US20020006665A1

US20020006665A1 - Ketogulonigenium endogenous plasmids

Info

Publication number: US20020006665A1
Application number: US09/826,191
Authority: US
Inventors: John D'Elia; Steven Stoddard
Original assignee: Archer Daniels Midland Co
Current assignee: Archer Daniels Midland Co
Priority date: 2000-04-05
Filing date: 2001-04-05
Publication date: 2002-01-17
Also published as: WO2001077348A8; US7030233B2; WO2001077348A3; US7053197B2; US7053196B2; AU2001251342A1; WO2001077348A2; US20030087440A1; US20030077830A1; US20030073224A1

Abstract

The present invention relates, in general, to a novel genus of bacteria known as Ketogulonigenium. The present invention further relates to transformed Ketogulonigenium, and methods of transforming Ketogulonigenium. The present invention also relates to nucleic acid molecules, and vectors.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application is related to provisional application No. 60/194,627, filed Apr. 5, 2000, the content of which is incorporated herein by reference.[0001]

BACKGROUND OF THE INVENTION

1. Field of the Invention

2. Background Art

The exploitation of microorganisms to synthesize vitamin C or its chemical pathway intermediates has both economic and ecological advantages.

One key intermediate in vitamin C synthesis is 2-keto-L-gulonic acid (2-KLG), which is easily converted chemically to L-ascorbic acid (vitamin C) by esterification followed by lactonization (Delic, V. et al., “Microbial reactions for the synthesis of vitamin C (L-ascorbic acid,” in Biotechnology of Vitamins, Pigments and Growth Factors, Vandamme, E. J., ed., Elsevier Applied Science (London & New York) pp. 299-336 (1989)). Members of a number of bacterial genera have been identified that produce 2-KLG from the oxidation of L-sorbose or sorbitol. Such 2-KLG producing genera include the acidogenic, alpha-proteobacteria Gluconobacter and Acetobacter, the gamma-proteobacteria Pseudomonas, Escherichia, Klebsiella, Serratia and Xanthmonas and the Gram positive Bacillus, Micrococcus and Pseudogluconobacter (Imai, K. et al., U.S. Pat. No. 4,933,289 (1990), Sugisawa, H. et al., “Microbial production of 2-keto-L-gulonic acid from L-sorbose and D-sorbitol by Gluconobacter melanogenus,” Agric. Biol. Chem. 54:1201-1209 (1990), Yin, G. et al., U.S. Pat. No. 4,935,359 (1990), Shirafuji, et al., U.S. Pat. No. 4,876,195 (1989) and Nogami, I. et al., U.S. Pat. No. 5,474,924 (1995)).

To aid in increasing the yield of bacterial products, attempts have been made to exploit endogenous plasmids within microorganism strains. (Beppu, T. et al., U.S. Pat. No. 5,580,782 (1996), Fujiwara, A. et al., U.S. Pat. No. 5,399,496 (1995); Tonouchi et al, U.S. Pat. No. 6,127,174 (2000), Hoshino, T. et at., U.S. Pat. No. 6,127,156 (2000)).

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention provides an isolated or purified nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a nucleotide sequence of a Ketogulonigenium plasmid replicon found on the endogenous plasmid contained in NRRL Deposit No. B-21627 and at least one exogenous nucleotide sequence.

Further embodiments of the invention include an isolated or purified nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical, to any of the above nucleotide sequences, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence as in the above. The polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

The present invention relates, in general, to a novel genus of bacteria known as Ketogulonigenium. The present invention further relates to Ketogulonigenium comprising a transgene (recombinant DNA), comprising an endogenous plasmid. The invention also relates to a method for transforming Ketogulonigenium comprising conjugative transfer of a vector from E. coli to Ketogulonigenium, and to a method for transforming Ketogulonigenium comprising electroporation.

The invention provides a nucleic acid molecule comprising a nucleotide sequence at least 95% identical to a Ketogulonigenium endogenous plasmid contained in NRRL Deposit No. B-21627. The invention also provides a nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a Ketogulonigenium replicon found on an endogenous plasmid contained in NRRL Deposit No. B-21627. The invention further provides a vector comprising a nucleic acid molecule comprising a nucleotide sequence of a Ketogulonigenium replicon found on an endogenous plasmid contained in NRRL Deposit No. B-21627.

Further advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. [0013] 1A-1E show the nucleotide (SEQ ID NO:1) sequence of an endogenous plasmid, determined by sequencing of the endogenous plasmid (pADMX6L1), contained in NRRL Deposit No. B-21627. The nucleotide has a sequence of about 7029 nucleic acid residues.
FIGS. [0014] 2A-2C show the nucleotide (SEQ ID NO:2) sequence of an endogenous plasmid, determined by sequencing of the endogenous plasmid (pADMX6L2), contained in NRRL Deposit No. B-21627. The nucleotide has a sequence of about 4005 nucleic acid residues.
FIGS. [0015] 3A-3N show the nucleotide (SEQ ID NO:3) sequence of an endogenous plasmid, determined by sequencing of the endogenous plasmid (pADMX6L3), contained in NRRL Deposit No. B-21627. The nucleotide has a sequence of about 19,695 nucleic acid residues.
FIGS. [0016] 4A-4C show the nucleotide (SEQ ID NO:4) sequence of an endogenous plasmid, determined by sequencing of the endogenous plasmid (pADMX6L4), contained in NRRL Deposit No. B-21627. The nucleotide has a sequence of about 4211 nucleic acid residues.
FIGS. [0017] 5A-5B show the nucleotide (SEQ ID NO:5) sequence of the replicon on an endogenous plasmid (pADMX6L1), determined by homology of amino acid sequences encoded by the endogenous plasmid to known replication proteins, contained in NRRL Deposit No. B-21627. The nucleotide has a sequence of about 1456 nucleic acid residues.
FIGS. [0018] 6A-6B show the nucleotide (SEQ ID NO:6) sequence of the the replicon on an endogenous plasmid (pADMX6L2),determined by homology of amino acid sequences encoded by the endogenous plasmid to known replication proteins, contained in NRRL Deposit No. B-21627. The nucleotide has a sequence of about 2401 nucleic acid residues.
FIGS. [0019] 7A-7B show the nucleotide (SEQ ID NO:7) sequence of the the replicon on an endogenous plasmid (pADMX6L3), determined by homology of amino acid sequences encoded by the endogenous plasmid to known replication proteins, contained in NRRL Deposit No. B-21627. The nucleotide has a sequence of about 2029 nucleic acid residues.
FIG. 8 shows the amino acid sequence (SEQ ID NO:8) of a replication protein encoded by an endogenous plasmid (pADMX6L1) determined from the nucleotide sequence of pADMX6L1 contained in NRRL B-21627. The polypeptide has a sequence of about 151 amino acids in length. [0020]
FIG. 9 shows the amino acid sequence (SEQ ID NO:9) of a replication protein encoded by an endogenous plasmid (pADMX6L2) determined from the nucleotide sequence of pADMX6L2 contained in NRRL B-21627. The polypeptide has a sequence of about 466 amino acids in length. [0021]
FIG. 10 shows the amino acid sequence (SEQ ID NO:10) of a replication protein encoded by an endogenous plasmid (pADMX6L3) determined from the nucleotide sequence of pADMX6L3 contained in NRRL B-21627. The polypeptide has a sequence of about 342 amino acids in length. [0022]

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the ABI Prism 3700). Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. [0023]
Unless otherwise indicated, each “nucleotide sequence” set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T). However, by “nucleotide sequence” of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T) in the specified deoxynucleotide sequence in is replaced by the ribonucleotide uridine (U). For instance, reference to an RNA molecule having the sequence of SEQ ID NO:1 set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxynucleotide A, G or C of SEQ ID NO:1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxynucleotide T has been replaced by a ribonucleotide U. [0024]
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. [0025]
By “isolated” nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. [0026]
For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. [0027]
Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. [0028]
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, in the endogenous plasmids contained in NRRL Deposit No. B-21627. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 ° C. By a polynucleotide which hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers. [0029]
Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide (e.g., the deposited endogenous plasmid), for instance, a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide, also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the deposited DNA or the nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1). By a portion of a polynucleotide of “at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide, (e.g., the deposited DNA or the nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1)). As indicated, such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, the entire disclosure of which is hereby incorporated herein by reference. [0030]
By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. [0031]
As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in FIG. 1 or to the nucleotide sequence of the deposited endogenous plasmid can be determined conventionally using known computer programs such as the FastA program. FastA does a Pearson and Lipman search for similarity between a query sequence and a group of sequences of the same type nucleic acid. Professor William Pearson of the University of Virginia Department of Biochemistry wrote the FASTA program family (FastA, TFastA, FastX, TFastX and SSearch). In collaboration with Dr. Pearson, the programs were modified and documented for distribution with GCG Version 6.1 by Mary Schultz and Irv Edelman, and for [0032] Versions 8 through 10 by Sue Olson.
The present invention provides Ketogulonigenium, comprising a transgene (recombinant DNA) comprising an endogenous Ketogulonigenium plasmid. Preferably, the endogenous Ketogulonigenium plasmid is contained in Deposit No. NRRL B-21627. As used herein, a transgene is defined as a transplanted nucleotide sequence which is exogenous, or non-native, to the host. An exogenous nucleotide sequence, as used in the current context, is a nucleotide sequence which is not found in Deposit No. NRRL B-21627. Thus, the term exogenous nucleotide sequence is meant to encompass a nucleotide sequence that is foreign to Deposit No. NRRL B-21627, as well as a nucleotide sequence endogenous, or native, to Deposit No. NRRL B-21627 that has been modified. Modification of the endogenous nucleotide sequence may include, for instance, mutation of the native nucleotide sequence or any of its regulatory elements. As used herein, mutation is defined as any change in the wild-type sequence of genomic or plasmid DNA. An additional form of modification may also include fusion of the endogenous nucleotide sequence to a nucleotide sequence that is normally not present, in relation to the endogenous nucleotide sequence. The transgene may be regulated by its normal promoter, or more commonly, by a promoter that normally regulates a different gene. The invention also provides a method for producing transformed Ketogulonigenium, comprising transforming Ketogulonigenium with a transgene, comprising, part or all of an endogenous Ketogulonigenium plasmid. Preferably, the endogenous Ketogulonigenium plasmid is contained in Deposit No. NRRL B-21627. [0033]
The term replicon as used herein is meant to encompass a DNA sequence comprising those genes and gene expression control elements such as promoters and terminators, other DNA sequence features such as short sequence repeats (iterons), origins of plasmid replication (ori or oriV sites), or other DNA sequence features that are required to support the autonomous replication of a circular DNA molecule in a bacterial host (Chapter 122, pp. 2295-2324, in [0034] Escherichia coli and Salmonella: Cellular and Molecular Biology, 2^ndEdition, Frederick C. Neidhardt, Ed., ASM Press (1996)). The requirements of a replicon can vary from as little as a short ori sequence in the case of plasmids that do not require their own replication proteins, to larger sequences containing one or more plasmid-borne replication genes.
The definition of a transformed cell, as used herein, is a cell where DNA has been inserted into a bacterial cell. The transformation of Ketogulonigenium may be transient or stable. Preferably, the invention provides a method for producing stably transformed Ketogulonigenium. As used herein, a stably transformed cell is a cell wherein a transgene is transmitted to every successive generation. A preferred embodiment of the present invention is that Ketogulonigenium is transformed via electroporation. An additional preferred embodiment of the present invention is that Ketogulonigenium is transformed by the process of conjugation, including, for instance, bi-parental and tri-parental conjugation. Conjugation, as used herein, is the process by which bacteria transfer DNA from a donor cell to a recipient cell through cell-to cell contact. [0035]
The present invention relates to a novel genus of bacteria, comprising the ADMX6L strain, designated as Ketogulonigenium. The present inventors have discovered novel strains of bacteria, not belonging to any known genera, that produce 2-keto-L-gulonic acid from sorbitol. [0036]
Ketogulonigenium (Ke.to.gu.lo.ni.gen'.i.um. M.L. n. acidum ketogulonicum ketogulonic acid; Gr. V. gennaio to produce; M.L. n. ketogulonigenium ketogulonic acid producing) is gram negative, facultatively anaerobic, motile or non-motile, has ovoid to rod-shaped cells, 0.8-1.3 μm long, 0.5-0.7 μm in diameter, with tapered ends, occurring as single cells, pairs and occasionally short chains. Some strains form elongated cells (up to 30 μm in length) on TSB. Flagella and fimbrae have been observed. Colonies are tan colored, smooth, circular, entire, raised to convex, 1-2 mm in diameter with a diffusable brown pigment after 48 hrs incubation. Oxidase and catalase reactions are positive. Optimum temperature range is 27 to 31° C., optimum pH range is 7.2 to 8.5 and optimum Na[0037] ⁺ concentration is 117-459 mM. Chemoorganotrophic. Carbon sources utilized include arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, sorbitol, sorbose, sucrose, trehalose, pyruvate and succinate. Favored carbon sources are inositol, mannitol, glycerol, sorbitol, lactose and arabinose. All strains examined produce 2-keto-L-gulonic acid from L-sorbose. Major cellular fatty acids are 16:0 and 18:1 ω7c/ω(ω9t/ω12t and the mol % DNA G+C is 52.1 to 54.0 percent. Small subunit rDNA sequence analysis place this genus in the alpha subgroup of the Proteobacteria. All strains isolated in the present study group originated in soil. DNA reassociation studies divide the genus into two species. K. vulgarae and K. robustum, of which K. vulgarae is the designated type species. A group of bacteria having the above-mentioned properties does not belong to any known genera as described in Bergey's Manual of Systematic Bacteriology, and therefore belongs to a new genus.
Strain ADMX6L of Ketogulonigenium was isolated and deposited at the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, on Oct. 1, 1996, under the provisions of the Budapest Treaty, and assigned accession numbers NRRL B-21627. [0038]
The present invention provides a method for conjugative transfer of a vector from [0039] E. coli to Ketogulonigenium comprising culturing the E. coli with the Ketogulonigenium under such conditions such that the E. coli transfers the vector to the Ketogulonigenium. The method of conjugative transfer relies on the ability of the vector to replicate in both organisms, and thus requires that the vector contain replicons that are functional in both organisms. A replicon is a nucleotide sequence, typically several hundred to several thousand base pairs long, that is vital to plasmid DNA replication. Preferably, the method comprises using any vector that contains a replicon that is functional in E. coli, as well a replicon that is functional in Ketogulonigenium. More preferably, the method of the invention comprises the vectors pDELIA8 and pXH2/K5. Given that the preferred method comprises using any vector that contains a replicon that is functional in E. coli, as well as a replicon that is functional in Ketogulonigenium, it would also be possible to transfer a vector, via conjugation, from Ketogulonigenium to E. coli.
The present invention also provides a method for transforming Ketogulonigenium comprising inserting a vector into the Ketogulonigenium through the process of electroporation. Preferably, the vector used in electroporation of Ketogulonigenium is pMF1014-α. [0040]
The present invention provides isolated or purified nucleic acid molecules comprising the polynucleotides, or their complements, of endogenous plasmids that have been isolated and purified from a strain, NRRL Deposit No. B-21627 (ADMX6L), of this novel genus. Four endogenous plasmids have been isolated from strain NRRL Deposit No. B-21627. The nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) is the nucleotide sequence of plasmid pADMX6L1 as determined by automated sequencing. The nucleotide sequence shown in FIG. 2 (SEQ ID NO:2) is the nucleotide sequence of plasmid pADMX6L2 as determined by automated sequencing. The nucleotide sequence shown in FIG. 3 (SEQ ID NO:3) is the nucleotide sequence of plasmid pADMX6L3 as determined by automated sequencing. The nucleotide sequence shown in FIG. 4 (SEQ ID NO:4) is the nucleotide sequence of plasmid pADMX6L4 as determined by automated sequencing. [0041]
The endogenous plasmids contained within NRRL B-21627 (ADMX6L) have been isolated and ligated into pUC19 or pJND1000. Specifically, plasmid pADMX6L1, corresponding to SEQ ID NO:1, and pJND1000 are digested with BamHI and ligated to each other using T4 ligase. The [0042] pXB 1 plasmid construct was then introduced into E. coli DH5αMCR and the culture collection was deposited at the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, on Feb. 23, 2001, under the provisions of the Budapest Treaty, and assigned accession numbers NRRL B-30418. Plasmid pADMX6L2, corresponding to SEQ ID NO:2, and pUC19 were digested with HinDIII and ligated to one another using T4 ligase. The pXH2 plasmid construct was then introduced into E. coli DH5αMCR and the culture collection was deposited at the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, on Feb. 23, 2001, under the provisions of the Budapest Treaty, and assigned accession numbers NRRL B-30419. Plasmid pADMX6L4, corresponding to SEQ ID NO:4, and pUC19 are digested with SspI and SmaI, respectively, and ligated to one another using T4 ligase. The pXB4 plasmid construct was then introduced into E. coli DH5αMCR and the culture collection is deposited at the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, on Mar. 12, 2001, under the provisions of the Budapest Treaty, and assigned accession number NRRL B-30435.
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the ABI Prism 3700). Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. [0043]
Unless otherwise indicated, each “nucleotide sequence” set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T). However, by “nucleotide sequence” of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T) in the specified deoxynucleotide sequence is replaced by the ribonucleotide uridine (U). For instance, reference to an RNA molecule having the sequence of SEQ ID NO:1 set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxynucleotide A, G or C of SEQ ID NO:1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxynucleotide T has been replaced by a ribonucleotide U. [0044]
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. [0045]
By “isolated” nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. [0046]
One aspect of the invention provides an isolated or purified nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; (b) a nucleotide sequence of a plasmid contained in NRRL Deposit No. B-30418, NRRL Deposit No. B-30419, and NRRL Deposit No. B-30435; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b) above. [0047]
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical, to any of the Ketogulonigenium nucleotide sequences in (a), (b) or (c) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to the Ketogulonigenium portion of a nucleotide sequence in (a), (b) or (c), above. The polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. [0048]
A polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. [0049]
As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 97%, 98% or 99% identical to, for instance, the nucleotide sequences shown in FIGS. 1, 2, [0050] 3, 4, 5, 6, or 7, or to the nucleotide sequences of the deposited plasmids can be determined conventionally using known computer programs such as the FastA program. FastA does a Pearson and Lipman search for similarity between a query sequence and a group of sequences of the same type nucleic acid. Professor William Pearson of the University of Virginia Department of Biochemistry wrote the FASTA program family (FastA, TFastA, FastX, TFastX and SSearch). In collaboration with Dr. Pearson, the programs were modified and documented for distribution with GCG Version 6.1 by Mary Schultz and Irv Edelman, and for Versions 8 through 10 by Sue Olson.
The present application is directed to nucleic acid molecules at least 95%, 97%, 98% or 99% identical to the nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:3), and FIG. 4 (SEQ ID NO:4); or to the Ketogulonigenium portion of the nucleic acid sequence of the deposited plasmids. [0051]
The present application is also directed to nucleic acid molecules at least 95%, 97%, 98% or 99% identical to the nucleic acid sequences shown in FIG. 5 (SEQ ID NO:5), FIG. 6 (SEQ ID NO:6), and FIG. 7 (SEQ ID NO:7). [0052]
One aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide of a nucleic acid molecule of the invention described above, for instance, in the plasmid contained in NRRL B-30418, NRRL B-30419, or NRRL B-30435, or in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. By a polynucleotide which hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers. [0053]
Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotides (e.g., the deposited endogenous plasmids), for instance, a portion 15-750 nt in length, or even to the entire length of the reference polynucleotide, are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the Ketogulonigenium portion of the nucleotide sequence of the deposited DNA or the nucleotide sequence as shown in FIGS. [0054] 1 (SEQ ID NO:1), 2 (SEQ ID:NO:2), 3 (SEQ ID NO:3), 4 (SEQ ID NO:4), 5 (SEQ ID NO:5), 6 (SEQ ID NO:6), and 7 (SEQ ID NO:7). A portion of a polynucleotide of, at least 15 nt in length, for example, is intended to mean 15 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide, (e.g., the deposited DNA or the nucleotide sequences as shown in FIGS. 1 (SEQ ID NO:1), 2 (SEQ ID:NO:2), 3 (SEQ ID NO:3), 4 (SEQ ID NO:4), 5 (SEQ ID NO:5), 6 (SEQ ID NO:6), 7 (SEQ ID NO:7)). As indicated, such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, the entire disclosure of which is hereby incorporated herein by reference.
One embodiment of the present invention is a vector comprising the nucleic acid molecules contained in the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, corresponding to endogenous plasmids contained in Deposit No. NRRL B-21627 (ADMX6L). A preferred embodiment of the present invention is that polynucleotides of interest can be joined to the nucleic acid molecules of the present invention, which may contain a selectable marker. An additional preferred embodiment is that the vectors comprising the nucleic acid molecules also contain a transcription terminator, a promoter and a polylinker site. As used herein, a polylinker site is defined to be a discrete series of restriction endonuclease sites that occur between the promoter and the terminator. The vector can optionally contain its native expression vector and/or expression vectors which include chromosomal-, and episomal-derived vectors, e.g., vectors derived from bacterial exogenous plasmids, bacteriophage, and vectors derived from combinations thereof, such as cosmids and phagemids. [0055]
A DNA insert of interest should be operatively linked to an appropriate promoter, such as its native promoter or a host-derived promoter, the phage lambda P[0056] _Lpromoter, the phage lambda P_Rpromoter, the E. coli lac promoters, such as the lacI and lacZ promoters, tip and tac promoters, the T3 and T7 promoters and the gpt promoter to name a few. Other suitable promoters will be known to the skilled artisan.
The expression constructs will preferably contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs can include a translation initiating codon at the beginning and a termination codon appropriately positioned at the end of the coding sequence to be translated. [0057]
As indicated, the expression vectors will preferably include at least one marker capable of being selected or screened for. Preferably the selectable marker comprises a nucleotide sequence which confers antibiotic resistance in a host cell population. Such markers include amikacin, ampicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, penicillin, spectinomycin, streptomycin, or tetracycline resistance genes. Other suitable markers will be readily apparent to the skilled artisan. [0058]
The translated polypeptide encoded by the DNA in the vector may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. [0059]
The translated protein encoded by the DNA contained in the vector can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. [0060]
The present invention also provides vectors comprising nucleic acid molecules comprising a nucleotide sequence of a replicon found on an endogenous plasmid contained in Deposit No. NRRL B-21627. Preferably, the vector of the present invention comprises a replicon selected from the group of nucleotide sequences comprising SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. The replicon is a nucleotide sequence that is typically several hundred to several thousand base pairs long and encodes functions controlling the replication of plasmid DNA. The nucleotide sequence in FIG. 5 (SEQ ID NO:5) is the nucleotide sequence comprising a replicon on plasmid pADMX6L1. The nucleotide sequence in FIG. 6 (SEQ ID NO:6) is the nucleotide sequence comprising a replicon on plasmid pADMX6L2. The nucleotide sequence in FIG. 7 (SEQ ID NO:7) is the nucleotide sequence comprising a replicon on plasmid pADMX6L3. [0061]
The present invention further provides a vector which has an replicon that is functional in [0062] Escherichia coli (E. coli ) as well as a replicon that is functional in Ketogulonigenium. The present invention also provides a vector which has a replicon that is functional in Ketogulonigenium, as well as a replicon that is functional in any of the genera comprising the group consisting of Acetobacter, Corynebacterium, Rhodobacter, Paracoccus, Roseobacter, Pseudomonas, Pseudogluconobacter, Gluconobacter, Serratia, Mycobacterium, Streptomyces and Bacillus. Utilizing the fact that the vector comprises a functional replicon in Ketogulonigenium, and preferably also comprises a replicon functional in E. coli, the present invention provides for a transformed cell of the genus Ketogulonigenium, comprising the vector. The present invention also provides for a transformed E. coli, comprising the vector. E. coli is known to be an efficient host for amplification of a vector DNA and manipulation of recombinant DNA by simple and rapid methods. On the other hand, Ketogulonigenium can be used as a host for expression of Ketogulonigenium genes. Since the vectors of the present invention are such functional constructs, they enable cloning of certain genes of Ketogulonigenium in E. coli and thereafter the effective expression of the genes in Ketogulonigenium. Furthermore, it is favorable that such functional constructs also contain a DNA region necessary for conjugal transfer (mob site). Hence the vectors of the present invention can first be assembled in E. coli and then directly introduced into Ketogulonigenium by conjugal mating without isolation of plasmid DNA from E. coli.
The present invention further relates to (a) nucleic acid sequences comprising at least 95% functional homology with those encoding polypeptides derived from the four endogenous plasmids from Ketogulonigenium strain ADMX6L, and (b) to amino acid sequences comprising at least 95% functional homology with those encoded within the plasmids. The invention also relates to nucleic acid sequences comprising at least 95% functional homology with, pADMX6L1 rep ORF (bases 2255- 2710 of FIG. 1 (SEQ ID NO:1), pADMX6L2 rep ORF (reverse compliment of bases 3960- 2562 of FIG. 2 (SEQ ID NO:2), and pADMX6L3 rep ORF (reverse compliment of bases 5031-4003 of FIG. 3 (SEQ ID NO:3)). The invention further relates to amino acid sequences comprising at least 95% functional homology with those set forth in FIGS. 8, 9 or [0063] 10.
Methods used and described herein are well known in the art and are more particularly described, for example, in R. F. Schleif and P. C. Wensink, [0064] Practical Methods in Molecular Biology, Springer-Verlag (1981); J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); M. Singer and P. Berg, Genes & Genomes, University Science Books, Mill Valley, Calif. (1991); J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); P. B. Kaufman et al., Handbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton, Fla. (1995); Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J. E. Thompson, eds., CRC Press, Boca Raton, Fla. (1993); P. F. Smith-Keary, Molecular Genetics of Escherichia coli, The Guilford Press, New York, N.Y. (1989); Plasmids: A Practical Approach, 2nd Edition, Hardy, K. D., ed., Oxford University Press, New York, N.Y. (1993); Vectors: Essential Data, Gacesa, P., and Ramji, D. P., eds., John Wiley & Sons Pub., New York, N.Y. (1994); Guide to Electroporation and electrofusions, Chang, D., et al., eds., Academic Press, San Diego, Calif. (1992); Promiscuous Plasmids of Gram-Negative Bacteria, Thomas, C. M., ed., Academic Press, London (1989); The Biology of Plasmids, Summers, D. K., Blackwell Science, Cambridge, Mass. (1996); Understanding DNA and Gene Cloning: A Guide for the Curious, Drlica, K., ed., John Wiley and Sons Pub., New York, N.Y. (1997); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Rodriguez, R. L., et al., eds., Butterworth, Boston, Mass. (1988); Bacterial Conjugation, Clewell, D. B., ed., Plenum Press, New York, N.Y. (1993); Del Solar, G., et al., “Replication and control of circular bacterial plasmids,” Microbiol. Mol. Biol. Rev. 62:434-464 (1998); Meijer, W. J., et al., “Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria,” FEMS Microbiol. Rev. 21:337-368 (1998); Khan, S. A., “Rolling-circle replication of bacterial plasmids,” Microbiol. Mol. Biol. Rev. 61:442-455 (1997); Baker, R. L., “Protein expression using ubiquitin fusion and cleavage,” Curr. Opin. Biotechnol. 7:541-546 (1996); Makrides, S. C., “Strategies for achieving high-level expression of genes in Escherichia coli,” Microbiol. Rev. 60:512-538 (1996); Alonso, J. C., et al., “Site-specific recombination in gram-positive theta-replicating plasmids,” FEMS Microbiol. Lett. 142:1-10 (1996); Miroux, B., et al., “Over-production of protein in Escherichia coli: mutant hosts that allow synthesis of some membrane protein and globular protein at high levels,” J. Mol. Biol. 260:289-298 (1996); Kurland, C. G., and Dong, H., “Bacterial growth inhibited by overproduction of protein,” Mol. Microbiol. 21:1-4 (1996); Saki, H., and Komano, T., “DNA replication of IncQ broad-host-range plasmids in gram-negative bacteria,” Biosci. Biotechnol. Biochem. 60:377-382 (1996); Deb, J. K., and Nath, N., “Plasmids of corynebacteria,” FEMS Microbiol. Lett. 175:11-20 (1999); Smith, G. P., “Filamentous phages as cloning vectors,” Biotechnol. 10:61-83 (1988); Espinosa, M., et al., “Plasmid rolling cicle replication and its control,” FEMS Microbiol. Lett. 130:111-120 (1995); Lanka, E., and Wilkins, B. M., “DNA processing reaction in bacterial conjugation,” Ann. Rev. Biochem. 64:141-169 (!995); Dreiseikelmann, B., “Translocation of DNA across bacterial membranes,” Microbiol. Rev. 58:293-316 (1994); Nordstrom, K., and Wagner, E. G., “Kinetic aspects of control of plasmid replication by antisense RNA,” Trends Biochem. Sci. 19:294-300 (1994); Frost, L. S., et al., “Analysis of the sequence gene products of the transfer region of the F sex factor,” Microbiol. Rev. 58:162-210 (1994); Drury, L., “Transformation of bacteria by electroporation,” Methods Mol. Biol. 58:249-256 (1996); Dower, W. J., “Electroporation of bacteria: a general approach to genetic transformation,” Genet. Eng. 12:275-295 (1990); Na, S., et al., “The factors affecting transformation efficiency of coryneform bacteria by electroporation,” Chin. J. Biotechnol. 11:193-198 (1995); Pansegrau, W., “Covalent association of the traI gene product of plasmid RP4 with the 5′-terminal nucleotide at the relaxation nick site,” J. Biol. Chem. 265:10637-10644 (1990); and Bailey, J. E., “Host-vector interactions in Escherichia coli,” Adv. Biochem. Eng. Biotechnol. 48:29-52 (1993).

EXAMPLES

The following examples are illustrative only and are not intended to limit the scope of the invention as defined by the appended claims. [0065]

Example 1

Preparation of Plasmid DNA from Ketogulonigenium and E. coli Strains

A portion of a frozen culture of [0066] Ketogulonigenium robustum ADMX6L (NRRL B-12627) was seeded into 10 ml ×6L medium (2% mannitol, 1% yeast extract, 1% soytone, 0.5% malt extract, 0.5% NaCl, 0.25% K₂HPO₄, pH 7.8) and grown overnight at 30° C. A portion of a frozen culture of Escherichia coli transformed with pJND1000 was seeded into 10 ml of Luria Broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) and grown overnight at 37° C. Both cultures were used to prepare plasmid DNA.
DNA was isolated using the Promega Wizard Plus Midipreps DNA Purification System (Madison, Wis.). The culture was centrifuged at 10,000×g for 10 minutes at 4° C. in a Sorval RC-5B centrifuge using the SS-34 rotor. The pellet was suspend in 3 ml of 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 100 μg/ml RnaseA. Three ml of cell lysis solution (0.2M NaOH, 1% SDS) was added and mixed by inverting the tube, then three ml of neutralization solution (1.32M potassium acetate, pH4.8) was added and mixed by inverting the tube. The lysate was centrifuged at 14,000×g for 15 minutes at 4° C. in a SS-34 rotor, then the supernatant was carefully decanted to a new centrifuge tube. Ten ml of resuspension resin (40% isopropanol, 4.2M guanidine hydrochloride) was added to the supernatant fluid and mixed by swirling, then the mixture was transferred into the Promega Wizard Midicolumn, which was connected a vacuum manifold. Vacuum was applied to pull the resin/DNA mixture completely into the midicolumn. The column was washed twice with 15 ml of column wash solution (95% ethanol, 80 mM potassium acetate, 8.3 mM Tris-HCl, pH 7.5, 40 μM EDTA. The reservoir was removed from the midicolumn with a scissors, then the column was placed in a microcentrifuge tube and centrifuged at 10,000×g for 2 minutes to remove any residual solution. The midicolumn was transferred to a new microcentrifuge tube and 300 μl of sterile dH[0067] ₂O was applied. The tube was microcentrifuged at 10,000×g for 20 seconds to elute to the DNA into solution. A similar procedure would be employed for other plasmids from Ketogulonigenium or E. coli, except that choice and concentration of selective antibiotics would be altered as suitable for the plasmid being isolated.

Example 2

Transformation of a Ketogulonigenium Host with a Plasmid using Electroporation

[0068] Ketoguonigenium robustum strain ADMX6L (NRRL B-21627) was transformed with plasmid pME1014-a using the electroporation method. Plasmid pMF1014-α was described in a Ph.D. thesis at MIT (M. T. Follettie, “DNA Technology for Corynebacterium glutamicum: Isolation and Characterization of Amino Acid Biosynthetic Genes,” Ph.D. Dissertation, Massachusetts Institute of Technology, Cambridge, Mass. (1989)). pMF1014αencodes kanamycin resistance and can replicate and be maintained in both E. coli and in Ketogulonigenium.
Competent Ketogulonigenium cells were prepared by seeding a single colony of ADMX6L into 10 ml of X6L medium (1% soytone, 1% yeast extract, 0.5% malt extract, 0.5% NaCl, 0.25% K[0069] ₂HPO₄, 2% mannitol, pH 7.8). The culture was shaken at 300 rpm at 30 C. until reaching an optical density of 0.8 units at 600 nm wavelength. Five ml of this culture was used to seed 500 ml of fresh X6L medium in a 2 L baffled erlenmeyer flask, which was shaken at 300 rpm at 30° C. until reaching an optical density of 0.8 units. The culture was chilled in an ice-water bath 10 minutes, then transferred to a pre-chilled centrifuge bottle and centrifuged at 5,000 rpm in a Sorvall 5C-RB Refrigerated Centrifuge for 15 minutes at 4° C. Cells were maintained at 2-4° C. for all steps to follow. The supernatent was decanted and the cell pellet suspended in 5 ml ice-cold Milli-Q water, then additional cold Milli-Q water was was added to a volume of 500 ml. The cells were centrifuged as before, then rewashed in 500 ml of Milli-Q water as before and recentrifuged. The twice-washed cell pellet was suspended in 40 ml of ice-cold 10% glycerol then centrifuged again as before. The supernatent was decanted, then the cells were suspended in a volume of chilled 10% glycerol approximately equal to the volume of the cell pellet. The competent Ketogulonigenium cells were aliquoted to microcentrifuge tubes (40 μl per tube) and stored at −80° C.
A BioRad “Gene Pulser II” electroporator device was set to 1.5 kV, 25 uF. The pulse controller was set to 200 Ohms. One μl of pMF1014-α DNA prepared as in Example 1 was added to 40 μl of thawed chilled competent Ketogulonigenium cells on ice. The cell-DNA mixture was transferred to a pre-chilled electroporation cuvette, which was then transferred to the electroporation device and the pulse was applied. One ml of X6L medium was added to the cuvette, then the mixture was removed, transferred to a 10-ml test tube, and incubated for 2 hours with orbital shaking at 300 rpm and 30° C. The incubated cells (approximately 1.4 ml) were placed in a microcentrifuge tube and spun at 13,000 rpm for 2 minutes, after which 0.9 ml of clear supernatent was carefully removed. The cell pellet was suspended in the remaining supernatent, then the cells were spread onto the surface of an X6L medium agar plate (1.2% Difco Bacto Agar) containing 50 μg/ml of kanamycin, and the plate was incubated for 2-3 days at 30° C. Colonies that formed on this plate were Ketogulonigenium transformed with plasmid vector pMF1014-α. [0070]

Example 3

Transfer of Plasmids from E. coli to Ketogulonigenium, Deposit No. NRRL B-21627 by Bi-parental or Tri-parental Mating

(A) Bi-parental Mating [0071]
Plasmid pDELIA8 was transferred into Ketogulonigenium from [0072] E. coli by conjugation. To make pDELIA8, the AflIII-SphI fragment from plasmid pFD288 (GenBank Accession No. U30830), which contains the cloned mob region of plasmid RK2 (GenBank Accession No. L27758), was isolated from agarose gels and ligated to the large AflIII-SphI fragment from plasmid pUC19 (GenBank Accession No. M77789) to make the intermediate plasmid pUC19/oriT. pUC19/oriT DNA was digested with SspI and DraI and the large (2527 bp) fragment was purified from an agarose gel. Plasmid pMF1014-α (M. T. Follettie, “DNA Technology for Corynebacterium glutamicum: Isolation and Characterization of Amino Acid Biosynthetic Genes”, Ph.D. Dissertation, Massachusetts Institute of Technology, Cambridge, Mass. (1989)) was digested with BamHI and PstI, made blunt ended with Klenow fragment, and the large fragment was isolated from an agarose gel. The gel purified, blunt-ended fragments from pUC19/oriT and pMF1014-α were ligated together to make pDELIA8. pDELIA8 carries a gene for kanamycin resistance, a plasmid replication gene, the replicon that is functional in E. coli and in Ketogulonigenium, lacZα and a polylinker.
A 5 ml culture of [0073] E. coli S17-1 (ATCC 47055) was transformed with pDELIA8 using calcium chloride mediated transformation. The S 17-7/pDELIA8 strain was grown in Luria Broth containing 50 μg/ml kanamycin at 37° C. overnight. Ketogulonigenium robustum strain ADMX6L01 was grown in X6L medium containing 50 μg/ml of nalidixic acid at 30° C. overnight. ADMX6L01 is a nalidixic acid resistant mutant of strain ADMX6L, derived by conventional mutagenesis of ADMX6L followed by selection of strains showing resistance to 50 μg/ml of nalidixic acid.
Fifty microliters of the fresh S 17-1/pDELIA8 culture was transferred into 3 ml of Luria Broth containing 50 μg/ml of kanamycin and 5 μg/ml of tetracycline and grown at 37° C. until reaching an OD[0074] ₆₀₀of 0.2 to 0.4 absorption units at 600 nm. Two hundred microliters of the fresh ADMX6L01 culture was transferred into 3 ml of X6L medium containing nalidixic acid and grown at 30° C. until reaching an OD₆₀₀of 0.8 absorption units. 400 μl of S17-1/pDELIA8 culture was placed in a 1.5 ml microcentrifuge tube and microcentrifuged for 2 minutes. The pellet was resuspended in X6L medium without antibiotics and spun down again. This pellet was resuspended in 1 ml of the fresh ADMX6L01 culture to achieve a suspension of mixed cells. The mixed cells were centrifuged to a pellet, resuspended in 1 ml of fresh X6L medium without antibiotics, then pelleted and washed once again in 1 ml of X6L medium. The final pellet of mixed cells was then resuspended in 100 μl of X6L medium without antibiotics and spotted onto a a sterilized GN-6 metrical 0.45 μm×25 mm filter (Gelman Sciences, Product No.63068) resting on the surface of an X6L agar medium plate without antibiotics, which was then incubated overnight at 30° C. The cell biomass on the filter was resuspended in 3 ml of ×6L medium and 50 μl of this suspension was plated onto X6L agar medium containing kanamycin and nalidixic acid at concentration 50 μg/ml each. Colonies that formed on this plate after 2-3 days incubation at 30° C. were Ketogulonigenium transconjugants transformed with pDELIA8.
(B) Tri-parental Mating [0075]
A 5 ml culture of [0076] E. coli HB101 (ATCC33694) harboring plasmid pDELIA8 was grown in Luria Broth (LB) containing 50 μg/ml of kanamycin at 37 degrees overnight. A 5 ml culture of E. coli HB101 harboring plasmid RP1 (GenBank Accession No. L27758) was grown in Luria Broth containing 5 μg/ml of tetracycline at 37 degrees overnight. A 5 ml culture of Ketogulonigenium robustum ADMX6L01 was grown in X6L medium containing 50 μg/ml of nalidixic acid at 30 degrees overnight. Fifty microliters each of the fresh E. coli HB101/pDELIA8 and HB 101/RP1 cultures were transferred to 3 ml of LB with 50 μg/ml of kanamycin or 5 μg/ml of tetracycline, respectively, and were grown to an OD_600nmof 0.2 to 0.4. Two hundred microliters of the fresh Ketogulonigenium robustum ADMX6L01 culture was transferred to 3 ml of X6L medium containing 50 μg/ml of nalidixic acid and grown to an OD₆₀₀of 0.8. Four hundred microliters each of the E. coli cultures was combined and pelleted in a 1.5 ml microfuge tube, then decanted, resuspended in 1 ml of X6L medium without antibiotics and spun down again. The supernatant was removed and the E. coli pellet was resuspended in 1 ml of the fresh ADMX6L01 culture. The three-strain mixture was centrifuged to a pellet, then resuspended again 1 ml of X6L medium without antibiotics and recentrifuged. The pellet from this wash was resuspended in 100 μl of X6L medium without antibiotics and spotted onto a sterilized GN-6 metrical 0.45 μm×25 mm filter (Gelman Sciences, Product No. 63068) resting on the surface of an X6L agar medium plate without antibiotics, which was then incubated overnight at 30° C. The cell biomass on the filter was resuspended in 3 ml of X6L medium and 50 μl of this suspension was plated onto X6L agar medium containing kanamycin and nalidixic acid at concentration 50 μg/ml each. Colonies that formed on this plate after 2-3 days incubation at 30° C. were Ketogulonigenium transconjugants transformed with plasmid pDELIA8.

Example 4

Construction of Plasmid Vector pJND1000

pJND1000 was constructed from segments of plasmids pUC19 (GenBank Accession No. M77789), pUC4K (GenBank Accession No. X06404), pFD288 (GenBank Accession No. U30830), and pFC5 (David M. Lonsdale et al., “pFC1 to pFC7: A novel family of combinatorial cloning vectors.”, [0077] Plant Molecular Biology Reporter 13(4):343-345 (1995)).
The ampicillin resistance gene (amp[0078] ^R) was removed from pUC19 by digesting pUC19 DNA with restriction enzymes DraI and SspI, separating the 1748 bp vector fragment from the smaller amp^Rfragment and other fragments by gel electrophoresis, then recovering the 1748 bp fragment from a gel slice. A kanamycin resistance gene (kan^R) fragment from pUC4K was prepared by digesting pUC4K with restriction enzyme PstI, treating the mixture with Klenow Fragment to produce blunt ends, separating the fragments by gel electrophoresis, then purifying the 1240 bp kan^Rfragment from a gel slice. The isolated fragments from pUC19 and pUC4K were mixed and ligated with T4 ligase following the protocol of GibcoBRL technical bulletin 15244-2 (Rockville, Md.) to produce “intermediate p1”, an intermediate plasmid carrying pUC19 features and a kan^Rgene. Strain E. coli DH5αMCR was transformed with the ligation mixture following an established protocol (“Fresh Competent E. coli prepared using CaCl₂”, pp. 1.82-1.84, in J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989)). Transformants were selected on Luria Broth plates containing 50μg/ml of kanamycin.
An oriT site for conjugative transfer was obtained from plasmid pFD288 by restricting it with HaeII, converting the single stranded ends to blunt ends by treating the mixture with Klenow Fragment, separating the fragments by agarose gel electrophoresis, then purifying the 778 bp oriT fragment from a gel slice. The intermediate p1 plasmid was opened at a single site with restriction enzyme SapI, then treated with Klenow Fragment to convert the single stranded ends to blunt ends. The SapI digested, blunt ended p1 intermediate was mixed with the purified oriT fragment, then treated with T4 ligase as above to create “intermediate p2”, a plasmid which carries oriT in addition to kan[0079] ^Rand other pUC19-derived features. Strain E. coli DH5αMCR was transformed with the ligation mixture as above. Intermediate p2 was confirmed by restriction digestion analysis.
The polylinker in intermediate plasmid p2 was replaced with the polylinker from pFC5. To do this the two plasmids were separately restricted with PvuII, the fragments from each digestion reaction were separated by gel electrophoresis, then the pFC5-derived polylinker fragment (531 bp), and the larger non-polylinker fragment from intermediate p2 were purified from gel slices. The recovered p2 fragment and the pFC5-derived polylinker fragment were mixed and joined using T4 ligase as above to make plasmid pJND1000. The structure of pJND1000 was confirmed by restriction digestion analysis. PJND1000 replicates in [0080] E. coli, has a functioning kanamycin resistance gene, an RK2-derived oriT site to permit conjugative transfer into Ketogulonigenium and other hosts, a polylinker for DNA cloning, forward and reverse M13 primers to facilitate DNA sequencing reactions into the polylinker, and permits screening for cloned inserts by inactivation of lacZα using Xgal indicator plates.

Example 5

Isolation of Plasmid pXB1 Containing Linearized Ketogulonigenium Plasmid pADMX6L1

The DNA sequence of plasmid pADMX6L1 (SEQ ID NO:1) is about 7029 bp long and contains a single BamHI restriction site. The BamHI site was utilized to clone the pADMX6L1 sequence into the[0081] E. coli vector pJND1000. A DNA prep containing a mixture of the endogenous plasmids from Ketogulonigenium robustum ADMX6L, and purified pJND1000 DNA from E. coli, were made as in Example A and separately digested with restriction enzyme BamHI. A Wizard DNA Clean-Up kit was used to separate the digested DNA from enzyme and salts in preparation for the next step, with a final DNA suspension volume of 50 μl. DNA from the two digestions (3 μl of pJND1000 DNA and 10 μl of Ketogulonigenium plasmid DNA) was mixed and ligated overnight at room temperature using T4 ligase with the protocol of GibcoBRL technical bulletin 15244-2 (Rockville, Md.). Strain E. coli DH5αMCR was transformed with the ligation mixture following an established protocol (“Fresh Competent E. coli prepared using CaCl₂”, pp. 1.82-1.84, in J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989)). Transformants were spread on Luria Broth agar plates containing 50 μg/ml of kanamycin and 40 μg/ml of Xgal and grown at 37 deg C. Colonies that were white, indicating insertion of DNA into the pJND1000 BamHI site, were picked and cultured for further processing. Digestion of plasmid DNA from transformants using BamHI, EcoRV, and XhoI separately, yielded the expected fragment sizes for sucessful cloning of pADMX6L1 into the pJND1000 E. coli vector. The identity of the pADMX6L1 insert was further confirmed by partial DNA sequencing into the pADMX6L1 DNA region using the M13 sequencing primer regions of the pJND1000 vector. The chimeric plasmid containing linearized pADMX6L1 cloned into pJND1000 was named pXB1. An E. coli host transformed with pXB 1 was deposited in the patent collection of the National Regional Research Laboratories in Peoria, Ill., U.S.A. under the terms of the Budapest Treaty as NRRL B-30418.

Example 6

Isolation of Plasmid pXH2 Containing Linearized Ketogulonigenium Plasmid pADMX6L2

The DNA sequence of plasmid pADMX6L2 (SEQ ID NO:2) is about 4005 bp long and contains a single HinDIII restriction site. The HinDIII site was utilized to clone the pADMX6L2 sequence into the [0082] E. coli vector pUC19. The procedure was the same as in Example 5, except that prior to the ligation step the plasmid DNAs were digested with HinDIII instead of with BamHI, and transformants were plated on LB agar plates containing Xgal and 100 μg/ml of ampicillin instead of the kanamycin. Plasmid DNA from transformants giving white colonies was isolated and digested with HinDIII and NdeI, giving the expected fragment sizes for correct insertion of linearized pADMX6L2 DNA into the pUC19 vector. The identity of the pADMX6L2 insert was further confirmed by partial DNA sequencing into the pADMX6L2 region using the M13 primer regions of pUC19. The chimeric plasmid containing linearized pADMX6L2 cloned into pUC19 was named pXH2. An E. coli host transformed with pXH2 was deposited in the patent collection of the National Regional Research Laboratories in Peoria, Ill., U.S.A. under the terms of the Budapest Treaty as NRRL B-30419.

Example 7

Construction of E. coli Ketogulonigenium Shuttle Plasmid pXH2/K5

Since kanamycin resistance can be expressed in Ketogulonigenium, it was desirable to replace the amp[0083] ^Rresistance gene in pXH2 with a kanamycin resistance gene, thereby allowing the selective isolation of Ketogulonigenium strains transformed with a plasmid. In vitro transposition was used to move a kanamycin resistance gene into pXH2 and simultaneously inactivate ampicillin resistance. Insertion of a kanamycin resistance gene into the ampicillin resistance gene of pXH2 was achieved using Epicentre technologies EZ::TN Insertion System (Madison, Wis.). 0.05 pmoles of pXH2 was combined with 0.05 pmoles of the <KAN-1> Transposon, 1 μl of EZ::TN 10X Reaction Buffer, 1 μl of EZ::TN Transposase, and 4 ul of sterile water giving a total volume of 10 μl in the transposition reaction. This mixture was incubated at 37° C. for 2 hours, then stopped by adding 1 μl of EZ::TN 10X Stop Solution, mixing, and incubation at 70° C. in a heat block for 10 minutes. E. coli DH5αMCR was transformed with the DNA mixture and transformants were selected on Luria Broth agar plates containing 50 μg/ml of kanamycin. Colonies recovered from these plates were patched onto two LB agar plates, one with 50 μg/ml of kanamycin and the other with 100 μg/ml of ampicillin. Only those that grew on the kanamycin plates were saved. Plasmid DNA from an ampicillin sensitive, kanamycin resistant colony was isolated as “pXH2/K5”. The proper insertion of the kanamycin resistance transposon into the vector ampicillin resistance gene was confirmed by analyzing pXH2/K2 DNA with various restriction endonucleases. To demonstrate the viability of pXH2/K5 as an E. coli /Ketogulonigenium shuttle vector, Ketogulonigenium robustum strain ADMX6L was transformed with pXH2/K5 DNA using the electroporation technique of Example 2. Stable, kanamycin resistant transformants were obtained from which pXH2/K5 DNA can be reisolated.

Example 8

Isolation of Plasmid pXB4 Containing Linearized Ketogulonigenium Plasmid pADMX6IA

The DNA sequence of plasmid pADMX6L4 (SEQ ID NO:4) is about 4005 bp long and contains a single SspI restriction site. The SspI site was utilized to clone the pADMX6L2 plasmid into the [0084] E. coli vector pUC19. The procedure was the same as in Example 6, except that prior to the ligation step the plasmid DNA from strain ADMX6L was digested with SspI and pUC 19 was digested with SmaI. Plasmid DNA from transformants giving white colonies was isolated and double digested with EcoRI and BamHI, and separately with PstI and SphI, in each case giving the expected fragment sizes for correct insertion of linearized pADMX6L4 DNA into pUC19. Partial DNA sequencing into the pADMX6L4 region using the M13 primers of pUC19 further confirmed the identity of the pADMX6L4 insert. The chimeric plasmid containing linearized pADMX6L4 cloned into pUC19 was named pXB4. An E. coli host transformed with pXB4 was deposited in the patent collection of the National Regional Research Laboratories in Peoria, Ill., U.S.A. under the terms of the Budapest Treaty as NRRL B-30435.

Example 9

Definition of DNA Sequences Comprising Replication Functions of Ketogulonigenium Plasmids

Various programs of the Wisconsin Package version 10.1 (Genetics Computer Group, Inc. Madison, Wis.) were used to analyze the DNA sequences of the four endogenous plasmids from Ketogulonigenium strain ADMX6L. The analysis revealed multiple open reading frames (ORFs), nucleotide sequences having protein-encoding potential, on each plasmid. The predicted amino acid sequences of these ORFs was obtained, and a similarity search against PIR and SWISS-PROT protein databases was conducted using the GCG implementations of the FASTA ( Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988)) and BLAST (Altschul et al., Nucleic Acids Research 25:3389-3402 (1997)) methods. Based on sequence similarity to known plasmid-encoded replication proteins, plasmids pADMX6L1, pADMX6L2, and pADMX6L3 were found to encode potential plasmid replication proteins. The genes (ORFs) for these functions have the following endpoints:



pADMX6L1	bases 2255-2710 of FIG. 1	(SEQ ID NO:1)
rep ORF:
pADMX6L2	reverse compliment of bases	(SEQ ID NO:2)
rep ORF:	3960-2562 of

pADMX6L3	reverse compliment of bases	(SEQ ID NO:3).
rep ORF:	5031-4003 of

A region of DNA sequence upstream and downstream of the regions defined by these ORFs, perhaps 500 bp in each direction, is likely to contain transcriptional promoters, terminators, and other sequences required for proper expression of the replication proteins and control of plasmid replication. Therefore a region comprising part or all of the plasmid replicon for these three plasmids could be defined by the following DNA sequences:



	pADMX6L1 replicon:	bases 1755-3210 of the FIG. 1
		sequence, also
		represented by
		SEQ ID NO:5
	pADMX6L2 replicon:	bases 455-2060 of the FIG. 2
		sequence, also
		represented by SEQ ID NO:6
	pADMX6L3 replicon:	bases 3503-5531 of the FIG. 3
		sequence, also
		represented by SEQ ID NO:7

All publications mentioned herein above are hereby incorporated in their entirety by reference. All publications cited in the annotations to the Genbank accession numbers or in ATCC strain descriptions cited herein are hereby incorporated in their entirety by reference. [0087]
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims. [0088]
1 10 1 7029 DNA Ketogulonigenium misc_feature pADMX6L1 1 gccatttctg cgctgcactt cgctaagggt tcaagggaaa cgcagggttc ccttgcccac 60 acaaacgcgc agcgtttgta taagtgggca cttcgtgttt gacacgctat ccactacgcg 120 gcacaaattc aactcttgta acgaggaagg gcggtagaat ggcgcgcacc atcgaccagc 180 agatcgcaga tgcgcaagcg aagctggcgc ggctcaaaac ccgtcagaaa gccagcgaca 240 cccgccgaaa gatcatcgtc ggcgccatcg tcaccaccga ggccctgaaa gaccccaaga 300 tttccaaatg gctggcatct accctgcgca agaacgcaac ccgggacgtg gaccagaagg 360 aaatcgccgg gctgctggcc gacctcgatg ccagggcgca aagcgccggg gcgggtgagg 420 catgagcggc agcaccgatc cgtttctggt tctggtcgat gatattggcg cgctgcgccg 480 ccagatcgag aacctgcaac gcaccagcct cgacagggac gaggccgaac atctcaacgc 540 gaccatcgcc cagagcctcg acaacatggc gcaaaccgga aaacggctgg aacagcgcct 600 tgagggccag ttgcagctcg ccaccgccaa aacccacagg gacgccatag aagccgctca 660 gggggccgcc agagcggcta tcagggaatc ccatgccgag atcctccaaa cggccaggag 720 cctctcacag gccgcaggag aggcccgcag agaggcgtgg cgctggttcg gcgggttctg 780 ggtctggctg gcctcgatcg gggccgcagg ggcgcttgtc ggcgcgctgg ccgtgttctg 840 gctccagggc cgcgccgatg ccaaagcctt cggacagtat cccagcatct actgcaccac 900 cgcaggcggg gcattcgccg atcagcgcga cggaagccga tactgcatct tcatgatttc 960 accgccgaca cagccagacg gggaatgacg gcttacgcgc cgggctggat cgagactttc 1020 agcccgagcg ccttagcgac cttcatcacc gtggacagcg tagggttccc atccccggat 1080 agcgccttgt tcagccccac ccggctcatg ccaacctcac gggccagcgc ggtcatgttc 1140 cgtgcgcggg caaccactcc aagggcgcgg gcaacatagg cgggatcgtc gccgccatct 1200 tccatgaccg cttcgagata ggccgcaata tcttcctcgg tcttgaggta gtcggcggaa 1260 tcgtagcggg cgaatttttc ttccggcatc gcttagccct tccactctgc ggccagcacc 1320 ttggcctgtt tgatgtcttt gctctgcgtg gacttgtcgc cgccacaaag caggatcacg 1380 agaaccggcc cgcgctggat gaaatacacc cggtagcccg gcccgtagtt gatccgcagt 1440 tccgaaacac cctctccgac cggctccaca tcgccggggt tccccgccgc aaggcggtcc 1500 agtctggcag tgatgcgcgc aaccgccctg cgatcccgca aaccggaaag ccaggtatcg 1560 aaggttccgc ttcggattaa ctcgatcatt cgacaactat agttatcatg tgggtgtctg 1620 acaaccagag ttatcacttc cttgttctaa gcaaatccga ggccagccac gggcgtagcc 1680 ggagcatcat ccctcccccg cacccccacc cgtcacgcgc acacatgcgc ggaatcgtcc 1740 actcggccca caaggggcct tgcatccgat ggcaagcaaa aactacccag tccgtccgta 1800 ggcggggggt cgccagccct gtgggtgggc gcttcccccc ggcccgcaag cgggcccgga 1860 atgggcattt tttgcctgcc ctaagatcat aagaagggca aaaaaaacat cgtttcaaaa 1920 cagcgtgtta ccacccccct ataggacacc agagtccggg gtagaggact ctggtgtcct 1980 cttaggccat ttatgtccaa gaatgtgaca gccagccgag cggaggtaga ggactctggt 2040 gtcctatgct taggccattt atgtccaaaa acttgacaag ggccacattc ctgccaaatc 2100 tgtccagaat ttggaaaaat tcgccggata gtagacagtg gcaaagcctc cccccattcc 2160 cgcaaagcgc ccgctcggca cttgggttca aactgaccgg gaagcccacg aggcgtgggc 2220 gatactggca aaaaagcctg ctgccagcgc tgtgatgcac attctgtgcg ccaacctcgg 2280 tgagcataat gccgtggtca tcagccagga caccatcgcc aagctgtgcg gcctttccac 2340 acggtccgtc aggcgcgcca tcgtcgatct ggccgaaggc cgatggatcg aggttcgcca 2400 acttggcgcg accagccaga ccaatgccta tgtcgtcaac gaccgggtgg catggcaggg 2460 atcacgggac ggactgcgct acagcctgtt tagtgcggct atagtcgtgt ccgaggagga 2520 gcaacccgac cgcgctgaac tcgaccagca agcccccctg cgacacctgc cacgcatcag 2580 cgaggggcag atacccaccg gccccggcct gccgccacct tcgcaaccgt ttctaaaaga 2640 catggagcca gacttgccca ccattgaccg ggcaacatca cccaactttg accagcagga 2700 acaggggtga aaaaggtgga caaactttcc atacgcgagg cggtaaaaca cttcgatgtt 2760 tcccggccaa ccctgcaaaa agcccttaaa tctggcaaga tttcaggtgt tcaggatgga 2820 caaggaacgt ggacaataga cccctcagag atggcaagag tttaccagcc aaggcaagat 2880 gaggtggtaa aggatggtgg ccaagaacat gaaaatttgt ccgccaagaa caccccttta 2940 catggtcaag ttgaggttct gaaagagcgg cttgcagatg ctgaaaaacg ggtggcgata 3000 gccgaggcac tggccgaaga acgtggaaaa cacatcgagg atctacgccg gatgctgcct 3060 gcaccggaag ccggtcagcc ccgccgccgc tggtggccat ggtaaggtca gctatgcggg 3120 accaagccgc agctcgcaag tgcggcaaca gaatcagacc cgcttcggac agagagctca 3180 agctggtgga aaaccgcctg tgaagctgct gggtggcagc cgctttactc agcgaagcag 3240 ccattcgggc ggatcgcagc gagattgccc catccagtcc taagggccgt gtaaacagca 3300 cttagtgcag cagcatcgca ggcgcggaaa tctgcctgcc cttcctcgcc acctctgagg 3360 cgtcagcgat ctgccccaaa cctgcctgcc actttggcgc agcatgttgc ttgagaacac 3420 ggagctgccg atctctgcgc taagcagaat ttcctgataa tacgcacaga tgcaaataat 3480 attgcggcat caatgcgttt ccgtttaccc ctaggcagtt gcctcatcat gctgtcgttc 3540 gctgtccaag ccgcaacgac ctgcgaccga accttcactg catcttactt tcggcccctg 3600 cgttcgcccg tgaccgcagc gcggcccaaa gcgcgaccgc cgccaaggcg ggtaggaaca 3660 gccccattgc catcgggaac gggctgtcgg tcccggccaa ggccacgcag gtcgaaatcg 3720 tgaaggccat cccgaactgc accgcaccca agagtgccga tccagttccg gcggcctcgg 3780 tcgcggcggc catggcaagc gaggtcgcat tggccgaaag cagcccgacc atgccgatcg 3840 cgatccagag cggaatgata aacatccata ccgaacccgt ggcagaggcc aggacggctg 3900 ccagcgccgc aagaccatag aacggcagtc cccggttcag catgtcccgg ggcgtaaagc 3960 ggtccaggag gcggctgttg atctgcgcga agacaaacag cgcggctgcg atgagcgcaa 4020 atatcaaccc gtagttgagt gcgctcatac cgaagaagcc ctggaacacc ccggacgatc 4080 cggtgatgaa ggcgaacatc ccgccctgga ccaaccctgc caccagcacg ggggcaaggt 4140 aggcgggctg tctgacaagc cgcaggccgt tccttgtcgc gcttcggaag ggctgcgcga 4200 cacggcgctc tggcgacagc gtctcgggca ccaccagttt cgacaggatg agggcaggca 4260 gaccgaccag caccattgtc acgaagatcg accgccagcc gaaggcctcg agcagcaggc 4320 tgccaagtgt cggcgcgatg accggaccta tggtcatcag catcaccagc aaggtcatca 4380 ccttggccgc cttttgcccc gagtagagat cggcgacgat ggccctgctg acgaccatcc 4440 ccacgcaggc cccgatcgcc tgcaaaaagc gcagcgcgtt gaagacaacg atattgtcga 4500 ccagcggcag cgcaattgat gtgacggtga agatgaagac gccgatcaga agcggccgct 4560 tgcgcccgta tccatcggtc agcggcccga cgatcagctg cccgaggcaa aggcccagaa 4620 agaacagcga cagtgaaagc tcggtcgccg catggctggt attcagatcc tcggcgagaa 4680 tgccgatggc cgagagatac atgtcggtcg ccagtggcgg aaagatcgac aaaagggcga 4740 gcacggcgat caaggcaccg gctcgggcag ggatgagggg gcggtccagc atgatatcct 4800 cttggcggga tggtgcgatt ccatttgtag atcgcagtct actaatgtaa aattatactc 4860 aagtctataa ttcaagtcgc ttggccggaa tcgcaggaga agacaggaat gcaggaggta 4920 cggagcggac cgggacggcc aaaagacccc gtggttgccg aagcgatccg caaggcggcc 4980 ctgcggctgg tgcgcgaaag gggataccgg aacgtaagca tcggcgcgat cgcgcaagcc 5040 gctggagtgg cgcggcaaac gctttacaat cgctggcacg cgaaggccga cctcatcctt 5100 gatgccgttt tcgaagagac cgggcggcgc gccgacgatc aattaccgct ggaaacaggg 5160 gacgcgtccc gtgatcggct ggaacggctt cttataggtg tgttcaatca cctccgggcg 5220 gatggcgata cactgcgcgc attgatcgcc gccgcgcagg aggacagcga gtttcgtgag 5280 gccttccggg aacggttcgt cgcaccgcgg gaaaccatcg tcaccgacat tctggccgaa 5340 gccctgcgcc gtggcgagct ttcccgggaa gccgacccgg ataccttgtc gaccatgatc 5400 cacggcgcat tctggtatcg cctcctgaac ggacgcgagc tcgatcatga actcgcccga 5460 tcaatagcgc ggagcgtgtt tccctgagcc aagacgaaac ggaaacggac ccaaatcatc 5520 ggaatgtata aagtcgcgca tcgttaccat cctgaggggg tgatcgctgc ggagcgagac 5580 gaatggcagc taccgggaat gcggccacgc cgcattggtc gaggtcggcc agggcaaagg 5640 gctggccttc acctatacaa gcgacaggcc tgccatgcgg cgccctgccc aagtgtcagc 5700 gatgggctca aaccgtcgat aggccagccc cggcccgctc gttccatcgc gggctgtagg 5760 gtcggggctg gccggatcag tgagtaagcc cgccatcccg gtcgtggtca cgggcttctc 5820 gctcccgcaa ccgttcctgc tgccgctctt gctccttcag gcgttcggcc tcctgcaccc 5880 gctggcgttc ctcggcctcc cggctttcct gcaaccgcgc tgccgcctcg gccagcgtgc 5940 cccggtcgat cccctgcgcc gcctcccgta gccgctcggc caggctgcgg gaggattccg 6000 gttccggggt ttgtccaggt gcagctatgg catcggacgc ggtgcggctc tggcgggcct 6060 cccatgcctc gcgcaaccgc gcggccaggt cctgcctccc ctcccggcca tcccgatccg 6120 cagcgccggc ataagccaga ccctgcgccg ccccctcttg cgtcaacggc ccgaggcgat 6180 ccacagctcg cccgatccag tcgcgcacct gaccggccag aacctccgcc acacgcgcaa 6240 cctcggccgc ctgcgccttg acctcgcgcc acacccggac ggccccgacc tcgataccgc 6300 gttccttcat ctgccacgcc ccggcagaca gttgcggcaa cggatcccgg tccagttcca 6360 ccgcccgcac cgtttgacgc agcgcctcgg cctcgtcgcc gcgatcatgc gccgcgctgg 6420 cccgctcctg cgcctcgatc cgctgcgcct ctagcgtccg gtggtcgatc cgttcctcat 6480 ggccgcatcg ctcaagggcg cggttgctgt cccgcgccca tgcctcacgc catccttcca 6540 gcatctcgac cgcgttccag tcccggttct tcgccccgaa cccctcgggg ccgatttcgc 6600 gggtggtcag caggatatgg gcatggtggt tgcgatcatc gccggtccgt cccggcgcat 6660 gaagggcaat gtcggccacc atgccacggg ccacgaactc gcgctggcag aattcgcgca 6720 ccagctccac gcgctgtccg tggtccagct cggcgggcag agccacccgg atttcgcggg 6780 cgacctgcga attcttccgg gtctctgccg cctcgaccgc gttccacagc gcctcccggt 6840 cctgcaccca tgcgggggcg ttggcagggg cgagggtttc gacgtgatcg acaccgccac 6900 gcgcacggta atcgaaggtc agcccggtgc ggtgatcctc gatccgttcg cccacgcggt 6960 aggccgcagc cgccgtggcg ctacgaccgg aagagcgaga tatcatcgtg gcccgaaggt 7020 gatagatcg 7029 2 4005 DNA Ketogulonigenium misc_feature pADMX6L2 2 gcgtctgtgc gcatactatc ctccgtgttc atcaggctca cgcctgatct gattagggct 60 cttgtctctg cttgtatcgt cgccaaacta tactttaagc agcgtctaga gcctgatgaa 120 cgatctaaga agcccgcccc ctgaaaggcg ggcttttttg atctgtgcca gatgttgtta 180 catcggcgct caaaaatcaa gttttttctt gactttcaat aatttgcatt atgcacatta 240 ttatcgtttg ataataagag ccagaacagc acagaatagt tgtgcgatag ctatgaataa 300 tagcagatcc atccctgttt cctttcttac taaatacaat gcgaaactgc tcgcatctgt 360 tttatttagt tgaatcggaa actccaaatc ggccggattc aaaaaaaata tagactatct 420 ttaaagtagc aacgccgccg ctcgcgcgac ggcattgcgg gaaatgcgat agcaacaaaa 480 ccacttattg tcgtatatcc ccactttcaa gcgaagcgcc ccgcgaagcg atcaaaaaag 540 aaagaaagaa gggggcgctg cccccgtcat gcgcaagcgc atgactcccc cgcccctcac 600 tcaccaaagc agcagctacg aagcaggacg ggatcggcgt ttgtgcatca cgtgctcgca 660 tcaccaacac aaatagacgt atccccatga atggggcccc acgtcgattt gtgttggcgc 720 tactgcgcgc gcaatgccct gcagcctcac ccgaccggct ccgaagcagc agcttttaag 780 acagtgagtg acacaagaaa acatgcgcaa gcgcatgtgt gcatcgcgtg cgcgcgatgc 840 acgggaaggt tgccttcgtt gtatataata atatagcctg cacgctgtgc gcacagcgtg 900 catgagaggc aaaaacatgt caaaaatgtt gactactgca caagttgcac agcgctttgg 960 actgtcacga tcgacagtta gcagagcgct taaaaacggc gacttgcgtg gcatccgcga 1020 caatagaggc gtgtggaaaa ttgccgaaga tgatgcgcat aaatggcgca gcgacgccgt 1080 gcatgaacag cgtgcgcaca gcgtgcatga cagtgcatta cgcacacgtg ctgaagttgc 1140 ggaagctcgt gcaacagcgc tagaaatgca cgtgtctgac ttgcaaagcg agcgtgacga 1200 cttgcgaaag cagcgcgacg agctgcaagc gaagttagac ggtcggcccg tcgaaactgt 1260 cagtatcagc cagcttttcg ggcgcctttt tcggcgctga catggcgcgt agaagcgcga 1320 cagtctcagc tttcgttcgc ttcatctgcg gcatgatctt cacagcatca gtgcgtgctg 1380 cgcgtgtctc ttcgacctct tttctagagc cgagccgcaa tgcaatctga atagatctgc 1440 gtattcgcgg caagctttta cgcacttctg aaatcgcttc atcgagcgac ttcaagcgct 1500 ctacaagaat gccgcgcaag ccagaaactc gcttgcgctc agccctgacg gcttcgacag 1560 cgacagactt ttgacgctca aaatcgactt gtgcagcatc gagctttgca cgctctgcat 1620 cgagctgctc gcgttgtgtt ttaagctctg tgcgctctgt ctcgacagct ttaaaagcct 1680 cagtttctaa gcgatccggc gcgccgtccg acttctcacg cttaggctct agcttgatgt 1740 tattccgacg ctcgaaaaac gcagcaaact cgctttgcag cgcaataccg accgcgcgcg 1800 gcgcgctata gtttggcgct gccccagaat gccgccgctg gatctcttcg cgatgtttat 1860 cggccagctc gcgaccaaat tttgtggcgc tcgaccacat ttcgccaggc tgatccggcg 1920 gcgttctttt cgtgcgcttt tcataaactg gcgaagcaaa aacatcgacg atgctctcgc 1980 ccgcctcgtc tctgtctagc ctcgctgcaa agacagcgtt accgccgtgc gtttcgttta 2040 taaactccac tgcttggcgc accatggcgc gctgcagctc ttctttgctt ctgttggttc 2100 ttctctcgtc aagaagctca ggcggaaacc gcactataaa gtgcagaaca ggcttcttcg 2160 ccgctttgtt ttgcctaacg ccttttgtgt gcgcgtcata tgccgaccga aggtctagcg 2220 tcttataaac gagcggagag gcatctctta cgacgcgttt cgcacttgtt ttgtcttgtc 2280 gtttcgcgtg cttttcggct gctgacaatc cagccatatc taacgcacta catctcaccg 2340 ctgctttcat tttcaatccc tcatatatta ccttttctgt tgtttttgcg aaaaacgcaa 2400 aactcgcttt gcgttttttg ttcggcacct gcggcacctc caaaaaacac gcttgctctc 2460 cgagggtctg gcaggagcct aagagggggc attctgcccc tgatcgaccc cattgaggct 2520 cgatctaaac agacccccca caggggcgtc tgtgggccgc tagtgcggcc ttccgccatc 2580 ttcgagccat ctgatctctg caattagaga tgcatggcgc atctcccatt ccagctcaga 2640 tatcccgtat ctgtcgatga cggtgtcata caattcgtcg agccttgctt cgcgctcttc 2700 ttttgtcatg ccagttgcat cgcgttcttc catctcctcg ttctcctcta tgatgaaatc 2760 attttcgtcc tcgtcgtttg gcccgctttc tgctgccttg accgcaacag cgcctccgcg 2820 ctctgcggcc tcgagcaagt tcacgcgttt agcgcgcact tgcttccacc cttcattgtc 2880 ccactccgcg acaatttcag ggccgctttg ctctgctgca tcaacttcag ccatagcttc 2940 atcgtcatcc agctcgacca aaccgcattc ttctttcaag ccttggctcc acaccaattg 3000 ccgcctacgc ttgccgctcg ttgcattgaa atattcgagc caaagcccgt catcgcccgc 3060 ctgaagtagc tgccttggcg tgcgtccttt gcgttttccg ctcttcgagc ttgaaagcgt 3120 caactcttcg gcagcgcccc acttcgctac gtagtcgccc gcattggcag ccccgcgaac 3180 gtcaaacgcc gcatcgttgc cccacatgcc ataccccttc agacatgcac gccacgcatc 3240 gcctagacgt tgcatcagat gcagcgcttc gctttcatcg ccagctctta gcaagacaat 3300 ttcgtgaaag tgcgggtgcc acccatttgc atagctatga gtaatttcag ttgatgtgac 3360 tgacccaaca aatggtaaat cgcgccactc gcggcgctga cgcaaccgct gtttcgcctt 3420 cttcatgttt tggagaagat caaaaagcga atcacctgct ttgtgctggg ctgtcagagt 3480 tatgagcacc ggcacaaacc cgttgtcgcg cgcccacgcg agcaagtgat tcatttcaga 3540 acggcgaatt tgcgcgatgc gagcgctaca aactgcgcag ccccacacat tccggcactg 3600 tgctagacct gaaaagaatg cccgacgccc gccatcctcg cccacgtttt gcacgtttag 3660 ctcaactgtc ggagacactt ttacgtgccg acatttcgca acttggtggg gcttgttttt 3720 gttcagattc aacagaatcc gagcggctga acgcagatct gcataaagct gccgcctact 3780 gaataacctg ttgttttctt tgtttttttc attcggttga cccccattct ggtcaaccga 3840 tttacggtat ataccaaggg gggtctgccg accccctgaa aaagcgtcat cgccgcgcgc 3900 ctgcgcaccc gcgttcttct tcgatttctg aactgaatgt gatgctagtt tgtgagacat 3960 ggccgcaaac cccgacgggt gcggcgacta ttctcttgat tttct 4005 3 19695 DNA Ketogulonigenium misc_feature pADMX6L3 3 cactttcgcc acaagatcag gcgcatgatc tttctcagcg ttcaacacac atttgagctc 60 ctgcgcctga tcgtcagtaa ggtaatatcg ttcttgcatc aatttctcct tagttggatt 120 ggttgcactg gatctttgcc ctgacagcct catgcctcga ggcctcggcg atgtcgtggt 180 gcattgtatt ctcgcggcac tgcatcgact tcttctcggt ttccttttgt tcgcactggg 240 tacatggatg tcgttgctgg tcacacacac cagagcacac ctacagctct cccatgcgac 300 cgccttggcc gcaccgagat acatcgcctt gtgtggttcg cgacttcggc agcaatcgcc 360 gtggcagctg gattggagca tcatcaggca agggcttcgc tgacagcgtt cgtcgtgaac 420 gcctaggggc tgtaaaccta aaactgcccc cattgaccgc cgccagaaga atgatccatg 480 tctagggtgc cccatgaatt acccgcgata tccggtgtgc cgtcgcctgc aacagtaggc 540 agaccgctag caggattgat cgagtgtatg ccaccaaatg tgccccctga aatatccagg 600 gtgctcaatt catccattgt atgcgctcca gaggttggga ttgtgctaaa ggattgaaca 660 gtatggtctg gagtacccca ccagttcaga ttcaaaaaac ttcctatcat tttgaacatg 720 attggcttcc ttttcgatta agttttcaac aaacagaatt aatctacggg ccagttgtgt 780 gcagaagagc gacggcaaat tgctgttata gtatttacga aaatggtcgc caaaaaatat 840 ctgtcgatca agtgttttct gtaaccatct gaactttttg ataaatatca aggtgtgagt 900 ggtttatgcg atgctttaca tctgacatct ggtaaccgcc cctgttttct cagggtgtag 960 aatgcagatg acactgaaaa tacaatgaag tgaatgagag ccgttgccct gcccagccaa 1020 ggcgaagatt tcattatccg ctgagcaaat cttgtaccaa agagggcttg cgttcaatga 1080 gggccagtag gacttgagct ggcccttgag gcatacggcg cttttgctcc cagttgagaa 1140 gagtgccctt cgccacgccg atactacgtg caaactccgt ttgagatagg ccggtctggg 1200 cgcgaatgcg cgcgacatct atctcaggca agctgatctc atgcacagcg gcaccggcct 1260 tttgcccagt cgaaaaggcg cgagcttctt ccaagccctg catgatactg tcgaacgcgc 1320 tcattgtttg ttgctccaaa tggcgatgat ctctttgctc atttcgaccg cggccgcctg 1380 ttctgtcggg gttaggttcg ccttttcgtt cttggcgaaa acggtgatca gaaatatcgg 1440 catgtggcgg ccgccaaaca cgtagatcgt tctgaacccc ccactcttgc cggcccctgc 1500 cctaggaatg cggacctttc tcaatccacc acccaaagcg atgccggcct ctggattttc 1560 tgcaatccaa gcaattgcag cgtcacgttc cgcatcggtc atgatcgatc gtgagcggcg 1620 ctggaactct ggcagttcaa ctacggtctg caggcttgtc atttaggtat acttcaatgg 1680 cgcataagtc aatggcgcac tacggcttct tgagctgctc aaggagcgca acagcgtgtt 1740 cgagggtttc accgaggccc caaccgttgg catctgctat tctgtaaaaa gattctatcg 1800 tttcaggcgt cgccttaatg ttgaactgcg cgtttcggcc agtcctacgg cgccgctgcg 1860 gcttggaaac agggggcgat ttaggctccc tgctcacgaa gccagacgcc tctgccgctt 1920 gttcagtgct gcggtcttta ggtgctatct tcggagcagg cgcgaagctg ctaagatcgt 1980 ttagtgcatc accaaaacca aggtttgcgc gttgcttgct catcactgat cctcttggct 2040 acgcttcagc tttccaacca cttcgcccgc aaattgccgc gcgttctcga ttgccttatc 2100 gacgttgctg acctgcgacg gatcaaggtc agacaaggtt ccgccatagt cgaacaagtc 2160 acgataggcc gcacgctcga caatcgatgt ttcgcatata tcgatgccac cgctcatgag 2220 ctgctcgtga acgtttttca gggcgcgtga gcgaaccgcg gcacttgttc gggtcagcac 2280 aacgcagtgc ggtatggcac gccgcgccat cttcgcttgg ttgctgatca atcgaatggt 2340 ttttgcgcca cctctcgcat ccatagacga gccctgtatc gggatcagta ccaggtctga 2400 cataccgata gcgtttgcga ccatcaggct cgcagtcccc tctaagtcga cgatcacgaa 2460 ttgagacgtg ccggatgccg cctcaatctg gtcaacaatg ccgtcttctg tcacgccgct 2520 tacgatggaa atattttcag gctttccggg taggctcgcc cattgggaga tccagcgttc 2580 tggatcggcg tcgatgattg tgacgcttgc cccgccctcc gaaagctgcg tggccaagat 2640 caaggctgat gttgtcttcc cagccccacc ctttggattg gcgaatgata tgacaggcat 2700 gtgtgatcgt ctccgcgctt ttaggtacaa tatcagatag ctaacagata tccatatata 2760 agttacagta tctagtagct atcggatatc atacaatact aaaatggtac tatttggtat 2820 catatgccaa atatcttgac ttcatgacca cttggttctg cggcaggtcc gacgaaagag 2880 cccggcgagg acgcttactt tttctttggc tgccattttt ttgcgagctt taaaacgtta 2940 cgacggagct catcgccatc ggggatatgt gggtcgaatg gaccttctac ccattgggtg 3000 agttcctcgt gttcagggtg tgcggcgtcg ccgattgcct cgatgaagtt ttcatatccg 3060 ggcaggccgc cgacgtcctc aggcggacat ctaccgacaa catcgacaag ccgtgggtag 3120 aggttttcgg gaatcggatc gtcgacgctc tccgtctcaa tcagatgaac ccagtaatct 3180 ccgaaatcgt aaacatatcg gatcgggtcc gctacggcag tgttgatgat gtcagctagg 3240 atgtccgctt cggaggtatc ctcgtagacg cggatcagtg cgaaattggc cttgagcgcg 3300 gccttgtcct tcaacttgtc ccgttcaatc actttgcgcg cactggcgct gtttttgccg 3360 tggtggcccg gcccgtcata ttcgatgatg gcgcgcacaa agccgtcctt gctccaaatc 3420 acgaaatccg cgcgctttgc gacaaggccc cgccacagat tgccttgagc ttggatgaag 3480 gcaccgtatg gcacctgcgg cgagagaata agatcaggtc tgttctcgtg ccgccagcgg 3540 ttaagcaggt tatagagccg aaattcactc ttattcatca attgcttgct gctgtagagc 3600 gattttcgcc cttcaacgaa ccagtagaca cccccgacaa gggcgatgac caccagaaat 3660 ccaattatca ttgtgaaatt aatgtcggtc atgactgcac tccctcaagg ccgaagtcag 3720 agattgcatc ttgacgcaga gcgacgagtt caacgccgtc gatgcggttt tgggtggcgg 3780 tagccgggct ggcgcggata cgcctaccgt tggcacggtt tctaggcgac atactgaggc 3840 aaaccggatt tccagtgggc tgcgtgcttt gttgtaagcg cgccatttca tccccccgag 3900 ggcaggggaa cggctcacaa cttccaaagg ccccgaaaaa ttctcgcgat gaccccacca 3960 cgggatttgt gcaacacaat cggcgcgcgg gacaggctca tatcaacttg gataagcgcc 4020 gcgttttttg caaaatgcga gaaagtgctt atccgcatct ttcacgttga tggccttgtc 4080 atgcacccaa gaacgccact cttcttcaag ggagtaaaca tcccacccag gagcaagttc 4140 acgggcagta tcgcgtgtgt cgggatcgtt gaacggcaag gcctgaaaag tggttgatgc 4200 tatagtttcc accaccttgg gtcggaagac agcgttctct ccttcgatac tcatgctgta 4260 gtcagggaag tggtcgtgcg cggtgtcatc ctcaatgatc ttcgaaagca ggcgacggaa 4320 ctctttttta gttgagccgg aaccgcactt gtttcgcaag agctccaagc tacacatcca 4380 ttttgactgc gcgccacaat gctttcgccc tatctcatac aaccgccttt cgaggggctt 4440 tctgagcagg aaataccccc ggcttagagt gaggacatgg ttgttctcga tcgcatcaaa 4500 gacccagtca gagagcgtta tttcgacatc aagcattcgg ccgtcgcggg ttacgcgcac 4560 gatctcggct gattcaatca ggccaaatac cttgaagtat tctttcccac cttgacgaat 4620 attcgtttca atttgggttc cctgcagccg ccgaagggca tctttgagca gctgataacc 4680 ctgaccggat gtctgacggt tcgttgccac aagcagatca taagccttga aacgcatcga 4740 tctacttatt ttctgcccct cattaatggc cgccatgcac tggctgatgc agtagatcag 4800 cacatcacgg tcatgaacgg tagcaaggcc atagcgtgaa ggggaaacct cgatccaatt 4860 gtcgttgttc tgatagcggc gtggcttcat gtccggcttt gtagacaggg tgaacatagg 4920 gtgctccatc gaagccatat cccccttggg aaccgcatca acgatgtcgc aaacgaaaag 4980 gtctttttgt ggatgacgat ccggcaaaag cggtgatcgt aggttcgtca tttcacacac 5040 tcccgcgcca agtaaaaatt cgtcatttca cacaccgtac aagactatcg tcatttcaca 5100 caccactcgt caaggctcgt catttcacac acccaaggag gctgtggata actcgagccg 5160 ctatcgtcat ttcacacacc atctttcgtc atttcacaca ccatctttcg tcatttcaca 5220 caccaggtgt tatttttttt atagttatat caattgatta cgagctgttt tcggagctgt 5280 aactctattc taactctatt ctaactctca tagcttgcca aaatggcacc tcatatcccg 5340 gatatccggt ttcattatga aaccaatcaa caacatttac ggtgtttttt gaggagcaac 5400 actgtcccaa cgcaggttca aaccgatcac gccgaatctg caaagaaagg ggcagtgcta 5460 tgttcatttg gtcactcgag gatcacccga accacaggta agccctcaca tgctatgttg 5520 atacctccag ggaagtagca aaggtcctca cgaccaccac ctgcaggatc tccatcagcg 5580 aagacaactg tcgcattgaa cgctggcgcc ggcttgtacg tactcggtat gggtgagaag 5640 tcaggcatac ctgggagaac acgacgaagg acaccgtggt tctcgaggat cccgtagaag 5700 atatcgggcg tcccattatc cacataagcc gcaccatagc gatcctcccc gtagagatac 5760 tcacggaaag tttgcatatg ccgccagcga gagcgaaaaa ggggaacccg aatagcgaga 5820 tcctgatcgc ccaaccaaat gtcagccatg atgaacatgg aatattgatt gttccacgac 5880 gacaaagccg caaattccat gaaccgtctg accgcatgca ggtcagagcg ctttggctcc 5940 atacgatgaa gagggtttac atctcgttca acactgcgac gatccgcctt cttcaccgcg 6000 ctcacaaatg gactgcgatg taatcggccc tcaaggtagc cttcaagctg ttccgcatag 6060 gcctcatcac gcttagagac actctccaag aggcagtcag aaacgacctg acgccagtac 6120 ccattcccac gcgctgtgtc gtgacctatg acgggcctct gattgtatcg cagggagagg 6180 tgcgtaaccg gagactcgaa gctctgagac cattcccaga ggctctgagc catgggatcc 6240 cacgaaggaa ggtccgtctc tggttgctct atctcgccaa aacctaaatc ctgcagctgc 6300 acattcgcag gatagacttt gaacatcgtt cgcaccttgc cgaggtcatg aacaaagccc 6360 gcgataaaag ccgcaagatc gcccgcctct gaggggacgg ccttactgaa ctcactgttg 6420 aagaaatcta ccgcaagccc agctgtttcc agggagtgat agaatagccc accggctgtg 6480 cggtggtgat gtgtagatga agctgggagg cacagcatat agtcgtggcc tccgcgcaac 6540 atttgcctca ccaagagtgt ttctttgtga gagaggaagg ggagtttttt gagctgctcc 6600 ttgatcagct gctcatagct tccaaacgtc acatgcgggt gcaatatccg gaactgagcg 6660 ttgcgcaggt agcgcccgta agacaggatg atgcgatcgt cataaaacat tggcggtttc 6720 ctcaatcgat atttgctctt agttggtagg tgcccgtatg ggcgcggcac aggtttctca 6780 gctgtgccag gcggtcagtg cgatagcgga tccaggcgaa gttggcctcg cacagaagca 6840 gttctctacg gatggcgtct ggaaagcgcg cgaagatccg atcgctgtat ttgtactttc 6900 ctgacatgcg gatctgttcg gttggggtga acttgtcacc tcccatccgg tagcgggcct 6960 tgccttggaa tttgacccag taaatgcgcg ggcccaatgt gtcctcgcgg attcgaacga 7020 caatgcccaa attcagcgcc tcggcgcccg cgtccatttt gcgataggcc ttcagggctt 7080 cgacagcccg atcctgccaa tagcgcgccc atccttccaa gtcataaccc atcgctggaa 7140 tggatcgcaa aagctcaggc tcccaatcgg agtcgaaatc atgaaagcgc acttcgtgcg 7200 aacagctctc ttgggggtgt tcttcagaca tgctgtgctc cgaccaaatg gcgcgccgac 7260 caaaacaaga aggttgggtt caatgccggg atgtttctgt ctgcgttcag ataaagccca 7320 tgtttcctaa tgtgacggga tggtagctcc gtcacattag ggggcgacgc attttgcgcg 7380 gcacatggcg cggagagccc gcaggcaggc ggctggacgg acgaaaatgg gcaggggccg 7440 cgctgagatc ccttgaaagc ccatgggcct aatgaatggc gctcacacag cctgtcatgc 7500 gtgtacttta cggggaggtc tcgcgatgag gtgagcgccc gcggtctctc gtcttggtcg 7560 tgtctgaccg tatgtgtccg tcgaataaac gtcatgatga ggtcctgaga taagaacgaa 7620 caggatcgtt gacggagggc atccgtctga ctgtcgtctg atgttgatgg aaagtcgccg 7680 gctctaggcg gccggtctca atttggtggt gagcagctct ttgcgctggg gggcattacg 7740 atgcagatca gccgtccagt ttaaatcggg aggggtgagg gaggtcgggg cggccgtgca 7800 caagacaatg gcatccgaaa gccggtcaat cattgggcgt atttgtccta cccatctgtg 7860 ccaattcata gcaaaaatcc cgaaccatcc gtttcgtcac acctattggc tagggggagg 7920 tggggatggc cgtcaatgaa aaatatcagg gattgagttt tataattaac tgaaattgct 7980 aaatattttt ggaaggaata ttccttccgt agctcgtcta ctcatttatc ccttttcgaa 8040 aaaagactta acagcgggcc aactgtttcg catgatgcgg acaagaaaaa ttggctatca 8100 tgggagtgtt cgggtggcgc gtgggactaa gacagccaaa agcaaaaatg ctgatccaat 8160 tgtagcagaa tgcttaaaac tgattcgcga agacagtggg tatgagagag atgaattcgc 8220 cgaacttcta ggggtgcagc ataaaacgta tcgcaactac gaaggctgta tatatccgct 8280 accgttaaaa gtggtaaaga cgatacgtga gaagttgggc tatgatcttg cggatcctga 8340 tctgacttca gacgcgatca tcaccaaaat tgcagagcag cgacacgatg ttgcagccgc 8400 tcctgatctt gccgcgacag agcaagtagc gaagggcgtt agttgccctc agaggatacg 8460 tacctgcctt caagctttcc ggcaggaact cattggtgtc cagagcaagc gcaagcatga 8520 tattcgagac gcagtttttg tcggagcagc tgcattgttt gctttctgct tggtggtact 8580 acgcactgaa ccgcaaaata taaggttaga gtctatctat acccttatgc tatccgtgtc 8640 cttcttggtt gcggcgtcaa ttgtcccgtt tcaggctata catatgatcc aagccgccta 8700 tcggtcgcga cgttgaccaa caatactagc cctccccgta tgcccgtctc acagggaggg 8760 ccaaagctac cttggcgaac gactgtcagt gcacgaaacg ggtgattaaa cgcaaagtgc 8820 tgactttttt tcccccgata ccgatggttc atcaaagata tatcgactga gcagatcggt 8880 gtccggggca cgcgtgtgca ttgcttctag gccagaaact gtgaagttct cttccacgtc 8940 ggattccggc acaaggaggt aatgccaggg cttctcaccg ctctttgcac cggcttgcgt 9000 cgcaatatgg caccacaggc ttgccgcatc tgctttacgc agaacctcgg tggcggtcat 9060 ttccgattgg cgttttacct cgatgatcag cttgcctgta tcggtttcga caacgaaatc 9120 gggctgatac ggcgcaccgt gattatcgaa aattttgaac tggttggggc caggtttcat 9180 ccaaaggcgg accgagctgt ctttttcaag gatgatcgcc agcttgcgtt ctggattaga 9240 gtcgaacttc gcatactgat agcagccctt cacgaagcca gtgaagatat acttccggat 9300 ctcctgcttt tgatctggcg gtgttttgaa atcccggatg aagctcgtac cggcggtatc 9360 gaaggtctgc ggacgcagct cgccgaatgc agaggtcaga gaaacacggt agttcgtgtg 9420 ctcacgccac atattctgct ttatttgcga gaaaatgctt tcggccatcg ccttcgcatg 9480 cccccggacg acactgcgag tttgctcatc gtcgtccgcg aagcggttgc gggtttgggt 9540 gactgcctgc ccagccagat catacaagat ggcagcatgg gcgtcatagt cgatctcagg 9600 gtagtcgatc agccgcgcga cgatgtaatt ctccagccgg tttgcggtgt cccccttatc 9660 ctcgctggaa atacgcgatg tcttctctgt acgcagagct tggatcatga gctcattgga 9720 caacggctgg aagttccagc tcttcatgtc caggtcgaag cgcttgaagc cgaaggaaac 9780 ttgttgctgc ggagtgatcg tgagagcagg gatcgcaagg gtccgctcca cgaaattctt 9840 gcacagctcc tgagcgaccg cgacggcctt ttccttcgtg atgctgggaa ggaaaccttc 9900 ttcgggtttc tgtgctgcaa tggctgcttc cgcgatgcgt tcgatcacct tggggtcgtt 9960 gagatcgcgg atcgaggaca cttccttgct gagctggggg atcacaacac tgagcaccgt 10020 gcgggcgacc ttcagctctt caggcgtgct gaagctgaaa ggcgcttggg tcggtgccgc 10080 agacgtcggc ttggcgacta gctgcacagg cgcgtccgaa gctttttcgt catccgcgac 10140 gacgactgtc ggctctgtct tggcctctgc ctgagcaagc atctggtcga ggatggaggg 10200 tgccgagacc gaaacaggct tcgagggcgg cacgtcacca ccctcaccaa tcgtcacctg 10260 cttcagcttg cgcgtgacgc cgttctcttc cttggctttc tcgatcagct cgttgaagcg 10320 ttcgtgggca atcacggtca gcgtgtcaac cacttcgacg ccggttcgct tgccataggg 10380 caggcgcaga ccgcgaccga gcgtctgttc ggttaggatg tccgaggccg aggcgcgcag 10440 cggaacgatg gtgaacaggt tcgagacgtc ccagccttcc ttcagcttgt tgacgtggat 10500 gacgatgtcc gtgtcgccgg ccttctcgat attcagcaaa cgctgtgcgt tctcgtcgct 10560 ttcctcgccg gtcagcttgg agtggatctc ggccacgcgc cccttgtagc gtccgccgaa 10620 gaactcgtcc gactggacga actcattcac ctgccgggca tgggtcgtgt cctgggtaac 10680 aaccagcatg aagggccgca ccaccttcac gtcattctgg cgggcatagg tttccagcgc 10740 caccttgacg tgctcgtggt agtggatgcc gtcttccagc ttgatccgtt ccagcgtgtc 10800 ttcgtcgacg gtcttcgggt tgaagttcgc gcgggtgccg acagccggtt ccttaacata 10860 gccgtcttcc atcgcgtccg gcagatcgta gcggtagacg acgttcttga agggctgcga 10920 gcgcgcgcca acggtcttcg gggtcgccgt cacctcaagg ccgaggatcg gtttcagctc 10980 ggcaatcgcc cgcgccccgg ccgaaccgcg atagcggtgc gcctcgtcca tcagcaggac 11040 caggtcatcc agttcggaga ggtaggagaa gtagctctcg ccgatatatt cctgaaggcg 11100 cttgatccgg ggggcgttgc cgccgcgtgc ttcggagttg atcttcgaga cgttgaagat 11160 gttgatgatc gcgccttctt ggccgaagag gtcggtgcca cgcacaccgc ggccttcctc 11220 gtagttctcg gcgttgacga tcagcggcgc gttatgggca aagacttcga tgccacggaa 11280 gacgtatttc gggctcgacg gctggaagtc ggacagcagc ttttcataga tcgtcaggtt 11340 cggggccaga acgaagaagt tccggctttt gccgatcatg tagaggtagc tgatgaaggc 11400 gcccatcagg cgcgtcttgc ccacgccggt cgccagcgca aagcagacgc tcgggaagtc 11460 gcgctcgaac tcctcgaagg tcgcgtcggc caggtcgcca tagacctcgc gcacggcggc 11520 gcgggccatg tcgatatcgg cttccttcgc ggggccgacg aggtcgacga tgtcatcgag 11580 ccgccgcagc gcctcggcct gcggcttgcg cagcgacagg cgttggttga tctgaaggac 11640 ggcgcgtttc gggtcggtcg tctgcatggc tcagttctcc tctgcgccaa acagatcggg 11700 tgtgtctgcg gctttggcct tggccgattt cttcggcgcg ggggtgtctt cgatgtcgtc 11760 cggctcatcc tctgccatcg gcagggcgtt gatcttcaag gaatagtcat cctgccccca 11820 ttcgcagcgg tcaagcacca cgcgcgggat cttcttcagg gtcaggttgc tcaggctttc 11880 gccctgggcc tcataggcca tgcagcagat cagcaggctg cggtcctcgc ccacctcgtc 11940 cgagatggcg cggagctgtt cgatggtcag gctggccgtt gtcacataga tgaaggcgtt 12000 ctccgaagcc tgaccgtgca tccagtaggc ctcggttgag ggcgcgtaga catagttgaa 12060 atgcttgcac atggcctcgg ccagcatctc ggcattgtag tccttggaga tgacccagtt 12120 gccccagaca tccttctgaa gcagagatgg cgcaagacgg aagaaacggt agccgccgcc 12180 gcctctccag tttgtggcct cggtcacgcc gcccttatcg gtgccgttga tgactttttg 12240 cagccgaggc gcgacatggg ttttggcatg gtcccccagc tcgaccatga tccagcggcg 12300 gcccatcttg tgggccactg cacctgtcgt gccagaacca gcgaacgagt ctatgacaag 12360 gtcgcccggt tgagttccta tctgtataat acgctccagc agtttttctg gcttcggtgt 12420 agcgaacgta gcgtctccca aaccaagaag cgtgcgcagg tcattggttg cttcacgggt 12480 tgtaccagct tcttcaccga accaaatgct ctcaggtacg cggccttctt gatcgcagag 12540 atagatcttc cgaagaacac gcgtgtcctc gtttaagaat gtaatttccc cggtctctac 12600 tttgctggca aaggtctcct ttgaccacct ccaccctttc tctggcggcg gaatgatctt 12660 cccagaaggg gtagtaacat cgaacatgag attttctctg tagttcggac tacgtacatc 12720 tcctgctcgc caaggtcctt ttggatcttt gtctggatta ttatagtttt tgttgtgctc 12780 aggctttctg ggcagaaggt tgcgcgagaa cgcctctgtt ttccgatatg ctagaacgta 12840 attatgatgg agactgacgg tcttggcgtc atttttccct tgaacgctat gctgccagat 12900 gatcgagccg aagaagttcg atcggccgaa aatctcgtcg cacataacct tgaggtaatg 12960 cacttcgttg tcgtcaattg tcatccaaat ggagccgtct tcagaaagta ggttccgaag 13020 aatttctaac cgatctcgca tcattgtcag ccaaagcgag tgctccagcc cgtcatcata 13080 atgctcgaag gccgacccgg tgttataggg cgggtcgatg aagatgcact taaccttgcc 13140 ccgcacggca gggtcggttt ccagcgcctt gagtgccagc aggttgtcgc cgtggatcag 13200 catgttgtcg aagatgtcgc cctcgcgccg cgtgctggcg tgatgcgaga attccggttc 13260 ttcgatcaga atgcggggct caagccgcgg gcggttgttc ttgccgatcc aggtcagttc 13320 aagtttggtt ttggcggcca tcaggcaagg gtccatctta cagtgaatag ggtcgtcatt 13380 tcgggggtca ggttcagctg atctgcgatg tcatcaagca tccgctcgcg ctcagcgtcg 13440 atttcgcgca ggcgggtgta aagctggtgc tgaaggtcat ccacctgccg ctgcaaggcc 13500 ttggcctcgc gttgcagggt gaccttttct tccagcccga tcgtggcacg ggccagtttc 13560 ttcttctcgg tcgcttcctt gttcagcgcc ttgatctgct ggtcgaagga caccttggca 13620 tcctcgcgcc aggcatcgag ccgctcttcc tcctcgttga ggaaggagct gagccgatcc 13680 tgtgccatgc cgatgatcgc ggtctggcgc gcgttcaggg tttgggtcag gtcgctttcc 13740 ggcagggtgt ccgcgccaag gccctcggtc gtcgccggga catagagcat ccgagaggcc 13800 gtttccgggt cgatggctgt tccgccgtcg ctgaaggctg cgaagaccag ctcgtcatag 13860 accttggctg gggttttcag tcggacccgc gccacgcgca tccagcccga ctgccccctg 13920 agctgggcca cgtcgcccat gttgccctga taggcgctat agtcgaggcg cagcatggca 13980 gggaccagat cgcgcgactt ggccttctgg accagctgat cggccagccc ctcgtcgcca 14040 aggcggaaga aacgccagcc gcgctcgtca gcctcgggcc attcgctgga ccaggtttcg 14100 cccccgtaat cgaagcgctg cgcatggtcg tcgtggaagc gggcctccgg cagctcggcc 14160 cgcgcgacac caaggagggc gcgcttgaag tcaccgatgg cggaatgcac ggcgtctttc 14220 cggccaagaa gccgctcgat caccttgtcg tccatctcgg ccagcagctg atctcggacg 14280 ttcttcttcg cctcgtcgat ctcgacgctg aactcttcct gaaggcggtc gaaggcggcg 14340 tcgatctggt ccgtggtgcg gcaggactgg acgatgtcga ggatgcgccg ctcgatgtcg 14400 acgccggatt caatgacgcc cagaacctcg tcggaggagc cgaacacacc ctcgaaaagc 14460 ttgaacttct gttccagaag ctggtggatg cgcgcttcag cgtggttctt ccggttcagg 14520 aagttgatga cggtcacgtc gatcttctgg ccgtagcgat ggcaccggcc aatgcgctgt 14580 tcgacccgct gcgggttcca gggcaggtcg taattgatca gcagggagca gaactggagg 14640 ttgatgccct cggcgccgga ctcggtcgcg atcagaatgg tgcgatcgtt gcggaaggca 14700 tcgacgatgg cggccttcat atcggcggtc ttggagcccg agaccacatt ggtgtcgccg 14760 tgcttgtcga gccaagcctt gtagagcgct ttgctgtctg cgtccgagtt ggagccattg 14820 aggacgacgg tctgtccctc gaagccgctt tgttcgagca gatcgcgcag ataggtttgc 14880 gtccgcacgg attccgtgaa gatcacggcc ttgcgctggc cgcccttgga gacgatctcg 14940 tcgagcacgt tcggcaggca gtccaggagc gccttgcctt ttgcgttgtc cgagatggat 15000 gcggccagat cgcggtactg ggtgagccgc ttgatttccg cttcgagctg agcgggatcg 15060 acgcgctcag catcttcggt gtcgtcttcg atcgcgtcgg cttccgaacc ggaatccgcc 15120 tcgcgccagt cctcggcttc gtcgctgaaa ccatcaaggt catcgagcgt gtctgccccg 15180 acaacacgct tcgcctcaag cctgcggatc atcttgtcga gggtctgcat cacggcgaag 15240 gaggaagatc cgaggatctt gcgcagcatg agcgtcacaa gatgacgccc gttctgcccc 15300 aaggcgatgg tgctggggtc ttggagatac tcggaaacct tttcgtagag atcggtttcc 15360 agtcgaccag gcgtgaagtc gaaggtcttg gggagacggt tggtatagtt gatcaggcca 15420 gcgcgttgga cttggcggcg cagggttcgt ttgcagatag gctcaagacg tttcgcgagc 15480 agggcctgag aggtcaggcc ttctcgtccg ccaaactcgc ttcggaaggc ttgttctgaa 15540 ccgaagtagg tttcgtcgat gatgctgatg agtccgtaca gctccatcag gttgttctga 15600 agcggagtcg ccgtcagaag gagtttctga cgcccagcca gtgcctttcg cagaaccgaa 15660 gcccgagagt tttcggcggc cttgtagacg ttacgcagct tatgagcttc gtcgaatacc 15720 acgaggctcc aaggcgtgcg tcgcagagtg tctgcaattc gtgcggcgta ctcgtaggag 15780 acgatgataa tgccttctcc gcgccctacc gggtgcggag tgccttcgtt ctcaaggtct 15840 ttgacgcgct tggcatcaag aatgaacgat ggcagcgaga atttttcacg cagctcggtc 15900 gcccactgct tgcgcagaga agcagggacg atgaggagaa tgtttctctc acgttcccac 15960 cagcgctggc taatgaccag cgcagcttcg attgtcttcc ccaagcctac ttcatcggcc 16020 agaagaacgc ccttcgacaa tggtgagcgt agggcaaatg tcgcagcatc cacttggtga 16080 gggttgaggt ccaccttggc tgccgagagg gactgcgtca gagcgtcttc ttcctgaatg 16140 ccttcggatg tcaggtggtg cgcaaagaac tttgattgat attcagaaaa ctggaagagc 16200 aataggtcag gctccaaggc atattagtta agggaaggtt gttctaaatg ctgtcaagtt 16260 atggcctgcg gcttgatcgg ccagcacgaa ggactccctt ctgcccagga tattgggcct 16320 tctcgttatc acgccactga aatgtcacct gtactgcgat accatgcttg tcggctattc 16380 tggaaacttc cttccaagcc tcctgggctg attgcccttg gatagagatg ccgaggtcag 16440 ggtaatatcg gaatccttcc tcttcgtagc acgccgtctt ggtaggaatc tgtatggcct 16500 cagctaggtg atctccggat aggcccttag ccttaagaga actcactgcg ccgagaagaa 16560 ttgctgccca gtttggcttg tcgatcttct tgtgatcaac ctccgcagat aagacttttg 16620 taaatgaaag gcctggagtg gatttgaact ccatgaagct gctgatttcc gacgtgggat 16680 caatgctgat ctcgatgtca cgctctaggt caagcgctgc catcttctcc cgtaccagca 16740 gcatgatcgt ttctgatggg gtttctgtcc ccatccaagt cgagatgcac ttcagatcga 16800 caaaagttgg gtcgttcagt ctgactacgg gcataggcgt catccttcta tattcatata 16860 gaatatgtta catattcaat attatggtgt caatgctagc gtttgattgg caacctgtcc 16920 taaaacggtc gtttttgggc atagtgggat gaagtggtca aaatataggc cttgataatt 16980 ttgaacatgc ggctactttt ggacagggtt ttttgacacg gagggccata cattgacaga 17040 gcaaggccgt gtttttgcct atgtacgtgt ctcgacactc gggcagacag tcgctggaca 17100 gatgcaggaa attgcggcgg ctggctttca gcccccagcg tatcgtgtcg tttcggaaac 17160 catctctggc agtgtcccgg cgatgcggcg accagagttc gcgcgcctag ttgatcgctt 17220 ggaacctggc gacattctag tcgtgtcgaa gcttgaccgt ctcggacgcg acgcaatcga 17280 cgttacggag acagtcgctg cgctctcgca gatacctgtc cgagttcatt gcctcgcttt 17340 aggtggcacg gatttgacta gctcgtctgg acagttgacg atgaacgtcc tcagcgccgt 17400 tgcgcaattt gagcgcgatt tactccgtga gcggaccagt gcaggccttg cagctgcaaa 17460 agccaaggga aaacgactag gtcgtcctaa agtgctgacc gaggataacg agagcgaagc 17520 acgagcggct ctcgccagag gggaaactgt ttctgggatt gcaaagcgtt tcaacgtcag 17580 tcgggcgacg atcggccgcc taagagatag ttaggctatc ttgcctatgt cgtcgtgacg 17640 agtcctccaa atacacggat ggcttcacgc tcccatgcac gattaaacca agtaacatgg 17700 cggcttactt ggtccttaat ctcagggcat cccatgatgt ccgctgcctt gtaagcatac 17760 cgcaagaaat ttcggcgttt tccaagatgg gtataacgcg ccaggctttc tcgcttgaat 17820 acttcaaacg acggtacgtt tttcgccttt gcggcgatca tcttggcatg aatgccccgg 17880 cgcatttttc ctcggtaggc atcgagtgac gaaggtcgga tcttcgtttt ctgcccgtcg 17940 aaggtgaaac ccaaatactg gataggtgtg gcagaggcca acagtccgtc cttgaagtcg 18000 gcagtgtctg ttttgtcgat ggacatggaa aggcaaaaat ctgccagcat tttctctact 18060 actgcaacca catgatgcac cttcgcgcct agaggcagag tgaccgcgat gtcatcagag 18120 tatcttcggt aagatccccc tgccctagag caccaagcga tcatttcacg gtcaaaagtt 18180 cgaagataga tatttgcata caggccggac accggggtgc cctgcgggat accgaacgtc 18240 tggtcatgct tccgaatgag gccatccttt cggcctcgga catgatccga aaagtctgaa 18300 ggtgagcata tccttccgtg cccatttcgc tttcgaccaa gaagtttgtc tagatcttca 18360 gtctcaaccc atgagtagcg ggtcacgttt ttccaaacgc ttgcatggtg cccttccagt 18420 cttgtttcgc ctatcaaatc ggcgatctca tcgcggagca aggtgtgatc gagacagtca 18480 aagaatccgg aaatgtccat tgcgaagacc gtgcaatctc cacgagactt aatctcgtcg 18540 aacaacgcct ttgcatggtg aatattagtg ccgcccccac ggcgataggc tagcacggag 18600 tctgatgtgc cgtcgcgcca tagcgcccgc tcgtacattc tattgagatg tcccgcgtag 18660 gcttgcaggt aggctgcatc ctcatgactg gcgaagcgga tcggtcgctc tttcactttt 18720 acctcacggg cgccatcctt gttcctgaca taccttctgt tgacgtcagt aaacccaagt 18780 aaaggtagaa atctgtgagg cttatcttcg accgagaagt caaacgacag ctcccgatcg 18840 actaacggaa gatcgaaatg cttgtatttg cgttccttgg atatggccgg aagcacaaag 18900 tcgtcggttg atggatcaaa ctcgttttca gacggcaggt aaaacccggg catagcgact 18960 ccgttcgtca aatgcccggg ctattgaacc tagctcccat caacaggcac cgaggtgccc 19020 tccggtacga gaaaacctgg cgtggtatgt tgtaaccacg atgtgcgaag gatgcgccat 19080 gaccgaaatc acgaccaccc ggtgccgctg ggcgcgggta gcgcttgcgg gccttaacac 19140 tggtcatccg ggtccacgcc ccggggcaaa atatcaatta ggtgctacct aattgaatgc 19200 aaggggacgc accaagaata aaaggacttt ttcagcttta gtggcgcctt atagtggtca 19260 tctggaacca ggacaaagag acaaccttct aggtaagatc ctcgtaagtt cgtatcagtt 19320 acggtgtcag atagtagcag ataacgcacg gcgttctgct ggcggagggc gagagaaatc 19380 tggttcgctg tgagcggtat ttcttgccca caacgcggcc cttattccag tctagtacag 19440 gcttggcagc caattgccgc gactggcata gcatcgtgct ccttgattac atgctaaaga 19500 ggtactttga ggtcttagcg gtcattcgac gattttgctc agataacagc gtaagtaaac 19560 gctgccccaa gtttcaggat ctagcgccca acgatcttat cacaagagac catgttaatg 19620 cgtctattac attaggatgg ttcaaagttg ggttcgggat catctggtat tggggttcca 19680 aagaccaatt cgtgc 19695 4 4211 DNA Ketogulonigenium misc_feature pADMX6L4 4 gcgtggccct tcccgccgag ctggacgccg aagcccaagc ccggcttgtc cgcacctggg 60 cacgggacca cctggccgcc gccggcatcg tcgcggacat cgccattcac gagccgagcc 120 gcgaaggcga cgaccgcaac acccatgccc acatcatgac caccctgcgg cgtttcgacg 180 gggcaaccgt ggacggatgg gcgaagggtg ccgcccgcga cctgaacgac aaggcgttcc 240 tcgagaacct gcgggcatcg tgggagaccg cccagaacgc cgcactggag gccgccgggt 300 caaccgcacg ggttgaccac cgcacccttg ccgcacagca tgaggacgcc ttggccgcag 360 gtgatgactt actggccgcc gtcctcaacc gccccccgga acctcacctg ggcgtgtcgg 420 cccaagcgat tgaccgccgg gccgggcgtc cggtcagcaa ccggggccgg gccttggcgg 480 aggtcaggga gacccgcacc cggctcatga cggcatacga cacggcacgg agggccgccg 540 tgttcatcgc aacgaccacc gccgggcttg ttgaccgggt gtccggtgcc cggtcagaga 600 ccggacaccc cattgcccga atgttcggcc ttggccgtca tgcggcaact gttgccgaca 660 ccgtgccaga accgtcccgc ccatcgccgg acgacgatac cccgtccccg tcctgacccg 720 cctcaaaccg tgccgatgca gcggcctctt taagcccact ctgtgcctct tcggcggccc 780 tgtgctgtct ggccatccag aaatccagaa tggccgcacc acgcatggct tcggcctcgg 840 cgtcccgtgc ctcgtccaga cctgcccgga ccaccaccgc ctcagcggtt ccgtgcatgt 900 ccagatgtgc ggccaccatg tccaacacgg cggccctgac ttcctcgggc gacacctcgg 960 cccccatcca atcgtggccg aacttctcgg ccacgatgcg tgacaccgcc gccttgcaga 1020 aggtccgggc atactgctcc agaccgtatg cagccttgtc ctcgaccgtc cgagcgtcag 1080 gcgccttgat gaccctctga actgccattg atgccccttt ctcgacttcg attagtgccg 1140 caatttgctc gaccatgcta cgaacccaat ggacggtccc cagcagacgc cacgccccat 1200 tccgtgccag acctgcggcc ccggccttct tgtccgattt gggcgaaaac cggaactcaa 1260 cccgcacaag atgcgggtcc gcgtcctcga tggccaactt gccgtccgcc acccgttcaa 1320 ggtctttctg ataaaccttc acggacgcct cacccttgcc ccaatagaag gtccggcccg 1380 tgtcgctctc gatgacacgc ggggccgcca tcttggacgc cttggacatg cggcgggcat 1440 agtccagcaa ggcgtccatc aaaccctctt ggctatggtc catcgacacg tcagcacgag 1500 ccagcaggga gggaccaaag gcgttcaggg cctgcggggc cagcagcgca caacgaccat 1560 caccaccggg tatttcaagg ctcggcatgt ttctggcatg ccctgcccgc accgtagccc 1620 ggcgttcctc atcaatgggt gagtccgcgt aatgcaaggc acccgcgaaa ccgtccgtcc 1680 cgttgcccac gcgttgagtg tgcagaccag ccagcacaga ccagagacaa aaggcatccc 1740 gtgcctctct tgcctcttcc tcgcccatcc tgtcctcgta gtgaatgaca cgcatggcct 1800 caacgcgttc agtgcccgtt aagcgggcag cctcggcaac ctccgacggg tcagccctgc 1860 ggcgttcgcc cttgccgttc cgcccgttag gcaacgtgat tgtgagccag tcgaaaaacc 1920 acgccgtttg cagccatttt tccatgaccg ggctggtttt cttgcccttc cttcgcttct 1980 ccgaaggtgg ggggcacttt ggggcacaaa ccgccggttt tacgccgttc ggggcctgct 2040 tttcacattg aaaatcaggt gtttgccccg aactaccccc cctgttagca tgtggggggt 2100 tttgggcctt gcggcggacc tcatcgagag ccgcgaccgc ctgctgtgaa agggttttac 2160 cgccgtggac catcatcgcg tctcccgttg agggtggacg cggtctcaag catgttgatt 2220 cttgcgaatg acatggctat atctctccaa agttctgagg acgacgcggg gaccgcgaaa 2280 tcacagcgtt cgtccaggtt aagggctccg gttaatcccg gagcctttcc atttcatatg 2340 aacgacatcc ggaaaaatca acatattgat gccgctggac gatgtgcggc caagatgtgc 2400 tgcagtattc ccggcgggcg gaaccgttcc gatatttgaa gtctccacgc ttccgcccgc 2460 ctaatgtcgt tctgtcgttg aactttcata aaggtgaaac cctgctggtt tcccccggcg 2520 gcgaagccgc ctcccccttc ctgcctcaca aaaaaaggga aaaggcccgt ttcaaccgat 2580 cgacgcaatc tcctttcacg gtatccagat accttttctg gtgaagccat ccgaaacaaa 2640 ggccgcgagt cgtcacacct gggcttgacc gacgcgaatc actcggacag tgtccggtca 2700 tggaccacat gctgacccca aaacaggcgg cggcccgtgc gggctgcggg cgttcctcta 2760 tcatgagggc gttaaagtcc caatctttgc ccgctatacg cgataatgag aaccgctggc 2820 agattgaccc ggacgccctt gaccgttggg ccggtcacag gccggacaat gaccggtcta 2880 tgaccgaaca cggaccggcc acaccttcgg acacccaaac ggacaccccg gagaccttgg 2940 cacggctcgc cgttgccgag gcccggttga gtgatgccct gtcccgcgtc gaagatttgc 3000 agagagaacg ggatgaatgg cgggcacagg cacaagcctt gacccgtcag cccggttggc 3060 tggaccgcct actgggtcga acctgacccc ccatcgtcac ctttggcctc accagcggat 3120 tgcttgtgaa ccaaggccag cagttccttg acctgttcca cgctacccgc ttcggcttca 3180 atatcgccca tcttgacgcg gacctttcgg ccttgtctcg cagccaccca tccaccgata 3240 gcaccaatca cagccggacc gagggttgtg gccatcatcg tgaactcacc caaaaaagtg 3300 atgccaccag agccagccga gtccctgatg aacgctcgct ccgaatattt tatgtcctgt 3360 tctttcagga tggtgcggaa ctccccggca tcttctttcg ggaaaaccgc gatttctaaa 3420 tcactcatgg aatgctcctt ttgcccatga ggtaataatg ccctgccaat gagcaccgag 3480 caaatcagcc gggttgctct ttcgaaacgg tctcgggctt tggaagtgtc cacccctcga 3540 acagtgcccg gtctctctct ggcatcgagg caatcgccat ccgcagcacc ttgacccatt 3600 gaggatcttt tgcagcttgc ccgataaccg caccgcccag gacgattttc cgccgtgcgt 3660 ccttttttcg gtcctctgcc cgaagttttg ccccggcttg tcgtatctgg gcctctgccc 3720 tcgccttggc ctctttggcc cgctctaact gcctttctac tgaagttctt gccattttcc 3780 gtcctccttc atagggccta tatgaacgga gagggaacga agcaagaagt tctcaagaac 3840 ccgaagggcg cacttacaca aactctgggg agtttgtttc gggcgcccta ccgggggtca 3900 tctcgccgat ggggcgtctc cgacggtaaa aagaggaggg gccgccgtgg cgatatatca 3960 cctgtccgcg tccattatcg ggcgaagcga tggccggtca gccgttgccg cgtcagcata 4020 tcgggccggg gccgacatga ccgacccgga caccgggacg cggcacgact acacccgaaa 4080 gcgaggcgtc cgggcaacct tcatggagtt gcccgaaggt gcccccgatt gggccaccga 4140 ccgcccgagc ctctggaacg ccgtccacgc gaaggagacg cggaagaact cgcggctttc 4200 gcgtgagatc c 4211 5 1458 DNA Ketogulonigenium misc_feature replicon pADMX6L1 5 abgggccttg catccgatgg caagcaaaaa ctacccagtc cgtccgtagg cggggggtcg 60 ccagccctgt gggtgggcgc ttccccccgg cccgcaagcg ggcccggaat gggcattttt 120 tgcctgccct aagatcataa gaagggcaaa aaaaacatcg tttcaaaaca gcgtgttacc 180 acccccctat aggacaccag agtccggggt agaggactct ggtgtcctct taggccattt 240 atgtccaaga atgtgacagc cagccgagcg gaggtagagg actctggtgt cctatgctta 300 ggccatttat gtccaaaaac ttgacaaggg ccacattcct gccaaatctg tccagaattt 360 ggaaaaattc gccggatagt agacagtggc aaagcctccc cccattcccg caaagcgccc 420 gctcggcact tgggttcaaa ctgaccggga agcccacgag gcgtgggcga tactggcaaa 480 aaagcctgct gccagcgctg tgatgcacat tctgtgcgcc aacctcggtg agcataatgc 540 cgtggtcatc agccaggaca ccatcgccaa gctgtgcggc ctttccacac ggtccgtcag 600 gcgcgccatc gtcgatctgg ccgaaggccg atggatcgag gttcgccaac ttggcgcgac 660 cagccagacc aatgcctatg tcgtcaacga ccgggtggca tggcagggat cacgggacgg 720 actgcgctac agcctgttta gtgcggctat agtcgtgtcc gaggaggagc aacccgaccg 780 cgctgaactc gaccagcaag cccccctgcg acacctgcca cgcatcagcg aggggcagat 840 acccaccggc cccggcctgc cgccaccttc gcaaccgttt ctaaaagaca tggagccaga 900 cttgcccacc attgaccggg caacatcacc caactttgac cagcaggaac aggggtgaaa 960 aaggtggaca aactttccat acgcgaggcg gtaaaacact tcgatgtttc ccggccaacc 1020 ctgcaaaaag cccttaaatc tggcaagatt tcaggtgttc aggatggaca aggaacgtgg 1080 acaatagacc cctcagagat ggcaagagtt taccagccaa ggcaagatga ggtggtaaag 1140 gatggtggcc aagaacatga aaatttgtcc gccaagaaca cccctttaca tggtcaagtt 1200 gaggttctga aagagcggct tgcagatgct gaaaaacggg tggcgatagc cgaggcactg 1260 gccgaagaac gtggaaaaca catcgaggat ctacgccgga tgctgcctgc accggaagcc 1320 ggtcagcccc gccgccgctg gtggccatgg taaggtcagc tatgcgggac caagccgcag 1380 ctcgcaagtg cggcaacaga atcagacccg cttcggacag agagctcaag ctggtggaaa 1440 accgcctgtg aagctgct 1458 6 2401 DNA Ketogulonigenium 6 caccatggcg cgctgcagct cttctttgct tctgttggtt cttctctcgt caagaagctc 60 aggcggaaac cgcactataa agtgcagaac aggcttcttc gccgctttgt tttgcctaac 120 gccttttgtg tgcgcgtcat atgccgaccg aaggtctagc gtcttataaa cgagcggaga 180 ggcatctctt acgacgcgtt tcgcacttgt tttgtcttgt cgtttcgcgt gcttttcggc 240 tgctgacaat ccagccatat ctaacgcact acatctcacc gctgctttca ttttcaatcc 300 ctcatatatt accttttctg ttgtttttgc gaaaaacgca aaactcgctt tgcgtttttt 360 gttcggcacc tgcggcacct ccaaaaaaca cgcttgctct ccgagggtct ggcaggagcc 420 taagaggggg cattctgccc ctgatcgacc ccattgaggc tcgatctaaa cagacccccc 480 acaggggcgt ctgtgggccg ctagtgcggc cttccgccat cttcgagcca tctgatctct 540 gcaattagag atgcatggcg catctcccat tccagctcag atatcccgta tctgtcgatg 600 acggtgtcat acaattcgtc gagccttgct tcgcgctctt cttttgtcat gccagttgca 660 tcgcgttctt ccatctcctc gttctcctct atgatgaaat cattttcgtc ctcgtcgttt 720 ggcccgcttt ctgctgcctt gaccgcaaca gcgcctccgc gctctgcggc ctcgagcaag 780 ttcacgcgtt tagcgcgcac ttgcttccac ccttcattgt cccactccgc gacaatttca 840 gggccgcttt gctctgctgc atcaacttca gccatagctt catcgtcatc cagctcgacc 900 aaaccgcatt cttctttcaa gccttggctc cacaccaatt gccgcctacg cttgccgctc 960 gttgcattga aatattcgag ccaaagcccg tcatcgcccg cctgaagtag ctgccttggc 1020 gtgcgtcctt tgcgttttcc gctcttcgag cttgaaagcg tcaactcttc ggcagcgccc 1080 cacttcgcta cgtagtcgcc cgcattggca gccccgcgaa cgtcaaacgc cgcatcgttg 1140 ccccacatgc catacccctt cagacatgca cgccacgcat cgcctagacg ttgcatcaga 1200 tgcagcgctt cgctttcatc gccagctctt agcaagacaa tttcgtgaaa gtgcgggtgc 1260 cacccatttg catagctatg agtaatttca gttgatgtga ctgacccaac aaatggtaaa 1320 tcgcgccact cgcggcgctg acgcaaccgc tgtttcgcct tcttcatgtt ttggagaaga 1380 tcaaaaagcg aatcacctgc tttgtgctgg gctgtcagag ttatgagcac cggcacaaac 1440 ccgttgtcgc gcgcccacgc gagcaagtga ttcatttcag aacggcgaat ttgcgcgatg 1500 cgagcgctac aaactgcgca gccccacaca ttccggcact gtgctagacc tgaaaagaat 1560 gcccgacgcc cgccatcctc gcccacgttt tgcacgttta gctcaactgt cggagacact 1620 tttacgtgcc gacatttcgc aacttggtgg ggcttgtttt tgttcagatt caacagaatc 1680 cgagcggctg aacgcagatc tgcataaagc tgccgcctac tgaataacct gttgttttct 1740 ttgttttttt cattcggttg acccccattc tggtcaaccg atttacggta tataccaagg 1800 ggggtctgcc gaccccctga aaaagcgtca tcgccgcgcg cctgcgcacc cgcgttcttc 1860 ttcgatttct gaactgaatg tgatgctagt ttgtgagaca tggccgcaaa ccccgacggg 1920 tgcggcgact attctcttga ttttctgcgt ctgtgcgcat actatcctcc gtgttcatca 1980 ggctcacgcc tgatctgatt agggctcttg tctctgcttg tatcgtcgcc aaactatact 2040 ttaagcagcg tctagagcct gatgaacgat ctaagaagcc cgccccctga aaggcgggct 2100 tttttgatct gtgccagatg ttgttacatc ggcgctcaaa aatcaagttt tttcttgact 2160 ttcaataatt tgcattatgc acattattat cgtttgataa taagagccag aacagcacag 2220 aatagttgtg cgatagctat gaataatagc agatccatcc ctgtttcctt tcttactaaa 2280 tacaatgcga aactgctcgc atctgtttta tttagttgaa tcggaaactc caaatcggcc 2340 ggattcaaaa aaaatataga ctatctttaa agtagcaacg ccgccgctcg cgcgacggca 2400 t 2401 7 2029 DNA Ketogulonigenium misc_feature replicon pADMX6L3 7 agagaataag atcaggtctg ttctcgtgcc gccagcggtt aagcaggtta tagagccgaa 60 attcactctt attcatcaat tgcttgctgc tgtagagcga ttttcgccct tcaacgaacc 120 agtagacacc cccgacaagg gcgatgacca ccagaaatcc aattatcatt gtgaaattaa 180 tgtcggtcat gactgcactc cctcaaggcc gaagtcagag attgcatctt gacgcagagc 240 gacgagttca acgccgtcga tgcggttttg ggtggcggta gccgggctgg cgcggatacg 300 cctaccgttg gcacggtttc taggcgacat actgaggcaa accggatttc cagtgggctg 360 cgtgctttgt tgtaagcgcg ccatttcatc cccccgaggg caggggaacg gctcacaact 420 tccaaaggcc ccgaaaaatt ctcgcgatga ccccaccacg ggatttgtgc aacacaatcg 480 gcgcgcggga caggctcata tcaacttgga taagcgccgc gttttttgca aaatgcgaga 540 aagtgcttat ccgcatcttt cacgttgatg gccttgtcat gcacccaaga acgccactct 600 tcttcaaggg agtaaacatc ccacccagga gcaagttcac gggcagtatc gcgtgtgtcg 660 ggatcgttga acggcaaggc ctgaaaagtg gttgatgcta tagtttccac caccttgggt 720 cggaagacag cgttctctcc ttcgatactc atgctgtagt cagggaagtg gtcgtgcgcg 780 gtgtcatcct caatgatctt cgaaagcagg cgacggaact cttttttagt tgagccggaa 840 ccgcacttgt ttcgcaagag ctccaagcta cacatccatt ttgactgcgc gccacaatgc 900 tttcgcccta tctcatacaa ccgcctttcg aggggctttc tgagcaggaa atacccccgg 960 cttagagtga ggacatggtt gttctcgatc gcatcaaaga cccagtcaga gagcgttatt 1020 tcgacatcaa gcattcggcc gtcgcgggtt acgcgcacga tctcggctga ttcaatcagg 1080 ccaaatacct tgaagtattc tttcccacct tgacgaatat tcgtttcaat ttgggttccc 1140 tgcagccgcc gaagggcatc tttgagcagc tgataaccct gaccggatgt ctgacggttc 1200 gttgccacaa gcagatcata agccttgaaa cgcatcgatc tacttatttt ctgcccctca 1260 ttaatggccg ccatgcactg gctgatgcag tagatcagca catcacggtc atgaacggta 1320 gcaaggccat agcgtgaagg ggaaacctcg atccaattgt cgttgttctg atagcggcgt 1380 ggcttcatgt ccggctttgt agacagggtg aacatagggt gctccatcga agccatatcc 1440 cccttgggaa ccgcatcaac gatgtcgcaa acgaaaaggt ctttttgtgg atgacgatcc 1500 ggcaaaagcg gtgatcgtag gttcgtcatt tcacacactc ccgcgccaag taaaaattcg 1560 tcatttcaca caccgtacaa gactatcgtc atttcacaca ccactcgtca aggctcgtca 1620 tttcacacac ccaaggaggc tgtggataac tcgagccgct atcgtcattt cacacaccat 1680 ctttcgtcat ttcacacacc atctttcgtc atttcacaca ccaggtgtta ttttttttat 1740 agttatatca attgattacg agctgttttc ggagctgtaa ctctattcta actctattct 1800 aactctcata gcttgccaaa atggcacctc atatcccgga tatccggttt cattatgaaa 1860 ccaatcaaca acatttacgg tgttttttga ggagcaacac tgtcccaacg caggttcaaa 1920 ccgatcacgc cgaatctgca aagaaagggg cagtgctatg ttcatttggt cactcgagga 1980 tcacccgaac cacaggtaag ccctcacatg ctatgttgat acctccagg 2029 8 151 PRT Ketogulonigenium 8 Met His Ile Leu Cys Ala Asn Leu Gly Glu His Asn Ala Val Val Ile 1 5 10 15 Ser Gln Asp Thr Ile Ala Lys Leu Cys Gly Leu Ser Thr Arg Ser Val 20 25 30 Arg Arg Ala Ile Val Asp Leu Ala Glu Gly Arg Trp Ile Glu Val Arg 35 40 45 Gln Leu Gly Ala Thr Ser Gln Thr Asn Ala Tyr Val Val Asn Asp Arg 50 55 60 Val Ala Trp Gln Gly Ser Arg Asp Gly Leu Arg Tyr Ser Leu Phe Ser 65 70 75 80 Ala Ala Ile Val Val Ser Glu Glu Glu Gln Pro Asp Arg Ala Glu Leu 85 90 95 Asp Gln Gln Ala Pro Leu Arg His Leu Pro Arg Ile Ser Glu Gly Gln 100 105 110 Ile Pro Thr Gly Pro Gly Leu Pro Pro Pro Ser Gln Pro Phe Leu Lys 115 120 125 Asp Met Glu Pro Asp Leu Pro Thr Ile Asp Arg Ala Thr Ser Pro Asn 130 135 140 Phe Asp Gln Gln Glu Gln Gly 145 150 9 466 PRT Ketogulonigenium 9 Met Ser His Lys Leu Ala Ser His Ser Val Gln Lys Ser Lys Lys Asn 1 5 10 15 Ala Gly Ala Gln Ala Arg Gly Asp Asp Ala Phe Ser Gly Gly Arg Gln 20 25 30 Thr Pro Leu Gly Ile Tyr Arg Lys Ser Val Asp Gln Asn Gly Gly Gln 35 40 45 Pro Asn Glu Lys Asn Lys Glu Asn Asn Arg Leu Phe Ser Arg Arg Gln 50 55 60 Leu Tyr Ala Asp Leu Arg Ser Ala Ala Arg Ile Leu Leu Asn Leu Asn 65 70 75 80 Lys Asn Lys Pro His Gln Val Ala Lys Cys Arg His Val Lys Val Ser 85 90 95 Pro Thr Val Glu Leu Asn Val Gln Asn Val Gly Glu Asp Gly Gly Arg 100 105 110 Arg Ala Phe Phe Ser Gly Leu Ala Gln Cys Arg Asn Val Trp Gly Cys 115 120 125 Ala Val Cys Ser Ala Arg Ile Ala Gln Ile Arg Arg Ser Glu Met Asn 130 135 140 His Leu Leu Ala Trp Ala Arg Asp Asn Gly Phe Val Pro Val Leu Ile 145 150 155 160 Thr Leu Thr Ala Gln His Lys Ala Gly Asp Ser Leu Phe Asp Leu Leu 165 170 175 Gln Asn Met Lys Lys Ala Lys Gln Arg Leu Arg Gln Arg Arg Glu Trp 180 185 190 Arg Asp Leu Pro Phe Val Gly Ser Val Thr Ser Thr Glu Ile Thr His 195 200 205 Ser Tyr Ala Asn Gly Trp His Pro His Phe His Glu Ile Val Leu Leu 210 215 220 Arg Ala Gly Asp Glu Ser Glu Ala Leu His Leu Met Gln Arg Leu Gly 225 230 235 240 Asp Ala Trp Arg Ala Cys Leu Lys Gly Tyr Gly Met Trp Gly Asn Asp 245 250 255 Ala Ala Phe Asp Val Arg Gly Ala Ala Asn Ala Gly Asp Tyr Val Ala 260 265 270 Lys Trp Gly Ala Ala Glu Glu Leu Thr Leu Ser Ser Ser Lys Ser Gly 275 280 285 Lys Arg Lys Gly Arg Thr Pro Arg Gln Leu Leu Gln Ala Gly Asp Asp 290 295 300 Gly Leu Trp Leu Glu Tyr Phe Asn Ala Thr Ser Gly Lys Arg Arg Arg 305 310 315 320 Gln Leu Val Trp Ser Gln Gly Leu Lys Glu Glu Cys Gly Leu Val Glu 325 330 335 Leu Asp Asp Asp Glu Ala Met Ala Glu Val Asp Ala Ala Glu Gln Ser 340 345 350 Gly Pro Glu Ile Val Ala Glu Trp Asp Asn Glu Gly Trp Lys Gln Val 355 360 365 Arg Ala Lys Arg Val Asn Leu Leu Glu Ala Ala Glu Arg Gly Gly Ala 370 375 380 Val Ala Val Lys Ala Ala Glu Ser Gly Pro Asn Asp Glu Asp Glu Asn 385 390 395 400 Asp Phe Ile Ile Glu Glu Asn Glu Glu Met Glu Glu Arg Asp Ala Thr 405 410 415 Gly Met Thr Lys Glu Glu Arg Glu Ala Arg Leu Asp Glu Leu Tyr Asp 420 425 430 Thr Val Ile Asp Arg Tyr Gly Ile Ser Glu Leu Glu Trp Glu Met Arg 435 440 445 His Ala Ser Leu Ile Ala Glu Ile Arg Trp Leu Glu Asp Gly Gly Arg 450 455 460 Pro His 465 10 342 PRT Ketogulonigenium 10 Met Thr Asn Leu Arg Ser Pro Leu Leu Pro Asp Arg His Pro Gln Lys 1 5 10 15 Asp Leu Phe Val Cys Asp Ile Val Asp Ala Val Pro Lys Gly Asp Met 20 25 30 Ala Ser Met Glu His Pro Met Phe Thr Leu Ser Thr Lys Pro Asp Met 35 40 45 Lys Pro Arg Arg Tyr Gln Asn Asn Asp Asn Trp Ile Glu Val Ser Pro 50 55 60 Ser Arg Tyr Gly Leu Ala Thr Val His Asp Arg Asp Val Leu Ile Tyr 65 70 75 80 Cys Ile Ser Gln Cys Met Ala Ala Ile Asn Glu Gly Gln Lys Ile Ser 85 90 95 Arg Ser Met Arg Phe Lys Ala Tyr Asp Leu Leu Val Ala Thr Asn Arg 100 105 110 Gln Thr Ser Gly Gln Gly Tyr Gln Leu Leu Lys Asp Ala Leu Arg Arg 115 120 125 Leu Gln Gly Thr Gln Ile Glu Thr Asn Ile Arg Gln Gly Gly Lys Glu 130 135 140 Tyr Phe Lys Val Phe Gly Leu Ile Glu Ser Ala Glu Ile Val Arg Val 145 150 155 160 Thr Arg Asp Gly Arg Met Leu Asp Val Glu Ile Thr Leu Ser Asp Trp 165 170 175 Val Phe Asp Ala Ile Glu Asn Asn His Val Leu Thr Leu Ser Arg Gly 180 185 190 Tyr Phe Leu Leu Arg Lys Pro Leu Glu Arg Arg Leu Tyr Glu Ile Gly 195 200 205 Arg Lys His Cys Gly Ala Gln Ser Lys Trp Met Cys Ser Leu Glu Leu 210 215 220 Leu Arg Asn Lys Cys Gly Ser Gly Ser Thr Lys Lys Glu Phe Arg Arg 225 230 235 240 Leu Leu Ser Lys Ile Ile Glu Asp Asp Thr Ala His Asp His Phe Pro 245 250 255 Asp Tyr Ser Met Ser Ile Glu Gly Glu Asn Ala Val Phe Arg Pro Lys 260 265 270 Val Val Glu Thr Ile Ala Ser Thr Thr Phe Gln Ala Leu Pro Phe Asn 275 280 285 Asp Pro Asp Thr Arg Asp Thr Ala Arg Glu Leu Ala Pro Gly Trp Asp 290 295 300 Val Tyr Ser Leu Glu Glu Glu Trp Arg Ser Trp Val His Asp Lys Ala 305 310 315 320 Ile Asn Val Lys Asp Ala Asp Lys His Phe Leu Ala Phe Cys Lys Lys 325 330 335 Arg Gly Ala Tyr Pro Ser 340

Claims

What is claimed is:

1. Ketogulonigenium comprising a transgene comprising a DNA sequence from an endogenous Ketogulonigenium plasmid.

2. The Ketogulonigenium of claim 1, wherein said endogenous Ketogulonigenium plasmid is contained in Deposit Number NRRL B-21627.

3. A method for producing the Ketogulonigenium of claim 1, comprising:

(a) transforming a Ketogulonigenium strain with a transgene comprising a DNA sequence from an endogenous Ketogulonigenium plasmid; and

(b) obtaining a stably transformed strain of Ketogulonigenium.

4. The method of claim 3, wherein said transforming comprises conjugation.

5. The method of claim 3, wherein said transforming comprises electroporation.

6. The method of claim 3, wherein said DNA sequence is derived from an endogenous plasmid contained in Deposit Number NRRL B-21627.

7. A method for conjugative transfer of a vector from E. coli to Ketogulonigenium comprising culturing said E. coli with said Ketogulonigenium under such conditions wherein said E. coli transfers said vector to said Ketogulonigenium.

8. The method of claim 7, wherein said vector comprises pDELIA8.

9. A method for transforming Ketogulonigenium comprising the process of inserting a vector into said Ketogulonigenium, wherein said insertion comprises electroporation, wherein said vector comprises pMF1014-α.

10. An isolated or purified nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of

the nucleotide sequence in SEQ ID NO:1,

the nucleotide sequence in SEQ ID NO:2,

the nucleotide sequence in SEQ ID NO:3,

the nucleotide sequence in SEQ ID NO:4,

a nucleotide sequence of the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30418,

a nucleotide sequence of the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30419,

a nucleotide sequence of the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30435,

a nucleotide sequence complementary to SEQ ID NO:1,

a nucleotide sequence complementary to SEQ ID NO:2,

a nucleotide sequence complementary to SEQ ID NO:3,

a nucleotide sequence complementary to SEQ ID NO:4,

a nucleotide sequence complementary to the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30418,

a nucleotide sequence complementary to the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30419, and

a nucleotide sequence complementary to the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30435.

11. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to said nucleotide sequence of claim 10, wherein said polynucleotide which hybridizes, does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

12. A vector comprising the molecule of claim 10 and at least one marker gene.

13. A vector comprising:

(a) the nucleic acid molecule of claim 10;

(b) a terminator of transcription;

(c) a promoter; and

(d) a discrete series of restriction endonuclease recognition sites, said series being between said promoter and said terminator.

14. An isolated or purified nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a nucleotide sequence of a Ketogulonigenium plasmid replicon found on any one of the endogenous plasmids contained in Deposit No. NRRL B-21627.

15. The isolated or purified nucleic acid molecule of claim 14 comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of

the nucleotide sequence in SEQ ID NO:5,

the nucleotide sequence in SEQ ID NO:6,

the nucleotide sequence in SEQ ID NO:7,

a nucleotide sequence complementary to SEQ ID NO:5,

a nucleotide sequence complementary to SEQ ID NO:6, and

a nucleotide sequence complementary to SEQ ID NO:7.

16. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to said nucleotide sequence of claim 14, wherein said polynucleotide which hybridizes, does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

17. An isolated or purified vector comprising the nucleic acid molecule of claim 14.

18. The vector of claim 16, comprising a replicon functional in E. Coli.

19. The vector of claim 16, comprising a replicon functional in an organism selected from the genera comprising Acetobacter, Corynebacterium, Rhodobacter, Paracoccus, Roseobacter, Pseudomonas, Pseudo gluconobacter, Gluconobacter, Serratia, Mycobacterium, Streptomyces and Bacillus.

20. A transformed cell of E. coli comprising the vector of claim 17.

21. A transformed cell of the genus Ketogulonigenium comprising the vector of claim 17.

22. The vector of claim 17, wherein said Ketogulonigenium replicon is selected from the group comprising the nucleotide sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.