185 190 gca caa ggc gag tat tat ttg
ccg gat gtc ata gag att ctt aaa aat 624Ala Gln Gly Glu Tyr Tyr Leu
Pro Asp Val Ile Glu Ile Leu Lys Asn 195
200 205 gaa ggc gaa act gtt gcc gct
tac cag act ggt aat ttc caa gaa acg 672Glu Gly Glu Thr Val Ala Ala
Tyr Gln Thr Gly Asn Phe Gln Glu Thr 210 215
220 ctc gga gtt aat gat aga gtt
gct ctt tct cag gca gaa caa ttt atg 720Leu Gly Val Asn Asp Arg Val
Ala Leu Ser Gln Ala Glu Gln Phe Met 225 230
235 240 aaa gag cgc att aat aaa cgg
cat atg caa aat ggc gtg acg ttg att 768Lys Glu Arg Ile Asn Lys Arg
His Met Gln Asn Gly Val Thr Leu Ile 245
250 255 gac ccg atg aat acg tat att
tct cct gac gct gtt atc gga agc gat 816Asp Pro Met Asn Thr Tyr Ile
Ser Pro Asp Ala Val Ile Gly Ser Asp 260
265 270 act gtg att tac cct gga act
gtg att aaa ggt gag gtg caa atc gga 864Thr Val Ile Tyr Pro Gly Thr
Val Ile Lys Gly Glu Val Gln Ile Gly 275
280 285 gaa gat acg att att ggc cct
cat acg gag att atg aat agt gcc att 912Glu Asp Thr Ile Ile Gly Pro
His Thr Glu Ile Met Asn Ser Ala Ile 290 295
300 ggc agc cgt acg gtt att aaa
caa tcg gta gtc aat cac agt aaa gtg 960Gly Ser Arg Thr Val Ile Lys
Gln Ser Val Val Asn His Ser Lys Val 305 310
315 320 ggg aat gat gta aac ata gga
cct ttt gct cac atc aga cct gat tct 1008Gly Asn Asp Val Asn Ile Gly
Pro Phe Ala His Ile Arg Pro Asp Ser 325
330 335 gtc atc ggg aat gaa gtg aag
atc ggg aat ttt gta gaa att aaa aag 1056Val Ile Gly Asn Glu Val Lys
Ile Gly Asn Phe Val Glu Ile Lys Lys 340
345 350 act caa ttc gga gac cga agc
aag gca tct cat cta agc tat gtc ggc 1104Thr Gln Phe Gly Asp Arg Ser
Lys Ala Ser His Leu Ser Tyr Val Gly 355
360 365 gat gct gag gta ggc act gat
gta aac ctg ggc tgc ggt tca att act 1152Asp Ala Glu Val Gly Thr Asp
Val Asn Leu Gly Cys Gly Ser Ile Thr 370 375
380 gtc aat tat gat gga aag aat
aag tat ttg aca aaa att gaa gat ggc 1200Val Asn Tyr Asp Gly Lys Asn
Lys Tyr Leu Thr Lys Ile Glu Asp Gly 385 390
395 400 gcg ttt atc ggc tgc aat tcc
aac ttg gtt gcc cct gtc aca gtc gga 1248Ala Phe Ile Gly Cys Asn Ser
Asn Leu Val Ala Pro Val Thr Val Gly 405
410 415 gaa ggc gct tat gtg gcg gca
ggt tca act gtt acg gaa gat gta cct 1296Glu Gly Ala Tyr Val Ala Ala
Gly Ser Thr Val Thr Glu Asp Val Pro 420
425 430 gga aaa gca ctt gct att gcc
aga gcg aga caa gta aat aaa gac gat 1344Gly Lys Ala Leu Ala Ile Ala
Arg Ala Arg Gln Val Asn Lys Asp Asp 435
440 445 tat gtg aaa aat att cat aaa
aaa 1368Tyr Val Lys Asn Ile His Lys
Lys 450 455
30456PRTBacillus subtilis
30Met Asp Lys Arg Phe Ala Val Val Leu Ala Ala Gly Gln Gly Thr Arg 1
5 10 15 Met Lys Ser Lys
Leu Tyr Lys Val Leu His Pro Val Cys Gly Lys Pro 20
25 30 Met Val Glu His Val Val Asp Glu Ala
Leu Lys Leu Ser Leu Ser Lys 35 40
45 Leu Val Thr Ile Val Gly His Gly Ala Glu Glu Val Lys Lys
Gln Leu 50 55 60
Gly Asp Lys Ser Glu Tyr Ala Leu Gln Ala Lys Gln Leu Gly Thr Ala 65
70 75 80 His Ala Val Lys Gln
Ala Gln Pro Phe Leu Ala Asp Glu Lys Gly Val 85
90 95 Thr Ile Val Ile Cys Gly Asp Thr Pro Leu
Leu Thr Ala Glu Thr Met 100 105
110 Glu Gln Met Leu Lys Glu His Thr Gln Arg Glu Ala Lys Ala Thr
Ile 115 120 125 Leu
Thr Ala Val Ala Glu Asp Pro Thr Gly Tyr Gly Arg Ile Ile Arg 130
135 140 Ser Glu Asn Gly Ala Val
Gln Lys Ile Val Glu His Lys Asp Ala Ser 145 150
155 160 Glu Glu Glu Arg Leu Val Thr Glu Ile Asn Thr
Gly Thr Tyr Cys Phe 165 170
175 Asp Asn Glu Ala Leu Phe Arg Ala Ile Asp Gln Val Ser Asn Asp Asn
180 185 190 Ala Gln
Gly Glu Tyr Tyr Leu Pro Asp Val Ile Glu Ile Leu Lys Asn 195
200 205 Glu Gly Glu Thr Val Ala Ala
Tyr Gln Thr Gly Asn Phe Gln Glu Thr 210 215
220 Leu Gly Val Asn Asp Arg Val Ala Leu Ser Gln Ala
Glu Gln Phe Met 225 230 235
240 Lys Glu Arg Ile Asn Lys Arg His Met Gln Asn Gly Val Thr Leu Ile
245 250 255 Asp Pro Met
Asn Thr Tyr Ile Ser Pro Asp Ala Val Ile Gly Ser Asp 260
265 270 Thr Val Ile Tyr Pro Gly Thr Val
Ile Lys Gly Glu Val Gln Ile Gly 275 280
285 Glu Asp Thr Ile Ile Gly Pro His Thr Glu Ile Met Asn
Ser Ala Ile 290 295 300
Gly Ser Arg Thr Val Ile Lys Gln Ser Val Val Asn His Ser Lys Val 305
310 315 320 Gly Asn Asp Val
Asn Ile Gly Pro Phe Ala His Ile Arg Pro Asp Ser 325
330 335 Val Ile Gly Asn Glu Val Lys Ile Gly
Asn Phe Val Glu Ile Lys Lys 340 345
350 Thr Gln Phe Gly Asp Arg Ser Lys Ala Ser His Leu Ser Tyr
Val Gly 355 360 365
Asp Ala Glu Val Gly Thr Asp Val Asn Leu Gly Cys Gly Ser Ile Thr 370
375 380 Val Asn Tyr Asp Gly
Lys Asn Lys Tyr Leu Thr Lys Ile Glu Asp Gly 385 390
395 400 Ala Phe Ile Gly Cys Asn Ser Asn Leu Val
Ala Pro Val Thr Val Gly 405 410
415 Glu Gly Ala Tyr Val Ala Ala Gly Ser Thr Val Thr Glu Asp Val
Pro 420 425 430 Gly
Lys Ala Leu Ala Ile Ala Arg Ala Arg Gln Val Asn Lys Asp Asp 435
440 445 Tyr Val Lys Asn Ile His
Lys Lys 450 455 3126DNABacillus subtilis
31ggatcctttc tatggataaa agggat
263231DNABacillus subtilis 32gttaacagga ttatttttta tgaatatttt t
313320DNABacillus subtilis 33cagagacgat
ggaacagatg
203420DNABacillus subtilis 34ggagttaatg atagagttgc
203520DNABacillus subtilis 35gaagatcggg
aattttgtag
203620DNABacillus subtilis 36catctgttcc atcgtctctg
203720DNABacillus subtilis 37gcaactctat
cattaactcc
203820DNABacillus subtilis 38ctacaaaatt cccgatcttc
203954DNAStreptococcus equisimilis 39gtgtcggaac
attcattaca tgcttaagca cccgctgtcc ttcttgttat ctcc
54401203DNAStreptococcus equisimilisCDS(1)..(1203) 40gtg aaa att tct gta
gca ggc tca gga tat gtc ggc cta tcc ttg agt 48Val Lys Ile Ser Val
Ala Gly Ser Gly Tyr Val Gly Leu Ser Leu Ser 1 5
10 15 att tta ctg gca caa
cat aat gac gtc act gtt gtt gat att att gat 96Ile Leu Leu Ala Gln
His Asn Asp Val Thr Val Val Asp Ile Ile Asp 20
25 30 gaa aag gtg aga ttg
atc aat caa ggc ata tct cca atc aag gat gct 144Glu Lys Val Arg Leu
Ile Asn Gln Gly Ile Ser Pro Ile Lys Asp Ala 35
40 45 gat att gag gag tat
tta aaa aat gcg ccg cta aat ctc aca gcg acc 192Asp Ile Glu Glu Tyr
Leu Lys Asn Ala Pro Leu Asn Leu Thr Ala Thr 50
55 60 ctt gat ggc gca agc
gct tat agc aat gca gac ctt att atc att gct 240Leu Asp Gly Ala Ser
Ala Tyr Ser Asn Ala Asp Leu Ile Ile Ile Ala 65
70 75 80 act ccg aca aat tat
gac agc gaa cgc aac tac ttt gac aca agg cat 288Thr Pro Thr Asn Tyr
Asp Ser Glu Arg Asn Tyr Phe Asp Thr Arg His 85
90 95 gtt gaa gag gtc att
gag cag gtc cta gac cta aat gcg tca gca acc 336Val Glu Glu Val Ile
Glu Gln Val Leu Asp Leu Asn Ala Ser Ala Thr 100
105 110 att att atc aaa tca
acc ata cca cta ggc ttt atc aag cat gtt agg 384Ile Ile Ile Lys Ser
Thr Ile Pro Leu Gly Phe Ile Lys His Val Arg 115
120 125 gaa aaa tac cag aca
gat cgt att att ttt agc cca gaa ttt tta aga 432Glu Lys Tyr Gln Thr
Asp Arg Ile Ile Phe Ser Pro Glu Phe Leu Arg 130
135 140 gaa tca aaa gcc tta
tac gat aac ctt tac cca agt cgg atc att gtt 480Glu Ser Lys Ala Leu
Tyr Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145
150 155 160 tct tat gaa aag gac
gac tca cca agg gtt att cag gct gct aaa gcc 528Ser Tyr Glu Lys Asp
Asp Ser Pro Arg Val Ile Gln Ala Ala Lys Ala 165
170 175 ttt gct ggt ctt tta
aag gaa gga gcc aaa agc aag gat act ccg gtc 576Phe Ala Gly Leu Leu
Lys Glu Gly Ala Lys Ser Lys Asp Thr Pro Val 180
185 190 tta ttt atg ggc tca
cag gag gct gag gcg gtc aag cta ttt gcg aat 624Leu Phe Met Gly Ser
Gln Glu Ala Glu Ala Val Lys Leu Phe Ala Asn 195
200 205 acc ttt ttg gct atg
cgg gtg tct tac ttt aat gaa tta gac acc tat 672Thr Phe Leu Ala Met
Arg Val Ser Tyr Phe Asn Glu Leu Asp Thr Tyr 210
215 220 tcc gaa agc aag ggt
cta gat gct cag cgc gtg att gaa gga gtc tgt 720Ser Glu Ser Lys Gly
Leu Asp Ala Gln Arg Val Ile Glu Gly Val Cys 225
230 235 240 cat gat cag cgc att
ggt aac cat tac aat aac cct tcc ttt gga tat 768His Asp Gln Arg Ile
Gly Asn His Tyr Asn Asn Pro Ser Phe Gly Tyr 245
250 255 ggc ggc tat tgc ctg
cca aag gac agc aaa cag ctg ttg gca aat tat 816Gly Gly Tyr Cys Leu
Pro Lys Asp Ser Lys Gln Leu Leu Ala Asn Tyr 260
265 270 aga ggc att ccc cag
tcc ttg atg tca gcg att gtt gag tcc aac aag 864Arg Gly Ile Pro Gln
Ser Leu Met Ser Ala Ile Val Glu Ser Asn Lys 275
280 285 ata cga aaa tcc tat
tta gct gaa caa ata tta gac aga gcc tct agt 912Ile Arg Lys Ser Tyr
Leu Ala Glu Gln Ile Leu Asp Arg Ala Ser Ser 290
295 300 caa aag cag gct ggt
gta cca tta acg att ggc ttt tac cgc ttg att 960Gln Lys Gln Ala Gly
Val Pro Leu Thr Ile Gly Phe Tyr Arg Leu Ile 305
310 315 320 atg aaa agc aac tct
gat aat ttc cga gaa agc gcc att aaa gat att 1008Met Lys Ser Asn Ser
Asp Asn Phe Arg Glu Ser Ala Ile Lys Asp Ile 325
330 335 att gat atc atc aac
gac tat ggg gtt aat att gtc att tac gaa ccc 1056Ile Asp Ile Ile Asn
Asp Tyr Gly Val Asn Ile Val Ile Tyr Glu Pro 340
345 350 atg ctt ggc gag gat
att ggc tac agg gtt gtc aag gac tta gag cag 1104Met Leu Gly Glu Asp
Ile Gly Tyr Arg Val Val Lys Asp Leu Glu Gln 355
360 365 ttc aaa aac gag tct
aca atc att gtg tca aat cgc ttt gag gac gac 1152Phe Lys Asn Glu Ser
Thr Ile Ile Val Ser Asn Arg Phe Glu Asp Asp 370
375 380 cta gga gat gtc att
gat aag gtt tat acg aga gat gtc ttt gga aga 1200Leu Gly Asp Val Ile
Asp Lys Val Tyr Thr Arg Asp Val Phe Gly Arg 385
390 395 400 gac
1203Asp
41401PRTStreptococcus
equisimilis 41Val Lys Ile Ser Val Ala Gly Ser Gly Tyr Val Gly Leu Ser Leu
Ser 1 5 10 15 Ile
Leu Leu Ala Gln His Asn Asp Val Thr Val Val Asp Ile Ile Asp
20 25 30 Glu Lys Val Arg Leu
Ile Asn Gln Gly Ile Ser Pro Ile Lys Asp Ala 35
40 45 Asp Ile Glu Glu Tyr Leu Lys Asn Ala
Pro Leu Asn Leu Thr Ala Thr 50 55
60 Leu Asp Gly Ala Ser Ala Tyr Ser Asn Ala Asp Leu Ile
Ile Ile Ala 65 70 75
80 Thr Pro Thr Asn Tyr Asp Ser Glu Arg Asn Tyr Phe Asp Thr Arg His
85 90 95 Val Glu Glu Val
Ile Glu Gln Val Leu Asp Leu Asn Ala Ser Ala Thr 100
105 110 Ile Ile Ile Lys Ser Thr Ile Pro Leu
Gly Phe Ile Lys His Val Arg 115 120
125 Glu Lys Tyr Gln Thr Asp Arg Ile Ile Phe Ser Pro Glu Phe
Leu Arg 130 135 140
Glu Ser Lys Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145
150 155 160 Ser Tyr Glu Lys Asp
Asp Ser Pro Arg Val Ile Gln Ala Ala Lys Ala 165
170 175 Phe Ala Gly Leu Leu Lys Glu Gly Ala Lys
Ser Lys Asp Thr Pro Val 180 185
190 Leu Phe Met Gly Ser Gln Glu Ala Glu Ala Val Lys Leu Phe Ala
Asn 195 200 205 Thr
Phe Leu Ala Met Arg Val Ser Tyr Phe Asn Glu Leu Asp Thr Tyr 210
215 220 Ser Glu Ser Lys Gly Leu
Asp Ala Gln Arg Val Ile Glu Gly Val Cys 225 230
235 240 His Asp Gln Arg Ile Gly Asn His Tyr Asn Asn
Pro Ser Phe Gly Tyr 245 250
255 Gly Gly Tyr Cys Leu Pro Lys Asp Ser Lys Gln Leu Leu Ala Asn Tyr
260 265 270 Arg Gly
Ile Pro Gln Ser Leu Met Ser Ala Ile Val Glu Ser Asn Lys 275
280 285 Ile Arg Lys Ser Tyr Leu Ala
Glu Gln Ile Leu Asp Arg Ala Ser Ser 290 295
300 Gln Lys Gln Ala Gly Val Pro Leu Thr Ile Gly Phe
Tyr Arg Leu Ile 305 310 315
320 Met Lys Ser Asn Ser Asp Asn Phe Arg Glu Ser Ala Ile Lys Asp Ile
325 330 335 Ile Asp Ile
Ile Asn Asp Tyr Gly Val Asn Ile Val Ile Tyr Glu Pro 340
345 350 Met Leu Gly Glu Asp Ile Gly Tyr
Arg Val Val Lys Asp Leu Glu Gln 355 360
365 Phe Lys Asn Glu Ser Thr Ile Ile Val Ser Asn Arg Phe
Glu Asp Asp 370 375 380
Leu Gly Asp Val Ile Asp Lys Val Tyr Thr Arg Asp Val Phe Gly Arg 385
390 395 400 Asp
42900DNAStreptococcus equisimilisCDS(1)..(900) 42atg aca aag gtc aga aaa
gcc att atc cca gcc gcc ggc cta ggc act 48Met Thr Lys Val Arg Lys
Ala Ile Ile Pro Ala Ala Gly Leu Gly Thr 1 5
10 15 cgc ttc cta ccc gcc acc
aag gca ctg gcc aag gaa atg ctc cca atc 96Arg Phe Leu Pro Ala Thr
Lys Ala Leu Ala Lys Glu Met Leu Pro Ile 20
25 30 gtc gat aag cca acc att
caa ttc atc gtc gag gaa gct cta aag gcc 144Val Asp Lys Pro Thr Ile
Gln Phe Ile Val Glu Glu Ala Leu Lys Ala 35
40 45 ggt atc gag gag att ctt
gtc gtc acc ggc aag gcc aaa cgc tct att 192Gly Ile Glu Glu Ile Leu
Val Val Thr Gly Lys Ala Lys Arg Ser Ile 50
55 60 gaa gac cac ttt gac tcc
aac ttc gag ctc gaa tac aat ctc caa gcc 240Glu Asp His Phe Asp Ser
Asn Phe Glu Leu Glu Tyr Asn Leu Gln Ala 65 70
75 80 aag ggc aaa acc gag ctg
ctc aag ctc gtt gat gag acc act gcc atc 288Lys Gly Lys Thr Glu Leu
Leu Lys Leu Val Asp Glu Thr Thr Ala Ile 85
90 95 aac ctg cac ttc att cgt
cag agc cac cct aga gga cta ggg gac gct 336Asn Leu His Phe Ile Arg
Gln Ser His Pro Arg Gly Leu Gly Asp Ala 100
105 110 gtc ctc cag gcc aag gcc
ttt gtg ggc aat gag ccc ttt gtg gtc atg 384Val Leu Gln Ala Lys Ala
Phe Val Gly Asn Glu Pro Phe Val Val Met 115
120 125 ctg ggg gat gac ctc atg
gat att acc aat cct agt gcc aag ccc ttg 432Leu Gly Asp Asp Leu Met
Asp Ile Thr Asn Pro Ser Ala Lys Pro Leu 130
135 140 gcc aag cag ctc att gag
gat tat gat tgc aca cac gcc tca acg att 480Ala Lys Gln Leu Ile Glu
Asp Tyr Asp Cys Thr His Ala Ser Thr Ile 145 150
155 160 gca gtg atg agg gtg ccg
cat gag gag gtt tcc aat tat ggc gtg att 528Ala Val Met Arg Val Pro
His Glu Glu Val Ser Asn Tyr Gly Val Ile 165
170 175 gca ccg caa ggg aag gct
gtt aag ggc ttg tat agt gtg gag acc ttt 576Ala Pro Gln Gly Lys Ala
Val Lys Gly Leu Tyr Ser Val Glu Thr Phe 180
185 190 gtt gag aag cca agt cca
gat gag gca ccg agt gac tta gcg att att 624Val Glu Lys Pro Ser Pro
Asp Glu Ala Pro Ser Asp Leu Ala Ile Ile 195
200 205 ggt cga tat ttg ttg acg
cct gag att ttt gcc ata ttg gag aat cag 672Gly Arg Tyr Leu Leu Thr
Pro Glu Ile Phe Ala Ile Leu Glu Asn Gln 210
215 220 gcg cct ggg gct ggc aat
gag gta cag cta gcc gat gcg att gac aag 720Ala Pro Gly Ala Gly Asn
Glu Val Gln Leu Ala Asp Ala Ile Asp Lys 225 230
235 240 ctc aac aag act cag cgg
gtt ttt gcg agg gag ttt aag gga gag cgg 768Leu Asn Lys Thr Gln Arg
Val Phe Ala Arg Glu Phe Lys Gly Glu Arg 245
250 255 tat gat gtt ggg gac aag
ttt ggc ttt atg aag acc tca ctt gac tat 816Tyr Asp Val Gly Asp Lys
Phe Gly Phe Met Lys Thr Ser Leu Asp Tyr 260
265 270 gct ctc aag cac cct cag
gtc aag gac gac ctc act gac tac att ata 864Ala Leu Lys His Pro Gln
Val Lys Asp Asp Leu Thr Asp Tyr Ile Ile 275
280 285 aag ctc agt aag caa ctg
aac aag gac gtt aaa aaa 900Lys Leu Ser Lys Gln Leu
Asn Lys Asp Val Lys Lys 290
295 300 43300PRTStreptococcus
equisimilis 43Met Thr Lys Val Arg Lys Ala Ile Ile Pro Ala Ala Gly Leu Gly
Thr 1 5 10 15 Arg
Phe Leu Pro Ala Thr Lys Ala Leu Ala Lys Glu Met Leu Pro Ile
20 25 30 Val Asp Lys Pro Thr
Ile Gln Phe Ile Val Glu Glu Ala Leu Lys Ala 35
40 45 Gly Ile Glu Glu Ile Leu Val Val Thr
Gly Lys Ala Lys Arg Ser Ile 50 55
60 Glu Asp His Phe Asp Ser Asn Phe Glu Leu Glu Tyr Asn
Leu Gln Ala 65 70 75
80 Lys Gly Lys Thr Glu Leu Leu Lys Leu Val Asp Glu Thr Thr Ala Ile
85 90 95 Asn Leu His Phe
Ile Arg Gln Ser His Pro Arg Gly Leu Gly Asp Ala 100
105 110 Val Leu Gln Ala Lys Ala Phe Val Gly
Asn Glu Pro Phe Val Val Met 115 120
125 Leu Gly Asp Asp Leu Met Asp Ile Thr Asn Pro Ser Ala Lys
Pro Leu 130 135 140
Ala Lys Gln Leu Ile Glu Asp Tyr Asp Cys Thr His Ala Ser Thr Ile 145
150 155 160 Ala Val Met Arg Val
Pro His Glu Glu Val Ser Asn Tyr Gly Val Ile 165
170 175 Ala Pro Gln Gly Lys Ala Val Lys Gly Leu
Tyr Ser Val Glu Thr Phe 180 185
190 Val Glu Lys Pro Ser Pro Asp Glu Ala Pro Ser Asp Leu Ala Ile
Ile 195 200 205 Gly
Arg Tyr Leu Leu Thr Pro Glu Ile Phe Ala Ile Leu Glu Asn Gln 210
215 220 Ala Pro Gly Ala Gly Asn
Glu Val Gln Leu Ala Asp Ala Ile Asp Lys 225 230
235 240 Leu Asn Lys Thr Gln Arg Val Phe Ala Arg Glu
Phe Lys Gly Glu Arg 245 250
255 Tyr Asp Val Gly Asp Lys Phe Gly Phe Met Lys Thr Ser Leu Asp Tyr
260 265 270 Ala Leu
Lys His Pro Gln Val Lys Asp Asp Leu Thr Asp Tyr Ile Ile 275
280 285 Lys Leu Ser Lys Gln Leu Asn
Lys Asp Val Lys Lys 290 295 300
441380DNAStreptococcus equisimilisCDS(1)..(1380) 44atg aaa aac tac gcc
att atc cta gca gct gga aag gga acc cgc atg 48Met Lys Asn Tyr Ala
Ile Ile Leu Ala Ala Gly Lys Gly Thr Arg Met 1 5
10 15 aat tca ggg ctt tcc
aag gtg ctg cac aag gta tca ggc cta agc atg 96Asn Ser Gly Leu Ser
Lys Val Leu His Lys Val Ser Gly Leu Ser Met 20
25 30 ctg gag cat gtc ctc
aag agc gtc tca gcc cta gct cct caa aag caa 144Leu Glu His Val Leu
Lys Ser Val Ser Ala Leu Ala Pro Gln Lys Gln 35
40 45 ctc aca gtg atc ggt
cat cag gca gag caa gta cgt gcc gtc cta ggt 192Leu Thr Val Ile Gly
His Gln Ala Glu Gln Val Arg Ala Val Leu Gly 50
55 60 gat caa tta ctg aca
gtg gtg caa gag gag cag cta gga aca ggc cat 240Asp Gln Leu Leu Thr
Val Val Gln Glu Glu Gln Leu Gly Thr Gly His 65
70 75 80 gca gtc atg atg gca
gaa gag gag cta tct ggc tta gaa ggg cag acc 288Ala Val Met Met Ala
Glu Glu Glu Leu Ser Gly Leu Glu Gly Gln Thr 85
90 95 cta gtg att gca ggt
gac acc ccc ttg atc aga gga gaa agc ctc aag 336Leu Val Ile Ala Gly
Asp Thr Pro Leu Ile Arg Gly Glu Ser Leu Lys 100
105 110 gct ctg cta gac tat
cat atc aga gaa aag aat gtg gca acc att ctc 384Ala Leu Leu Asp Tyr
His Ile Arg Glu Lys Asn Val Ala Thr Ile Leu 115
120 125 aca gcc aat gcc aag
gat ccc ttt ggc tac ggc cga atc att cgc aat 432Thr Ala Asn Ala Lys
Asp Pro Phe Gly Tyr Gly Arg Ile Ile Arg Asn 130
135 140 gca gca gga gag gtg
gtc aac atc gtt gaa caa aag gac gct aat gag 480Ala Ala Gly Glu Val
Val Asn Ile Val Glu Gln Lys Asp Ala Asn Glu 145
150 155 160 gca gag caa gag gtc
aag gag atc aac aca ggg acc tat atc ttt gac 528Ala Glu Gln Glu Val
Lys Glu Ile Asn Thr Gly Thr Tyr Ile Phe Asp 165
170 175 aat aag cgc ctc ttt
gag gct cta aag cat ctc acg act gat aat gcc 576Asn Lys Arg Leu Phe
Glu Ala Leu Lys His Leu Thr Thr Asp Asn Ala 180
185 190 caa ggg gaa tat tac
cta acc gat gtg atc agt att ttc aag gcc agc 624Gln Gly Glu Tyr Tyr
Leu Thr Asp Val Ile Ser Ile Phe Lys Ala Ser 195
200 205 caa gaa aag gtt gga
gct tac ctg ctg aag gat ttt gat gaa agc cta 672Gln Glu Lys Val Gly
Ala Tyr Leu Leu Lys Asp Phe Asp Glu Ser Leu 210
215 220 ggg gtt aat gat cgc
cta gct cta gcc cag gct gag gtg atc atg cag 720Gly Val Asn Asp Arg
Leu Ala Leu Ala Gln Ala Glu Val Ile Met Gln 225
230 235 240 gag cgg atc aac aag
cag cac atg ctt aat ggg gtg acc ctg caa aac 768Glu Arg Ile Asn Lys
Gln His Met Leu Asn Gly Val Thr Leu Gln Asn 245
250 255 cct gca gct acc tat
atc gaa agc agt gta gag att gcg ccg gac gtc 816Pro Ala Ala Thr Tyr
Ile Glu Ser Ser Val Glu Ile Ala Pro Asp Val 260
265 270 ttg att gaa gct aat
gtg acc cta aag gga cag act aga att ggc agc 864Leu Ile Glu Ala Asn
Val Thr Leu Lys Gly Gln Thr Arg Ile Gly Ser 275
280 285 aga agt gtt ata acc
aat ggg agc tat atc ctt gat tca agg ctt ggt 912Arg Ser Val Ile Thr
Asn Gly Ser Tyr Ile Leu Asp Ser Arg Leu Gly 290
295 300 gag ggc gta gtg gtg
agc cag tca gtg att gag ggc tca gtc cta gca 960Glu Gly Val Val Val
Ser Gln Ser Val Ile Glu Gly Ser Val Leu Ala 305
310 315 320 gat ggt gtg aca gta
ggg ccc tat gca cac att cgc ccg gac tct cag 1008Asp Gly Val Thr Val
Gly Pro Tyr Ala His Ile Arg Pro Asp Ser Gln 325
330 335 ctc gat gag tgt gtt
cat att ggg aac ttt gta gag gtt aag ggg tct 1056Leu Asp Glu Cys Val
His Ile Gly Asn Phe Val Glu Val Lys Gly Ser 340
345 350 cat cta ggg gcc aat
acc aag gca ggg cat ttg act tat ctg ggg aat 1104His Leu Gly Ala Asn
Thr Lys Ala Gly His Leu Thr Tyr Leu Gly Asn 355
360 365 gcc gag att ggc tca
gag gtt aat att ggt gca gga agc att acg gtt 1152Ala Glu Ile Gly Ser
Glu Val Asn Ile Gly Ala Gly Ser Ile Thr Val 370
375 380 aat tat gat ggt caa
cgg aaa tac cag aca gtg att ggc gat cac gct 1200Asn Tyr Asp Gly Gln
Arg Lys Tyr Gln Thr Val Ile Gly Asp His Ala 385
390 395 400 ttt att ggg agt cat
tcg act ttg ata gct ccg gta gag gtt ggg gag 1248Phe Ile Gly Ser His
Ser Thr Leu Ile Ala Pro Val Glu Val Gly Glu 405
410 415 aat gct tta aca gca
gca ggg tct acg ata gcc cag tcg gtg cca gca 1296Asn Ala Leu Thr Ala
Ala Gly Ser Thr Ile Ala Gln Ser Val Pro Ala 420
425 430 gac agt gtg gct ata
ggg cgt agc cgt cag gtg gtg aag gaa ggc tat 1344Asp Ser Val Ala Ile
Gly Arg Ser Arg Gln Val Val Lys Glu Gly Tyr 435
440 445 gcc aag agg cta cca
cat cac ccg gat cag ccc cag 1380Ala Lys Arg Leu Pro
His His Pro Asp Gln Pro Gln 450
455 460
45460PRTStreptococcus equisimilis 45Met Lys Asn Tyr Ala Ile Ile Leu Ala
Ala Gly Lys Gly Thr Arg Met 1 5 10
15 Asn Ser Gly Leu Ser Lys Val Leu His Lys Val Ser Gly Leu
Ser Met 20 25 30
Leu Glu His Val Leu Lys Ser Val Ser Ala Leu Ala Pro Gln Lys Gln
35 40 45 Leu Thr Val Ile
Gly His Gln Ala Glu Gln Val Arg Ala Val Leu Gly 50
55 60 Asp Gln Leu Leu Thr Val Val Gln
Glu Glu Gln Leu Gly Thr Gly His 65 70
75 80 Ala Val Met Met Ala Glu Glu Glu Leu Ser Gly Leu
Glu Gly Gln Thr 85 90
95 Leu Val Ile Ala Gly Asp Thr Pro Leu Ile Arg Gly Glu Ser Leu Lys
100 105 110 Ala Leu Leu
Asp Tyr His Ile Arg Glu Lys Asn Val Ala Thr Ile Leu 115
120 125 Thr Ala Asn Ala Lys Asp Pro Phe
Gly Tyr Gly Arg Ile Ile Arg Asn 130 135
140 Ala Ala Gly Glu Val Val Asn Ile Val Glu Gln Lys Asp
Ala Asn Glu 145 150 155
160 Ala Glu Gln Glu Val Lys Glu Ile Asn Thr Gly Thr Tyr Ile Phe Asp
165 170 175 Asn Lys Arg Leu
Phe Glu Ala Leu Lys His Leu Thr Thr Asp Asn Ala 180
185 190 Gln Gly Glu Tyr Tyr Leu Thr Asp Val
Ile Ser Ile Phe Lys Ala Ser 195 200
205 Gln Glu Lys Val Gly Ala Tyr Leu Leu Lys Asp Phe Asp Glu
Ser Leu 210 215 220
Gly Val Asn Asp Arg Leu Ala Leu Ala Gln Ala Glu Val Ile Met Gln 225
230 235 240 Glu Arg Ile Asn Lys
Gln His Met Leu Asn Gly Val Thr Leu Gln Asn 245
250 255 Pro Ala Ala Thr Tyr Ile Glu Ser Ser Val
Glu Ile Ala Pro Asp Val 260 265
270 Leu Ile Glu Ala Asn Val Thr Leu Lys Gly Gln Thr Arg Ile Gly
Ser 275 280 285 Arg
Ser Val Ile Thr Asn Gly Ser Tyr Ile Leu Asp Ser Arg Leu Gly 290
295 300 Glu Gly Val Val Val Ser
Gln Ser Val Ile Glu Gly Ser Val Leu Ala 305 310
315 320 Asp Gly Val Thr Val Gly Pro Tyr Ala His Ile
Arg Pro Asp Ser Gln 325 330
335 Leu Asp Glu Cys Val His Ile Gly Asn Phe Val Glu Val Lys Gly Ser
340 345 350 His Leu
Gly Ala Asn Thr Lys Ala Gly His Leu Thr Tyr Leu Gly Asn 355
360 365 Ala Glu Ile Gly Ser Glu Val
Asn Ile Gly Ala Gly Ser Ile Thr Val 370 375
380 Asn Tyr Asp Gly Gln Arg Lys Tyr Gln Thr Val Ile
Gly Asp His Ala 385 390 395
400 Phe Ile Gly Ser His Ser Thr Leu Ile Ala Pro Val Glu Val Gly Glu
405 410 415 Asn Ala Leu
Thr Ala Ala Gly Ser Thr Ile Ala Gln Ser Val Pro Ala 420
425 430 Asp Ser Val Ala Ile Gly Arg Ser
Arg Gln Val Val Lys Glu Gly Tyr 435 440
445 Ala Lys Arg Leu Pro His His Pro Asp Gln Pro Gln
450 455 460 4629DNABacillus subtilis
46gcggccgcgg tacctgtgtt acacctgtt
294738DNABacillus subtilis 47gtcaagctta attctcatgt ttgacagctt atcatcgg
384818DNABacillus subtilis 48catgggagag acctttgg
184917DNABacillus
subtilis 49gtcggtcttc catttgc
175017DNABacillus subtilis 50cgaccactgt atcttgg
175117DNABacillus subtilis 51gagatgccaa
acagtgc
175216DNABacillus subtilis 52catgtccatc gtgacg
165317DNABacillus subtilis 53caggagcatt tgatacg
175416DNABacillus
subtilis 54ccttcagatg tgatcc
165517DNABacillus subtilis 55gtgttgacgt caactgc
175618DNABacillus subtilis 56gttcagcctt
tcctctcg
185718DNABacillus subtilis 57gctaccttct ttcttagg
185818DNABacillus subtilis 58cgtcaatatg atctgtgc
185917DNABacillus
subtilis 59ggaaagaagg tctgtgc
176017DNABacillus subtilis 60cagctatcag ctgacag
176120DNABacillus subtilis 61gctcagctat
gacatattcc
206217DNABacillus subtilis 62gatcgtcttg attaccg
176316DNABacillus subtilis 63agctttatcg gtgacg
166416DNABacillus
subtilis 64tgagcacgat tgcagg
166517DNABacillus subtilis 65cattgcggag acattgc
176626DNABacillus subtilis 66tagacaattg
gaagagaaaa gagata
266720DNABacillus subtilis 67ccgtcgctat tgtaaccagt
206829DNABacillus subtilis 68ggaattccaa
agctgcagcg gccggcgcg
296932DNABacillus subtilis 69gaagatctcg tatacttggc ttctgcagct gc
327031DNABacillus subtilis 70gaagatctgg
tcaacaagct ggaaagcact c
317133DNABacillus subtilis 71cccaagcttc gtgacgtaca gcaccgttcc ggc
337250DNABacillus subtilis 72ccttaagggc
cgaatattta tacggagctc cctgaaacaa caaaaacggc
507334DNABacillus subtilis 73ggtgttctct agagcggccg cggttgcggt cagc
347427DNABacillus subtilis 74gtccttcttg
gtacctggaa gcagagc
277539DNABacillus subtilis 75gtataaatat tcggccctta aggccagtac cattttccc
397613DNABacillus subtilis 76gggccggatc cgc
137720DNABacillus
subtilis 77attcccggcc taggcgccgg
207819DNABacillus subtilis 78ggaaattatc gtgatcaac
197921DNABacillus subtilis 79gcacgagcac
tgataaatat g
218021DNABacillus subtilis 80catatttatc agtgctcgtg c
218117DNABacillus subtilis 81tcgtagacct catatgc
178217DNABacillus
subtilis 82gtcgttaaac cgtgtgc
178339DNABacillus subtilis 83ctagaggatc cccgggtacc gtgctctgcc
ttttagtcc 398437DNABacillus subtilis
84gtacatcgaa ttcgtgctca ttattaatct gttcagc
378520DNABacillus subtilis 85aactattgcc gatgataagc
20861260DNABacillus subtilisCDS(1)..(1260) 86atg
aaa aaa gtg atg tta gct acg gct ttg ttt tta gga ttg act cca 48Met
Lys Lys Val Met Leu Ala Thr Ala Leu Phe Leu Gly Leu Thr Pro 1
5 10 15 gct
ggc gcg aac gca gct gat tta ggc cac cag acg ttg gga tcc aat 96Ala
Gly Ala Asn Ala Ala Asp Leu Gly His Gln Thr Leu Gly Ser Asn
20 25 30 gat
ggc tgg ggc gcg tac tcg acc ggc acg aca ggc gga tca aaa gca 144Asp
Gly Trp Gly Ala Tyr Ser Thr Gly Thr Thr Gly Gly Ser Lys Ala
35 40 45 tcc
tcc tca aat gtg tat acc gtc agc aac aga aac cag ctt gtc tcg 192Ser
Ser Ser Asn Val Tyr Thr Val Ser Asn Arg Asn Gln Leu Val Ser
50 55 60 gca
tta ggg aag gaa acg aac aca acg cca aaa atc att tat atc aag 240Ala
Leu Gly Lys Glu Thr Asn Thr Thr Pro Lys Ile Ile Tyr Ile Lys 65
70 75 80 gga
acg att gac atg aac gtg gat gac aat ctg aag ccg ctt ggc cta 288Gly
Thr Ile Asp Met Asn Val Asp Asp Asn Leu Lys Pro Leu Gly Leu
85 90 95 aat
gac tat aaa gat ccg gag tat gat ttg gac aaa tat ttg aaa gcc 336Asn
Asp Tyr Lys Asp Pro Glu Tyr Asp Leu Asp Lys Tyr Leu Lys Ala
100 105 110 tat
gat cct agc aca tgg ggc aaa aaa gag ccg tcg gga aca caa gaa 384Tyr
Asp Pro Ser Thr Trp Gly Lys Lys Glu Pro Ser Gly Thr Gln Glu
115 120 125 gaa
gcg aga gca cgc tct cag aaa aac caa aaa gca cgg gtc atg gtg 432Glu
Ala Arg Ala Arg Ser Gln Lys Asn Gln Lys Ala Arg Val Met Val
130 135 140 gat
atc cct gca aac acg acg atc gtc ggt tca ggg act aac gct aaa 480Asp
Ile Pro Ala Asn Thr Thr Ile Val Gly Ser Gly Thr Asn Ala Lys 145
150 155 160 gtc
gtg gga gga aac ttc caa atc aag agt gat aac gtc att att cgc 528Val
Val Gly Gly Asn Phe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg
165 170 175 aac
att gaa ttc cag gat gcc tat gac tat ttt ccg caa tgg gat ccg 576Asn
Ile Glu Phe Gln Asp Ala Tyr Asp Tyr Phe Pro Gln Trp Asp Pro
180 185 190 act
gac gga agc tca ggg aac tgg aac tca caa tac gac aac atc acg 624Thr
Asp Gly Ser Ser Gly Asn Trp Asn Ser Gln Tyr Asp Asn Ile Thr
195 200 205 ata
aac ggc ggc aca cac atc tgg att gat cac tgt aca ttt aat gac 672Ile
Asn Gly Gly Thr His Ile Trp Ile Asp His Cys Thr Phe Asn Asp
210 215 220 ggt
tcg cgt ccg gac agc aca tca ccg aaa tat tat gga aga aaa tat 720Gly
Ser Arg Pro Asp Ser Thr Ser Pro Lys Tyr Tyr Gly Arg Lys Tyr 225
230 235 240 cag
cac cat gac ggc caa acg gat gct tcc aac ggt gct aac tat atc 768Gln
His His Asp Gly Gln Thr Asp Ala Ser Asn Gly Ala Asn Tyr Ile
245 250 255 acg
atg tcc tac aac tat tat cac gat cat gat aaa agc tcc att ttc 816Thr
Met Ser Tyr Asn Tyr Tyr His Asp His Asp Lys Ser Ser Ile Phe
260 265 270 gga
tca agt gac agc aaa acc tcc gat gac ggc aaa tta aaa att acg 864Gly
Ser Ser Asp Ser Lys Thr Ser Asp Asp Gly Lys Leu Lys Ile Thr
275 280 285 ctg
cat cat aac cgc tat aaa aat att gtc cag cgc gcg ccg aga gtc 912Leu
His His Asn Arg Tyr Lys Asn Ile Val Gln Arg Ala Pro Arg Val
290 295 300 cgc
ttc ggg caa gtg cac gta tac aac aac tat tat gaa gga agc aca 960Arg
Phe Gly Gln Val His Val Tyr Asn Asn Tyr Tyr Glu Gly Ser Thr 305
310 315 320 agc
tct tca agt tat cct ttt agc tat gca tgg gga atc gga aag tca 1008Ser
Ser Ser Ser Tyr Pro Phe Ser Tyr Ala Trp Gly Ile Gly Lys Ser
325 330 335 tct
aaa atc tat gcc caa aac aat gtc att gac gta ccg gga ctg tca 1056Ser
Lys Ile Tyr Ala Gln Asn Asn Val Ile Asp Val Pro Gly Leu Ser
340 345 350 gct
gct aaa acg atc agc gta ttc agc ggg gga acg gct tta tat gac 1104Ala
Ala Lys Thr Ile Ser Val Phe Ser Gly Gly Thr Ala Leu Tyr Asp
355 360 365 tcc
ggc acg ttg ctg aac ggc aca cag atc aac gca tcg gct gca aac 1152Ser
Gly Thr Leu Leu Asn Gly Thr Gln Ile Asn Ala Ser Ala Ala Asn
370 375 380 ggg
ctg agc tct tct gtc ggc tgg acg ccg tct ctg cat gga tcg att 1200Gly
Leu Ser Ser Ser Val Gly Trp Thr Pro Ser Leu His Gly Ser Ile 385
390 395 400 gat
gct tct gct aat gtg aaa tca aat gtt ata aat caa gcg ggt gcg 1248Asp
Ala Ser Ala Asn Val Lys Ser Asn Val Ile Asn Gln Ala Gly Ala
405 410 415 ggt
aaa tta aat 1260Gly
Lys Leu Asn
420
87420PRTBacillus subtilis 87Met Lys Lys Val Met Leu Ala Thr Ala Leu Phe
Leu Gly Leu Thr Pro 1 5 10
15 Ala Gly Ala Asn Ala Ala Asp Leu Gly His Gln Thr Leu Gly Ser Asn
20 25 30 Asp Gly
Trp Gly Ala Tyr Ser Thr Gly Thr Thr Gly Gly Ser Lys Ala 35
40 45 Ser Ser Ser Asn Val Tyr Thr
Val Ser Asn Arg Asn Gln Leu Val Ser 50 55
60 Ala Leu Gly Lys Glu Thr Asn Thr Thr Pro Lys Ile
Ile Tyr Ile Lys 65 70 75
80 Gly Thr Ile Asp Met Asn Val Asp Asp Asn Leu Lys Pro Leu Gly Leu
85 90 95 Asn Asp Tyr
Lys Asp Pro Glu Tyr Asp Leu Asp Lys Tyr Leu Lys Ala 100
105 110 Tyr Asp Pro Ser Thr Trp Gly Lys
Lys Glu Pro Ser Gly Thr Gln Glu 115 120
125 Glu Ala Arg Ala Arg Ser Gln Lys Asn Gln Lys Ala Arg
Val Met Val 130 135 140
Asp Ile Pro Ala Asn Thr Thr Ile Val Gly Ser Gly Thr Asn Ala Lys 145
150 155 160 Val Val Gly Gly
Asn Phe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg 165
170 175 Asn Ile Glu Phe Gln Asp Ala Tyr Asp
Tyr Phe Pro Gln Trp Asp Pro 180 185
190 Thr Asp Gly Ser Ser Gly Asn Trp Asn Ser Gln Tyr Asp Asn
Ile Thr 195 200 205
Ile Asn Gly Gly Thr His Ile Trp Ile Asp His Cys Thr Phe Asn Asp 210
215 220 Gly Ser Arg Pro Asp
Ser Thr Ser Pro Lys Tyr Tyr Gly Arg Lys Tyr 225 230
235 240 Gln His His Asp Gly Gln Thr Asp Ala Ser
Asn Gly Ala Asn Tyr Ile 245 250
255 Thr Met Ser Tyr Asn Tyr Tyr His Asp His Asp Lys Ser Ser Ile
Phe 260 265 270 Gly
Ser Ser Asp Ser Lys Thr Ser Asp Asp Gly Lys Leu Lys Ile Thr 275
280 285 Leu His His Asn Arg Tyr
Lys Asn Ile Val Gln Arg Ala Pro Arg Val 290 295
300 Arg Phe Gly Gln Val His Val Tyr Asn Asn Tyr
Tyr Glu Gly Ser Thr 305 310 315
320 Ser Ser Ser Ser Tyr Pro Phe Ser Tyr Ala Trp Gly Ile Gly Lys Ser
325 330 335 Ser Lys
Ile Tyr Ala Gln Asn Asn Val Ile Asp Val Pro Gly Leu Ser 340
345 350 Ala Ala Lys Thr Ile Ser Val
Phe Ser Gly Gly Thr Ala Leu Tyr Asp 355 360
365 Ser Gly Thr Leu Leu Asn Gly Thr Gln Ile Asn Ala
Ser Ala Ala Asn 370 375 380
Gly Leu Ser Ser Ser Val Gly Trp Thr Pro Ser Leu His Gly Ser Ile 385
390 395 400 Asp Ala Ser
Ala Asn Val Lys Ser Asn Val Ile Asn Gln Ala Gly Ala 405
410 415 Gly Lys Leu Asn 420
8826DNABacillus subtilis 88actagtaatg atggctgggg cgcgta
268926DNABacillus subtilis 89gtcgacatgt tgtcgtattg
tgagtt 269052DNABacillus
subtilis 90gagctctaca acgcttatgg atccgcggcc gcggcggcac acacatctgg at
529126DNABacillus subtilis 91gacgtcagcc cgtttgcagc cgatgc
26921257DNAStreptococcus
pyogenesCDS(1)..(1257) 92gtg cct att ttt aaa aaa act tta att gtt tta tcc
ttt att ttt ttg 48Val Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser
Phe Ile Phe Leu 1 5 10
15 ata tct atc ttg att tat cta aat atg tat cta ttt
gga aca tca act 96Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe
Gly Thr Ser Thr 20 25
30 gta gga att tat gga gta ata tta ata acc tat cta
gtt att aaa ctt 144Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu
Val Ile Lys Leu 35 40
45 gga tta tct ttc ctt tat gag cca ttt aaa gga aag
cca cat gac tat 192Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Lys
Pro His Asp Tyr 50 55 60
aaa gtt gct gct gta att cct tct tat aat gaa gat
gcc gag tca tta 240Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp
Ala Glu Ser Leu 65 70 75
80 tta gaa act ctt aaa agt gtg tta gca cag acc tat
ccg tta tca gaa 288Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr
Pro Leu Ser Glu 85 90
95 att tat att gtt gat gat ggg agt tca aac aca gat
gca ata caa tta 336Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp
Ala Ile Gln Leu 100 105
110 att gaa gag tat gta aat aga gaa gtg gat att tgt
cga aac gtt atc 384Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys
Arg Asn Val Ile 115 120
125 gtt cac cgt tcc ctt gtc aat aaa gga aaa cgc cat
gct caa gcg tgg 432Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His
Ala Gln Ala Trp 130 135 140
gca ttt gaa aga tct gac gct gac gtt ttt tta acc
gta gat tca gat 480Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr
Val Asp Ser Asp 145 150 155
160 act tat atc tat cca aat gcc tta gaa gaa ctc cta
aaa agc ttc aat 528Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu
Lys Ser Phe Asn 165 170
175 gat gag aca gtt tat gct gca aca gga cat ttg aat
gct aga aac aga 576Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn
Ala Arg Asn Arg 180 185
190 caa act aat cta tta acg cga ctt aca gat atc cgt
tac gat aat gcc 624Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg
Tyr Asp Asn Ala 195 200
205 ttt ggg gtg gag cgt gct gct caa tca tta aca ggt
aat att tta gtt 672Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly
Asn Ile Leu Val 210 215 220
tgc tca gga cca ttg agt att tat cga cgt gaa gtg
att att cct aac 720Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val
Ile Ile Pro Asn 225 230 235
240 tta gag cgc tat aaa aat caa aca ttc cta ggt tta
cct gtt agc att 768Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu
Pro Val Ser Ile 245 250
255 ggg gat gat cga tgt tta aca aat tat gct att gat
tta gga cgc act 816Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp
Leu Gly Arg Thr 260 265
270 gtc tac caa tca aca gct aga tgt gat act gat gta
cct ttc caa tta 864Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val
Pro Phe Gln Leu 275 280
285 aaa agt tat tta aag caa caa aat cga tgg aat aaa
tct ttt ttt aaa 912Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys
Ser Phe Phe Lys 290 295 300
gaa tct att att tct gtt aaa aaa att ctt tct aat
ccc atc gtt gcc 960Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn
Pro Ile Val Ala 305 310 315
320 tta tgg act att ttc gaa gtc gtt atg ttt atg atg
ttg att gtc gca 1008Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met
Leu Ile Val Ala 325 330
335 att ggg aat ctt ttg ttt aat caa gct att caa tta
gac ctt att aaa 1056Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu
Asp Leu Ile Lys 340 345
350 ctt ttt gcc ttt tta tcc atc atc ttt atc gtt gct
tta tgt cgt aat 1104Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala
Leu Cys Arg Asn 355 360
365 gtt cat tat atg atc aaa cat cct gct agt ttt ttg
tta tct cct ctg 1152Val His Tyr Met Ile Lys His Pro Ala Ser Phe Leu
Leu Ser Pro Leu 370 375 380
tat gga ata tta cac ttg ttt gtc tta cag ccc cta
aaa ctt tat tct 1200Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu
Lys Leu Tyr Ser 385 390 395
400 tta tgc acc att aaa aat acg gaa tgg gga aca cgt
aaa aag gtc act 1248Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg
Lys Lys Val Thr 405 410
415 att ttt aaa
1257Ile Phe Lys
93419PRTStreptococcus pyogenes 93Val Pro Ile Phe
Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu 1 5
10 15 Ile Ser Ile Leu Ile Tyr Leu Asn Met
Tyr Leu Phe Gly Thr Ser Thr 20 25
30 Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile
Lys Leu 35 40 45
Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Lys Pro His Asp Tyr 50
55 60 Lys Val Ala Ala Val
Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu 65 70
75 80 Leu Glu Thr Leu Lys Ser Val Leu Ala Gln
Thr Tyr Pro Leu Ser Glu 85 90
95 Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln
Leu 100 105 110 Ile
Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile 115
120 125 Val His Arg Ser Leu Val
Asn Lys Gly Lys Arg His Ala Gln Ala Trp 130 135
140 Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu
Thr Val Asp Ser Asp 145 150 155
160 Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn
165 170 175 Asp Glu
Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg 180
185 190 Gln Thr Asn Leu Leu Thr Arg
Leu Thr Asp Ile Arg Tyr Asp Asn Ala 195 200
205 Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly
Asn Ile Leu Val 210 215 220
Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn 225
230 235 240 Leu Glu Arg
Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile 245
250 255 Gly Asp Asp Arg Cys Leu Thr Asn
Tyr Ala Ile Asp Leu Gly Arg Thr 260 265
270 Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro
Phe Gln Leu 275 280 285
Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Lys 290
295 300 Glu Ser Ile Ile
Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala 305 310
315 320 Leu Trp Thr Ile Phe Glu Val Val Met
Phe Met Met Leu Ile Val Ala 325 330
335 Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu
Ile Lys 340 345 350
Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn
355 360 365 Val His Tyr Met
Ile Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu 370
375 380 Tyr Gly Ile Leu His Leu Phe Val
Leu Gln Pro Leu Lys Leu Tyr Ser 385 390
395 400 Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg
Lys Lys Val Thr 405 410
415 Ile Phe Lys 942916DNAPasteurella multocidaCDS(1)..(2916) 94atg
aat aca tta tca caa gca ata aaa gca tat aac agc aat gac tat 48Met
Asn Thr Leu Ser Gln Ala Ile Lys Ala Tyr Asn Ser Asn Asp Tyr 1
5 10 15 caa
tta gca ctc aaa tta ttt gaa aag tcg gcg gaa atc tat gga cgg 96Gln
Leu Ala Leu Lys Leu Phe Glu Lys Ser Ala Glu Ile Tyr Gly Arg
20 25 30 aaa
att gtt gaa ttt caa att acc aaa tgc caa gaa aaa ctc tca gca 144Lys
Ile Val Glu Phe Gln Ile Thr Lys Cys Gln Glu Lys Leu Ser Ala
35 40 45 cat
cct tct gtt aat tca gca cat ctt tct gta aat aaa gaa gaa aaa 192His
Pro Ser Val Asn Ser Ala His Leu Ser Val Asn Lys Glu Glu Lys
50 55 60 gtc
aat gtt tgc gat agt ccg tta gat att gca aca caa ctg tta ctt 240Val
Asn Val Cys Asp Ser Pro Leu Asp Ile Ala Thr Gln Leu Leu Leu 65
70 75 80 tcc
aac gta aaa aaa tta gta ctt tct gac tcg gaa aaa aac acg tta 288Ser
Asn Val Lys Lys Leu Val Leu Ser Asp Ser Glu Lys Asn Thr Leu
85 90 95 aaa
aat aaa tgg aaa ttg ctc act gag aag aaa tct gaa aat gcg gag 336Lys
Asn Lys Trp Lys Leu Leu Thr Glu Lys Lys Ser Glu Asn Ala Glu
100 105 110 gta
aga gcg gtc gcc ctt gta cca aaa gat ttt ccc aaa gat ctg gtt 384Val
Arg Ala Val Ala Leu Val Pro Lys Asp Phe Pro Lys Asp Leu Val
115 120 125 tta
gcg cct tta cct gat cat gtt aat gat ttt aca tgg tac aaa aag 432Leu
Ala Pro Leu Pro Asp His Val Asn Asp Phe Thr Trp Tyr Lys Lys
130 135 140 cga
aag aaa aga ctt ggc ata aaa cct gaa cat caa cat gtt ggt ctt 480Arg
Lys Lys Arg Leu Gly Ile Lys Pro Glu His Gln His Val Gly Leu 145
150 155 160 tct
att atc gtt aca aca ttc aat cga cca gca att tta tcg att aca 528Ser
Ile Ile Val Thr Thr Phe Asn Arg Pro Ala Ile Leu Ser Ile Thr
165 170 175 tta
gcc tgt tta gta aac caa aaa aca cat tac ccg ttt gaa gtt atc 576Leu
Ala Cys Leu Val Asn Gln Lys Thr His Tyr Pro Phe Glu Val Ile
180 185 190 gtg
aca gat gat ggt agt cag gaa gat cta tca ccg atc att cgc caa 624Val
Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile Ile Arg Gln
195 200 205 tat
gaa aat aaa ttg gat att cgc tac gtc aga caa aaa gat aac ggt 672Tyr
Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln Lys Asp Asn Gly
210 215 220 ttt
caa gcc agt gcc gct cgg aat atg gga tta cgc tta gca aaa tat 720Phe
Gln Ala Ser Ala Ala Arg Asn Met Gly Leu Arg Leu Ala Lys Tyr 225
230 235 240 gac
ttt att ggc tta ctc gac tgt gat atg gcg cca aat cca tta tgg 768Asp
Phe Ile Gly Leu Leu Asp Cys Asp Met Ala Pro Asn Pro Leu Trp
245 250 255 gtt
cat tct tat gtt gca gag cta tta gaa gat gat gat tta aca atc 816Val
His Ser Tyr Val Ala Glu Leu Leu Glu Asp Asp Asp Leu Thr Ile
260 265 270 att
ggt cca aga aaa tac atc gat aca caa cat att gac cca aaa gac 864Ile
Gly Pro Arg Lys Tyr Ile Asp Thr Gln His Ile Asp Pro Lys Asp
275 280 285 ttc
tta aat aac gcg agt ttg ctt gaa tca tta cca gaa gtg aaa acc 912Phe
Leu Asn Asn Ala Ser Leu Leu Glu Ser Leu Pro Glu Val Lys Thr
290 295 300 aat
aat agt gtt gcc gca aaa ggg gaa gga aca gtt tct ctg gat tgg 960Asn
Asn Ser Val Ala Ala Lys Gly Glu Gly Thr Val Ser Leu Asp Trp 305
310 315 320 cgc
tta gaa caa ttc gaa aaa aca gaa aat ctc cgc tta tcc gat tcg 1008Arg
Leu Glu Gln Phe Glu Lys Thr Glu Asn Leu Arg Leu Ser Asp Ser
325 330 335 cct
ttc cgt ttt ttt gcg gcg ggt aat gtt gct ttc gct aaa aaa tgg 1056Pro
Phe Arg Phe Phe Ala Ala Gly Asn Val Ala Phe Ala Lys Lys Trp
340 345 350 cta
aat aaa tcc ggt ttc ttt gat gag gaa ttt aat cac tgg ggt gga 1104Leu
Asn Lys Ser Gly Phe Phe Asp Glu Glu Phe Asn His Trp Gly Gly
355 360 365 gaa
gat gtg gaa ttt gga tat cgc tta ttc cgt tac ggt agt ttc ttt 1152Glu
Asp Val Glu Phe Gly Tyr Arg Leu Phe Arg Tyr Gly Ser Phe Phe
370 375 380 aaa
act att gat ggc att atg gcc tac cat caa gag cca cca ggt aaa 1200Lys
Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro Pro Gly Lys 385
390 395 400 gaa
aat gaa acc gat cgt gaa gcg gga aaa aat att acg ctc gat att 1248Glu
Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile Thr Leu Asp Ile
405 410 415 atg
aga gaa aag gtc cct tat atc tat aga aaa ctt tta cca ata gaa 1296Met
Arg Glu Lys Val Pro Tyr Ile Tyr Arg Lys Leu Leu Pro Ile Glu
420 425 430 gat
tcg cat atc aat aga gta cct tta gtt tca att tat atc cca gct 1344Asp
Ser His Ile Asn Arg Val Pro Leu Val Ser Ile Tyr Ile Pro Ala
435 440 445 tat
aac tgt gca aac tat att caa cgt tgc gta gat agt gca ctg aat 1392Tyr
Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp Ser Ala Leu Asn
450 455 460 cag
act gtt gtt gat ctc gag gtt tgt att tgt aac gat ggt tca aca 1440Gln
Thr Val Val Asp Leu Glu Val Cys Ile Cys Asn Asp Gly Ser Thr 465
470 475 480 gat
aat acc tta gaa gtg atc aat aag ctt tat ggt aat aat cct agg 1488Asp
Asn Thr Leu Glu Val Ile Asn Lys Leu Tyr Gly Asn Asn Pro Arg
485 490 495 gta
cgc atc atg tct aaa cca aat ggc gga ata gcc tca gca tca aat 1536Val
Arg Ile Met Ser Lys Pro Asn Gly Gly Ile Ala Ser Ala Ser Asn
500 505 510 gca
gcc gtt tct ttt gct aaa ggt tat tac att ggg cag tta gat tca 1584Ala
Ala Val Ser Phe Ala Lys Gly Tyr Tyr Ile Gly Gln Leu Asp Ser
515 520 525 gat
gat tat ctt gag cct gat gca gtt gaa ctg tgt tta aaa gaa ttt 1632Asp
Asp Tyr Leu Glu Pro Asp Ala Val Glu Leu Cys Leu Lys Glu Phe
530 535 540 tta
aaa gat aaa acg cta gct tgt gtt tat acc act aat aga aac gtc 1680Leu
Lys Asp Lys Thr Leu Ala Cys Val Tyr Thr Thr Asn Arg Asn Val 545
550 555 560 aat
ccg gat ggt agc tta atc gct aat ggt tac aat tgg cca gaa ttt 1728Asn
Pro Asp Gly Ser Leu Ile Ala Asn Gly Tyr Asn Trp Pro Glu Phe
565 570 575 tca
cga gaa aaa ctc aca acg gct atg att gct cac cac ttt aga atg 1776Ser
Arg Glu Lys Leu Thr Thr Ala Met Ile Ala His His Phe Arg Met
580 585 590 ttc
acg att aga gct tgg cat tta act gat gga ttc aat gaa aaa att 1824Phe
Thr Ile Arg Ala Trp His Leu Thr Asp Gly Phe Asn Glu Lys Ile
595 600 605 gaa
aat gcc gta gac tat gac atg ttc ctc aaa ctc agt gaa gtt gga 1872Glu
Asn Ala Val Asp Tyr Asp Met Phe Leu Lys Leu Ser Glu Val Gly
610 615 620 aaa
ttt aaa cat ctt aat aaa atc tgc tat aac cgt gta tta cat ggt 1920Lys
Phe Lys His Leu Asn Lys Ile Cys Tyr Asn Arg Val Leu His Gly 625
630 635 640 gat
aac aca tca att aag aaa ctt ggc att caa aag aaa aac cat ttt 1968Asp
Asn Thr Ser Ile Lys Lys Leu Gly Ile Gln Lys Lys Asn His Phe
645 650 655 gtt
gta gtc aat cag tca tta aat aga caa ggc ata act tat tat aat 2016Val
Val Val Asn Gln Ser Leu Asn Arg Gln Gly Ile Thr Tyr Tyr Asn
660 665 670 tat
gac gaa ttt gat gat tta gat gaa agt aga aag tat att ttc aat 2064Tyr
Asp Glu Phe Asp Asp Leu Asp Glu Ser Arg Lys Tyr Ile Phe Asn
675 680 685 aaa
acc gct gaa tat caa gaa gag att gat atc tta aaa gat att aaa 2112Lys
Thr Ala Glu Tyr Gln Glu Glu Ile Asp Ile Leu Lys Asp Ile Lys
690 695 700 atc
atc cag aat aaa gat gcc aaa atc gca gtc agt att ttt tat ccc 2160Ile
Ile Gln Asn Lys Asp Ala Lys Ile Ala Val Ser Ile Phe Tyr Pro 705
710 715 720 aat
aca tta aac ggc tta gtg aaa aaa cta aac aat att att gaa tat 2208Asn
Thr Leu Asn Gly Leu Val Lys Lys Leu Asn Asn Ile Ile Glu Tyr
725 730 735 aat
aaa aat ata ttc gtt att gtt cta cat gtt gat aag aat cat ctt 2256Asn
Lys Asn Ile Phe Val Ile Val Leu His Val Asp Lys Asn His Leu
740 745 750 aca
cca gat atc aaa aaa gaa ata cta gcc ttc tat cat aaa cat caa 2304Thr
Pro Asp Ile Lys Lys Glu Ile Leu Ala Phe Tyr His Lys His Gln
755 760 765 gtg
aat att tta cta aat aat gat atc tca tat tac acg agt aat aga 2352Val
Asn Ile Leu Leu Asn Asn Asp Ile Ser Tyr Tyr Thr Ser Asn Arg
770 775 780 tta
ata aaa act gag gcg cat tta agt aat att aat aaa tta agt cag 2400Leu
Ile Lys Thr Glu Ala His Leu Ser Asn Ile Asn Lys Leu Ser Gln 785
790 795 800 tta
aat cta aat tgt gaa tac atc att ttt gat aat cat gac agc cta 2448Leu
Asn Leu Asn Cys Glu Tyr Ile Ile Phe Asp Asn His Asp Ser Leu
805 810 815 ttc
gtt aaa aat gac agc tat gct tat atg aaa aaa tat gat gtc ggc 2496Phe
Val Lys Asn Asp Ser Tyr Ala Tyr Met Lys Lys Tyr Asp Val Gly
820 825 830 atg
aat ttc tca gca tta aca cat gat tgg atc gag aaa atc aat gcg 2544Met
Asn Phe Ser Ala Leu Thr His Asp Trp Ile Glu Lys Ile Asn Ala
835 840 845 cat
cca cca ttt aaa aag ctc att aaa act tat ttt aat gac aat gac 2592His
Pro Pro Phe Lys Lys Leu Ile Lys Thr Tyr Phe Asn Asp Asn Asp
850 855 860 tta
aaa agt atg aat gtg aaa ggg gca tca caa ggt atg ttt atg acg 2640Leu
Lys Ser Met Asn Val Lys Gly Ala Ser Gln Gly Met Phe Met Thr 865
870 875 880 tat
gcg cta gcg cat gag ctt ctg acg att att aaa gaa gtc atc aca 2688Tyr
Ala Leu Ala His Glu Leu Leu Thr Ile Ile Lys Glu Val Ile Thr
885 890 895 tct
tgc cag tca att gat agt gtg cca gaa tat aac act gag gat att 2736Ser
Cys Gln Ser Ile Asp Ser Val Pro Glu Tyr Asn Thr Glu Asp Ile
900 905 910 tgg
ttc caa ttt gca ctt tta atc tta gaa aag aaa acc ggc cat gta 2784Trp
Phe Gln Phe Ala Leu Leu Ile Leu Glu Lys Lys Thr Gly His Val
915 920 925 ttt
aat aaa aca tcg acc ctg act tat atg cct tgg gaa cga aaa tta 2832Phe
Asn Lys Thr Ser Thr Leu Thr Tyr Met Pro Trp Glu Arg Lys Leu
930 935 940 caa
tgg aca aat gaa caa att gaa agt gca aaa aga gga gaa aat ata 2880Gln
Trp Thr Asn Glu Gln Ile Glu Ser Ala Lys Arg Gly Glu Asn Ile 945
950 955 960 cct
gtt aac aag ttc att att aat agt ata act cta 2916Pro
Val Asn Lys Phe Ile Ile Asn Ser Ile Thr Leu
965 970
95972PRTPasteurella multocida 95Met Asn Thr Leu Ser Gln Ala Ile Lys Ala
Tyr Asn Ser Asn Asp Tyr 1 5 10
15 Gln Leu Ala Leu Lys Leu Phe Glu Lys Ser Ala Glu Ile Tyr Gly
Arg 20 25 30 Lys
Ile Val Glu Phe Gln Ile Thr Lys Cys Gln Glu Lys Leu Ser Ala 35
40 45 His Pro Ser Val Asn Ser
Ala His Leu Ser Val Asn Lys Glu Glu Lys 50 55
60 Val Asn Val Cys Asp Ser Pro Leu Asp Ile Ala
Thr Gln Leu Leu Leu 65 70 75
80 Ser Asn Val Lys Lys Leu Val Leu Ser Asp Ser Glu Lys Asn Thr Leu
85 90 95 Lys Asn
Lys Trp Lys Leu Leu Thr Glu Lys Lys Ser Glu Asn Ala Glu 100
105 110 Val Arg Ala Val Ala Leu Val
Pro Lys Asp Phe Pro Lys Asp Leu Val 115 120
125 Leu Ala Pro Leu Pro Asp His Val Asn Asp Phe Thr
Trp Tyr Lys Lys 130 135 140
Arg Lys Lys Arg Leu Gly Ile Lys Pro Glu His Gln His Val Gly Leu 145
150 155 160 Ser Ile Ile
Val Thr Thr Phe Asn Arg Pro Ala Ile Leu Ser Ile Thr 165
170 175 Leu Ala Cys Leu Val Asn Gln Lys
Thr His Tyr Pro Phe Glu Val Ile 180 185
190 Val Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile
Ile Arg Gln 195 200 205
Tyr Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln Lys Asp Asn Gly 210
215 220 Phe Gln Ala Ser
Ala Ala Arg Asn Met Gly Leu Arg Leu Ala Lys Tyr 225 230
235 240 Asp Phe Ile Gly Leu Leu Asp Cys Asp
Met Ala Pro Asn Pro Leu Trp 245 250
255 Val His Ser Tyr Val Ala Glu Leu Leu Glu Asp Asp Asp Leu
Thr Ile 260 265 270
Ile Gly Pro Arg Lys Tyr Ile Asp Thr Gln His Ile Asp Pro Lys Asp
275 280 285 Phe Leu Asn Asn
Ala Ser Leu Leu Glu Ser Leu Pro Glu Val Lys Thr 290
295 300 Asn Asn Ser Val Ala Ala Lys Gly
Glu Gly Thr Val Ser Leu Asp Trp 305 310
315 320 Arg Leu Glu Gln Phe Glu Lys Thr Glu Asn Leu Arg
Leu Ser Asp Ser 325 330
335 Pro Phe Arg Phe Phe Ala Ala Gly Asn Val Ala Phe Ala Lys Lys Trp
340 345 350 Leu Asn Lys
Ser Gly Phe Phe Asp Glu Glu Phe Asn His Trp Gly Gly 355
360 365 Glu Asp Val Glu Phe Gly Tyr Arg
Leu Phe Arg Tyr Gly Ser Phe Phe 370 375
380 Lys Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro
Pro Gly Lys 385 390 395
400 Glu Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile Thr Leu Asp Ile
405 410 415 Met Arg Glu Lys
Val Pro Tyr Ile Tyr Arg Lys Leu Leu Pro Ile Glu 420
425 430 Asp Ser His Ile Asn Arg Val Pro Leu
Val Ser Ile Tyr Ile Pro Ala 435 440
445 Tyr Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp Ser Ala
Leu Asn 450 455 460
Gln Thr Val Val Asp Leu Glu Val Cys Ile Cys Asn Asp Gly Ser Thr 465
470 475 480 Asp Asn Thr Leu Glu
Val Ile Asn Lys Leu Tyr Gly Asn Asn Pro Arg 485
490 495 Val Arg Ile Met Ser Lys Pro Asn Gly Gly
Ile Ala Ser Ala Ser Asn 500 505
510 Ala Ala Val Ser Phe Ala Lys Gly Tyr Tyr Ile Gly Gln Leu Asp
Ser 515 520 525 Asp
Asp Tyr Leu Glu Pro Asp Ala Val Glu Leu Cys Leu Lys Glu Phe 530
535 540 Leu Lys Asp Lys Thr Leu
Ala Cys Val Tyr Thr Thr Asn Arg Asn Val 545 550
555 560 Asn Pro Asp Gly Ser Leu Ile Ala Asn Gly Tyr
Asn Trp Pro Glu Phe 565 570
575 Ser Arg Glu Lys Leu Thr Thr Ala Met Ile Ala His His Phe Arg Met
580 585 590 Phe Thr
Ile Arg Ala Trp His Leu Thr Asp Gly Phe Asn Glu Lys Ile 595
600 605 Glu Asn Ala Val Asp Tyr Asp
Met Phe Leu Lys Leu Ser Glu Val Gly 610 615
620 Lys Phe Lys His Leu Asn Lys Ile Cys Tyr Asn Arg
Val Leu His Gly 625 630 635
640 Asp Asn Thr Ser Ile Lys Lys Leu Gly Ile Gln Lys Lys Asn His Phe
645 650 655 Val Val Val
Asn Gln Ser Leu Asn Arg Gln Gly Ile Thr Tyr Tyr Asn 660
665 670 Tyr Asp Glu Phe Asp Asp Leu Asp
Glu Ser Arg Lys Tyr Ile Phe Asn 675 680
685 Lys Thr Ala Glu Tyr Gln Glu Glu Ile Asp Ile Leu Lys
Asp Ile Lys 690 695 700
Ile Ile Gln Asn Lys Asp Ala Lys Ile Ala Val Ser Ile Phe Tyr Pro 705
710 715 720 Asn Thr Leu Asn
Gly Leu Val Lys Lys Leu Asn Asn Ile Ile Glu Tyr 725
730 735 Asn Lys Asn Ile Phe Val Ile Val Leu
His Val Asp Lys Asn His Leu 740 745
750 Thr Pro Asp Ile Lys Lys Glu Ile Leu Ala Phe Tyr His Lys
His Gln 755 760 765
Val Asn Ile Leu Leu Asn Asn Asp Ile Ser Tyr Tyr Thr Ser Asn Arg 770
775 780 Leu Ile Lys Thr Glu
Ala His Leu Ser Asn Ile Asn Lys Leu Ser Gln 785 790
795 800 Leu Asn Leu Asn Cys Glu Tyr Ile Ile Phe
Asp Asn His Asp Ser Leu 805 810
815 Phe Val Lys Asn Asp Ser Tyr Ala Tyr Met Lys Lys Tyr Asp Val
Gly 820 825 830 Met
Asn Phe Ser Ala Leu Thr His Asp Trp Ile Glu Lys Ile Asn Ala 835
840 845 His Pro Pro Phe Lys Lys
Leu Ile Lys Thr Tyr Phe Asn Asp Asn Asp 850 855
860 Leu Lys Ser Met Asn Val Lys Gly Ala Ser Gln
Gly Met Phe Met Thr 865 870 875
880 Tyr Ala Leu Ala His Glu Leu Leu Thr Ile Ile Lys Glu Val Ile Thr
885 890 895 Ser Cys
Gln Ser Ile Asp Ser Val Pro Glu Tyr Asn Thr Glu Asp Ile 900
905 910 Trp Phe Gln Phe Ala Leu Leu
Ile Leu Glu Lys Lys Thr Gly His Val 915 920
925 Phe Asn Lys Thr Ser Thr Leu Thr Tyr Met Pro Trp
Glu Arg Lys Leu 930 935 940
Gln Trp Thr Asn Glu Gln Ile Glu Ser Ala Lys Arg Gly Glu Asn Ile 945
950 955 960 Pro Val Asn
Lys Phe Ile Ile Asn Ser Ile Thr Leu 965
970 961206DNAStreptococcus pyogenesCDS(1)..(1206) 96atg aaa ata
gca gtt gct gga tca gga tat gtt gga tta tca cta gga 48Met Lys Ile
Ala Val Ala Gly Ser Gly Tyr Val Gly Leu Ser Leu Gly 1
5 10 15 gtt ctt tta
tca ctt caa aac gaa gtc act att gtt gat att ctt ccc 96Val Leu Leu
Ser Leu Gln Asn Glu Val Thr Ile Val Asp Ile Leu Pro
20 25 30 tct aaa gtt
gat aag att aat aat ggc tta tca cca att caa gat gaa 144Ser Lys Val
Asp Lys Ile Asn Asn Gly Leu Ser Pro Ile Gln Asp Glu 35
40 45 tat att gaa
tat tac tta aaa agt aag caa tta tct att aaa gca act 192Tyr Ile Glu
Tyr Tyr Leu Lys Ser Lys Gln Leu Ser Ile Lys Ala Thr 50
55 60 tta gat agc
aaa gca gct tat aaa gaa gcg gaa ctg gtc att att gcc 240Leu Asp Ser
Lys Ala Ala Tyr Lys Glu Ala Glu Leu Val Ile Ile Ala 65
70 75 80 aca cct aca
aat tac aac agt aga att aat tat ttt gat aca cag cat 288Thr Pro Thr
Asn Tyr Asn Ser Arg Ile Asn Tyr Phe Asp Thr Gln His
85 90 95 gtt gaa aca
gtt atc aaa gag gta cta agc gtt aat agc cat gca act 336Val Glu Thr
Val Ile Lys Glu Val Leu Ser Val Asn Ser His Ala Thr
100 105 110 ctt atc atc
aaa tca aca att cca ata ggt ttc att act gaa atg aga 384Leu Ile Ile
Lys Ser Thr Ile Pro Ile Gly Phe Ile Thr Glu Met Arg 115
120 125 cag aaa ttc
caa act gat cgt att atc ttc agc cct gaa ttt tta aga 432Gln Lys Phe
Gln Thr Asp Arg Ile Ile Phe Ser Pro Glu Phe Leu Arg 130
135 140 gaa tct aaa
gct tta tat gac aac tta tat cca agc cga att att gtt 480Glu Ser Lys
Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145
150 155 160 tct tgt gaa
gaa aac gat tct cca aaa gta aag gca gac gca gaa aaa 528Ser Cys Glu
Glu Asn Asp Ser Pro Lys Val Lys Ala Asp Ala Glu Lys
165 170 175 ttt gca ctt
tta tta aag tct gca gct aaa aaa aat aat gta cca gta 576Phe Ala Leu
Leu Leu Lys Ser Ala Ala Lys Lys Asn Asn Val Pro Val
180 185 190 ctt att atg
gga gct tca gaa gct gaa gca gta aaa cta ttt gcc aat 624Leu Ile Met
Gly Ala Ser Glu Ala Glu Ala Val Lys Leu Phe Ala Asn 195
200 205 act tat tta
gcg tta agg gta gct tat ttt aat gag tta gac act tac 672Thr Tyr Leu
Ala Leu Arg Val Ala Tyr Phe Asn Glu Leu Asp Thr Tyr 210
215 220 gca gaa tcg
aga aaa tta aat agt cac atg att att caa gga att tct 720Ala Glu Ser
Arg Lys Leu Asn Ser His Met Ile Ile Gln Gly Ile Ser 225
230 235 240 tat gat gat
cga ata gga atg cat tat aat aac cca tca ttt ggt tat 768Tyr Asp Asp
Arg Ile Gly Met His Tyr Asn Asn Pro Ser Phe Gly Tyr
245 250 255 gga ggt tat
tgt cta cct aaa gat acg aag caa tta ttg gca aat tac 816Gly Gly Tyr
Cys Leu Pro Lys Asp Thr Lys Gln Leu Leu Ala Asn Tyr
260 265 270 aat aat att
cct caa acg cta att gaa gct atc gtt tca tca aat aat 864Asn Asn Ile
Pro Gln Thr Leu Ile Glu Ala Ile Val Ser Ser Asn Asn 275
280 285 gtg cgc aag
tcc tat att gct aag caa att atc aac gtc tta gaa gag 912Val Arg Lys
Ser Tyr Ile Ala Lys Gln Ile Ile Asn Val Leu Glu Glu 290
295 300 cgg gag tcc
cca gta aaa gta gtc ggg gtt tac cgt tta att atg aaa 960Arg Glu Ser
Pro Val Lys Val Val Gly Val Tyr Arg Leu Ile Met Lys 305
310 315 320 agt aac tca
gat aat ttt aga gaa agt gct atc aaa gat gtt att gac 1008Ser Asn Ser
Asp Asn Phe Arg Glu Ser Ala Ile Lys Asp Val Ile Asp
325 330 335 att ctt aaa
agt aaa gac att aag ata att att tat gag cca atg tta 1056Ile Leu Lys
Ser Lys Asp Ile Lys Ile Ile Ile Tyr Glu Pro Met Leu
340 345 350 aac aaa ctt
gaa tct gaa gat caa tct gta ctt gta aat gat tta gag 1104Asn Lys Leu
Glu Ser Glu Asp Gln Ser Val Leu Val Asn Asp Leu Glu 355
360 365 aat ttc aag
aaa caa gca aat att atc gta act aat cgc tat gat aat 1152Asn Phe Lys
Lys Gln Ala Asn Ile Ile Val Thr Asn Arg Tyr Asp Asn 370
375 380 gaa tta caa
gat gtt aaa aat aaa gtt tac agt aga gat att ttt aat 1200Glu Leu Gln
Asp Val Lys Asn Lys Val Tyr Ser Arg Asp Ile Phe Asn 385
390 395 400 aga gac
1206Arg Asp
97402PRTStreptococcus pyogenes 97Met Lys Ile Ala Val Ala Gly Ser Gly Tyr
Val Gly Leu Ser Leu Gly 1 5 10
15 Val Leu Leu Ser Leu Gln Asn Glu Val Thr Ile Val Asp Ile Leu
Pro 20 25 30 Ser
Lys Val Asp Lys Ile Asn Asn Gly Leu Ser Pro Ile Gln Asp Glu 35
40 45 Tyr Ile Glu Tyr Tyr Leu
Lys Ser Lys Gln Leu Ser Ile Lys Ala Thr 50 55
60 Leu Asp Ser Lys Ala Ala Tyr Lys Glu Ala Glu
Leu Val Ile Ile Ala 65 70 75
80 Thr Pro Thr Asn Tyr Asn Ser Arg Ile Asn Tyr Phe Asp Thr Gln His
85 90 95 Val Glu
Thr Val Ile Lys Glu Val Leu Ser Val Asn Ser His Ala Thr 100
105 110 Leu Ile Ile Lys Ser Thr Ile
Pro Ile Gly Phe Ile Thr Glu Met Arg 115 120
125 Gln Lys Phe Gln Thr Asp Arg Ile Ile Phe Ser Pro
Glu Phe Leu Arg 130 135 140
Glu Ser Lys Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145
150 155 160 Ser Cys Glu
Glu Asn Asp Ser Pro Lys Val Lys Ala Asp Ala Glu Lys 165
170 175 Phe Ala Leu Leu Leu Lys Ser Ala
Ala Lys Lys Asn Asn Val Pro Val 180 185
190 Leu Ile Met Gly Ala Ser Glu Ala Glu Ala Val Lys Leu
Phe Ala Asn 195 200 205
Thr Tyr Leu Ala Leu Arg Val Ala Tyr Phe Asn Glu Leu Asp Thr Tyr 210
215 220 Ala Glu Ser Arg
Lys Leu Asn Ser His Met Ile Ile Gln Gly Ile Ser 225 230
235 240 Tyr Asp Asp Arg Ile Gly Met His Tyr
Asn Asn Pro Ser Phe Gly Tyr 245 250
255 Gly Gly Tyr Cys Leu Pro Lys Asp Thr Lys Gln Leu Leu Ala
Asn Tyr 260 265 270
Asn Asn Ile Pro Gln Thr Leu Ile Glu Ala Ile Val Ser Ser Asn Asn
275 280 285 Val Arg Lys Ser
Tyr Ile Ala Lys Gln Ile Ile Asn Val Leu Glu Glu 290
295 300 Arg Glu Ser Pro Val Lys Val Val
Gly Val Tyr Arg Leu Ile Met Lys 305 310
315 320 Ser Asn Ser Asp Asn Phe Arg Glu Ser Ala Ile Lys
Asp Val Ile Asp 325 330
335 Ile Leu Lys Ser Lys Asp Ile Lys Ile Ile Ile Tyr Glu Pro Met Leu
340 345 350 Asn Lys Leu
Glu Ser Glu Asp Gln Ser Val Leu Val Asn Asp Leu Glu 355
360 365 Asn Phe Lys Lys Gln Ala Asn Ile
Ile Val Thr Asn Arg Tyr Asp Asn 370 375
380 Glu Leu Gln Asp Val Lys Asn Lys Val Tyr Ser Arg Asp
Ile Phe Asn 385 390 395
400 Arg Asp 98912DNAStreptococcus pyogenesCDS(1)..(912) 98atg acc aaa gtc
aga aaa gcc att att cct gct gca ggt cta gga aca 48Met Thr Lys Val
Arg Lys Ala Ile Ile Pro Ala Ala Gly Leu Gly Thr 1
5 10 15 cgt ttt tta cct
gct acc aaa gct ctt gcc aaa gag atg ttg ccc atc 96Arg Phe Leu Pro
Ala Thr Lys Ala Leu Ala Lys Glu Met Leu Pro Ile 20
25 30 gtt gat aaa cca
acc atc cag ttt atc gtc gaa gaa gcg cta aaa tct 144Val Asp Lys Pro
Thr Ile Gln Phe Ile Val Glu Glu Ala Leu Lys Ser 35
40 45 ggc atc gag gaa
atc ctt gtg gtg acc gga aaa gct aaa cgc tct atc 192Gly Ile Glu Glu
Ile Leu Val Val Thr Gly Lys Ala Lys Arg Ser Ile 50
55 60 gag gac cat ttt
gat tca aac ttt gaa tta gaa tac aac ctc caa gct 240Glu Asp His Phe
Asp Ser Asn Phe Glu Leu Glu Tyr Asn Leu Gln Ala 65
70 75 80 aag ggg aaa aat
gaa ctg ttg aaa tta gtg gat gaa acc act gcc att 288Lys Gly Lys Asn
Glu Leu Leu Lys Leu Val Asp Glu Thr Thr Ala Ile
85 90 95 aac ctt cat ttt
atc cgt caa agc cac cca aga ggg ctg gga gat gct 336Asn Leu His Phe
Ile Arg Gln Ser His Pro Arg Gly Leu Gly Asp Ala 100
105 110 gtc tta caa gcc
aaa gcc ttt gtg ggc aat gaa ccc ttt gtg gtc atg 384Val Leu Gln Ala
Lys Ala Phe Val Gly Asn Glu Pro Phe Val Val Met 115
120 125 ctt gga gat gac
tta atg gac att aca aat gca tcc gct aaa cct ctc 432Leu Gly Asp Asp
Leu Met Asp Ile Thr Asn Ala Ser Ala Lys Pro Leu 130
135 140 acc aaa caa ctc
atg gag gac tat gac aag acg cat gca tcc act atc 480Thr Lys Gln Leu
Met Glu Asp Tyr Asp Lys Thr His Ala Ser Thr Ile 145
150 155 160 gct gtg atg aaa
gtt cct cat gaa gat gtg tct agc tat ggg gtt atc 528Ala Val Met Lys
Val Pro His Glu Asp Val Ser Ser Tyr Gly Val Ile
165 170 175 gct cct caa ggc
aag gct gtc aag ggc ctt tac agt gta gac acc ttt 576Ala Pro Gln Gly
Lys Ala Val Lys Gly Leu Tyr Ser Val Asp Thr Phe 180
185 190 gtt gaa aaa cca
caa cca gaa gat gcg cct agt gat ttg gct att att 624Val Glu Lys Pro
Gln Pro Glu Asp Ala Pro Ser Asp Leu Ala Ile Ile 195
200 205 ggt cgt tac ctc
cta acc cct gaa att ttt ggt att ttg gaa aga cag 672Gly Arg Tyr Leu
Leu Thr Pro Glu Ile Phe Gly Ile Leu Glu Arg Gln 210
215 220 acc cct gga gca
ggt aac gaa gtg caa ctc aca gat gct atc gat acc 720Thr Pro Gly Ala
Gly Asn Glu Val Gln Leu Thr Asp Ala Ile Asp Thr 225
230 235 240 ctc aat aaa act
cag cgt gtc ttt gca cga gaa ttt aaa ggc aat cgt 768Leu Asn Lys Thr
Gln Arg Val Phe Ala Arg Glu Phe Lys Gly Asn Arg
245 250 255 tac gat gtt ggg
gat aaa ttt gga ttc atg aaa aca tct atc gac tat 816Tyr Asp Val Gly
Asp Lys Phe Gly Phe Met Lys Thr Ser Ile Asp Tyr 260
265 270 gcc tta gaa cac
cca cag gtc aaa gag gac ttg aaa aat tac att atc 864Ala Leu Glu His
Pro Gln Val Lys Glu Asp Leu Lys Asn Tyr Ile Ile 275
280 285 aaa cta gga aaa
gct ttg gaa aaa agt aaa gta cca aca cat tca aag 912Lys Leu Gly Lys
Ala Leu Glu Lys Ser Lys Val Pro Thr His Ser Lys 290
295 300
99304PRTStreptococcus pyogenes 99Met Thr Lys Val Arg Lys Ala Ile Ile Pro
Ala Ala Gly Leu Gly Thr 1 5 10
15 Arg Phe Leu Pro Ala Thr Lys Ala Leu Ala Lys Glu Met Leu Pro
Ile 20 25 30 Val
Asp Lys Pro Thr Ile Gln Phe Ile Val Glu Glu Ala Leu Lys Ser 35
40 45 Gly Ile Glu Glu Ile Leu
Val Val Thr Gly Lys Ala Lys Arg Ser Ile 50 55
60 Glu Asp His Phe Asp Ser Asn Phe Glu Leu Glu
Tyr Asn Leu Gln Ala 65 70 75
80 Lys Gly Lys Asn Glu Leu Leu Lys Leu Val Asp Glu Thr Thr Ala Ile
85 90 95 Asn Leu
His Phe Ile Arg Gln Ser His Pro Arg Gly Leu Gly Asp Ala 100
105 110 Val Leu Gln Ala Lys Ala Phe
Val Gly Asn Glu Pro Phe Val Val Met 115 120
125 Leu Gly Asp Asp Leu Met Asp Ile Thr Asn Ala Ser
Ala Lys Pro Leu 130 135 140
Thr Lys Gln Leu Met Glu Asp Tyr Asp Lys Thr His Ala Ser Thr Ile 145
150 155 160 Ala Val Met
Lys Val Pro His Glu Asp Val Ser Ser Tyr Gly Val Ile 165
170 175 Ala Pro Gln Gly Lys Ala Val Lys
Gly Leu Tyr Ser Val Asp Thr Phe 180 185
190 Val Glu Lys Pro Gln Pro Glu Asp Ala Pro Ser Asp Leu
Ala Ile Ile 195 200 205
Gly Arg Tyr Leu Leu Thr Pro Glu Ile Phe Gly Ile Leu Glu Arg Gln 210
215 220 Thr Pro Gly Ala
Gly Asn Glu Val Gln Leu Thr Asp Ala Ile Asp Thr 225 230
235 240 Leu Asn Lys Thr Gln Arg Val Phe Ala
Arg Glu Phe Lys Gly Asn Arg 245 250
255 Tyr Asp Val Gly Asp Lys Phe Gly Phe Met Lys Thr Ser Ile
Asp Tyr 260 265 270
Ala Leu Glu His Pro Gln Val Lys Glu Asp Leu Lys Asn Tyr Ile Ile
275 280 285 Lys Leu Gly Lys
Ala Leu Glu Lys Ser Lys Val Pro Thr His Ser Lys 290
295 300 1001347DNAStreptococcus equi
zooepidemicusCDS(1)..(1347) 100atg tca cat att aca ttt gat tat tca aag
gtt ctt gag caa ttt gcc 48Met Ser His Ile Thr Phe Asp Tyr Ser Lys
Val Leu Glu Gln Phe Ala 1 5 10
15 gga cag cat gaa att gac ttt tta caa ggt
cag gta aca gag gct gat 96Gly Gln His Glu Ile Asp Phe Leu Gln Gly
Gln Val Thr Glu Ala Asp 20 25
30 cag gca cta cgt cag ggc act gga cct gga
tca gat ttc ttg ggc tgg 144Gln Ala Leu Arg Gln Gly Thr Gly Pro Gly
Ser Asp Phe Leu Gly Trp 35 40
45 ctt gag tta cct gaa aac tat gac aaa gaa
gaa ttt gct cgt atc ctt 192Leu Glu Leu Pro Glu Asn Tyr Asp Lys Glu
Glu Phe Ala Arg Ile Leu 50 55
60 aaa gca gct gag aag att aag gct gac agt
gac gtt ctt gtt gtg att 240Lys Ala Ala Glu Lys Ile Lys Ala Asp Ser
Asp Val Leu Val Val Ile 65 70
75 80 ggt att ggt ggc tct tac ctt ggt gct aag
gct gca att gac ttt ttg 288Gly Ile Gly Gly Ser Tyr Leu Gly Ala Lys
Ala Ala Ile Asp Phe Leu 85 90
95 aac agc cat ttt gcc aac cta caa aca gca
aaa gag cgc aaa gca cca 336Asn Ser His Phe Ala Asn Leu Gln Thr Ala
Lys Glu Arg Lys Ala Pro 100 105
110 caa att ctt tat gct ggt aac tcc atc tca
tca agc tat ctt gct gat 384Gln Ile Leu Tyr Ala Gly Asn Ser Ile Ser
Ser Ser Tyr Leu Ala Asp 115 120
125 ctt gtg gac tat gtt caa gat aaa gat ttc
tct gtt aac gtg att tct 432Leu Val Asp Tyr Val Gln Asp Lys Asp Phe
Ser Val Asn Val Ile Ser 130 135
140 aag tct ggt aca aca aca gag cct gca atc
gcc ttt cgt gtc ttt aaa 480Lys Ser Gly Thr Thr Thr Glu Pro Ala Ile
Ala Phe Arg Val Phe Lys 145 150
155 160 gaa tta ctt gtt aaa aag tac ggt caa gaa
gag gcc aac aag cgt atc 528Glu Leu Leu Val Lys Lys Tyr Gly Gln Glu
Glu Ala Asn Lys Arg Ile 165 170
175 tat gca acg act gat aag gtc aag ggt gct
gtt aag gtt gag gct gat 576Tyr Ala Thr Thr Asp Lys Val Lys Gly Ala
Val Lys Val Glu Ala Asp 180 185
190 gca aat cat tgg gaa acc ttt gtt gtg cca
gat aat gtt ggt ggc cgt 624Ala Asn His Trp Glu Thr Phe Val Val Pro
Asp Asn Val Gly Gly Arg 195 200
205 ttc tca gtg ctg aca gct gtg ggc ttg cta
cca att gca gca tca ggg 672Phe Ser Val Leu Thr Ala Val Gly Leu Leu
Pro Ile Ala Ala Ser Gly 210 215
220 gct gat att acc gcg ctg atg gaa gga gca
aat gca gct cgt aag gac 720Ala Asp Ile Thr Ala Leu Met Glu Gly Ala
Asn Ala Ala Arg Lys Asp 225 230
235 240 ctg tca tca gat aaa atc tca gaa aac atc
gct tac caa tat gct gtg 768Leu Ser Ser Asp Lys Ile Ser Glu Asn Ile
Ala Tyr Gln Tyr Ala Val 245 250
255 gtc cgc aat atc ctc tat cgc aaa ggc tat
gta act gaa att ttg gca 816Val Arg Asn Ile Leu Tyr Arg Lys Gly Tyr
Val Thr Glu Ile Leu Ala 260 265
270 aac tat gag cca tca ttg cag tat ttt agc
gaa tgg tgg aag caa ctg 864Asn Tyr Glu Pro Ser Leu Gln Tyr Phe Ser
Glu Trp Trp Lys Gln Leu 275 280
285 gct ggt gag tct gaa gga aag gac caa aag
ggt att tac cca act tca 912Ala Gly Glu Ser Glu Gly Lys Asp Gln Lys
Gly Ile Tyr Pro Thr Ser 290 295
300 gct aat ttc tcg aca gac ctg cat tct ctt
ggt caa ttt atc caa gaa 960Ala Asn Phe Ser Thr Asp Leu His Ser Leu
Gly Gln Phe Ile Gln Glu 305 310
315 320 ggc tac cgt aac ctc ttt gag aca gtg att
cgt gtg gac aag cca cgt 1008Gly Tyr Arg Asn Leu Phe Glu Thr Val Ile
Arg Val Asp Lys Pro Arg 325 330
335 caa aat gtg att atc cca gaa atg gct gag
gac ctt gat ggc ctt ggc 1056Gln Asn Val Ile Ile Pro Glu Met Ala Glu
Asp Leu Asp Gly Leu Gly 340 345
350 tac cta caa gga aaa gac gtt gac ttt gtc
aac aaa aaa gca aca gat 1104Tyr Leu Gln Gly Lys Asp Val Asp Phe Val
Asn Lys Lys Ala Thr Asp 355 360
365 ggt gtc ctt ctt gcc cat aca gat ggt ggt
gtg cca aat atg ttt atc 1152Gly Val Leu Leu Ala His Thr Asp Gly Gly
Val Pro Asn Met Phe Ile 370 375
380 acg ctt cca gag caa gac gaa ttt aca cta
ggc tat acg atc tac ttc 1200Thr Leu Pro Glu Gln Asp Glu Phe Thr Leu
Gly Tyr Thr Ile Tyr Phe 385 390
395 400 ttt gag ctt gct att gcc ctt tca ggc tac
ctc aac ggg gtc aat cca 1248Phe Glu Leu Ala Ile Ala Leu Ser Gly Tyr
Leu Asn Gly Val Asn Pro 405 410
415 ttt gat cag cca ggc gtt gag gct tac aag
aaa aac atg ttt gcc ctt 1296Phe Asp Gln Pro Gly Val Glu Ala Tyr Lys
Lys Asn Met Phe Ala Leu 420 425
430 ctt ggt aag cca ggc ttt gaa gag cta gga
gca gcg ctc aac gca cgc 1344Leu Gly Lys Pro Gly Phe Glu Glu Leu Gly
Ala Ala Leu Asn Ala Arg 435 440
445 ttg
1347Leu
101449PRTStreptococcus equi
zooepidemicus 101Met Ser His Ile Thr Phe Asp Tyr Ser Lys Val Leu Glu Gln
Phe Ala 1 5 10 15
Gly Gln His Glu Ile Asp Phe Leu Gln Gly Gln Val Thr Glu Ala Asp
20 25 30 Gln Ala Leu Arg Gln
Gly Thr Gly Pro Gly Ser Asp Phe Leu Gly Trp 35
40 45 Leu Glu Leu Pro Glu Asn Tyr Asp Lys
Glu Glu Phe Ala Arg Ile Leu 50 55
60 Lys Ala Ala Glu Lys Ile Lys Ala Asp Ser Asp Val Leu
Val Val Ile 65 70 75
80 Gly Ile Gly Gly Ser Tyr Leu Gly Ala Lys Ala Ala Ile Asp Phe Leu
85 90 95 Asn Ser His Phe
Ala Asn Leu Gln Thr Ala Lys Glu Arg Lys Ala Pro 100
105 110 Gln Ile Leu Tyr Ala Gly Asn Ser Ile
Ser Ser Ser Tyr Leu Ala Asp 115 120
125 Leu Val Asp Tyr Val Gln Asp Lys Asp Phe Ser Val Asn Val
Ile Ser 130 135 140
Lys Ser Gly Thr Thr Thr Glu Pro Ala Ile Ala Phe Arg Val Phe Lys 145
150 155 160 Glu Leu Leu Val Lys
Lys Tyr Gly Gln Glu Glu Ala Asn Lys Arg Ile 165
170 175 Tyr Ala Thr Thr Asp Lys Val Lys Gly Ala
Val Lys Val Glu Ala Asp 180 185
190 Ala Asn His Trp Glu Thr Phe Val Val Pro Asp Asn Val Gly Gly
Arg 195 200 205 Phe
Ser Val Leu Thr Ala Val Gly Leu Leu Pro Ile Ala Ala Ser Gly 210
215 220 Ala Asp Ile Thr Ala Leu
Met Glu Gly Ala Asn Ala Ala Arg Lys Asp 225 230
235 240 Leu Ser Ser Asp Lys Ile Ser Glu Asn Ile Ala
Tyr Gln Tyr Ala Val 245 250
255 Val Arg Asn Ile Leu Tyr Arg Lys Gly Tyr Val Thr Glu Ile Leu Ala
260 265 270 Asn Tyr
Glu Pro Ser Leu Gln Tyr Phe Ser Glu Trp Trp Lys Gln Leu 275
280 285 Ala Gly Glu Ser Glu Gly Lys
Asp Gln Lys Gly Ile Tyr Pro Thr Ser 290 295
300 Ala Asn Phe Ser Thr Asp Leu His Ser Leu Gly Gln
Phe Ile Gln Glu 305 310 315
320 Gly Tyr Arg Asn Leu Phe Glu Thr Val Ile Arg Val Asp Lys Pro Arg
325 330 335 Gln Asn Val
Ile Ile Pro Glu Met Ala Glu Asp Leu Asp Gly Leu Gly 340
345 350 Tyr Leu Gln Gly Lys Asp Val Asp
Phe Val Asn Lys Lys Ala Thr Asp 355 360
365 Gly Val Leu Leu Ala His Thr Asp Gly Gly Val Pro Asn
Met Phe Ile 370 375 380
Thr Leu Pro Glu Gln Asp Glu Phe Thr Leu Gly Tyr Thr Ile Tyr Phe 385
390 395 400 Phe Glu Leu Ala
Ile Ala Leu Ser Gly Tyr Leu Asn Gly Val Asn Pro 405
410 415 Phe Asp Gln Pro Gly Val Glu Ala Tyr
Lys Lys Asn Met Phe Ala Leu 420 425
430 Leu Gly Lys Pro Gly Phe Glu Glu Leu Gly Ala Ala Leu Asn
Ala Arg 435 440 445
Leu 1021251DNAStreptococcus uberisCDS(1)..(1251) 102atg gaa aaa cta aaa
aat ctc att aca ttt atg act ttt att ttc ctg 48Met Glu Lys Leu Lys
Asn Leu Ile Thr Phe Met Thr Phe Ile Phe Leu 1 5
10 15 tgg ctc ata att att
ggg ctt aat gtt ttt gta ttt gga act aaa gga 96Trp Leu Ile Ile Ile
Gly Leu Asn Val Phe Val Phe Gly Thr Lys Gly 20
25 30 agt cta aca gtg tat
ggg att att cta tta acc tat ttg tcg ata aaa 144Ser Leu Thr Val Tyr
Gly Ile Ile Leu Leu Thr Tyr Leu Ser Ile Lys 35
40 45 atg gga tta tct ttt
ttt tat cgt ccc tat aaa gga agt gta ggt caa 192Met Gly Leu Ser Phe
Phe Tyr Arg Pro Tyr Lys Gly Ser Val Gly Gln 50
55 60 tat aag gta gca gct
att atc cca tct tat aat gag gat ggt gtc ggt 240Tyr Lys Val Ala Ala
Ile Ile Pro Ser Tyr Asn Glu Asp Gly Val Gly 65
70 75 80 tta cta gaa act cta
aag agt gtt caa aaa caa aca tat cca att gca 288Leu Leu Glu Thr Leu
Lys Ser Val Gln Lys Gln Thr Tyr Pro Ile Ala 85
90 95 gaa att ttc gta att
gac gat ggg tca gta gat aaa aca ggt ata aaa 336Glu Ile Phe Val Ile
Asp Asp Gly Ser Val Asp Lys Thr Gly Ile Lys 100
105 110 ttg gtc gaa gac tat
gtg aag tta aat ggc ttt gga gac caa gtt atc 384Leu Val Glu Asp Tyr
Val Lys Leu Asn Gly Phe Gly Asp Gln Val Ile 115
120 125 gtt cat cag atg cct
gaa aat gtt ggt aaa aga cat gct cag gct tgg 432Val His Gln Met Pro
Glu Asn Val Gly Lys Arg His Ala Gln Ala Trp 130
135 140 gca ttt gaa agg tct
gat gct gat gtt ttc tta aca gtg gat tca gat 480Ala Phe Glu Arg Ser
Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp 145
150 155 160 acc tac atc tat cct
gat gct ctt gaa gaa tta tta aag aca ttt aat 528Thr Tyr Ile Tyr Pro
Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe Asn 165
170 175 gat cca gag gtc tac
gct gca act ggt cat tta aat gca aga aat aga 576Asp Pro Glu Val Tyr
Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg 180
185 190 caa act aat ctc tta
act aga ctg act gat att cgt tac gat aat gca 624Gln Thr Asn Leu Leu
Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala 195
200 205 ttt ggt gta gaa cgt
gct gct cag tct gtt acg gga aat att ttg gtt 672Phe Gly Val Glu Arg
Ala Ala Gln Ser Val Thr Gly Asn Ile Leu Val 210
215 220 tgt tcc gga cct tta
agt att tat aga cgt tcc gtc ggt att cca aat 720Cys Ser Gly Pro Leu
Ser Ile Tyr Arg Arg Ser Val Gly Ile Pro Asn 225
230 235 240 ctt gaa cgc tat acc
tca caa aca ttt ctt ggt gtc cct gta agc ata 768Leu Glu Arg Tyr Thr
Ser Gln Thr Phe Leu Gly Val Pro Val Ser Ile 245
250 255 ggg gat gac cgt tgt
ttg aca aat tat gca act gat ttg gga aaa acg 816Gly Asp Asp Arg Cys
Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys Thr 260
265 270 gtt tat cag tca act
gca aga tgt gat act gac gtt cca gat aag ttt 864Val Tyr Gln Ser Thr
Ala Arg Cys Asp Thr Asp Val Pro Asp Lys Phe 275
280 285 aag gtt ttc atc aaa
caa caa aat cgt tgg aat aag tca ttt ttt agg 912Lys Val Phe Ile Lys
Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg 290
295 300 gag tct att atc tct
gtt aag aag tta tta gcc aca cca agt gtt gct 960Glu Ser Ile Ile Ser
Val Lys Lys Leu Leu Ala Thr Pro Ser Val Ala 305
310 315 320 gtt tgg act att aca
gaa gtt tcc atg ttc atc atg cta gtt tat tct 1008Val Trp Thr Ile Thr
Glu Val Ser Met Phe Ile Met Leu Val Tyr Ser 325
330 335 atc ttt agc tta ttg
ata gga gag gct caa gaa ttt aat ctc ata aaa 1056Ile Phe Ser Leu Leu
Ile Gly Glu Ala Gln Glu Phe Asn Leu Ile Lys 340
345 350 ctg gtt gct ttt tta
gtt att att ttc ata gta gct ctt tgt aga aat 1104Leu Val Ala Phe Leu
Val Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355
360 365 gtt cat tac atg gtt
aag cat cca ttt gct ttt tta ttg tca ccg ttt 1152Val His Tyr Met Val
Lys His Pro Phe Ala Phe Leu Leu Ser Pro Phe 370
375 380 tat gga ttg ata cat
cta ttc gtt ttg caa cct ctt aag ata tat tcg 1200Tyr Gly Leu Ile His
Leu Phe Val Leu Gln Pro Leu Lys Ile Tyr Ser 385
390 395 400 tta ttt act ata aga
aat gct aca tgg gga act cgt aaa aag aca agt 1248Leu Phe Thr Ile Arg
Asn Ala Thr Trp Gly Thr Arg Lys Lys Thr Ser 405
410 415 aaa
1251Lys
103417PRTStreptococcus uberis 103Met Glu Lys Leu Lys Asn Leu Ile Thr Phe
Met Thr Phe Ile Phe Leu 1 5 10
15 Trp Leu Ile Ile Ile Gly Leu Asn Val Phe Val Phe Gly Thr Lys
Gly 20 25 30 Ser
Leu Thr Val Tyr Gly Ile Ile Leu Leu Thr Tyr Leu Ser Ile Lys 35
40 45 Met Gly Leu Ser Phe Phe
Tyr Arg Pro Tyr Lys Gly Ser Val Gly Gln 50 55
60 Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn
Glu Asp Gly Val Gly 65 70 75
80 Leu Leu Glu Thr Leu Lys Ser Val Gln Lys Gln Thr Tyr Pro Ile Ala
85 90 95 Glu Ile
Phe Val Ile Asp Asp Gly Ser Val Asp Lys Thr Gly Ile Lys 100
105 110 Leu Val Glu Asp Tyr Val Lys
Leu Asn Gly Phe Gly Asp Gln Val Ile 115 120
125 Val His Gln Met Pro Glu Asn Val Gly Lys Arg His
Ala Gln Ala Trp 130 135 140
Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp 145
150 155 160 Thr Tyr Ile
Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe Asn 165
170 175 Asp Pro Glu Val Tyr Ala Ala Thr
Gly His Leu Asn Ala Arg Asn Arg 180 185
190 Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr
Asp Asn Ala 195 200 205
Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu Val 210
215 220 Cys Ser Gly Pro
Leu Ser Ile Tyr Arg Arg Ser Val Gly Ile Pro Asn 225 230
235 240 Leu Glu Arg Tyr Thr Ser Gln Thr Phe
Leu Gly Val Pro Val Ser Ile 245 250
255 Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly
Lys Thr 260 265 270
Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Asp Lys Phe
275 280 285 Lys Val Phe Ile
Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg 290
295 300 Glu Ser Ile Ile Ser Val Lys Lys
Leu Leu Ala Thr Pro Ser Val Ala 305 310
315 320 Val Trp Thr Ile Thr Glu Val Ser Met Phe Ile Met
Leu Val Tyr Ser 325 330
335 Ile Phe Ser Leu Leu Ile Gly Glu Ala Gln Glu Phe Asn Leu Ile Lys
340 345 350 Leu Val Ala
Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355
360 365 Val His Tyr Met Val Lys His Pro
Phe Ala Phe Leu Leu Ser Pro Phe 370 375
380 Tyr Gly Leu Ile His Leu Phe Val Leu Gln Pro Leu Lys
Ile Tyr Ser 385 390 395
400 Leu Phe Thr Ile Arg Asn Ala Thr Trp Gly Thr Arg Lys Lys Thr Ser
405 410 415 Lys
1041203DNAStreptococcus uberisCDS(1)..(1203) 104gtg aaa att gca gtt gca
ggt tct ggc tat gtt ggc cta tca tta agt 48Val Lys Ile Ala Val Ala
Gly Ser Gly Tyr Val Gly Leu Ser Leu Ser 1 5
10 15 gta tta tta gca cag aaa
aat cct gtt aca gtt gta gat att att gag 96Val Leu Leu Ala Gln Lys
Asn Pro Val Thr Val Val Asp Ile Ile Glu 20
25 30 aag aaa gta aat ctc ata
aat caa aaa caa tca cca atc cag gat gtt 144Lys Lys Val Asn Leu Ile
Asn Gln Lys Gln Ser Pro Ile Gln Asp Val 35
40 45 gat att gaa aac tat tta
aaa gaa aaa aag tta caa tta aga gct act 192Asp Ile Glu Asn Tyr Leu
Lys Glu Lys Lys Leu Gln Leu Arg Ala Thr 50
55 60 cta gac gcc gat caa gca
ttt agg gat gca gat ata cta att att gct 240Leu Asp Ala Asp Gln Ala
Phe Arg Asp Ala Asp Ile Leu Ile Ile Ala 65 70
75 80 aca cca acc aat tat gat
gtg gag aag aat ttt ttt gat act agt cat 288Thr Pro Thr Asn Tyr Asp
Val Glu Lys Asn Phe Phe Asp Thr Ser His 85
90 95 gtt gag act gta att gag
aaa gct tta gct tta aat agt cag gct ttg 336Val Glu Thr Val Ile Glu
Lys Ala Leu Ala Leu Asn Ser Gln Ala Leu 100
105 110 tta gtt att aaa tca acg
ata cca ctt ggt ttt att aaa aag atg cgt 384Leu Val Ile Lys Ser Thr
Ile Pro Leu Gly Phe Ile Lys Lys Met Arg 115
120 125 caa aaa tat cag aca gac
cgt att att ttt agt ccc gaa ttt ctt aga 432Gln Lys Tyr Gln Thr Asp
Arg Ile Ile Phe Ser Pro Glu Phe Leu Arg 130
135 140 gag tct aaa gct tta aaa
gat aat ctt tat cct agt cga ata att gtt 480Glu Ser Lys Ala Leu Lys
Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145 150
155 160 tcc ttt gaa gat gat gat
tct atg gaa gta ata gaa gca gca aag act 528Ser Phe Glu Asp Asp Asp
Ser Met Glu Val Ile Glu Ala Ala Lys Thr 165
170 175 ttt gct caa ttg tta aaa
gat ggt tct ttg gat aaa gat gtt cct gta 576Phe Ala Gln Leu Leu Lys
Asp Gly Ser Leu Asp Lys Asp Val Pro Val 180
185 190 ctt ttt atg ggt tca gca
gag gct gaa gca gta aaa tta ttt gcc aat 624Leu Phe Met Gly Ser Ala
Glu Ala Glu Ala Val Lys Leu Phe Ala Asn 195
200 205 acc tat tta gct atg cgt
gtc tcc tat ttt aat gag tta gat aca tat 672Thr Tyr Leu Ala Met Arg
Val Ser Tyr Phe Asn Glu Leu Asp Thr Tyr 210
215 220 gct gaa aag aat ggt tta
cgt gtg gat aat att att gag ggc gtt tgc 720Ala Glu Lys Asn Gly Leu
Arg Val Asp Asn Ile Ile Glu Gly Val Cys 225 230
235 240 cat gat cga cgc ata gga
att cat tat aat aac cct tct ttt ggc tat 768His Asp Arg Arg Ile Gly
Ile His Tyr Asn Asn Pro Ser Phe Gly Tyr 245
250 255 gga gga tac tgc tta cct
aaa gat acc aaa cag ttg cta gca ggc tat 816Gly Gly Tyr Cys Leu Pro
Lys Asp Thr Lys Gln Leu Leu Ala Gly Tyr 260
265 270 gat ggt att cct caa tcg
ctt ata aaa gca att gtt gat tct aat aaa 864Asp Gly Ile Pro Gln Ser
Leu Ile Lys Ala Ile Val Asp Ser Asn Lys 275
280 285 att cgt aaa gag tat atc
gca tca caa att tta caa caa ttg agt gat 912Ile Arg Lys Glu Tyr Ile
Ala Ser Gln Ile Leu Gln Gln Leu Ser Asp 290
295 300 att aat gta gat cct aaa
gat gca acg att ggt att tac cgc ctt atc 960Ile Asn Val Asp Pro Lys
Asp Ala Thr Ile Gly Ile Tyr Arg Leu Ile 305 310
315 320 atg aaa agt aac tct gat
aat ttc aga gag agt gca ata aaa gat att 1008Met Lys Ser Asn Ser Asp
Asn Phe Arg Glu Ser Ala Ile Lys Asp Ile 325
330 335 att gat cat att aag agc
tat caa att aat ata gtc ttg tat gag cca 1056Ile Asp His Ile Lys Ser
Tyr Gln Ile Asn Ile Val Leu Tyr Glu Pro 340
345 350 atg atg aat gaa gat ttt
gat tta cca atc att gat gat tta tct gac 1104Met Met Asn Glu Asp Phe
Asp Leu Pro Ile Ile Asp Asp Leu Ser Asp 355
360 365 ttc aaa gcc atg tca cat
att atc gtt tca aat aga tat gat tta gcc 1152Phe Lys Ala Met Ser His
Ile Ile Val Ser Asn Arg Tyr Asp Leu Ala 370
375 380 tta gaa gat gtt aaa gaa
aaa gtt tac acc aga gat att tac ggt gtg 1200Leu Glu Asp Val Lys Glu
Lys Val Tyr Thr Arg Asp Ile Tyr Gly Val 385 390
395 400 gat
1203Asp
105401PRTStreptococcus
uberis 105Val Lys Ile Ala Val Ala Gly Ser Gly Tyr Val Gly Leu Ser Leu Ser
1 5 10 15 Val Leu
Leu Ala Gln Lys Asn Pro Val Thr Val Val Asp Ile Ile Glu 20
25 30 Lys Lys Val Asn Leu Ile Asn
Gln Lys Gln Ser Pro Ile Gln Asp Val 35 40
45 Asp Ile Glu Asn Tyr Leu Lys Glu Lys Lys Leu Gln
Leu Arg Ala Thr 50 55 60
Leu Asp Ala Asp Gln Ala Phe Arg Asp Ala Asp Ile Leu Ile Ile Ala 65
70 75 80 Thr Pro Thr
Asn Tyr Asp Val Glu Lys Asn Phe Phe Asp Thr Ser His 85
90 95 Val Glu Thr Val Ile Glu Lys Ala
Leu Ala Leu Asn Ser Gln Ala Leu 100 105
110 Leu Val Ile Lys Ser Thr Ile Pro Leu Gly Phe Ile Lys
Lys Met Arg 115 120 125
Gln Lys Tyr Gln Thr Asp Arg Ile Ile Phe Ser Pro Glu Phe Leu Arg 130
135 140 Glu Ser Lys Ala
Leu Lys Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145 150
155 160 Ser Phe Glu Asp Asp Asp Ser Met Glu
Val Ile Glu Ala Ala Lys Thr 165 170
175 Phe Ala Gln Leu Leu Lys Asp Gly Ser Leu Asp Lys Asp Val
Pro Val 180 185 190
Leu Phe Met Gly Ser Ala Glu Ala Glu Ala Val Lys Leu Phe Ala Asn
195 200 205 Thr Tyr Leu Ala
Met Arg Val Ser Tyr Phe Asn Glu Leu Asp Thr Tyr 210
215 220 Ala Glu Lys Asn Gly Leu Arg Val
Asp Asn Ile Ile Glu Gly Val Cys 225 230
235 240 His Asp Arg Arg Ile Gly Ile His Tyr Asn Asn Pro
Ser Phe Gly Tyr 245 250
255 Gly Gly Tyr Cys Leu Pro Lys Asp Thr Lys Gln Leu Leu Ala Gly Tyr
260 265 270 Asp Gly Ile
Pro Gln Ser Leu Ile Lys Ala Ile Val Asp Ser Asn Lys 275
280 285 Ile Arg Lys Glu Tyr Ile Ala Ser
Gln Ile Leu Gln Gln Leu Ser Asp 290 295
300 Ile Asn Val Asp Pro Lys Asp Ala Thr Ile Gly Ile Tyr
Arg Leu Ile 305 310 315
320 Met Lys Ser Asn Ser Asp Asn Phe Arg Glu Ser Ala Ile Lys Asp Ile
325 330 335 Ile Asp His Ile
Lys Ser Tyr Gln Ile Asn Ile Val Leu Tyr Glu Pro 340
345 350 Met Met Asn Glu Asp Phe Asp Leu Pro
Ile Ile Asp Asp Leu Ser Asp 355 360
365 Phe Lys Ala Met Ser His Ile Ile Val Ser Asn Arg Tyr Asp
Leu Ala 370 375 380
Leu Glu Asp Val Lys Glu Lys Val Tyr Thr Arg Asp Ile Tyr Gly Val 385
390 395 400 Asp
106912DNAStreptococcus uberisCDS(1)..(912) 106atg act aaa gta aga aaa gcc
att att cca gct gcc gga ctt ggc aca 48Met Thr Lys Val Arg Lys Ala
Ile Ile Pro Ala Ala Gly Leu Gly Thr 1 5
10 15 cgt ttt tta cca gca aca aaa
gct ctc gct aag gaa atg ttg ccc atc 96Arg Phe Leu Pro Ala Thr Lys
Ala Leu Ala Lys Glu Met Leu Pro Ile 20
25 30 gtt gac aaa cca acc att caa
ttc atc gtg gaa gaa gct ttg cgt tct 144Val Asp Lys Pro Thr Ile Gln
Phe Ile Val Glu Glu Ala Leu Arg Ser 35
40 45 ggc att gaa gaa atc ttg gtc
gta aca gga aaa tca aaa cgc tcc att 192Gly Ile Glu Glu Ile Leu Val
Val Thr Gly Lys Ser Lys Arg Ser Ile 50 55
60 gaa gac cat ttt gat tcc aac
ttt gaa ctc gaa tat aat ttg caa gaa 240Glu Asp His Phe Asp Ser Asn
Phe Glu Leu Glu Tyr Asn Leu Gln Glu 65 70
75 80 aaa ggg aaa act gaa ctc tta
aaa tta gtt gat gaa acc act tct ata 288Lys Gly Lys Thr Glu Leu Leu
Lys Leu Val Asp Glu Thr Thr Ser Ile 85
90 95 aac ttg cat ttc att cgt caa
agt cat ccc aaa ggc tta ggg gat gct 336Asn Leu His Phe Ile Arg Gln
Ser His Pro Lys Gly Leu Gly Asp Ala 100
105 110 gtt tta caa gca aaa gct ttt
gta gga aat gaa ccc ttc att gtt atg 384Val Leu Gln Ala Lys Ala Phe
Val Gly Asn Glu Pro Phe Ile Val Met 115
120 125 ctt ggt gac gat ttg atg gac
att aca aat acc aaa gct gtc cca tta 432Leu Gly Asp Asp Leu Met Asp
Ile Thr Asn Thr Lys Ala Val Pro Leu 130 135
140 acc aaa caa tta atg gac gat
tat gaa aca aca cat gct tct aca ata 480Thr Lys Gln Leu Met Asp Asp
Tyr Glu Thr Thr His Ala Ser Thr Ile 145 150
155 160 gcc gta atg aaa gtt cct cac
gat gac gta tcc tct tat ggt gtc att 528Ala Val Met Lys Val Pro His
Asp Asp Val Ser Ser Tyr Gly Val Ile 165
170 175 gct cca aac ggc aaa gcc ttg
aat ggc tta tat agc gtg gat acc ttt 576Ala Pro Asn Gly Lys Ala Leu
Asn Gly Leu Tyr Ser Val Asp Thr Phe 180
185 190 gtt gaa aaa cca aaa cct gag
gac gca cca agt gac ctt gct atc att 624Val Glu Lys Pro Lys Pro Glu
Asp Ala Pro Ser Asp Leu Ala Ile Ile 195
200 205 gga cgt tat ctc tta aca cct
gaa att ttt gac att ctt gaa aat caa 672Gly Arg Tyr Leu Leu Thr Pro
Glu Ile Phe Asp Ile Leu Glu Asn Gln 210 215
220 gca cca ggt gcc gga aac gaa
gtc caa tta act gat gct atc gat acc 720Ala Pro Gly Ala Gly Asn Glu
Val Gln Leu Thr Asp Ala Ile Asp Thr 225 230
235 240 ctc aac aaa aca caa cgt gtt
ttt gct cgt gag ttt act ggc aaa cgc 768Leu Asn Lys Thr Gln Arg Val
Phe Ala Arg Glu Phe Thr Gly Lys Arg 245
250 255 tac gat gtt gga gac aag ttt
ggc ttc atg aaa aca tct atc gat tat 816Tyr Asp Val Gly Asp Lys Phe
Gly Phe Met Lys Thr Ser Ile Asp Tyr 260
265 270 gcc cta aaa cac cat caa gtc
aaa gat gac cta aaa gct tat att atc 864Ala Leu Lys His His Gln Val
Lys Asp Asp Leu Lys Ala Tyr Ile Ile 275
280 285 aag tta ggt aaa gaa tta gaa
aaa gca caa gat tcc aaa gaa agc aaa 912Lys Leu Gly Lys Glu Leu Glu
Lys Ala Gln Asp Ser Lys Glu Ser Lys 290 295
300 107304PRTStreptococcus
uberis 107Met Thr Lys Val Arg Lys Ala Ile Ile Pro Ala Ala Gly Leu Gly Thr
1 5 10 15 Arg Phe
Leu Pro Ala Thr Lys Ala Leu Ala Lys Glu Met Leu Pro Ile 20
25 30 Val Asp Lys Pro Thr Ile Gln
Phe Ile Val Glu Glu Ala Leu Arg Ser 35 40
45 Gly Ile Glu Glu Ile Leu Val Val Thr Gly Lys Ser
Lys Arg Ser Ile 50 55 60
Glu Asp His Phe Asp Ser Asn Phe Glu Leu Glu Tyr Asn Leu Gln Glu 65
70 75 80 Lys Gly Lys
Thr Glu Leu Leu Lys Leu Val Asp Glu Thr Thr Ser Ile 85
90 95 Asn Leu His Phe Ile Arg Gln Ser
His Pro Lys Gly Leu Gly Asp Ala 100 105
110 Val Leu Gln Ala Lys Ala Phe Val Gly Asn Glu Pro Phe
Ile Val Met 115 120 125
Leu Gly Asp Asp Leu Met Asp Ile Thr Asn Thr Lys Ala Val Pro Leu 130
135 140 Thr Lys Gln Leu
Met Asp Asp Tyr Glu Thr Thr His Ala Ser Thr Ile 145 150
155 160 Ala Val Met Lys Val Pro His Asp Asp
Val Ser Ser Tyr Gly Val Ile 165 170
175 Ala Pro Asn Gly Lys Ala Leu Asn Gly Leu Tyr Ser Val Asp
Thr Phe 180 185 190
Val Glu Lys Pro Lys Pro Glu Asp Ala Pro Ser Asp Leu Ala Ile Ile
195 200 205 Gly Arg Tyr Leu
Leu Thr Pro Glu Ile Phe Asp Ile Leu Glu Asn Gln 210
215 220 Ala Pro Gly Ala Gly Asn Glu Val
Gln Leu Thr Asp Ala Ile Asp Thr 225 230
235 240 Leu Asn Lys Thr Gln Arg Val Phe Ala Arg Glu Phe
Thr Gly Lys Arg 245 250
255 Tyr Asp Val Gly Asp Lys Phe Gly Phe Met Lys Thr Ser Ile Asp Tyr
260 265 270 Ala Leu Lys
His His Gln Val Lys Asp Asp Leu Lys Ala Tyr Ile Ile 275
280 285 Lys Leu Gly Lys Glu Leu Glu Lys
Ala Gln Asp Ser Lys Glu Ser Lys 290 295
300 1085158DNAStreptococcus equisimilis 108tcaatttatg
gctttttgct gatagcttac ctattagtca aaatgtcctt atcctttttt 60tacaagccat
ttaagggaag ggctgggcaa tataaggttg cagccattat tccctcttat 120aacgaagatg
ctgagtcatt gctagagacc ttaaaaagtg ttcagcagca aacctatccc 180ctagcagaaa
tttatgttgt tgacgatgga agtgctgatg agacaggtat taagcgcatt 240gaagactatg
tgcgtgacac tggtgaccta tcaagcaatg tcattgttca tcggtcagag 300aaaaatcaag
gaaagcgtca tgcacaggcc tgggcctttg aaagatcaga cgctgatgtc 360tttttgaccg
ttgactcaga tacttatatc taccctgatg ctttagagga gttgttaaaa 420acctttaatg
acccaactgt ttttgctgcg acgggtcacc ttaatgtcag aaatagacaa 480accaatctct
taacacgctt gacagatatt cgctatgata atgcttttgg cgttgaacga 540gctgcccaat
ccgttacagg taatatcctt gtttgctcag gtccgcttag cgtttacaga 600cgcgaggtgg
ttgttcctaa catagataga tacatcaacc agaccttcct gggtattcct 660gtaagtattg
gtgatgacag gtgcttgacc aactatgcaa ctgatttagg aaagactgtt 720tatcaatcca
ctgctaaatg tattacagat gttcctgaca agatgtctac ttacttgaag 780cagcaaaacc
gctggaacaa gtccttcttt agagagtcca ttatttctgt taagaaaatc 840atgaacaatc
cttttgtagc cctatggacc atacttgagg tgtctatgtt tatgatgctt 900gtttattctg
tggtggattt ctttgtaggc aatgtcagag aatttgattg gctcagggtt 960ttagcctttc
tggtgattat cttcattgtt gccctgtgtc ggaacattca ttacatgctt 1020aagcacccgc
tgtccttctt gttatctccg ttttatgggg tgctgcattt gtttgtccta 1080cagcccttga
aattatattc tctttttact attagaaatg ctgactgggg aacacgtaaa 1140aaattattat
aaaccaacta gacctaggtt ctgacaaggg agctaagcta gggataaaca 1200aagagttttg
atccgactcg agcagctcat aaacgaaagc tatcccactt gtaattgaag 1260ctaagagctt
ttagcttgca gctctataaa gacgaaccag aggctgagtg tcagctttgg 1320tgtgagggct
aggtcattat gatccttcag gtgtggcacc tgagctccgg cagtagctaa 1380ctgtactaag
gtatcaaagg aaaaaatgaa gtgaaaattt ctgtagcagg ctcaggatat 1440gtcggcctat
ccttgagtat tttactggca caacataatg acgtcactgt tgttgacatt 1500attgatgaaa
aggtgagatt gatcaatcaa ggcatatcgc caatcaagga tgctgatatt 1560gaggagtatt
taaaaaatgc gccgctaaat ctcacagcga cgcttgatgg cgcaagcgct 1620tatagcaatg
cagaccttat tatcattgct actccgacaa attatgacag cgaacgcaac 1680tactttgaca
caaggcatgt tgaagaggtc atcgagcagg tcctagacct aaatgcgtca 1740gcaaccatta
ttatcaaatc aaccatacca ctaggcttta tcaagcatgt tagggaaaaa 1800taccagacag
atcgtattat ttttagccca gaatttttaa gagaatcaaa agccttatac 1860gataaccttt
acccaagtcg gatcattgtt tcttatgaaa aggacgactc accaagggtt 1920attcaggctg
ctaaagcctt tgctggtctt ttaaaggaag gagccaaaag caaggatact 1980ccggtcttat
ttatgggctc acaggaggct gaggcggtca agctatttgc gaataccttt 2040ttggctatgc
gggtgtctta ctttaatgaa ttagacacct attccgaaag caagggtcta 2100gatgctcagc
gcgtgattga aggagtctgt catgatcagc gcattggtaa ccattacaat 2160aacccttcct
ttggatatgg cggctattgc ctgccaaagg acagcaagca gctgttggca 2220aattatagag
gcattcccca gtccttgatg tcagcgattg ttgaatccaa caagatacga 2280aaatcttatt
tggctgaaca aatattagac agagcctcta gtcaaaagca ggctggtgta 2340ccattaacga
ttggctttta ccgcttgatt atgaaaagca actctgataa tttccgagaa 2400agcgccatta
aagatattat tgatatcatc aacgactatg gggttaatat tgtcatttac 2460gaacccatgc
ttggcgagga tattggctac agggttgtca aggacttaga gcagttcaaa 2520aacgagtcta
caatcattgt gtcaaatcgc tttgaggacg acctaggaga tgtcattgat 2580aaggtttata
cgagagatgt ctttggaaga gactagtcag aaaacgaatg gcactcataa 2640ggaaccacaa
atcaaggagg aactcatgac aaaggtcaga aaagccatta tcccagccgc 2700cggcctaggc
actcgcttcc tgcccgccac caaggcactg gccaaggaaa tgctcccaat 2760cgtcgataag
ccaaccattc aattcatcgt cgaggaagcc ctaaaggcag gtatcgagga 2820gattcttgtc
gtcaccggca aggccaaacg ctctatcgag gaccactttg actccaactt 2880cgagctcgaa
tacaatctcc aagccaaggg caaaaccgag ctactcaagc tcgttgatga 2940gaccactgcc
atcaacctgc acttcattcg tcagagccac cctagaggac taggggacgc 3000tgtcctccaa
gccaaggcct ttgttggcaa tgagcccttt gtggtcatgc tgggggatga 3060cctcatggat
attaccaatc ctagtgccaa gcccttgacc aagcagctta ttgaggatta 3120tgattgcaca
cacgcctcaa cgattgcagt gatgagggtg ccgcatgagg aggtttccaa 3180ttatggtgtg
attgcaccgc aagggaaggc tgttaagggc ttgtatagtg tggagacctt 3240tgttgagaag
ccaagtccag atgaggcacc gagtgactta gcgattattg gtcgatattt 3300gttgacgcct
gagatttttg ccatattgga gaagcaggcg cctggagctg gcaatgaggt 3360acagctgacc
gatgcgattg acaagctcaa taagacacag cgggtttttg cgagggagtt 3420taagggagag
cggtatgatg ttggggacaa gtttggcttt atgaagacct cacttgacta 3480tgctctcaag
caccctcagg tcaaggacga cctcactgac tacattataa agctcagtaa 3540gcaactgaac
aaggacgtca agaaataggc gtttattgat cagctattgc agagctattt 3600aaaagcattt
agagctttaa ggtgggatac tagaggattg gtatctcact ttttaggctg 3660acttgtatta
ataccaaaag ccaaaactag gcagataagc ataaggaatt agattaaaaa 3720taaggaacca
aaacatgaaa aactacgcca ttatcctagc agctggaaag ggaacgcgca 3780tgaagtcagc
gcttcccaag gtgctgcaca aggtatcagg cctaagcatg ctggagcatg 3840tcctcaagag
tgtctcagcc ctagcccctc aaaagcagct cacagtgatc ggtcatcagg 3900cagagcaggt
gcgtgctgtc ctaggagagc aatcgctaac agtggtgcaa gaggagcagc 3960tagggacagg
ccatgcagtc atgatggcag aagaggagct atctggctta gaggggcaaa 4020ccctagtgat
tgcaggtgac acccccttga tcagaggaga aagcctcaag gctctgctag 4080actatcatat
cagagaaaag aatgtggcaa ccattctcac agccaatgcc aaggatccct 4140ttggctatgg
acgaatcatt cgcaatgcag caggagaggt ggtcaacatc gttgagcaaa 4200aggatgctaa
tgaggcagag caagaggtca aggagatcaa cacagggact tatatctttg 4260acaataagcg
cctttttgag gctctaaagc atctcacgac tgataatgcc caaggggagt 4320actacctaac
cgatgtgatc agtattttca aggctggcca agaaagggtt ggcgcttacc 4380tgctgaagga
ctttgatgag agcctagggg ttaatgatcg cttagctcta gcccaggccg 4440aggtgattat
gcaagagcgg atcaacaggc agcacatgct taatggggtg accctgcaaa 4500acccggcagc
tacctatatt gaaagcagtg tagagattgc accagacgtc ttgattgaag 4560ccaatgtgac
cttaaaggga cagactagaa ttggcagcag aagtgtcata agcaatggga 4620gctatatcct
tgattcgagg cttggtgagg gtgtagtggt tagccagtcg gtgattgagg 4680cttcagtctt
agcagatgga gtgacagtag ggccatatgc acacattcgc ccggactccc 4740agctcgatga
gtgtgttcat attgggaact ttgtagaggt taaggggtct catctagggg 4800ccaataccaa
ggcagggcat ttgacttacc tggggaatgc cgagattggc tcagaggtta 4860acattggtgc
aggaagcatt acggttaatt atgatggtca acggaaatac cagacagtga 4920ttggcgatca
cgcttttatt gggagtcatt cgactttgat agctccggta gaggttgggg 4980agaatgcttt
aacagcagca gggtctacga tagcccagtc agtgccggca gacagtgtgg 5040ctatagggcg
cagccgtcag gtggtgaagg aaggctatgc caagaggctg ccgcaccacc 5100caaatcaagc
ctaatcgctc aaccaaaaga ggcaggtgag aaaacctagg ccattaaa 5158
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