Nucleic acid coding for an .alpha.-acetolactate synthase from lactococcus and its applications

This application is the U.S. national stage application of PCT International Application No. PCT/GB93/02012, filed 27 Sep. 1993, which claims priority of French Patent Application 92/11470, filed 25 September 1992.
The invention relates to a nucleic acid coding for an .alpha.-acetolactate synthase, as well as to vectors containing this nucleic acid and to the use of these vectors for transforming microorganisms in which the production of .alpha.-acetolactate and diacetyl will be promoted.
Diacetyl is a flavouring sought in the production of food products such as butter, creme fraiche and some cheeses. Conversely, this flavouring is undesirable in the production of other food products such as beer.
A limited number of bacteria, such as Lactococcus lactis subsp. lactis biovar diacetylactis as well as bacteria of the genera Leuconostoc, Pediococcus, Lactobacillus, naturally produce diacetyl; this production takes place from a substrate present in milk in small amounts, citrate; these bacterial strains are capable of metabolizing citrate to pyruvate, which is then converted to acetoin and to diacetyl.
.alpha.-Acetolactate is also an intermediate of the pathway of synthesis of the branched amino acids, which are leucine, isoleucine and valine. This pathway has been very widely studied in bacteria, fungi and plants (1, 2, 3, 4). However, the sequences of the assembly of genes involved in this pathway in one and the same microorganism have never been reported.
The regulation of the expression of the genes involved in the pathway of synthesis of the branched amino acids is complex on account of the steps common to the synthesis of these three amino acids, and this pathway is often presented as a model for the study of organization and regulation.
The organization of these genes has been characterized in Escherichia coli (3), Salmonella typhimurium (3), Bacillus subtilis (5, 6, 7), Corynebacterium typhimurium (8) and Staphylococcus aureus (9).
On the E. coli chromosome, the genes are localized in three groups (10). The largest, located at 85 minutes, is organized in one large transcription unit and two small transcription units, comprising the ilvGMEDA, ilvY and ilvC genes, respectively (11, 12); another group, located at 2 minutes, is composed of two transcription units comprising the ilvH and leuACBD genes (13, 14), and the last group, at 82 min, groups together the ilvBN genes in a single transcription unit.
A similar organization is found in other Entero-bacteriaceae.
In B. subtilis, the ilvBN and leuACBD genes are encountered in one region of the chromosome (5) and the ilvAD genes in another region.
Three cloned nonadjacent chromosomal fragments of C. glutamicum carry five genes of the pathway of synthesis of the branched amino acids, ilvCBN, ilvA and ilvE (9).
In S. aureus, genetic mapping shows that eight genes are grouped together in the following order: ilvAB-CD, leuABCD.
The synthesis of the branched amino acids leucine, isoleucine and valine in L. lactis subsp. lactis is a complex pathway involving eight enzymes, four of which are common to the synthesis of the above three amino acids. The synthesis of valine involves only these four, whereas the synthesis of isoleucine and of leucine involves, respectively, one and four additional specific enzymes. In addition, this system is complicated by the fact that the excess of one of the three amino acids interferes in the synthesis of the other amino acids.
The present invention relates to the cloning, characterization and sequencing of the genes involved in the pathway of synthesis of the branched amino acids in L. lactis subsp. lactis. This work was carried out using the strain NCDO2118 (AFRC, Institute of Food Research Reading Laboratory, Shinfield, Reading, Berks, United Kingdom). The genes were characterized by cloning, complementation in E. coli and B. subtilis and sequence analysis. Nine structural genes are assembled on a 12-kb DNA fragment in the following order: leuABCDilvDBNCA.
Among these genes, the invention relates to nucleic acid sequence coding for two polypeptides forming the subunits of a protein possessing .alpha.-acetolactate synthase activity; this enzyme is of very special interest since it is responsible, in L. lactis subsp. lactis, for the direct conversion of pyruvate to .alpha.-acetolactate independently of the presence of citrate in the medium.
The techniques used for cloning and sequencing the nucleic acid of the invention will be described more particularly in the detailed description of the invention.

Reference will be made in what follows to the tables and figures, wherein:
Table I shows the bacterial strains, plasmids and phages used for cloning the L. lactis subsp. lactis genes involved in the pathway of synthesis of the branched amino acids;
Table II shows a comparison of proteins involved in the pathway of synthesis of the branched amino acids between L. lactis subsp. lactis and different microorganisms;
FIG. 1 shows the structure of the region of L. lactis subsp. lactis DNA carrying the genes involved in the pathway of synthesis of the branched amino acids;
FIGS. 2A-2F show the nucleotide sequence, together with the polypeptide sequence deduced from this nucleotide sequence, of the region of L. lactis subsp. lactis strain NCDO2118 DNA carrying the genes involved in the pathway of synthesis of the branched amino acids; nucleotides 8018 to 9742 of SEQ ID NO: 11 represent the ilvB gene (SEQ ID NO: 12) and encode 575 amino acids as shown (SEQ ID NO: 7); nucleotides 9738 to 10211 of SEQ ID NO: 11 represent the ilvN gene (SEQ ID NO: 13) and encode 158 amino acids as shown (SEQ ID NO: 8);
FIG. 3 shows the alignment of 6 ATP-binding proteins with ORF2 of L. lactis;
FIGS. 4A and 4B show the secondary structures of the major transcript serving as a transcription attenuator.

Research work carried out on the DNA of FIG. 2, and also on the polypeptide deduced from this DNA, leads to the following observations:
Ten genes are present in the region of L. lactis subsp. lactis DNA shown in FIG. 2:
the leuA gene of 1539 nucleotides, located between nucleotides 450 and 1988 of SEQ ID NO: 11 and coding for a polypeptide of 513 amino acids (SEQ ID NO: 1);
the leuB gene of 1041 nucleotides, located between nucleotides 2003 and 3037 of SEQ ID NO: 11 and coding for a polypeptide of 347 amino acids (SEQ ID No: 2 );
the leuC gene of 1380 nucleotides, located between nucleotides 3403 and 4782 of SEQ ID NO: 11 and coding for a polypeptide of 460 amino acids (SEQ ID NO: 3);
the leuD gene of 573 nucleotides, located between nucleotides 4805 and 5377 of SEQ ID NO: 11 and coding for a polypeptide of 191 amino acids (SEQ ID NO: 4);
the orf2 gene of 777 nucleotides, located between nucleotides 5394 and 6170 of SEQ ID NO: 11 and coding for a polypeptide of 259 amino acids (SEQ ID NO: 5);
the ilvD gene of 1710 nucleotides, located between nucleotides 6295 and 8004 of SEQ ID NO: 11 and coding for a polypeptide of 570 amino acids (SEQ ID NO: 6);
the ilvB gene of 1725 nucleotides (SEQ ID NO: 12 which includes the stop codon), located between nucleotides 8018 and 9742 of SEQ ID NO: 11 and coding for a polypeptide of 575 amino acids (SEQ ID NO: 7);
the ilvN gene of 474 nucleotides (SEQ ID NO: 13 which includes the stop codon), located between nucleotides 9738 and 10211 of SEQ ID NO: 11 and coding for a polypeptide of 158 amino acids (SEQ ID NO: 8);
the ilvC gene of 1032 nucleotides, located between nucleotides 10260 and 11291 of SEQ ID NO: 11 and coding for a polypeptide of 344 amino acids (SEQ ID NO: 9);
the ilvA gene of 1323 nucleotides, located between nucleotides 11337 and 12659 of SEQ ID NO: 11 and coding for a polypeptide of 441 amino acids (SEQ ID NO: 10).
The invention relates to any nucleic acid comprising all or part of the DNA sequence bounded by the nucleotides located at positions 8018 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for one or both subunits of a protein capable of converting pyruvate to .alpha.-acetolactate; and for any other novel polynucleotide, peptide or protein disclosed herein.
.alpha.-Acetolactate synthase of L. lactis subsp. lactis is a protein consisting of two subunits; the first subunit is a polypeptide of 575 amino acids (SEQ ID NO: 7) encoded by the fragment bounded by the nucleotides located at positions 8018 and 9742 of SEQ ID NO: 11 and as shown in FIG. 2, and corresponding to the ilvB gene (SEQ ID NO: 12); the second subunit is a polypeptide of 158 amino acids (SEQ ID NO: 8) encoded by the fragment bounded by the nucleotides located at positions 9738 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2, and corresponding to the ilvN gene (SEQ ID NO: 15). Consequently, the .alpha.-acetolactate synthase activity of L. lactis subsp. lactis is possible only if both of these genes are expressed.
The subject of the invention is, more especially, the fragments of the above nucleic acid coding for one or both subunits of a protein capable of possessing the enzymatic properties of .alpha.-acetolactate synthase of L. lactis subsp. lactis. More specifically, the invention relates to nucleic acids comprising one or other or both of the following segments:
the first segment corresponding to the ilvB gene (SEQ ID NO: 12), bounded by the nucleotides located at positions 8018 and 9742 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for the polypeptide of 575 amino acids (SEQ ID NO: 7) corresponding to one of the two subunits of .alpha.-acetolactate synthase of L. lactis subsp. lactis;
the second segment corresponding to the ilvN gene (SEQ ID NO: 13), bounded by the nucleotides located at positions 9738 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for the polypeptide of 158 amino acids (SEQ ID NO: 8) corresponding to the other subunit of .alpha.-acetolactate synthase of L. lactis subsp. lactis.
The invention also relates to the above nucleic acids in which the sequences are modified, provided the polypeptides encoded by these nucleic acids retain their enzymatic properties of the .alpha.-acetolactate synthase type.
Such modifications, without implied limitation, lead, for example, to variant nucleic acids which differ from the nucleic acid of the invention by the addition and/or deletion of one or more nucleotides and/or modification of one or more nucleotides.
Thus, the invention further relates to DNA sequences coding for .alpha.-acetolactate synthase of the pathway of synthesis of the branched amino acids of bacteria of the genus Lactococcus, such as L. plantarum, L. rafinolactis and L. lactis, for instance L. lactis subsp. cremoris; which DNA sequences display a strong homology with the nucleic acid sequence of ilvBN (SEQ ID NO: 14).
Advantageously, these DNA sequences are identified, and where appropriate selected, by genetic hybridization with one or more nucleic acid probes originating from the above nucleic acid sequences, under moderately stringent conditions.
As an example, a method for the identification of such a DNA sequence comprises the following steps:
extraction of the DNA from the bacterium;
digestion of this DNA with one or more restriction enzymes such as EcoRI;
transfer of the DNA fragments obtained onto a nitrocellulose membrane;
hybridization with the complete ilvBN sequence in a hybridization solution whose composition is: 6.times.SSC, approximately 10% formamide, 5.times.Denhardt, 100 ml of phosphate buffer pH 7.
The invention also relates to polypeptide sequences comprising all or part of the polypeptide sequences encoded, on the one hand by the DNA sequence bounded by the nucleotides located at positions 8018 and 9742 of SEQ ID NO: 11 and as shown in FIG. 2, and on the other hand by the DNA sequence bounded by the nucleotides located at positions 9738 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2; these two polypeptides constituting the subunits of a protein possessing the enzymatic properties of .alpha.-acetolactate synthase of L. lactis subsp. lactis.
The subject of the invention is also any polypeptide sequence originating from each of the above polypeptide sequences, provided the protein formed from these polypeptides possesses enzymatic properties of the type possessed by .alpha.-acetolactate synthase.
The above polypeptides may be modified provided they retain the enzymatic properties defined above. For example, without implied limitation, polypeptides lying within the scope of the invention can differ from those defined above by the addition and/or deletion of one or more amino acids and/or modification of one or more amino acids.
A person skilled in the art has at his disposal means enabling him to identify those polypeptides of shorter sequences which lie within the field of the invention. A general means enabling him to undertake this identification consists in treating each of the above polypeptides with a protease that cleaves the polypeptides at a specific site, and then separating the fragments obtained and testing them for their enzymatic activity with respect to pyruvate.
Another means enabling regions of the polypeptides which are essential to the .alpha.-acetolactate synthase activity to be identified consists in cleaving the nucleic acids corresponding to the ilvB and ilvN genes, for example using one or more restriction enzymes, before introducing them into the cell host or the expression vector used for the production of a protein displaying .alpha.-acetolactate synthase activity. The truncated nucleic acid may thus be tested for its capacity to express effectively a product possessing .alpha.-acetolactate synthase activity or, on the contrary, no longer possessing it.
The subject of the invention is also any recombinant nucleic acid comprising at least one nucleotide sequence of the abovementioned type coding for .alpha.-acetolactate synthase, combined with at least one promoter and/or one transcription terminator recognized by the polymerases of the host into which the said recombinant nucleic acid is introduced.
The introduction of the recombinant nucleic acid may be carried out using vectors of the plasmid type capable of replicating in the host microorganism and of permitting the expression therein of the sequence coding for the enzyme, or alternatively the recombinant nucleic acid may be introduced directly into the genome by genetic engineering techniques known to a person skilled in the art.
The host microorganisms into which the recombinant nucleic acid is capable of being introduced are preferably prokaryotic cells such as Lactococcus lactis, Leuconostoc lactis, Lactobacillus lactis, Lactobacillus plantarum, but may also be eukaryotes such as yeasts.
More especially, the invention relates to all microorganisms capable of being used as leaven in an agri-foodstuffs process, in which microorganisms the recombinant nucleic acid of the invention is introduced for the purpose of obtaining an overproduction of diacetyl in an agri-foodstuffs product such as dairy product derivatives, fermented wheys, soft white cheeses, and the like.
Advantageously, the introduction of the recombinant nucleic acid of the invention into L. lactis bacteria is combined with an inhibition of the enzyme responsible in these bacteria for the conversion of acetolactate to acetoin (.alpha.-acetolactate decarboxylase) and/or of the enzyme involved in the conversion of pyruvate to lactate (lactate dehydrogenase), in order to increase the acetolactate pool leading to diacetyl.
The inhibition of these enzymes may be achieved by all chemical or biological means that enable the metabolic flux towards diacetyl production to be increased; a first biological means consists in deleting all or part of the wild-type genes coding or these enzymes; a second biological means consists in replacing the wild-type genes coding for these enzymes by mutated genes incapable of expressing the enzymes in question.
The recombinant nucleic acid of the invention may also be introduced into other microorganisms, such as Bacillus, for the production of diacetyl for non-agri-foodstuffs purposes, for example in cosmetology and as an antibacterial.
Other features of the invention will become apparent in the description which follows, which relates on the one hand to the cloning, characterization and sequencing of the region of L. lactis subsp. lactis DNA carrying the genes involved in the pathway of synthesis of the branched amino acids, and on the other hand to the introduction of a nucleic acid according to the invention, coding for a polypeptide displaying .alpha.-acetolactate synthase activity, into a cell host.
I--CLONING OF THE GENES INVOLVED IN THE SYNTHESIS OF AMINO ACIDS IN L. LACTIS SUBSP. LACTIS
1) Materials and Methods
a) Bacterial strains, plasmids and culture media
The bacterial strains, plasmids and phages used for gene cloning are listed in Table I below.
TABLE I______________________________________ Bibliographic referencesStrains and orplasmids Characteristics sources______________________________________L. lactis subsp.lactisNCDO2118 natural isolate NCDOB. subtilisCU740 leuA5 trpC2 (SP.beta.) (7)CU741 leuC7 trpC2 (7)CU315 leuD117 trp2 (SP.beta.) (15)IL2685 leuB6 trpC2 ilvA r.sup.- /m.sup.- Cm.sup.RMT119 leuB6 trpC2 r.sup.- /m.sup.- (16)GSY184 ilvC1 trpC2 (17)IL3151 ilvD4 leuB6 r.sup.- /m.sup.+1012 leuA8.sup.a metb5 r.sup.- /m.sup.+ (18)GSY276 ilvD4 trpC2 (17)E. coliCU518 leuA371 (19)CU514 leuB401 (19)CU520 leuC171 (19)CUS26 leuD101 (19)AB1255 tonA2 lacY1 tsx-5 supE44 (20) gal-6 .lambda..sup.- hisG1 rpsL8 malA1 xyl-7 mtl-2 ilvA201 metB1FD1062 argH1 thi-1 ara-14 ilvI614 ilvH612 .lambda..sup.- glyA18 relA1 spoT1 ilVB619 bglR20 rbs-5::tn5JP58 ilvG468 (ilvG.sup.+ ) thi-1 (21) galK2 .lambda..sup.- rpSL704 xyl-5TG1 mtl-1 ilvC7 argE3 thi-1 (22) supE thi D (lac-proAB) hsdD5 F.sup.+ traD36 proAB lacI z.DELTA.15PlasmidspIL253 (23) Em.sup.R, 4.9-kbpHV438 (42) Hybrid between pBR322, the Cm.sup.R gene of pC194, thyB and the X segments of B. subtilis DNApIL371 9.2 kb Sau3A fragment of the L. lactis chromosome in pIL253pIL373 4.5-kb Sau3A fragment of the L. lactis chromosome in pIL253pIL374 8.5-kb Sau3 fragment of the L. lactis chromosome in pIL253pIL384 13-kb Sau3A fragment of the L. lactis chromosome in pIL253pIL389 7.5-kb left-hand Sau3A fragment of pIL384 in pBSpIL500 18.5-kb XbaI fragment of the L. lactis chromosome in pIL253pIL505 6.5-kb right-hand SmaI-Sau3A fragment of pIL384 in pBSpIL533 2.5-kb left-hand Sau3A-ExoIII fragment of pIL384pBluescript Ap.sup.R, M13 ori, pBR322 (Strata- ori gene)______________________________________
L. lactis subsp. lactis strain IL1403 was cultured at 37.degree. C. on M17 medium (25) in which lactose was replaced by glucose. Escherichia coli was cultured on Luria-Bertani (LB) medium or in M9 minimum medium (26) at 37.degree. C. Bacillus subtilis was cultured on a medium at 37.degree. C., on LB medium or in Spizizen-salt minimum medium supplemented with the appropriate amino acids. If necessary, erythromycin (5 .mu.g/ml for L. lactis subsp. lactis or 0.3 .mu.g/ml for B. subtilis), ampicillin (50 .mu.g/ml for E. coli) or tetracycline (10 .mu.g/ml for E. coli) is added to the medium.
b) Molecular cloning and DNA manipulation
The plasmids and the chromosomal DNA were prepared according to known techniques of the prior art (26, 27, 28, 29). Transformation of E. coli cells was carried out either by a standard CaCl.sub.2 procedure (36) or by electroporation (30). Induction of competence and transformation of B. subtilis cells were carried out as described by Anagnotopoulos and Spizizen (31), with a few modifications (32). Southern blotting and DNA hybridizations were carried out as described by Maniatis et al. (26). Nucleic acid probes were prepared using [.alpha.-.sup.32 P]-dCTP and a nick translation kit in accordance with the supplier's recommendation. The other molecular techniques were carried out as described by Maniatis et al. (26).
c) Analysis of the DNA sequence
The E. coli clones used for sequencing were obtained by subcloning DNA fragments into plasmids of the pBluescript family and using exonuclease III and mung bean nuclease from the company Stratagene in order to generate a series of clones containing overlapping DNA fragments.
The DNA was then sequenced using the "Taq DyeDeoxy Terminator Cycle Sequencing" kit and the "Sequencer 370A" marketed by the company Applied Biosystems. Nucleotide and polypeptide sequences were analysed with the University of Wisconsin's "BISANCE" and "GCG" softwares. Protein sequences were aligned with a "MULTALIN" software (33).
The nucleic acid sequences were determined on both strands. Restrictively, B. subtills strains mutated in the ilvA gene were prepared by transforming competent MT119 cells with pHV438 (34) to Cm.sup.R. Integration of this plasmid in the chromosome by double crossing-over replaces the ilvA gene by the Cm.sup.R gene (24).
A representative ilvA leuB6 r.sup.- /m.sup.- Cm.sup.R clone is designated IL2685.
An ilvD4 leuB6 double mutant of B. subtilis was constructed by transformation, using GSY276 DNA to transform competent 1012 cells to cells which were prototrophic for methionine. The transformed cells were then tested for their requirements for isoleucine and leucine and for the absence of restriction by titration of the rho phage.
An ilvD4 leuB6 r.sup.- /m.sup.+ clone was designated IL3151.
2) Gene Cloning
Total DNA of L. lactis subsp. lactis NCDO2118 was partially digested with the endonuclease Sau3A1. 20 .mu.g of DNA segments of size larger than 10 kb were ligated with 10 .mu.g of DNA of plasmid vector pIL253 cleaved with BamHI, at a final concentration of 500 .mu.g/ml.
The ligated mixture was used to transform competent cells of the mutant B. subtilis strain leuB6 ilV4, IL3151 to isoleucin-independent cells. Four transformants were also Em.sup.R, and contained 18-, 14.2-, 13.5- or 9.5-kb plasmids designated pIL384, pIL371, pIL374 and pIL373, respectively.
According to a similar experiment, L. lactis subsp. lactis DNA cleaved with XbaI was cloned into pIL253 using the mutant B. subtilis ilvA strain IL2685 as recipient. The transformants were selected on a minimum medium enriched with leucine and tryptophan but lacking isoleucine. Two Em.sup.R Ile.sup.+ clones, containing an apparently identical 23.5-kb plasmid, designated pIL500, were obtained.
3) Complementation Experiments
The cloned DNA segments were used to complement, in B. subtilis, leu and ilv mutants, and in E. coli after subcloning into pBluescript. The results relating to these studies are reported in FIG. 1.
FIG. 1 shows the structure of the DNA region carrying the genes for the enzymes involved in the pathway of synthesis of the branched amino acids in L. lactis. In this figure, the open bars represent the segments used for the complementation experiments in B. subtilis and E. Coli. These segments are carried by the plasmids indicated in the figure, which were constructed as described in Table I above. The results of the complementation experiments with the mutants listed in Table I are represented by (+) and (-), referring to growth or the absence of growth on the culture medium, lacking the corresponding amino acids. The organization of the DNA region carrying the genes involved in the pathway of synthesis of the branched amino acids (orf1, leuA, leuB, leuC, leuD, orf2, ilvD, ilvB, ilvN, ilvC, ilvA) is also shown at the bottom of FIG. 1; p1 and p2 indicate the transcription promoters and t1 the transcription terminator. The open reading frame between p1 and t1 represents the leader peptide.
In these complementation experiments, the E. coli nomenclature has been used; the leuB and leuC genes of E. coli correspond to the leuC and leuB genes, respectively, of B. subtils, and the three isoenzymes of acetolactate synthase and of acetohydroxide synthase encoded by ilvBN, ilvIH and ilvGN in E. coli correspond to a single enzyme encoded by ilvBN in B. subtilis. The leuA, leuB, leuC, ilvD and ilvA genes complement the corresponding mutations in B. subtilis ; the leuA, leuB, leuC, leuD and ilvC genes complement the corresponding mutations in E. coli.
These results are sufficient to identify nine genes involved in the pathway of biosynthesis of the branched amino acids in L. lactis subsp. lactis.
4) Nucleotide Sequences of the Genes
The nucleotide sequence of a region of 12,720 bp was determined. This sequence (SEQ ID NO: 11) is shown in FIG. 2. A computer analysis according to the method of Griskov et al. (35) discloses ten open reading frames (ORF) of size larger than 200 bp. Each ORF is preceded by a ribosome binding site, complementary to the 3' end of rRNA 163 of L. lactis subsp. lactis (36). All the ORFs begin with the codon ATG, except for the second which begins with the codon TTG; furthermore, four 72-bp repeat units are present between the second and the third ORF.
FIG. 2 shows the nucleotide sequence, and the polypeptide sequence deduced from this nucleotide sequence, of the DNA region coding for the genes involved in the pathway of synthesis of the branched amino acids of L. lactis strain NCDO2118; the numbers at the right indicate the position of the oligonucleotides; the names of the genes are indicated at the beginning of each amino acid sequence; the transcription stop codons and also the ribosome binding sites are indicated respectively by (*) and RBS; the consensus sequences "-10" and "-35" of the promoters are underlined; inverted repeat units, which can constitute "preamptors" and "terminators", are numbered (1,2) and (5, 6), respectively, and those capable of forming "antiterminators" are numbered (3and 5), (see FIG. 4 below); the arrow (.fwdarw.) indicates the 72-bp direct repeat unit, and the bold characters indicate the 6-bp inverted repeat units which it contains.
5) Assignment of the ORFs
The proteins deduced from the ten ORFs were compared with the GENPRO and NBRF protein databases. Significant homologies were identified for ten ORFs; nine of them correspond to the genes involved in the pathway of synthesis of the branched amino acids, as seen in Table II below.
TABLE II__________________________________________________________________________ %.sup.a of identical amino acids BibliographicMicroorganism LeuA LeuB LeuC LeuD IlvD IlvB IlvN IlvC IlvA references__________________________________________________________________________E. coli --.sup.b -- -- 46 42 42.sup.c <20.sup.d 34 36 (13, 11, 12, 37) 43.sup.e 37.sup.f (38) 40.sup.R <20.sup.h (11)Salmonella 41 -- 49 -- -- -- -- -- -- (39, 40)tryphenuriumBacillus -- 53 -- -- -- -- -- -- 50 Genbank (41)subtilisBacillus -- 53 -- -- -- -- -- -- -- (42)coagulansThermus -- 42 -- -- -- -- -- -- -- (43)aquaticusSaccharomyces 25 46 50.sup.i 52.sup.j -- 41 -- 33 38 (44, 45, 46, 47,cerevisiae 48, 49)Phycomyces -- -- 50.sup.i 49.sup.j -- -- -- -- -- (50)blakesleeanusMucor -- -- 49 -- -- -- -- -- -- (51)circinelloides__________________________________________________________________________ In Table II: .sup.a indicates that the percentage was calculated using a software (52) from the percentage identity by the size of the smallest protein; .sup.b indicates sequences not available; .sup.c indicates the comparison with IlvB nucleic acid; .sup.d indicates the comparison with IlvN nucleic acid; .sup.e indicates the comparison with IlvI nucleic acid; .sup.f indicates the comparison with IlvH nucleic acid; .sup.g indicates the comparison with IlvC nucleic acid; .sup.h indicates the comparison with IlvM nucleic acid; .sup.i Nterminal end of Leu1; .sup.j Cterminal end of Leu1.
In addition, two genes detected by complementation, the ilvB gene (homologous to ilvB, ilvI and ilvG of E. coli ) and the ilvN gene (homologous to ilvH of E. coli ), for which complementation data were not obtained, were identified.
Most of the proteins of L. lactis were similar in size to their homologues. However, three exceptions were observed. Relative to the proteins of E. coli, the lactococcal proteins IlvA and IlvC lack the C-terminal amino acids 73 and 147, respectively, and IlvD displays a deletion of 36 amino acids. One of the ORFs, designated ORF2, codes for a protein not displaying any homology with the enzymes of the pathway of synthesis of the branched amino acids, but carries two regions that are conserved in a superfamily of ATP-binding proteins (53).
FIG. 3 shows the alignment of 6 ATP-binding proteins with the ORF2 sequence of L. lactis. Needleman's software was used to obtain these results (54). In FIG. 3:
the binding domains (53) are represented by NB1 and NB2;
a short sequence shared by all the members of the ATP-dependent family is indicated by the symbols (#);
the symbols (.lambda.) indicate amino acids common to all the proteins;
the symbols (.) indicate conserved substitutions;
the amino acids shown in bold characters are present in at least five proteins;
the figures at the left indicate the position of the amino acids, and the figures in brackets indicate the size of the proteins;
the functions of the different proteins are shown in the following manner:
(.sup.1), nodulation in Rhizobium leguminosarum (55);
(.sup.2), maltose transport in E. coli (56);
(.sup.3), glutamine transport in E. coli (57);
(.sup.4), glycine, betaine and proline transport in E. coli (58);
(.sup.5), haemolysin secretion in E. coli (59);
(.sup.6), cyclolysine secretion in Bordetella pertussis (60).
The organization of the sequenced segments is presented in FIG. 1. All the genes, except for the first one, upstream of LeuA (ORF1), are transcribed in the same direction. The leu and ilv genes are grouped together, and the two groups are separated by 121 bp. The leu genes are separated by less than 19 bp, except for leuB and leuC which are separated by four 72-bp direct repeats. The distance between the ilv genes is between approximately 10 and 42 bp, except for ilvB and ilvN which are separated by 9 bp.
6) Transcription Signals
The sequences conforming to the consensus units of the lactococcus promoters were identified upstream of the assemblies of leu and ilv genes (see the positions of p1 and p2 in FIG. 1). The region between p1 and leuA strongly resembles the regulatory regions of the amino acid biosynthesis operons controlled by attenuation (61). The transcript initiated at p1 can follow two paths. One to the formation of a rho-independent transcription "terminator" (as shown in FIG. 4A). In addition, the transcript carries a 51-bp message, beginning with an ATG codon and ending with a TAG codon, which determines a leader peptide of 16 amino acids, four of which are a succession of leucine and isoleucine (as shown in FIG. 4B). The ribosome sited at the Leu and Ile codons prevents formation of the termination signal and leads to transcription of the leu genes downstream. Independent transcription "terminators" were not found between the two assemblies of genes or downstream of the ilvA gene.
7) Organization of the Ilv Operon
The genes involved in the pathway of synthesis of the branched amino acids in L. lactis subsp. lactis are organized in a large assembly divided into two units grouping together the leu and ilv genes. The two units are necessary for the synthesis of leucine, whereas only the second is required for the synthesis of isoleucine and valine. A transaminase, involved in the last step of the pathway of synthesis of the branched amino acids (product of the ilvE gene in E. coli ), is not encoded by the assembly. This implies that this reaction is implemented by a non-specific transaminase, or alternatively that the corresponding gene in L. lactis subsp. lactis occurs elsewhere on the chromosome.
The leu and ilv genes are both preceded by a promoter; however, they are not separated by an independent transcription "terminator", suggesting that they form a single operon. This operon extends beyond the last biosynthesis gene, ilvA.
8) Regulation of the Ilv Operon
Sequence analysis strongly suggests that the operon is regulated by an attenuation mechanism, the mediator of which is a leucine-rich leader peptide. This peptide very closely resembles the leader peptide of the leu operon of E. coli and of S. typhimurium (62, 63), but differs from that of the ilvBN and ilvGMEDA operons of E. coli.
FIG. 4 shows the secondary structures of the leader transcript capable of participating as a mediator in transcription attenuation:
at A, the termination topology is shown; units (1) and (2) are paired, and units (5) and (6) form a transcription terminator;
at B, the antitermination topology is shown; units (3) and (4) are paired, and mask units (5) and (6).
In FIG. 4, the nucleotides in bold characters indicate the sequence coding for the leader peptide, and the nucleotides in bold italics indicate the ribosome binding sites of the leader peptide and the leuA gene. Stalling of the ribosome at the successive Leu and Ile residues could mask unit (1) and favour this topology.
Current models propose that the strength of the attenuation is dependent on the tRNA.sup.lcu load during translation of the leader peptide. The presence of rare codons increases the response to leucine starvation by increasing the duration of ribosome stalling. In E. coli, the codon corresponding to the four leucine residues present in the leader peptide is CUA, which corresponds to only 2% of the leucine codons used for the proteins of this organism (64). In contrast, the leucine codon UUC, encountered three times in the leader transcript of the pathway of synthesis of the branched amino acids in L. lactis, corresponds to 24% of the leucine codons in the proteins of L. lactis (65). The isoleucine codon is also present in the leader peptide of L. lactis, following very closely the Leu codons, which is not the case in E. coli, and may hence affect the response of the operon to an arrest of the pathway of synthesis of the branched amino acids. Other studies are necessary to determine whether the model proposed for regulation in E. coli may be applied directly to L. lactis.
9) The Open Reading Frame ORF2
ORF2, inasmuch as it corresponds to a product belonging to the superfamily of ATP-binding proteins, is not a biosynthesis gene in the pathway of synthesis of the branched amino acids. The proteins of this superfamily are encountered both in prokaryotes and in eukaryotes, and share a similar base organization (66, 67). In prokaryotes, most of the members of this superfamily are components of the transport systems involving periplasmic binding proteins. These genes are generally cotranscribed with other genes (67), and have never been found in the biosynthesis operon. Other studies are necessary to establish the function of ORF2, but the latter is doubtless involved in the transport of the chain of synthesis of the branched amino acids, or in regulation of the genes of this pathway.
Isoleucine, leucine and valine represent 20% of the amino acids of L. lactis (68), whereas tryptophan and histidine, two other amino acids whose pathways of biosynthesis in L. lactis have been studied, represent less than 2%. This suggests that a fine regulation of the expression of the genes of the pathway of synthesis of the branched amino acids is necessary.
II--INTRODUCTION OF A NUCLEIC ACID CODING FOR AN .alpha.-ACETOLACTATE SYNTHASE INTO A HOST
The nucleic acid consisting of the DNA sequence bounded by the nucleotides located at positions 7977 and 10246 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for the IlvB and IlvN polypeptides, is amplified by the Taq polymerase chain reaction from the DNA of FIG. 2, and using two oligo-nucleotides located upstream and downstream of the nucleic acid sequence coding for IlvBN.
The following two oligonucleotides were used:
CTAGTGAAGGTTGCGTTACA (SEQ ID No: 15)
TGCCATTTTTGTTTCCTCTA (SEQ ID No: 16)
After the polymerase chain reaction, the product of the reaction is cloned into an expression vector, such as pIL252, pIL253, pWV01, containing a promoter such as the promoter of the lactose operon or of the tryptophan operon, and where appropriate a terminator.
After cloning of the amplified segment in the appropriate orientation, the plasmid obtained is introduced by transformation into a strain of L. lactis, such as IL1403 or MG1363, which expresses IlvBN at a high level. This results in diversion of a part of the pyruvate pool obtained after glycolysis towards .alpha.-acetolactate, a precursor of diacetyl.
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__________________________________________________________________________SEQUENCE LISTING__________________________________________________________________________LOCUS LACLEUILV 12720 bp ds-DNA BCT 02-NOV-1992DEFINITION Lactococcus lactis operon (leuA, leuB, leuC,leuD, ilvD, ilvB, ilvN, ilvC, ilvA) genes complete cds; completeORF2.ACCESSION M90761KEYWORDS ilvA gene; ilvB gene; ilvC gene; ilvD gene;ilvN gene; leuA gene; leuB gene; leuC gene; leuD gene; operon.SOURCE Lactococcus lactis (library: NCDO2118) DNA. ORGANISM Lactococcus lactis Prokaryotae; Firmicutes; Firmibacteria;Gram-positive cocci; Streptococcaceae.REFERENCE 1 (bases 1 to 12720) AUTHORS Godon, J.-J.J.-J., Chopin, M.-C.M. andEhrlich, D. S. TITLE Branched-chain amino acid biosythesis genes inLactococcus lactis subup. lactis. JOURNAL J. Bacteriol. 174, 6580-6589 (1992) STANDARD full automaticFEATURES Location/Qualifiers attenuator 260. .450 /note="putative" repeat.sub.-- region 3039. .3327 /function="unknown" /rpt.sub.-- type=DIRECT /note="putative" CDS 450. .1991 /EC.sub.-- number="4.1.3.12" /gene="leuA" /citation=[1] /note="putative" /codon.sub.-- start=1 / t r a n s l a -tion="MRKIEFFDTSLRDGEQTPGVSFSISEKVTIAKQLEKWRISVIEAGFSAASPDSFEAVKQIADSLNDTAVTALARCVISDIDKAVEAVKGAKYPQIHVFIATSPIHMKYKLKISPEEVLKNIDKCVRYARERVEVVEFSPEDATRTELNFLLEAVQTAVDAGATYINIPDTVGYTTPEEYGKIFKFLIDNTKSDREIIFSPHCHDDLGMAVANSLAAIKAGAGRVEGTVNGIGERAGNAALEEIAVALHIRKDFYQAQSPLKLSETAATAELISQFSGIAIPKNKAIVGANAFAHESGIHQDGVLKNAETYEIITPELVGIKHNSLPLGKLSGRHAFSEKLTELNIAYDDESLAILFEKFKKLADKKKEITDADIHALFTGETVKNLAGFILDNVQIDGHKALVQLKNQEEEIYVSQGEGSGSVDAIFKAIDKVFNHQLKLISYSVDAVTDGIDAQATTLVSVENLSTGTIFNAKGVDYDVLKGSAIAYMNANVLVQKENLQGKVEQIS AHDGI"CDS 2003. .3040 /gene="leuB" /note="putative" /codon.sub.-- start=1/translation="LSKKIVTLAGDGIGPEIMSAGLSVLKAVSKKIDFEYELEAKDFGGIAIDKHGHPLPEETLQAVKNADAILLAAIGHPKYNNAKVRPEQGLLALRKELGLYANVRPLKIYPALKKLSPIRNVENVDFLVIRELTGGIYFGQHELADDKARDVNDYSADEIRRILHFAFKSAQSRPRKLLTSVDKQNVLATSKLWRKMADEIADEYPDVRLEHQLVDSCAMLLITNPQQFDVIVTENLFGDILSDEASSLAGSLGVMPSSSHGFNGLALYEPIHGSAPDIAGKGIANPVSMILSIAMMLRESFGQEDGAAMIEKAVTQTFTDGILTKDLGGTATTK EXTEAILKNCQ"CDS 3403. .4785 /gene="leuC" /note="putative" /codon.sub.-- start=1/translation="MSGKTIFDKLWDQHVIAGNEGEPQLLYIDLHVIHEVTSPQAFQGLREAGRRVRRKDLTYGTLDHNVPTQNIFNIQDLISKKQIDTFTKNVKEFDVPAETHGGKGQGIVHMVAPESGRTQPGKTIVCGDSHTATNGAFGAIAFGIGTSEVEHVLATQTIWQVKPKRMKIEFQGHPQKGIYSKDFILALIAKYGVDAGVGYAVEYSGDAISDLSMEERMTICNMSIEFGAKIGLMNPDEKTYDYVKGREHAPKNFDEAVSKWEKLVSDSDAQYDKILSLDVSQLKPMVTWGTNPGMGLEFGEKFPEINNDLNYERAYQYMDLKPGQTASDIDLGYIFIGSCTNARLGDLEEAAKIIGDRHIADGLTGIVVPGSRPVKEAAEAQGLDKIFKEAGFEWREPGCSACLGMNPDQIPEYVHCASTSNRNFEGRQGHNARTHLCSPAMAAAAAIAGK FVDVRMLVTD"CDS 4805. .5380 /gene="leuD" /note="putative" /codon.sub.-- start=1/translation="MEKFTIYKGTSVPVMNDNIDTDQIIPKQFLKAIDKKGFGKNLFYEWRYLKDYDENPDFILNAPKYKKASLLISGDNFGSGSSREHAAWALSDYGFRAIIAGSYSDIFYNNALKNGLLPIKQPREVLNQLTKLSSQEEITIDLPHQLIITSLGDFHFEIDP IWKDKLINGLDDIGITLQYEEAISAYEQKNQ"CDS 5394. .6173 /function="unknown" /note="ORF2; putative" /codon.sub.-- start=1/translation="MTIINLKNVNLTRNKKEILKDITWKVNPGENWVILGLNGSGKSSLLKLILAEEWKTSGEITVLNTQFRNGEIPKLRKRISVVGSFIAERFQPNIKAENLVYTGKFNSSMLYKPYTDQELDEARQLLRQMGAKSLIGRNYASLSQGEKQVLLIARSLILKPELLILDEATNGLDLFAKEKLLKQLQQINQLKTAPTLIYISHHPDEITDIFTHLLLLREGKVIQSGKKENLLNEKILTDFYQEKVEVHRFEQKYFVIPAN"CDS 6295. .8007 /gene="ilvD" /note="putative" /codon.sub.-- start=1/translation="MEFKYNGKVESVELNKYSKTLTPRSTQPATQAMYYGIGFKDEDFKKAQVGIVSMDWDGNPCNMHLGTLGSKIKSSVNQTDGLIGLQFHTIGVSDGIANGKLGMRYSLVSREVIADSIETNAGAEYYDAIVAIPGCDKNMPGSIIGMARLNRPSIMVYGGTIEHGEYKGEKLNIVSAFEALGQKITGNISDEDYHGVICNAIPGQGACGGMYTANTLAAAIETLGMSLPYSSSNPAVSQEKQEECDDIGLAIKNLLEKDIKPSDIMTKEAFENAITIVMVLGGSTNAVLHIIAMANAIGVEITQDDFQRISDIIPVLGDFKPSGKYMMEDLHKIGGLPAVLKYLLKEGKLHGDCLTVTGKTLAENVETALDLDFDSQDIMRPLKNPIKATGHLQILYGNLAQGGSVAKISGKEGEFFKGTARVFDGEQHFIDGIESGRLHAGDVAVIRNIGPVGGPGMPEMLKPTSALIGAGLGKSCALITDGRFSGGTHGFVVGHIVPEAVEGGLIGLVEDDDIIEIDAVNNSISLKVSNEEIAKRRANYQKPTPKATRGVLAKFAKLTRPASEGC VTDL"CDS 8018. .9745 /gene="ilvB" /note="putative" /condon.sub.-- start=1/translation="MKKIKLEKPTSGSQLVLQTLKELGVEIIFGYPGGAMLPLYDAIHNFEGIQHILARHEQGATHEAEGYAKSSGKVGVVVVTSGPGATNAVTGIADAYLDSVPLLVFTGQVGRQSIGKDAFQEADTVGITAPITKYNYQIRETADIPRIVTEAYYLARTGRPGPVEIDLPKDVSTLEVTEINDPSLNLPHYHESEKATDEQLQELLTELSVSKKPVIIAGGGINYSGSVDIFRAFVEKYQIPVVSTLLGLGTLPISHELQLGMAGMHGSYAANMALVEADYIINLGSRFDDRVVSNPAKFAKNAVVAHIDIDAAELGKIVKTDIPILSDLKAALSRLLQLNKVRTDFNDWIKTVIENKEKAPFTYEPQNHDIRPQETIKLIGEYTQGDAIIVTDVGQHQMWVAQYYPYKNARQLITSGGMGTMGFGIPAAIGAKLAQPNKNVIVFVGDGGFQMTNQELALLNGYGIAIKVVLINNHSLGMVRQWQESFYEERRSQSVFDVEPNFQLLAEAYGIKHVKLDNPKTLADDLKIITEDEPMLIEVLISKSEHVLPMIPAGLHNDEMIGLHFT DKNEEIDNA"CDS 9738. .10214 /gene="ilvN" /note="putative" /condon.sub.-- start=1/translation="MRRMIIAKLHNVTGIMNRFTAVLNRRQVNILSITAGVTESQDLTHTTFVIEVDHLDEVEQIIKQLNRLIDVIEVADITDFPHVEREVVLIKVSAPPTIRAEIFTMIEPFRVNVVDVNLENVTIQLTGDSAKIEALIEVVSPYGILNMARTGSAGFERG"CDS 10260. .11294 /gene="ilvC" /note="putative" /condon.sub.-- start=1/translation="MAVTMYYEDDVEVSALAGKQIAVIGYGSQGHAHAQNLRDSGHNVIIGVRHGKSFDKAKEDGFETFEVGEAVAKADVIMVLAPDELQQSIYEEDIKPNLKAGSALGFAHGFNIHFGYIKVPEDVDVFMVAPKAPGHLVRRTYTEGFGTPALFVSHQNASGHAREIAMDWAKGIGCARVGIIETTFKEETEEDLFGEQAVLCGGLTALVEAGFETLTEAGYAGELAYFEVLHEMKLIVDLMYEGGFTKMRQSISNTAEFGDYVTGPRIITDEVKKNMKLVLADIQSGKFAQDFVDDFKAGRPKLIAYREAAKNLEIEKIGAEHVKQCHSHNLVMTM PLKSISNFSY"CDS 11337. .12662 /gene="ilvA" /note="putative" /condon.sub.-- start=1/translation="MISAKEVEDAYDLLKAVVTKTPLQLDPYLSNKYQANIYLKEVVTKTPLQLDPYLSNKYQANIYLKEENLQKVRSFKLRGAYYSISKLSDEQRSKGVVCASAGNHAQGVAFAANQLNISATIFMPVTTPNQKISQVKFFGESHVTIRLIGDTFDESARAAKAFSQDNDKPFIDPFDDENVIAGQGTVALEIFAQAKKQGISLDKIFVQIGGGGLIAGITAYSKERYPQTEIIGVEAKGATSMKAAYSAGQPVTLEHIDKFADGIAVATVGQKTYQLINDKVKQLLAVDEGLISQTILELYSKLGIVAEPAGATSVAALELIKDEIKGKNIVCIISGGNNDISRMQEIEERALVYEGLKHYFVINFPQRPGSLRTFVSDILGPNDDITRFEYIKRADKGKGPCLVGILLSDASDYDSLINRIERFDNRYVNLRGNDSLYELLV"BASE COUNT 4254 a 2046 c 2621 g 3799 tORIGINM90761 Length: 12720 April 6, 1993 18:44 Type: NCheck: 7819 . . 1 TAAAACTCGA TAATCTTGAG TCATAATTTC TCCTTAATCTTATTAGTACA 51 TTAGAATCCA TTATAATTTA ATCATTTTAT GTCTACCTAAAGCAACAAAA 101 TTGCTTGTAT ATTTTCTAAC AAGCTTAATT ATGTGGATTTAATTGAATAT 151 TAAAGGGAGA AGTTGTAATC TATTTGTTGT TAAATTCTTGTTAATACAAA 201 TAAATTTATT AAATATTATT ATTTTATTGA CAATTTAAAATATTAAGAGT 251 ATTATAATGT AAATTAACAA AAAAAAGAGG AACTTGAAATGACATACACA 301 CAATTTTCAT TGTTGTTGAT CAAGGTGGAC CTACATTAGCTTTTTTGGCT 351 AAAATATGTG GGTCCTGTTT GGCGATAGTC ATTTCGAGGACCGAGAGACG 401 TCCTCACGGG CGTCTTTTTT GTTTCTTAAT AAAAAATAGAGGTAATATTA 451 TGCGAAAAAT TGAATTCTTT GACACAAGTT TGAGAGATGGCGAACAGACA 501 CCGGGCGTTA GTTTCTCCAT TTCAGAAAAA GTAACGATTGCTAAACAACT 551 GGAAAAATGG AGGATTTCTG TCATAGAGGC TGGTTTTTCTGCGGCAAGTC 601 CAGATAGTTT TGAAGCAGTA AAGCAAATTG CTGATTCTTTGAATGATACG 651 GCTGTCACTG CATTAGCTCG CTGTGTTATT TCAGATATCGATAAAGCGGT 701 TGAAGCGGTA AAGGGGGCTA AATATCCGCA AATTCATGTTTTCATTGCAA 751 CTTCACCTAT TCACATGAAA TATAAACTTA AAATCAGTCCCGAAGAAGTT 801 TTGAAAAATA TTGATAAGTG TGTGAGATAC GCACGTGAACGGGTCGAGGT 851 TGTTGAGTTT TCTCCAGAGG ATGCAACAAG AACGGAGTTGAATTTTCTTT 901 TAGAGGCTGT TCAAACGGCT GTCGATGCTG GAGCAACTTATATTAATATT 951 CCTGACACTG TCGGTTATAC GACACCAGAA GAATATGGAAAAATTTTTAA 1001 ATTTTTGATT GATAATACTA AGTCTGACCG AGAAATTATTTTTAGTCCAC 1051 ATTGTCATGA TGATTTAGGA ATGGCTGTAG CTAATTCATTAGCTGCAATT 1101 AAAGCTGGGG CTGGGAGAGT TGAAGGAACT GTCAATGGTATTGGAGAGCG 1151 AGCTGGGAAT GCTGCTCTTG AAGAAATTGC TGTGGCACTACATATTCGTA 1201 AAGATTTTTA TCAGGCACAA AGTCCTTTAA AACTTTCAGAAACTGCTGCA 1251 ACGGCAGAAC TAATTTCACA ATTTTCAGGA ATTGCTATTCCAAAAAATAA 1301 AGCAATTGTT GGTGCTAATG CTTTTGCACA CGAATCAGGAATTCATCAAG 1351 ATGGTGTCCT TAAAAATGCT GAAACTTATG AAATTATTACACCAGAACTT 1401 GTCGGAATAA AGCATAATTC GTTGCCTTTA CGTAAACTTTCTGGTCGTCA 1451 TGCTTTTAGT GAAAAATTGA CGGAACTTAA TATTGCTTATGACGATGAAA 1501 GTCTTGCAAT TTTATTTGAA AAATTTAAAA AATTAGCTGACAAGAAAAAA 1551 GAAATTACTG ACGCAGATAT TCATGCCTTG TTTACAGGAGAAACGGTAAA 1601 AAATCTAGCT GGATTTATAC TTGATAATGT TCAAATTGATGGGCACAAGG 1651 CATTGGTGCA ACTAAAAAAT CAAGAAGAGG AAATTTATGTTAGCCAAGGA 1701 GAGGGGTCAG GTTCAGTGGA TGCAATTTTT AAAGCTATTGATAAAGTCTT 1751 TAATCATCAA CTAAAATTAA TTTCCTATTC AGTTGATGCTGTAACTGATG 1801 GAATTGATGC ACAAGCAACG ACTTTGGTTT CTGTTGAAAATCTATCTACA 1851 GGCACTATAT TTAATGCTAA AGGTGTTGAT TATGATGTATTGAAAGGAAG 1901 CGCCATTGCT TACATGAACG CTAATGTTTT AGTTCAAAAAGAAAATTTAC 1951 AAGGAAAGGT TGAACAAATT TCAGCTCATG ATGGAATTTAAGGTGAAAAA 2001 TATTGTCTAA AAAAATTGTG ACACTTGCGG GAGATGGAATTGGGCCAGAA 2051 ATTATGTCAG CTGGTTTAAG TGTTTTAAAA GCTGTCAGTAAAAAAATTGA 2101 TTTTGAGTAT GAATTAGAAG CTAAAGATTT TGGAGGAATTGCAATTGATA 2151 AGCATGGTCA TCCTTTACCA GAAGAAACTT TGCAAGCAGTTAAAAATGCT 2201 GACGCAATCT TGCTCGCTGC AATTGGTCAT CCTAAATACAACAATGCAAA 2251 AGTTAGACCA GAACAAGGGC TACTTGCTTT ACGAAAAGAATTAGGACTGT 2301 ATGCTAATGT TCGTCCATTA AAAATTTATC CGGCTCTAAAAAAACTTTCT 2351 CCCATACGAA ATGTTGAAAA TGTTGATTTC CTAGTGATTCGCGAACTTAC 2401 AGGGGGAATC TATTTCGGTC AGCATGAATT GGCAGATGATAAAGCACGAG 2451 ATGTCAATGA TTATTCTGCT GATGAAATAA GGAGAATTCTTCATTTTGCT 2501 TTCAAAAGTG CTCAAAGTCG GCCCAGAAAA TTACTGACTTCGGTTGATAA 2551 ACAAAATGTT CTTGCAACTT CTAAATTATG GCGAAAAATGGCTGATGAAA 2601 TTGCTGACGA ATATCCTGAT GTACGATTAG AGCACCAATTGGTCGATTCT 2651 TGTGCGATGT TACTGATTAC TAATCCGCAA CAATTTGATGTGATAGTCAC 2701 TGAAAATCTA TTTGGTGATA TTCTCTCTGA TGAAGCAAGTAGTTTGGCCG 2751 GTAGCTTAGG AGTGATGCCT TCGAGTTCGC ATGGATTTAACGGTTTAGCA 2801 CTCTATGAGC CAATTCATGG TTCGGCACCA GATATTGCAGGAAAAGGAAT 2851 TGCGAACCCT GTTTCGATGA TTCTATCAAT TGCCATGATGCTAAGAGAAT 2901 CTTTTGGGCA AGAAGATGGG GCTGCGATGA TTGAAAAAGCCGTAACCCAA 2951 ACTTTTACTG ACGGAATTTT GACTAAAGAT TTAGGTGGGACTGCAACAAC 3001 TAAAGAAATG ACAGAAGCAA TCCTGAAAAA TTGTCAGTAAAATGCGATTG 3051 AATAGTGAGC ATTTTAGTTG TAGATAAAAG AACCGTCAGCATAGCTGACA 3101 ATTCTGTCAG TAAATGCGAT TGAATAGTGA GCATTTTAGTTGTAGATAAA 3151 AGAACCGTCA GCATAGCTGA CAATTCTGTC AGTAATTGCGATTGAATAGT 3201 GAGCATTTTA GTTGTAGATA AAAGAACCGT CAGCATAGCTGACAATTCTG 3251 TCAGTAATTG CGATTGAATA GTGAGCATTT TAGTTGTAGATAAAAGAACT 3301 ATCAGCGTAA CTGACAATTC TGTCAGTAAA TATTACTGACAAAAAGTACA 3351 AAATTACTGA CAGAATTTGT CAGAATAAAT TTTTAAAAAAGGAAATAAAA 3401 AAATGTCAGG TAAAACAATA TTTGATAAAC TTTGGGATCAGCATGTGATT 3451 GCTGGAAATG AGGGAGAACC TCAACTGCTT TATATTGACCTTCATGTTAT 3501 TCATGAGGTT ACGAGTCCGC AAGCATTTCA GGGCTTACGTGAAGCAGGAC 3551 GTCGTGTTCG GAGAAAAGAT TTGACATACG GAACTCTTGACCACAATGTT 3601 CCAACACAAA ATATTTTTAA TATTCAAGAT TTGATTTCTAAAAAACAAAT 3651 TGATACTTTT ACTAAAAATG TCAAAGAATT TGATGTTCCAGCGGAGACTC 3701 ATGGTGGAAA AGGACAAGGA ATTGTTCACA TGGTAGCACCTGAATCTGGC 3751 AGAACTCAAC CGGGAAAAAC AATTGTTTGT GGCGATAGTCATACCGCAAC 3801 AAATGGAGCA TTTGGTGCAA TTGCTTTTGG AATTGGTACAAGTGAAGTTG 3851 AACATGTTCT TGCAACTCAA ACCATTTGGC AAGTTAAACCCAAGCGTATG 3901 AAAATTGAAT TTCAAGGTCA TCCACAAAAA GGAATTTATAGCAAAGACTT 3951 TATCCTCGCA TTAATTGCTA AATATGGTGT GGATGCAGGTGTAGGTTATG 4001 CGGTTGAATA TAGTGGGGAT GCTATCAGTG ATTTAAGCATGGAAGAACGG 4051 ATGACAATCT GTAACATGTC AATTGAATTT GGGGCAAAAATTGGCCTGAT 4101 GAATCCTGAT GAAAAAACTT ATGACTATGT CAAAGGGCGTGAACATGCAC 4151 CTAAAAACTT TGATGAAGCT GTCAGTAAAT GGGAAAAACTTGTCAGTGAT 4201 TCTGATGCAC AATACGATAA GATTTTAAGT CTTGATGTCAGCCAGTTGAA 4251 ACCAATGGTG ACATGGGGAA CAAATCCCGG AATGGGCCTAGAATTTGGCG 4301 AAAAGTTTCC GGAAATTAAC AATGATTTGA ATTATGAACGTGCTTATCAG 4351 TACATGGATT TAAAGCCAGG CCAAACCGCT TCTGACATAGATTTAGGCTA 4401 TATTTTCATT GGTTCTTGTA CGAATGCTAG ACTTGGTGATTTAGAAGAAG 4451 CTGCAAAAAT TATTGGAGAC AGACATATTG CTGATGGACTGACAGGAATT 4501 GTCGTCCCTG GAAGCAGACC TGTGAAAGAA GCGGCTGAAGCACAAGGGCT 4551 TGATAAAATT TTTAAAGAAG CTGGTTTTGA ATGGCGGGAACCGGGTTGCT 4601 CAGCCTGTCT TGGAATGAAT CCTGACCAAA TTCCAGAATACGTTCATTGT 4651 GCTTCAACCT CTAATCGAAA TTTTGAAGGT CGTCAAGGACATAATGCAAG 4701 AACGCACCTG TGCTCTCCAG CTATGGCTGC TGCCGCCGCAATCGCTGGTA 4751 AATTTGTAGA TGTTAGAATG CTCGTAACAG ATTAGTCTGTAGAAAGAAAA 4801 AAAGATGGAA AAATTCACGA TTTACAAAGG GACAAGTGTTCCAGTCATGA 4851 ACGATAATAT TGACACAGAC CAAATTATTC CTAAACAATTTTTGAAAGCA 4901 ATCGATAAAA AGGGCTTTGG GAAAAATTTA TTTTATGAATGGCGTTATCT 4951 TAAAGATTAC GATGAGAATC CTGATTTTAT TTTGAATGCTCCAAAATACA 5001 AAAAAGCTTC TCTGTTAATT TCAGGAGATA ATTTTGGTTCGGGTTCTTCA 5051 AGAGAACATG CGGCATGGGC CTTATCAGAT TACGGCTTTCGGGCAATTAT 5101 TGCTGGCTCT TACTCAGATA TTTTTTATAA TAATGCTTTAAAAAATGGCT 5151 TGTTACCAAT TAAACAACCA AGAGAAGTTC TAAATCAACTGACAAAACTG 5201 TCAAGTCAAG AAGAAATTAC AATTGATTTA CCCCATCAGCTAATCATCAC 5251 AAGCCTTGGT GACTTTCATT TTGAGATTGA CCCCATTTGGAAAGACAAAT 5301 TAATTAATGG CTTAGATGAT ATTGGAATAA CTTTGCAATATGAAGAAGCA 5351 ATCTCAGCTT ACGAACAAAA AAATCAATAA GAGCGAGCCTAAAATGACAA 5401 TTATTAATTT AAAGAATGTA AATCTTACTC GAAATAAAAAAGAAATTCTT 5451 AAAGATATTA CTTGGAAAGT AAATCCCGGC GAAAATTGGGTTATTCTGGG 5501 CCTCAACGGC TCTGGAAAAT CAAGTCTTTT GAAATTGATTTTAGCAGAAG 5551 AATGGAAAAC TTCTGGTGAA ATCACTGTTT TAAATACTCAATTTAGAAAT 5601 GGAGAAATTC CTAAGTTGAG AAAAAGAATC AGCGTAGTTGGCTCATTTAT 5651 TGCTGAAAGA TTTCAACCAA ATATTAAGGC TGAAAACCTTGTTTATACTG 5701 GGAAATTTAA TTCGAGCATG CTCTATAAAC CCTACACAGATCAGGAACTT 5751 GATGAGGCCC GTCAGCTTTT AAGACAAATG GGCGCAAAATCACTTATTGG 5801 CCGAAATTAT GCCAGCCTTT CTCAAGGGGA AAAGCAAGTTCTTCTTATTG 5851 CTAGGAGCTT AATTTTAAAG CCTGAGCTTT TAATTTTGGACGAAGCAACG 5901 AACGGTTTAG ATTTATTTGC TAAAGAAAAA TTATTAAAGCAACTGCAGCA 5951 GATTAATCAA TTAAAAACCG CACCAACACT AATTTATATTTCTCATCATC 6001 CCGATGAAAT CACTGATATT TTTACTCACC TTTTACTTTTAAGAGAAGGA 6051 AAAGTGATTC AATCAGGGAA AAAAGAAAAC TTATTAAATGAAAAGATACT 6101 TACTGATTTT TATCAAGAAA AAGTAGAAGT TCACCGTTTTGAGCAGAAAT 6151 ATTTTGTAAT TCCTGCTAAC TGAGAAAGGA AAGCAAAAGTATTTTATATA 6201 CTATATAGAA TATTCTGACA GATTATTGTA TTTTCATTTTTTTAGTGATA 6251 AAATAGCTCT ATGTAAATTT ACGGGGAGGT CAAAAAGATAACATATGGAA 6301 TTCAAATATA ACGGAAAAGT TGAATCAGTG GAACTCAATAAATATTCTAA 6351 GACATTGACT CCAAGATCAA CACAACCAGC GACTCAAGCGATGTACTACG 6401 GCATTGGTTT TAAAGATGAG GATTTCAAAA AAGCTCAGGTCGGAATCGTC 6451 AGCATGGATT GGGACGGAAA TCCATGTAAT ATGCACTTGGGAACACTTGG 6501 GAGTAAAATC AAAAGTTCTG TCAACCAAAC TGACGGATTGATTGGACTTC 6551 AATTTCATAC TATTGGAGTT TCTGATGGAA TTGCTAACGGAAAGCTTGGC 6601 ATGAGATATT CTTTGGTCAG TCGTGAAGTT ATTGCTGACAGCATCGAAAC 6651 CAACGCTGGC GCAGAATATT ATGATGCCAT CGTTGCCATTCCCGGTTGTG 6701 ATAAAAATAT GCCCGGGTCA ATTATCGGAA TGGCTCGCTTAAATCGTCCG 6751 TCAATTATGG TCTATGGTGG AACGATTGAA CATGGCGAATATAAAGGTGA 6801 AAAATTAAAT ATTGTTTCGG CCTTTGAAGC TCTGGGGCAAAAAATCACTG 6851 GAAATATTTC TGATGAAGAT TATCATGGCG TTATTTGCAATGCCATTCCA 6901 GGACAAGGTG CTTGCGGAGG AATGTACACT GCCAATACCCTGGCTGCTGC 6951 TATTGAAACT TTGGGAATGA GTTTACCTTA TTCCTCTTCCAATCCAGCAG 7001 TCAGTCAAGA AAAACAAGAA GAGTGTGATG ACATTGGTTTAGCCATCAAA 7051 AATTTATTAG AAAAAGATAT TAAACCAAGT GATATCATGACCAAAGAAGC 7101 TTTTGAAAAT GCCATAACAA TTGTCATGGT CCTTGGAGGCTCAACCAATG 7151 CTGTGCTTCA TATCATTGCA ATGGCAAATG CCATTGGTGTAGAAATTACG 7201 CAAGATGATT TCCAACGTAT TTCAGATATT ATCCCTGTTCTTGGCGATTT 7251 CAAACCGAGC GGAAAATATA TGATGGAAGA TCTGCACAAAATTGGTGGCC 7301 TTCCTGCTGT TTTGAAATAC CTACTTAAAG AAGGAAAACTTCACGGTGAT 7351 TGTTTGACCG TCACAGGTAA AACTTTGGCT GAAAATGTTGAAACAGCATT 7401 AGATTTGGAC TTTGACAGTC AAGATATTAT GCGACCACTAAAAAATCCAA 7451 TTAAAGCTAC TGGACATTTA CAAATTTTGT ACGGTAATCTTGCCCAAGGG 7501 GGTTCTGTTG CAAAAATTTC TGGTAAAGAA GGCGAATTTTTCAAAGGAAC 7551 AGCTCGTGTT TTTGACGGAG AACAACACTT TATCGATGGCATTGAGTCTG 7601 GCCGATTGCA TGCCGGTGAT GTTGCGGTCA TTAGAAATATTGGCCCAGTC 7651 GGAGGTCCGG GAATGCCAGA GATGTTAAAA CCAACCTCAGCATTAATTGG 7701 AGCAGGACTT GGAAAATCTT GTGCCCTAAT TACTGACGGAAGATTTTCTG 7751 GTGGCACACA CGGCTTTGTT GTGGGTCATA TCGTCCCTGAAGCAGTTGAA 7801 GGTGGGTTGA TTGGTTTAGT TGAAGATGAT GATATTATCGAAATTGATGC 7851 GGTGAATAAT AGTATTAGTT TAAAAGTTTC TAATGAAGAAATTGCTAAAC 7901 GACGTGCCAA TTATCAAAAA CCAACCCCTA AAGCAACGCGTGGTGTTCTT 7951 GCAAAATTTG CCAAACTTAC GCGCCCCGCT AGTGAAGGTTGCGTTACAGA 8001 TTTATAGAAA GGTTTGAATG AAAAAAATAA AGTTAGAAAAACCTACTTCC 8051 GGTTCCCAAC TTGTTCTCCA AACCTTAAAA GAACTTGGAGTAGAAATTAT 8101 TTTTGGTTAT CCTGGTGGGG CCATGCTCCC CTTGTATGATGCGATTCATA 8151 ATTTTGAAGG AATTCAACAT ATTTTAGCCC GTCATGAGCAAGGAGCAACG 8201 CATGAAGCCG AAGGTTACGC TAAATCGTCT GGTAAAGTTGGTGTCGTCGT 8251 TGTTACGTCA GGACCAGGAG CGACTAATGC AGTAACCGGAATTGCTGACG 8301 CTTATCTTGA TTCAGTCCCA TTGTTAGTTT TCACAGGTCAAGTTGGCCGT 8351 CAGTCAATTG GTAAAGATGC TTTTCAAGAA GCAGATACTGTTGGAATTAC 8401 AGCCCCAATT ACAAAATATA ATTATCAAAT TAGGGAAACCGCAGATATTC 8451 CAAGAATTGT TACAGAAGCC TATTATTTGG CAAGGACAGGACGTCCTGGA 8501 CCAGTAGAAA TTGATTTACC AAAAGATGTT TCCACCCTTGAAGTCACTGA 8551 AATTAATGAC CCAAGCTTGA ATCTTCCTCA TTATCACGAAAGTGAAAAAG 8601 CGACTGATGA ACAATTGCAA GAATTACTGA CAGAACTTTCTGTCAGTAAA 8651 AAACCAGTCA TTATTGCTGG CGGAGGAATT AATTATTCTGGCTCAGTTGA 8701 TATTTTCAGA GCATTTGTCG AAAAATATCA AATTCCAGTTGTTTCTACAT 8751 TGCTTGGCTT AGGAACATTA CCAATCAGCC ACGAATTGCAACTAGGAATG 8801 GCAGGAATGC ACGGTTCATA CGCTGCAAAT ATGGCTTTAGTTGAAGCTGA 8851 CTATATTATT AATTTGGGAT CACGTTTTGA CGATAGAGTTGTATCCAATC 8901 CTGCAAAATT TGCTAAAAAT GCTGTCGTTG CTCATATTGATATTGACGCT 8951 GCTGAACTTG GCAAAATTGT AAAAACCGAT ATTCCAATCCTTTCTGATTT 9001 GAAAGCGGCT TTAAGCAGAC TTTTGCAATT AAATAAGGTCAGGACTGACT 9051 TTAATGATTG GATTAAAACT GTCATTGAAA ATAAAGAGAAAGCACCATTT 9101 ACTTATGAGC CCCAAAACCA TGATATCCGT CCACAGGAAACAATTAAATT 9151 AATTGGAGAA TACACTCAAG GAGATGCAAT CATTGTAACTGACGTTGGGC 9201 AACATCAAAT GTGGGTGGCG CAATATTATC CTTATAAAAATGCAAGGCAA 9251 CTTATTACTT CTGGGGGAAT GGGAACGATG GGCTTTGGCATTCCTGCAGC 9301 AATCGGTGCA AAGCTGGCAC AGCCAAATAA AAATGTCATTGTTTTTGTTG 9351 GCGATGGTGG CTTTCAAATG ACTAATCAAG AATTAGCATTACTTAATGGC 9401 TACGGTATTG CAATCAAAGT TGTGCTGATT AATAATCATTCATTGGGAAT 9451 GGTACGTCAA TGGCAAGAAT CATTCTATGA AGAGCGACGTTCACAATCGG 9501 TTTTTGATGT TGAACCCAAT TTTCAATTGT TAGCCGAAGCTTATGGCATC 9551 AAACATGTTA AGTTAGATAA TCCAAAAACT TTGGCTGATGATTTAAAAAT 9601 TATTACAGAA GATGAGCCAA TGCTTATTGA AGTTCTAATTTCAAAATCTG 9651 AGCATGTTTT ACCAATGATA CCAGCTGGAT TACACAATGACGAAATGATT 9701 GGACTTCATT TTACTGATAA GAATGAGGAG ATAGATAATGCGTAGAATGA 9751 TTATCGCAAA ACTTCATAAC GTGACAGGAA TTATGAATCGATTTACCGCC 9801 GTTCTCAATC GAAGGCAAGT GAACATTCTC TCAATTACCGCTGGAGTTAC 9851 AGAAAGTCAA GACTTAACTC ATACCACTTT TGTTATTGAAGTTGATCATC 9901 TTGATGAAGT AGAACAAATC ATTAAACAAT TAAATCGCTTAATAGATGTA 9951 ATTGAAGTAG CTGATATTAC TGATTTTCCT CATGTAGAACGTGAAGTCGT 10001 CTTGATTAAA GTATCAGCTC CACCGACCAT TAGGGCAGAAATTTTTACAA 10051 TGATTGAACC TTTTAGAGTA AATGTAGTTG ATGTCAATCTGGAAAATGTC 10101 ACCATTCAAT TAACGGGTGA TTCAGCAAAA ATCGAAGCACTTATTGAGGT 10151 TGTTAGTCCT TATGGCATTC TAAATATGGC TCGGACAGGTAGTGCAGGTT 10201 TTGAGCGTGG CTAAATTTAA ATAAGTTAAC AAATAAATAGAAAAATAGAG 10251 GAAACAAAAA TGGCAGTTAC AATGTATTAT GAAGATGATGTAGAAGTATC 10301 AGCACTTGCT GGAAAGCAAA TTGCAGTAAT CGGTTATGGTTCACAAGGAC 10351 ATGCTCACGC ACAGAATTTG CGTGATTCTG GTCACAACGTTATCATTGGT 10401 GTGCGCCACG GAAAATCTTT TGATAAAGCA AAAGAAGATGGCTTTGAAAC 10451 ATTTGAAGTA GGAGAAGCAG TAGCTAAAGC TGATGTTATTATGGTTTTGG 10501 CACCAGATGA ACTTCAACAA TCCATTTATG AAGAGGACATCAAACCAAAC 10551 TTGAAAGCAG GTTCAGCACT TGGTTTTGCT CACGGATTTAATATCCATTT 10601 TGGCTATATT AAAGTACCAG AAGACGTTGA CGTCTTTATGGTTGCGCCTA 10651 AGGCTCCAGG TCACCTTGTC CGTCGGACTT ATACTGAAGGTTTTGGTACA 10701 CCAGCTTTGT TTGTTTCACA CCAAAATGCA AGTGGTCATGCGCGTGAAAT 10751 CGCAATGGAT TGGGCCAAAG GAATTGGTTG TGCTCGAGTGGGAATTATTG 10801 AAACAACTTT TAAAGAAGAA ACAGAAGAAG ATTTGTTTGGAGAACAAGCT 10851 GTTCTATGTG GAGGTTTGAC AGCACTTGTT GAAGCCGGTTTTGAAACACT 10901 GACAGAAGCT GGATACGCTG GCGAATTGGC TTACTTTGAAGTTTTGCACG 10951 AAATGAAATT GATTGTTGAC CTCATGTATG AAGGTGGTTTTACTAAAATG 11001 CGTCAATCCA TCTCAAATAC TGCTGAGTTT GGCGATTATGTGACTGGTCC 11051 ACGGATTATT ACTGACGAAG TTAAAAAGAA TATGAAGCTTGTTTTGGCTG 11101 ATATTCAATC TGGAAAATTT GCTCAAGATT TCGTTGATGACTTCAAAGCG 11151 GGGCGTCCAA AATTAATAGC CTATCGCGAA GCTGCAAAAAATCTTGAAAT 11201 TGAAAAAATT GGGGCAGAGC ACGTCAAGCA ATGCCATTCACACAATCTGG 11251 TGATGACGAT GCCTTTAAAA TCTATCAGTA ATTTCTCTTATTGATTGAAC 11301 AAAAACATAA AAGCATTTTA TGGAGGAATG ACATAAATGATAAGTGCCAA 11351 AGAGGTTGAA GATGCCTATG ATTTGTTAAA AGCAGTTGTCACTAAAACAC 11401 CTTTACAATT AGACCCTTAC CTTTCCAATA AATATCAAGCAAATATTTAC 11451 TTAAAAGAAG TTGTCACTAA AACACCTTTA CAATTAGACCCTTACCTTTC 11501 CAATAAATAT CAAGCAAATA TTTACTTAAA AGAAGAAAACTTACAGAAAG 11551 TTCGTTCTTT TAAATTACGA GGAGCTTATT ATTCTATCAGTAAATTATCT 11601 GATGAGCAAC GCTCTAAAGG AGTGGTTTGT GCCTCAGCAGGAAATCATGC 11651 ACAAGGGGTT GCTTTTGCTG CAAATCAATT AAATATTTCTGCGACAATTT 11701 TTATGCCCGT TACCACACCT AACCAAAAAA TTTCACAAGTTAAATTTTTT 11751 GGCGAAAGTC ACGTAACAAT TCGTTTAATT GGTGATACTTTTGATGAATC 11801 AGCCAGAGCA GCAAAAGCTT TTTCTCAAGA TAATGACAAACCATTTATAG 11851 ACCCTTTTGA TGATGAAAAT GTAATTGCTG GTCAAGGGACAGTGGCTTTA 11901 GAAATTTTTG CGCAAGCTAA AAAACAAGGA ATAAGTTTAGATAAGATTTT 11951 TGTACAGATT GGTGGAGGTG GTTTAATTGC AGGAATTACTGCCTACAGTA 12001 AGGAGCGCTA TCCCCAAACT GAAATTATCG GAGTTGAAGCAAAAGGGGCA 12051 ACAAGTATGA AAGCTGCCTA CTCTGCTGGT CAGCCCGTCACCTTGGAACA 12101 CATTGATAAA TTTGCTGACG GAATTGCGGT TGCGACTGTCGGTCAGAAAA 12151 CTTACCAACT TATTAATGAC AAAGTGAAAC AATTGCTTGCGGTTGATGAA 12201 GGTTTAATTT CTCAAACCAT ACTCGAATTG TATTCAAAATTAGGAATTGT 12251 CGCCGAGCCA GCAGGTGCAA CATCTGTTGC CGCACTTGAACTTATTAAAG 12301 ATGAAATCAA GGGTAAAAAT ATCGTCTGTA TCATCAGCGGCGGAAATAAT 12351 GATATTAGTC GAATGCAAGA AATTGAAGAA AGAGCTTTGGTTTATGAAGG 12401 TCTAAAACAT TATTTTGTCA TTAACTTTCC TCAAAGACCAGGATCCTTAC 12451 GAACTTTTGT CAGTGATATT TTAGGGCCAA ATGATGATATCACCCGATTT 12501 GAGTACATCA AAAGGGCTGA TAAAGGTAAA GGACCTTGTCTTGTTGGGAT 12551 TTTACTTTCA GATGCTAGTG ATTATGATTC ATTGATTAATCGGATTGAAA 12601 GATTTGATAA TCGTTATGTT AACTTACGTG GAAATGATAGTTTATACGAA 12651 CTTTTGGTCT AACTAACCAA TTGGTTTGAG CCATTTTCTAGTTTCAATTC 12701 TCTTTAAATC ACTAGAAATTilvb Length: 1728 September 22, 1993 11:08 Type: NCheck: 9885 . . 1 ATGAAAAAAA TAAAGTTAGA AAAACCTACT TCCGGTTCCCAACTTGTTCT 51 CCAAACCTTA AAAGAACTTG GAGTAGAAAT TATTTTTGGTTATCCTGGTG 101 GGGCCATGCT CCCCTTGTAT GATGCGATTC ATAATTTTGAAGGAATTCAA 151 CATATTTTAG CCCGTCATGA GCAAGGAGCA ACGCATGAAGCCGAAGGTTA 201 CGCTAAATCG TCTGGTAAAG TTGGTGTCGT CGTTGTTACGTCAGGACCAG 251 GAGCGACTAA TGCAGTAACC GGAATTGCTG ACGCTTATCTTGATTCAGTC 301 CCATTGTTAG TTTTCACAGG TCAAGTTGGC CGTCAGTCAATTGGTAAAGA 351 TGCTTTTCAA GAAGCAGATA CTGTTGGAAT TACAGCCCCAATTACAAAAT 401 ATAATTATCA AATTAGGGAA ACCGCAGATA TTCCAAGAATTGTTACAGAA 451 GCCTATTATT TGGCAAGGAC AGGACGTCCT GGACCAGTAGAAATTGATTT 501 ACCAAAAGAT GTTTCCACCC TTGAAGTCAC TGAAATTAATGACCCAAGCT 551 TGAATCTTCC TCATTATCAC GAAAGTGAAA AAGCGACTGATGAACAATTG 601 CAAGAATTAC TGACAGAACT TTCTGTCAGT AAAAAACCAGTCATTATTGC 651 TGGCGGAGGA ATTAATTATT CTGGCTCAGT TGATATTTTCAGAGCATTTG 701 TCGAAAAATA TCAAATTCCA GTTGTTTCTA CATTGCTTGGCTTAGGAACA 751 TTACCAATCA GCCACGAATT GCAACTAGGA ATGGCAGGAATGCACGGTTC 801 ATACGCTGCA AATATGGCTT TAGTTGAAGC TGACTATATTATTAATTTGG 851 GATCACGTTT TGACGATAGA GTTGTATCCA ATCCTGCAAAATTTGCTAAA 901 AATGCTGTCG TTGCTCATAT TGATATTGAC GCTGCTGAACTTGGCAAAAT 951 TGTAAAAACC GATATTCCAA TCCTTTCTGA TTTGAAAGCGGCTTTAAGCA 1001 GACTTTTGCA ATTAAATAAG GTCAGGACTG ACTTTAATGATTGGATTAAA 1051 ACTGTCATTG AAAATAAAGA GAAAGCACCA TTTACTTATGAGCCCCAAAA 1101 CCATGATATC CGTCCACAGG AAACAATTAA ATTAATTGGAGAATACACTC 1151 AAGGAGATGC AATCATTGTA ACTGACGTTG GGCAACATCAAATGTGGGTG 1201 GCGCAATATT ATCCTTATAA AAATGCAAGG CAACTTATTACTTCTGGGGG 1251 AATGGGAACG ATGGGCTTTG GCATTCCTGC AGCAATCGGTGCAAAGCTGG 1301 CACAGCCAAA TAAAAATGTC ATTGTTTTTG TTGGCGATGGTGGCTTTCAA 1351 ATGACTAATC AAGAATTAGC ATTACTTAAT GGCTACGGTATTGCAATCAA 1401 AGTTGTGCTG ATTAATAATC ATTCATTGGG AATGGTACGTCAATGGCAAG 1451 AATCATTCTA TGAAGAGCGA CGTTCACAAT CGGTTTTTGATGTTGAACCC 1501 AATTTTCAAT TGTTAGCCGA AGCTTATGGC ATCAAACATGTTAAGTTAGA 1551 TAATCCAAAA ACTTTGGCTG ATGATTTAAA AATTATTACAGAAGATGAGC 1601 CAATGCTTAT TGAAGTTCTA ATTTCAAAAT CTGAGCATGTTTTACCAATG 1651 ATACCAGCTG GATTACACAA TGACGAAATG ATTGGACTTCATTTTACTGA 1701 TAAGAATGAG GAGATAGATA ATGCGTAGilvn Length: 477 September 22, 1993 11:09 Type: NCheck: 6061 . . 1 ATGCGTAGAA TGATTATCGC AAAACTTCAT AACGTGACAGGAATTATGAA 51 TCGATTTACC GCCGTTCTCA ATCGAAGGCA AGTGAACATTCTCTCAATTA 101 CCGCTGGAGT TACAGAAAGT CAAGACTTAA CTCATACCACTTTTGTTATT 151 GAAGTTGATC ATCTTGATGA AGTAGAACAA ATCATTAAACAATTAAATCG 201 CTTAATAGAT GTAATTGAAG TAGCTGATAT TACTGATTTTCCTCATGTAG 251 AACGTGAAGT CGTCTTGATT AAAGTATCAG CTCCACCGACCATTAGGGCA 301 GAAATTTTTA CAATGATTGA ACCTTTTAGA GTAAATGTAGTTGATGTCAA 351 TCTGGAAAAT GTCACCATTC AATTAACGGG TGATTCAGCAAAAATCGAAG 401 CACTTATTGA GGTTGTTAGT CCTTATGGCA TTCTAAATATGGCTCGGACA 451 GGTAGTGCAG GTTTTGAGCG TGGCTAAilvbn Length: 2231 September 22, 1993 11:12 Type: NCheck: 7704 . . 1 ATTTATAGAA AGGTTTGAAT GAAAAAAATA AAGTTAGAAAAACCTACTTC 51 CGGTTCCCAA CTTGTTCTCC AAACCTTAAA AGAACTTGGAGTAGAAATTA 101 TTTTTGGTTA TCCTGGTGGG GCCATGCTCC CCTTGTATGATGCGATTCAT 151 AATTTTGAAG GAATTCAACA TATTTTAGCC CGTCATGAGCAAGGAGCAAC 201 GCATGAAGCC GAAGGTTACG CTAAATCGTC TGGTAAAGTTGGTGTCGTCG 251 TTGTTACGTC AGGACCAGGA GCGACTAATG CAGTAACCGGAATTGCTGAC 301 GCTTATCTTG ATTCAGTCCC ATTGTTAGTT TTCACAGGTCAAGTTGGCCG 351 TCAGTCAATT GGTAAAGATG CTTTTCAAGA AGCAGATACTGTTGGAATTA 401 CAGCCCCAAT TACAAAATAT AATTATCAAA TTAGGGAAACCGCAGATATT 451 CCAAGAATTG TTACAGAAGC CTATTATTTG GCAAGGACAGGACGTCCTGG 501 ACCAGTAGAA ATTGATTTAC CAAAAGATGT TTCCACCCTTGAAGTCACTG 551 AAATTAATGA CCCAAGCTTG AATCTTCCTC ATTATCACGAAAGTGAAAAA 601 GCGACTGATG AACAATTGCA AGAATTACTG ACAGAACTTTCTGTCAGTAA 651 AAAACCAGTC ATTATTGCTG GCGGAGGAAT TAATTATTCTGGCTCAGTTG 701 ATATTTTCAG AGCATTTGTC GAAAAATATC AAATTCCAGTTGTTTCTACA 751 TTGCTTGGCT TAGGAACATT ACCAATCAGC CACGAATTGCAACTAGGAAT 801 GGCAGGAATG CACGGTTCAT ACGCTGCAAA TATGGCTTTAGTTGAAGCTG 851 ACTATATTAT TAATTTGGGA TCACGTTTTG ACGATAGAGTTGTATCCAAT 901 CCTGCAAAAT TTGCTAAAAA TGCTGTCGTT GCTCATATTGATATTGACGC 951 TGCTGAACTT GGCAAAATTG TAAAAACCGA TATTCCAATCCTTTCTGATT 1001 TGAAAGCGGC TTTAAGCAGA CTTTTGCAAT TAAATAAGGTCAGGACTGAC 1051 TTTAATGATT GGATTAAAAC TGTCATTGAA AATAAAGAGAAAGCACCATT 1101 TACTTATGAG CCCCAAAACC ATGATATCCG TCCACAGGAAACAATTAAAT 1151 TAATTGGAGA ATACACTCAA GGAGATGCAA TCATTGTAACTGACGTTGGG 1201 CAACATCAAA TGTGGGTGGC GCAATATTAT CCTTATAAAAATGCAAGGCA 1251 ACTTATTACT TCTGGGGGAA TGGGAACGAT GGGCTTTGGCATTCCTGCAG 1301 CAATCGGTGC AAAGCTGGCA CAGCCAAATA AAAATGTCATTGTTTTTGTT 1351 GGCGATGGTG GCTTTCAAAT GACTAATCAA GAATTAGCATTACTTAATGG 1401 CTACGGTATT GCAATCAAAG TTGTGCTGAT TAATAATCATTCATTGGGAA 1451 TGGTACGTCA ATGGCAAGAA TCATTCTATG AAGAGCGACGTTCACAATCG 1501 GTTTTTGATG TTGAACCCAA TTTTCAATTG TTAGCCGAAGCTTATGGCAT 1551 CAAACATGTT AAGTTAGATA ATCCAAAAAC TTTGGCTGATGATTTAAAAA 1601 TTATTACAGA AGATGAGCCA ATGCTTATTG AAGTTCTAATTTCAAAATCT 1651 GAGCATGTTT TACCAATGAT ACCAGCTGGA TTACACAATGACGAAATGAT 1701 TGGACTTCAT TTTACTGATA AGAATGAGGA GATAGATAATGCGTAGAATG 1751 ATTATCGCAA AACTTCATAA CGTGACAGGA ATTATGAATCGATTTACCGC 1801 CGTTCTCAAT CGAAGGCAAG TGAACATTCT CTCAATTACCGCTGGAGTTA 1851 CAGAAAGTCA AGACTTAACT CATACCACTT TTGTTATTGAAGTTGATCAT 1901 CTTGATGAAG TAGAACAAAT CATTAAACAA TTAAATCGCTTAATAGATGT 1951 AATTGAAGTA GCTGATATTA CTGATTTTCC TCATGTAGAACGTGAAGTCG 2001 TCTTGATTAA AGTATCAGCT CCACCGACCA TTAGGGCAGAAATTTTTACA 2051 ATGATTGAAC CTTTTAGAGT AAATGTAGTT GATGTCAATCTGGAAAATGT 2101 CACCATTCAA TTAACGGGTG ATTCAGCAAA AATCGAAGCACTTATTGAGG 2151 TTGTTAGTCC TTATGGCATT CTAAATATGG CTCGGACAGGTAGTGCAGGT 2201 TTTGAGCGTG GCTAAATTTA AATAAGTTAA C__________________________________________________________________________
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 16(2) INFORMATION FOR SEQ ID NO: 1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 513 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: LEUA(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:MetArgLysIleGluPhePheAspThrSerLeuArgAspGlyGluGln151015ThrProGlyValSerPheSerIleSerGluLysValThrIleAlaLys202530GlnLeuGluLysTrpArgIleSerValIleGluAlaGlyPheSerAla354045AlaSerProAspSerPheGluAlaValLysGlnIleAlaAspSerLeu505560AsnAspThrAlaValThrAlaLeuAlaArgCysValIleSerAspIle65707580AspLysAlaValGluAlaValLysGlyAlaLysTyrProGlnIleHis859095ValPheIleAlaThrSerProIleHisMetLysTyrLysLeuLysIle100105110SerProGluGluValLeuLysAsnIleAspLysCysValArgTyrAla115120125ArgGluArgValGluValValGluPheSerProGluAspAlaThrArg130135140ThrGluLeuAsnPheLeuLeuGluAlaValGlnThrAlaValAspAla145150155160GlyAlaThrTyrIleAsnIleProAspThrValGlyTyrThrThrPro165170175GluGluTyrGlyLysIlePheLysPheLeuIleAspAsnThrLysSer180185190AspArgGluIleIlePheSerProHisCysHisAspAspLeuGlyMet195200205AlaValAlaAsnSerLeuAlaAlaIleLysAlaGlyAlaGlyArgVal210215220GluGlyThrValAsnGlyIleGlyGluArgAlaGlyAsnAlaAlaLeu225230235240GluGluIleAlaValAlaLeuHisIleArgLysAspPheTyrGlnAla245250255GlnSerProLeuLysLeuSerGluThrAlaAlaThrAlaGluLeuIle260265270SerGlnPheSerGlyIleAlaIleProLysAsnLysAlaIleValGly275280285AlaAsnAlaPheAlaHisGluSerGlyIleHisGlnAspGlyValLeu290295300LysAsnAlaGluThrTyrGluIleIleThrProGluLeuValGlyIle305310315320LysHisAsnSerLeuProLeuGlyLysLeuSerGlyArgHisAlaPhe325330335SerGluLysLeuThrGluLeuAsnIleAlaTyrAspAspGluSerLeu340345350AlaIleLeuPheGluLysPheLysLysLeuAlaAspLysLysLysGlu355360365IleThrAspAlaAspIleHisAlaLeuPheThrGlyGluThrValLys370375380AsnLeuAlaGlyPheIleLeuAspAsnValGlnIleAspGlyHisLys385390395400AlaLeuValGlnLeuLysAsnGlnGluGluGluIleTyrValSerGln405410415GlyGluGlySerGlySerValAspAlaIlePheLysAlaIleAspLys420425430ValPheAsnHisGlnLeuLysLeuIleSerTyrSerValAspAlaVal435440445ThrAspGlyIleAspAlaGlnAlaThrThrLeuValSerValGluAsn450455460LeuSerThrGlyThrIlePheAsnAlaLysGlyValAspTyrAspVal465470475480LeuLysGlySerAlaIleAlaTyrMetAsnAlaAsnValLeuValGln485490495LysGluAsnLeuGlnGlyLysValGluGlnIleSerAlaHisAspGly500505510Ile(2) INFORMATION FOR SEQ ID NO: 2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 345 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: LEUB(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:LeuSerLysLysIleValThrLeuAlaGlyAspGlyIleGlyProGlu151015IleMetSerAlaGlyLeuSerValLeuLysAlaValSerLysLysIle202530AspPheGluTyrGluLeuGluAlaLysAspPheGlyGlyIleAlaIle354045AspLysHisGlyHisProLeuProGluGluThrLeuGlnAlaValLys505560AsnAlaAspAlaIleLeuLeuAlaAlaIleGlyHisProLysTyrAsn65707580AsnAlaLysValArgProGluGlnGlyLeuLeuAlaLeuArgLysGlu859095LeuGlyLeuTyrAlaAsnValArgProLeuLysIleTyrProAlaLeu100105110LysLysLeuSerProIleArgAsnValGluAsnValAspPheLeuVal115120125IleArgGluLeuThrGlyGlyIleTyrPheGlyGlnHisGluLeuAla130135140AspAspLysAlaArgAspValAsnAspTyrSerAlaAspGluIleArg145150155160ArgIleLeuHisPheAlaPheLysSerAlaGlnSerArgProArgLys165170175LeuLeuThrSerValAspLysGlnAsnValLeuAlaThrSerLysLeu180185190TrpArgLysMetAlaAspGluIleAlaAspGluTyrProAspValArg195200205LeuGluHisGlnLeuValAspSerCysAlaMetLeuLeuIleThrAsn210215220ProGlnGlnPheAspValIleValThrGluAsnLeuPheGlyAspIle225230235240LeuSerAspGluAlaSerSerLeuAlaGlySerLeuGlyValMetPro245250255SerSerSerHisGlyPheAsnGlyLeuAlaLeuTyrGluProIleHis260265270GlySerAlaProAspIleAlaGlyLysGlyIleAlaAsnProValSer275280285MetIleLeuSerIleAlaMetMetLeuArgGluSerPheGlyGlnGlu290295300AspGlyAlaAlaMetIleGluLysAlaValThrGlnThrPheThrAsp305310315320GlyIleLeuThrLysAspLeuGlyGlyThrAlaThrThrLysGluMet325330335ThrGluAlaIleLeuLysAsnCysGln340345(2) INFORMATION FOR SEQ ID NO: 3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 460 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: LEUC(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:MetSerGlyLysThrIlePheAspLysLeuTrpAspGlnHisValIle151015AlaGlyAsnGluGlyGluProGlnLeuLeuTyrIleAspLeuHisVal202530IleHisGluValThrSerProGlnAlaPheGlnGlyLeuArgGluAla354045GlyArgArgValArgArgLysAspLeuThrTyrGlyThrLeuAspHis505560AsnValProThrGlnAsnIlePheAsnIleGlnAspLeuIleSerLys65707580LysGlnIleAspThrPheThrLysAsnValLysGluPheAspValPro859095AlaGluThrHisGlyGlyLysGlyGlnGlyIleValHisMetValAla100105110ProGluSerGlyArgThrGlnProGlyLysThrIleValCysGlyAsp115120125SerHisThrAlaThrAsnGlyAlaPheGlyAlaIleAlaPheGlyIle130135140GlyThrSerGluValGluHisValLeuAlaThrGlnThrIleTrpGln145150155160ValLysProLysArgMetLysIleGluPheGlnGlyHisProGlnLys165170175GlyIleTyrSerLysAspPheIleLeuAlaLeuIleAlaLysTyrGly180185190ValAspAlaGlyValGlyTyrAlaValGluTyrSerGlyAspAlaIle195200205SerAspLeuSerMetGluGluArgMetThrIleCysAsnMetSerIle210215220GluPheGlyAlaLysIleGlyLeuMetAsnProAspGluLysThrTyr225230235240AspTyrValLysGlyArgGluHisAlaProLysAsnPheAspGluAla245250255ValSerLysTrpGluLysLeuValSerAspSerAspAlaGlnTyrAsp260265270LysIleLeuSerLeuAspValSerGlnLeuLysProMetValThrTrp275280285GlyThrAsnProGlyMetGlyLeuGluPheGlyGluLysPheProGlu290295300IleAsnAsnAspLeuAsnTyrGluArgAlaTyrGlnTyrMetAspLeu305310315320LysProGlyGlnThrAlaSerAspIleAspLeuGlyTyrIlePheIle325330335GlySerCysThrAsnAlaArgLeuGlyAspLeuGluGluAlaAlaLys340345350IleIleGlyAspArgHisIleAlaAspGlyLeuThrGlyIleValVal355360365ProGlySerArgProValLysGluAlaAlaGluAlaGlnGlyLeuAsp370375380LysIlePheLysGluAlaGlyPheGluTrpArgGluProGlyCysSer385390395400AlaCysLeuGlyMetAsnProAspGlnIleProGluTyrValHisCys405410415AlaSerThrSerAsnArgAsnPheGluGlyArgGlnGlyHisAsnAla420425430ArgThrHisLeuCysSerProAlaMetAlaAlaAlaAlaAlaIleAla435440445GlyLysPheValAspValArgMetLeuValThrAsp450455460(2) INFORMATION FOR SEQ ID NO: 4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 191 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: LEUD(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:MetGluLysPheThrIleTyrLysGlyThrSerValProValMetAsn151015AspAsnIleAspThrAspGlnIleIleProLysGlnPheLeuLysAla202530IleAspLysLysGlyPheGlyLysAsnLeuPheTyrGluTrpArgTyr354045LeuLysAspTyrAspGluAsnProAspPheIleLeuAsnAlaProLys505560TyrLysLysAlaSerLeuLeuIleSerGlyAspAsnPheGlySerGly65707580SerSerArgGluHisAlaAlaTrpAlaLeuSerAspTyrGlyPheArg859095AlaIleIleAlaGlySerTyrSerAspIlePheTyrAsnAsnAlaLeu100105110LysAsnGlyLeuLeuProIleLysGlnProArgGluValLeuAsnGln115120125LeuThrLysLeuSerSerGlnGluGluIleThrIleAspLeuProHis130135140GlnLeuIleIleThrSerLeuGlyAspPheHisPheGluIleAspPro145150155160IleTrpLysAspLysLeuIleAsnGlyLeuAspAspIleGlyIleThr165170175LeuGlnTyrGluGluAlaIleSerAlaTyrGluGlnLysAsnGln180185190(2) INFORMATION FOR SEQ ID NO: 5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 259 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ORF2(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:MetThrIleIleAsnLeuLysAsnValAsnLeuThrArgAsnLysLys151015GluIleLeuLysAspIleThrTrpLysValAsnProGlyGluAsnTrp202530ValIleLeuGlyLeuAsnGlySerGlyLysSerSerLeuLeuLysLeu354045IleLeuAlaGluGluTrpLysThrSerGlyGluIleThrValLeuAsn505560ThrGlnPheArgAsnGlyGluIleProLysLeuArgLysArgIleSer65707580ValValGlySerPheIleAlaGluArgPheGlnProAsnIleLysAla859095GluAsnLeuValTyrThrGlyLysPheAsnSerSerMetLeuTyrLys100105110ProTyrThrAspGlnGluLeuAspGluAlaArgGlnLeuLeuArgGln115120125MetGlyAlaLysSerLeuIleGlyArgAsnTyrAlaSerLeuSerGln130135140GlyGluLysGlnValLeuLeuIleAlaArgSerLeuIleLeuLysPro145150155160GluLeuLeuIleLeuAspGluAlaThrAsnGlyLeuAspLeuPheAla165170175LysGluLysLeuLeuLysGlnLeuGlnGlnIleAsnGlnLeuLysThr180185190AlaProThrLeuIleTyrIleSerHisHisProAspGluIleThrAsp195200205IlePheThrHisLeuLeuLeuLeuArgGluGlyLysValIleGlnSer210215220GlyLysLysGluAsnLeuLeuAsnGluLysIleLeuThrAspPheTyr225230235240GlnGluLysValGluValHisArgPheGluGlnLysTyrPheValIle245250255ProAlaAsn(2) INFORMATION FOR SEQ ID NO: 6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 570 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ILVD(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:MetGluPheLysTyrAsnGlyLysValGluSerValGluLeuAsnLys151015TyrSerLysThrLeuThrProArgSerThrGlnProAlaThrGlnAla202530MetTyrTyrGlyIleGlyPheLysAspGluAspPheLysLysAlaGln354045ValGlyIleValSerMetAspTrpAspGlyAsnProCysAsnMetHis505560LeuGlyThrLeuGlySerLysIleLysSerSerValAsnGlnThrAsp65707580GlyLeuIleGlyLeuGlnPheHisThrIleGlyValSerAspGlyIle859095AlaAsnGlyLysLeuGlyMetArgTyrSerLeuValSerArgGluVal100105110IleAlaAspSerIleGluThrAsnAlaGlyAlaGluTyrTyrAspAla115120125IleValAlaIleProGlyCysAspLysAsnMetProGlySerIleIle130135140GlyMetAlaArgLeuAsnArgProSerIleMetValTyrGlyGlyThr145150155160IleGluHisGlyGluTyrLysGlyGluLysLeuAsnIleValSerAla165170175PheGluAlaLeuGlyGlnLysIleThrGlyAsnIleSerAspGluAsp180185190TyrHisGlyValIleCysAsnAlaIleProGlyGlnGlyAlaCysGly195200205GlyMetTyrThrAlaAsnThrLeuAlaAlaAlaIleGluThrLeuGly210215220MetSerLeuProTyrSerSerSerAsnProAlaValSerGlnGluLys225230235240GlnGluGluCysAspAspIleGlyLeuAlaIleLysAsnLeuLeuGlu245250255LysAspIleLysProSerAspIleMetThrLysGluAlaPheGluAsn260265270AlaIleThrIleValMetValLeuGlyGlySerThrAsnAlaValLeu275280285HisIleIleAlaMetAlaAsnAlaIleGlyValGluIleThrGlnAsp290295300AspPheGlnArgIleSerAspIleIleProValLeuGlyAspPheLys305310315320ProSerGlyLysTyrMetMetGluAspLeuHisLysIleGlyGlyLeu325330335ProAlaValLeuLysTyrLeuLeuLysGluGlyLysLeuHisGlyAsp340345350CysLeuThrValThrGlyLysThrLeuAlaGluAsnValGluThrAla355360365LeuAspLeuAspPheAspSerGlnAspIleMetArgProLeuLysAsn370375380ProIleLysAlaThrGlyHisLeuGlnIleLeuTyrGlyAsnLeuAla385390395400GlnGlyGlySerValAlaLysIleSerGlyLysGluGlyGluPhePhe405410415LysGlyThrAlaArgValPheAspGlyGluGlnHisPheIleAspGly420425430IleGluSerGlyArgLeuHisAlaGlyAspValAlaValIleArgAsn435440445IleGlyProValGlyGlyProGlyMetProGluMetLeuLysProThr450455460SerAlaLeuIleGlyAlaGlyLeuGlyLysSerCysAlaLeuIleThr465470475480AspGlyArgPheSerGlyGlyThrHisGlyPheValValGlyHisIle485490495ValProGluAlaValGluGlyGlyLeuIleGlyLeuValGluAspAsp500505510AspIleIleGluIleAspAlaValAsnAsnSerIleSerLeuLysVal515520525SerAsnGluGluIleAlaLysArgArgAlaAsnTyrGlnLysProThr530535540ProLysAlaThrArgGlyValLeuAlaLysPheAlaLysLeuThrArg545550555560ProAlaSerGluGlyCysValThrAspLeu565570(2) INFORMATION FOR SEQ ID NO: 7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 575 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ILVB(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:MetLysLysIleLysLeuGluLysProThrSerGlySerGlnLeuVal151015LeuGlnThrLeuLysGluLeuGlyValGluIleIlePheGlyTyrPro202530GlyGlyAlaMetLeuProLeuTyrAspAlaIleHisAsnPheGluGly354045IleGlnHisIleLeuAlaArgHisGluGlnGlyAlaThrHisGluAla505560GluGlyTyrAlaLysSerSerGlyLysValGlyValValValValThr65707580SerGlyProGlyAlaThrAsnAlaValThrGlyIleAlaAspAlaTyr859095LeuAspSerValProLeuLeuValPheThrGlyGlnValGlyArgGln100105110SerIleGlyLysAspAlaPheGlnGluAlaAspThrValGlyIleThr115120125AlaProIleThrLysTyrAsnTyrGlnIleArgGluThrAlaAspIle130135140ProArgIleValThrGluAlaTyrTyrLeuAlaArgThrGlyArgPro145150155160GlyProValGluIleAspLeuProLysAspValSerThrLeuGluVal165170175ThrGluIleAsnAspProSerLeuAsnLeuProHisTyrHisGluSer180185190GluLysAlaThrAspGluGlnLeuGlnGluLeuLeuThrGluLeuSer195200205ValSerLysLysProValIleIleAlaGlyGlyGlyIleAsnTyrSer210215220GlySerValAspIlePheArgAlaPheValGluLysTyrGlnIlePro225230235240ValValSerThrLeuLeuGlyLeuGlyThrLeuProIleSerHisGlu245250255LeuGlnLeuGlyMetAlaGlyMetHisGlySerTyrAlaAlaAsnMet260265270AlaLeuValGluAlaAspTyrIleIleAsnLeuGlySerArgPheAsp275280285AspArgValValSerAsnProAlaLysPheAlaLysAsnAlaValVal290295300AlaHisIleAspIleAspAlaAlaGluLeuGlyLysIleValLysThr305310315320AspIleProIleLeuSerAspLeuLysAlaAlaLeuSerArgLeuLeu325330335GlnLeuAsnLysValArgThrAspPheAsnAspTrpIleLysThrVal340345350IleGluAsnLysGluLysAlaProPheThrTyrGluProGlnAsnHis355360365AspIleArgProGlnGluThrIleLysLeuIleGlyGluTyrThrGln370375380GlyAspAlaIleIleValThrAspValGlyGlnHisGlnMetTrpVal385390395400AlaGlnTyrTyrProTyrLysAsnAlaArgGlnLeuIleThrSerGly405410415GlyMetGlyThrMetGlyPheGlyIleProAlaAlaIleGlyAlaLys420425430LeuAlaGlnProAsnLysAsnValIleValPheValGlyAspGlyGly435440445PheGlnMetThrAsnGlnGluLeuAlaLeuLeuAsnGlyTyrGlyIle450455460AlaIleLysValValLeuIleAsnAsnHisSerLeuGlyMetValArg465470475480GlnTrpGlnGluSerPheTyrGluGluArgArgSerGlnSerValPhe485490495AspValGluProAsnPheGlnLeuLeuAlaGluAlaTyrGlyIleLys500505510HisValLysLeuAspAsnProLysThrLeuAlaAspAspLeuLysIle515520525IleThrGluAspGluProMetLeuIleGluValLeuIleSerLysSer530535540GluHisValLeuProMetIleProAlaGlyLeuHisAsnAspGluMet545550555560IleGlyLeuHisPheThrAspLysAsnGluGluIleAspAsnAla565570575(2) INFORMATION FOR SEQ ID NO: 8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 158 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ILVN(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:MetArgArgMetIleIleAlaLysLeuHisAsnValThrGlyIleMet151015AsnArgPheThrAlaValLeuAsnArgArgGlnValAsnIleLeuSer202530IleThrAlaGlyValThrGluSerGlnAspLeuThrHisThrThrPhe354045ValIleGluValAspHisLeuAspGluValGluGlnIleIleLysGln505560LeuAsnArgLeuIleAspValIleGluValAlaAspIleThrAspPhe65707580ProHisValGluArgGluValValLeuIleLysValSerAlaProPro859095ThrIleArgAlaGluIlePheThrMetIleGluProPheArgValAsn100105110ValValAspValAsnLeuGluAsnValThrIleGlnLeuThrGlyAsp115120125SerAlaLysIleGluAlaLeuIleGluValValSerProTyrGlyIle130135140LeuAsnMetAlaArgThrGlySerAlaGlyPheGluArgGly145150155(2) INFORMATION FOR SEQ ID NO: 9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 344 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ILVC(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:MetAlaValThrMetTyrTyrGluAspAspValGluValSerAlaLeu151015AlaGlyLysGlnIleAlaValIleGlyTyrGlySerGlnGlyHisAla202530HisAlaGlnAsnLeuArgAspSerGlyHisAsnValIleIleGlyVal354045ArgHisGlyLysSerPheAspLysAlaLysGluAspGlyPheGluThr505560PheGluValGlyGluAlaValAlaLysAlaAspValIleMetValLeu65707580AlaProAspGluLeuGlnGlnSerIleTyrGluGluAspIleLysPro859095AsnLeuLysAlaGlySerAlaLeuGlyPheAlaHisGlyPheAsnIle100105110HisPheGlyTyrIleLysValProGluAspValAspValPheMetVal115120125AlaProLysAlaProGlyHisLeuValArgArgThrTyrThrGluGly130135140PheGlyThrProAlaLeuPheValSerHisGlnAsnAlaSerGlyHis145150155160AlaArgGluIleAlaMetAspTrpAlaLysGlyIleGlyCysAlaArg165170175ValGlyIleIleGluThrThrPheLysGluGluThrGluGluAspLeu180185190PheGlyGluGlnAlaValLeuCysGlyGlyLeuThrAlaLeuValGlu195200205AlaGlyPheGluThrLeuThrGluAlaGlyTyrAlaGlyGluLeuAla210215220TyrPheGluValLeuHisGluMetLysLeuIleValAspLeuMetTyr225230235240GluGlyGlyPheThrLysMetArgGlnSerIleSerAsnThrAlaGlu245250255PheGlyAspTyrValThrGlyProArgIleIleThrAspGluValLys260265270LysAsnMetLysLeuValLeuAlaAspIleGlnSerGlyLysPheAla275280285GlnAspPheValAspAspPheLysAlaGlyArgProLysLeuIleAla290295300TyrArgGluAlaAlaLysAsnLeuGluIleGluLysIleGlyAlaGlu305310315320HisValLysGlnCysHisSerHisAsnLeuValMetThrMetProLeu325330335LysSerIleSerAsnPheSerTyr340(2) INFORMATION FOR SEQ ID NO: 10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 441 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ILVA(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:MetIleSerAlaLysGluValGluAspAlaTyrAspLeuLeuLysAla151015ValValThrLysThrProLeuGlnLeuAspProTyrLeuSerAsnLys202530TyrGlnAlaAsnIleTyrLeuLysGluValValThrLysThrProLeu354045GlnLeuAspProTyrLeuSerAsnLysTyrGlnAlaAsnIleTyrLeu505560LysGluGluAsnLeuGlnLysValArgSerPheLysLeuArgGlyAla65707580TyrTyrSerIleSerLysLeuSerAspGluGlnArgSerLysGlyVal859095ValCysAlaSerAlaGlyAsnHisAlaGlnGlyValAlaPheAlaAla100105110AsnGlnLeuAsnIleSerAlaThrIlePheMetProValThrThrPro115120125AsnGlnLysIleSerGlnValLysPhePheGlyGluSerHisValThr130135140IleArgLeuIleGlyAspThrPheAspGluSerAlaArgAlaAlaLys145150155160AlaPheSerGlnAspAsnAspLysProPheIleAspProPheAspAsp165170175GluAsnValIleAlaGlyGlnGlyThrValAlaLeuGluIlePheAla180185190GlnAlaLysLysGlnGlyIleSerLeuAspLysIlePheValGlnIle195200205GlyGlyGlyGlyLeuIleAlaGlyIleThrAlaTyrSerLysGluArg210215220TyrProGlnThrGluIleIleGlyValGluAlaLysGlyAlaThrSer225230235240MetLysAlaAlaTyrSerAlaGlyGlnProValThrLeuGluHisIle245250255AspLysPheAlaAspGlyIleAlaValAlaThrValGlyGlnLysThr260265270TyrGlnLeuIleAsnAspLysValLysGlnLeuLeuAlaValAspGlu275280285GlyLeuIleSerGlnThrIleLeuGluLeuTyrSerLysLeuGlyIle290295300ValAlaGluProAlaGlyAlaThrSerValAlaAlaLeuGluLeuIle305310315320LysAspGluIleLysGlyLysAsnIleValCysIleIleSerGlyGly325330335AsnAsnAspIleSerArgMetGlnGluIleGluGluArgAlaLeuVal340345350TyrGluGlyLeuLysHisTyrPheValIleAsnPheProGlnArgPro355360365GlySerLeuArgThrPheValSerAspIleLeuGlyProAsnAspAsp370375380IleThrArgPheGluTyrIleLysArgAlaAspLysGlyLysGlyPro385390395400CysLeuValGlyIleLeuLeuSerAspAlaSerAspTyrAspSerLeu405410415IleAsnArgIleGluArgPheAspAsnArgTyrValAsnLeuArgGly420425430AsnAspSerLeuTyrGluLeuLeuVal435440(2) INFORMATION FOR SEQ ID NO: 11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12720 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iii) ANTI-SENSE: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:TAAAACTCGATAATCTTGAGTCATAATTTCTCCTTAATCTTATTAGTACATTAGAATCCA60TTATAATTTAATCATTTTATGTCTACCTAAAGCAACAAAATTGCTTGTATATTTTCTAAC120AAGCTTAATTATGTGGATTTAATTGAATATTAAAGGGAGAAGTTGTAATCTATTTGTTGT180TAAATTCTTGTTAATACAAATAAATTTATTAAATATTATTATTTTATTGACAATTTAAAA240TATTAAGAGTATTATAATGTAAATTAACAAAAAAAAGAGGAACTTGAAATGACATACACA300CAATTTTCATTGTTGTTGATCAAGGTGGACCTACATTAGCTTTTTTGGCTAAAATATGTG360GGTCCTGTTTGGCGATAGTCATTTCGAGGACCGAGAGACGTCCTCACGGGCGTCTTTTTT420GTTTCTTAATAAAAAATAGAGGTAATATTATGCGAAAAATTGAATTCTTTGACACAAGTT480TGAGAGATGGCGAACAGACACCGGGCGTTAGTTTCTCCATTTCAGAAAAAGTAACGATTG540CTAAACAACTGGAAAAATGGAGGATTTCTGTCATAGAGGCTGGTTTTTCTGCGGCAAGTC600CAGATAGTTTTGAAGCAGTAAAGCAAATTGCTGATTCTTTGAATGATACGGCTGTCACTG660CATTAGCTCGCTGTGTTATTTCAGATATCGATAAAGCGGTTGAAGCGGTAAAGGGGGCTA720AATATCCGCAAATTCATGTTTTCATTGCAACTTCACCTATTCACATGAAATATAAACTTA780AAATCAGTCCCGAAGAAGTTTTGAAAAATATTGATAAGTGTGTGAGATACGCACGTGAAC840GGGTCGAGGTTGTTGAGTTTTCTCCAGAGGATGCAACAAGAACGGAGTTGAATTTTCTTT900TAGAGGCTGTTCAAACGGCTGTCGATGCTGGAGCAACTTATATTAATATTCCTGACACTG960TCGGTTATACGACACCAGAAGAATATGGAAAAATTTTTAAATTTTTGATTGATAATACTA1020AGTCTGACCGAGAAATTATTTTTAGTCCACATTGTCATGATGATTTAGGAATGGCTGTAG1080CTAATTCATTAGCTGCAATTAAAGCTGGGGCTGGGAGAGTTGAAGGAACTGTCAATGGTA1140TTGGAGAGCGAGCTGGGAATGCTGCTCTTGAAGAAATTGCTGTGGCACTACATATTCGTA1200AAGATTTTTATCAGGCACAAAGTCCTTTAAAACTTTCAGAAACTGCTGCAACGGCAGAAC1260TAATTTCACAATTTTCAGGAATTGCTATTCCAAAAAATAAAGCAATTGTTGGTGCTAATG1320CTTTTGCACACGAATCAGGAATTCATCAAGATGGTGTCCTTAAAAATGCTGAAACTTATG1380AAATTATTACACCAGAACTTGTCGGAATAAAGCATAATTCGTTGCCTTTAGGTAAACTTT1440CTGGTCGTCATGCTTTTAGTGAAAAATTGACGGAACTTAATATTGCTTATGACGATGAAA1500GTCTTGCAATTTTATTTGAAAAATTTAAAAAATTAGCTGACAAGAAAAAAGAAATTACTG1560ACGCAGATATTCATGCCTTGTTTACAGGAGAAACGGTAAAAAATCTAGCTGGATTTATAC1620TTGATAATGTTCAAATTGATGGGCACAAGGCATTGGTGCAACTAAAAAATCAAGAAGAGG1680AAATTTATGTTAGCCAAGGAGAGGGGTCAGGTTCAGTGGATGCAATTTTTAAAGCTATTG1740ATAAAGTCTTTAATCATCAACTAAAATTAATTTCCTATTCAGTTGATGCTGTAACTGATG1800GAATTGATGCACAAGCAACGACTTTGGTTTCTGTTGAAAATCTATCTACAGGCACTATAT1860TTAATGCTAAAGGTGTTGATTATGATGTATTGAAAGGAAGCGCCATTGCTTACATGAACG1920CTAATGTTTTAGTTCAAAAAGAAAATTTACAAGGAAAGGTTGAACAAATTTCAGCTCATG1980ATGGAATTTAAGGTGAAAAATATTGTCTAAAAAAATTGTGACACTTGCGGGAGATGGAAT2040TGGGCCAGAAATTATGTCAGCTGGTTTAAGTGTTTTAAAAGCTGTCAGTAAAAAAATTGA2100TTTTGAGTATGAATTAGAAGCTAAAGATTTTGGAGGAATTGCAATTGATAAGCATGGTCA2160TCCTTTACCAGAAGAAACTTTGCAAGCAGTTAAAAATGCTGACGCAATCTTGCTCGCTGC2220AATTGGTCATCCTAAATACAACAATGCAAAAGTTAGACCAGAACAAGGGCTACTTGCTTT2280ACGAAAAGAATTAGGACTGTATGCTAATGTTCGTCCATTAAAAATTTATCCGGCTCTAAA2340AAAACTTTCTCCCATACGAAATGTTGAAAATGTTGATTTCCTAGTGATTCGCGAACTTAC2400AGGGGGAATCTATTTCGGTCAGCATGAATTGGCAGATGATAAAGCACGAGATGTCAATGA2460TTATTCTGCTGATGAAATAAGGAGAATTCTTCATTTTGCTTTCAAAAGTGCTCAAAGTCG2520GCCCAGAAAATTACTGACTTCGGTTGATAAACAAAATGTTCTTGCAACTTCTAAATTATG2580GCGAAAAATGGCTGATGAAATTGCTGACGAATATCCTGATGTACGATTAGAGCACCAATT2640GGTCGATTCTTGTGCGATGTTACTGATTACTAATCCGCAACAATTTGATGTGATAGTCAC2700TGAAAATCTATTTGGTGATATTCTCTCTGATGAAGCAAGTAGTTTGGCCGGTAGCTTAGG2760AGTGATGCCTTCGAGTTCGCATGGATTTAACGGTTTAGCACTCTATGAGCCAATTCATGG2820TTCGGCACCAGATATTGCAGGAAAAGGAATTGCGAACCCTGTTTCGATGATTCTATCAAT2880TGCCATGATGCTAAGAGAATCTTTTGGGCAAGAAGATGGGGCTGCGATGATTGAAAAAGC2940CGTAACCCAAACTTTTACTGACGGAATTTTGACTAAAGATTTAGGTGGGACTGCAACAAC3000TAAAGAAATGACAGAAGCAATCCTGAAAAATTGTCAGTAAAATGCGATTGAATAGTGAGC3060ATTTTAGTTGTAGATAAAAGAACCGTCAGCATAGCTGACAATTCTGTCAGTAAATGCGAT3120TGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACCGTCAGCATAGCTGACAATTCTGTC3180AGTAATTGCGATTGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACCGTCAGCATAGCT3240GACAATTCTGTCAGTAATTGCGATTGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACT3300ATCAGCGTAACTGACAATTCTGTCAGTAAATATTACTGACAAAAAGTACAAAATTACTGA3360CAGAATTTGTCAGAATAAATTTTTAAAAAAGGAAATAAAAAAATGTCAGGTAAAACAATA3420TTTGATAAACTTTGGGATCAGCATGTGATTGCTGGAAATGAGGGAGAACCTCAACTGCTT3480TATATTGACCTTCATGTTATTCATGAGGTTACGAGTCCGCAAGCATTTCAGGGCTTACGT3540GAAGCAGGACGTCGTGTTCGGAGAAAAGATTTGACATACGGAACTCTTGACCACAATGTT3600CCAACACAAAATATTTTTAATATTCAAGATTTGATTTCTAAAAAACAAATTGATACTTTT3660ACTAAAAATGTCAAAGAATTTGATGTTCCAGCGGAGACTCATGGTGGAAAAGGACAAGGA3720ATTGTTCACATGGTAGCACCTGAATCTGGCAGAACTCAACCGGGAAAAACAATTGTTTGT3780GGCGATAGTCATACCGCAACAAATGGAGCATTTGGTGCAATTGCTTTTGGAATTGGTACA3840AGTGAAGTTGAACATGTTCTTGCAACTCAAACCATTTGGCAAGTTAAACCCAAGCGTATG3900AAAATTGAATTTCAAGGTCATCCACAAAAAGGAATTTATAGCAAAGACTTTATCCTCGCA3960TTAATTGCTAAATATGGTGTGGATGCAGGTGTAGGTTATGCGGTTGAATATAGTGGGGAT4020GCTATCAGTGATTTAAGCATGGAAGAACGGATGACAATCTGTAACATGTCAATTGAATTT4080GGGGCAAAAATTGGCCTGATGAATCCTGATGAAAAAACTTATGACTATGTCAAAGGGCGT4140GAACATGCACCTAAAAACTTTGATGAAGCTGTCAGTAAATGGGAAAAACTTGTCAGTGAT4200TCTGATGCACAATACGATAAGATTTTAAGTCTTGATGTCAGCCAGTTGAAACCAATGGTG4260ACATGGGGAACAAATCCCGGAATGGGCCTAGAATTTGGCGAAAAGTTTCCGGAAATTAAC4320AATGATTTGAATTATGAACGTGCTTATCAGTACATGGATTTAAAGCCAGGCCAAACCGCT4380TCTGACATAGATTTAGGCTATATTTTCATTGGTTCTTGTACGAATGCTAGACTTGGTGAT4440TTAGAAGAAGCTGCAAAAATTATTGGAGACAGACATATTGCTGATGGACTGACAGGAATT4500GTCGTCCCTGGAAGCAGACCTGTGAAAGAAGCGGCTGAAGCACAAGGGCTTGATAAAATT4560TTTAAAGAAGCTGGTTTTGAATGGCGGGAACCGGGTTGCTCAGCCTGTCTTGGAATGAAT4620CCTGACCAAATTCCAGAATACGTTCATTGTGCTTCAACCTCTAATCGAAATTTTGAAGGT4680CGTCAAGGACATAATGCAAGAACGCACCTGTGCTCTCCAGCTATGGCTGCTGCCGCCGCA4740ATCGCTGGTAAATTTGTAGATGTTAGAATGCTCGTAACAGATTAGTCTGTAGAAAGAAAA4800AAAGATGGAAAAATTCACGATTTACAAAGGGACAAGTGTTCCAGTCATGAACGATAATAT4860TGACACAGACCAAATTATTCCTAAACAATTTTTGAAAGCAATCGATAAAAAGGGCTTTGG4920GAAAAATTTATTTTATGAATGGCGTTATCTTAAAGATTACGATGAGAATCCTGATTTTAT4980TTTGAATGCTCCAAAATACAAAAAAGCTTCTCTGTTAATTTCAGGAGATAATTTTGGTTC5040GGGTTCTTCAAGAGAACATGCGGCATGGGCCTTATCAGATTACGGCTTTCGGGCAATTAT5100TGCTGGCTCTTACTCAGATATTTTTTATAATAATGCTTTAAAAAATGGCTTGTTACCAAT5160TAAACAACCAAGAGAAGTTCTAAATCAACTGACAAAACTGTCAAGTCAAGAAGAAATTAC5220AATTGATTTACCCCATCAGCTAATCATCACAAGCCTTGGTGACTTTCATTTTGAGATTGA5280CCCCATTTGGAAAGACAAATTAATTAATGGCTTAGATGATATTGGAATAACTTTGCAATA5340TGAAGAAGCAATCTCAGCTTACGAACAAAAAAATCAATAAGAGCGAGCCTAAAATGACAA5400TTATTAATTTAAAGAATGTAAATCTTACTCGAAATAAAAAAGAAATTCTTAAAGATATTA5460CTTGGAAAGTAAATCCCGGCGAAAATTGGGTTATTCTGGGCCTCAACGGCTCTGGAAAAT5520CAAGTCTTTTGAAATTGATTTTAGCAGAAGAATGGAAAACTTCTGGTGAAATCACTGTTT5580TAAATACTCAATTTAGAAATGGAGAAATTCCTAAGTTGAGAAAAAGAATCAGCGTAGTTG5640GCTCATTTATTGCTGAAAGATTTCAACCAAATATTAAGGCTGAAAACCTTGTTTATACTG5700GGAAATTTAATTCGAGCATGCTCTATAAACCCTACACAGATCAGGAACTTGATGAGGCCC5760GTCAGCTTTTAAGACAAATGGGCGCAAAATCACTTATTGGCCGAAATTATGCCAGCCTTT5820CTCAAGGGGAAAAGCAAGTTCTTCTTATTGCTAGGAGCTTAATTTTAAAGCCTGAGCTTT5880TAATTTTGGACGAAGCAACGAACGGTTTAGATTTATTTGCTAAAGAAAAATTATTAAAGC5940AACTGCAGCAGATTAATCAATTAAAAACCGCACCAACACTAATTTATATTTCTCATCATC6000CCGATGAAATCACTGATATTTTTACTCACCTTTTACTTTTAAGAGAAGGAAAAGTGATTC6060AATCAGGGAAAAAAGAAAACTTATTAAATGAAAAGATACTTACTGATTTTTATCAAGAAA6120AAGTAGAAGTTCACCGTTTTGAGCAGAAATATTTTGTAATTCCTGCTAACTGAGAAAGGA6180AAGCAAAAGTATTTTATATACTATATAGAATATTCTGACAGATTATTGTATTTTCATTTT6240TTTAGTGATAAAATAGCTCTATGTAAATTTACGGGGAGGTCAAAAAGATAACATATGGAA6300TTCAAATATAACGGAAAAGTTGAATCAGTGGAACTCAATAAATATTCTAAGACATTGACT6360CCAAGATCAACACAACCAGCGACTCAAGCGATGTACTACGGCATTGGTTTTAAAGATGAG6420GATTTCAAAAAAGCTCAGGTCGGAATCGTCAGCATGGATTGGGACGGAAATCCATGTAAT6480ATGCACTTGGGAACACTTGGGAGTAAAATCAAAAGTTCTGTCAACCAAACTGACGGATTG6540ATTGGACTTCAATTTCATACTATTGGAGTTTCTGATGGAATTGCTAACGGAAAGCTTGGC6600ATGAGATATTCTTTGGTCAGTCGTGAAGTTATTGCTGACAGCATCGAAACCAACGCTGGC6660GCAGAATATTATGATGCCATCGTTGCCATTCCCGGTTGTGATAAAAATATGCCCGGGTCA6720ATTATCGGAATGGCTCGCTTAAATCGTCCGTCAATTATGGTCTATGGTGGAACGATTGAA6780CATGGCGAATATAAAGGTGAAAAATTAAATATTGTTTCGGCCTTTGAAGCTCTGGGGCAA6840AAAATCACTGGAAATATTTCTGATGAAGATTATCATGGCGTTATTTGCAATGCCATTCCA6900GGACAAGGTGCTTGCGGAGGAATGTACACTGCCAATACCCTGGCTGCTGCTATTGAAACT6960TTGGGAATGAGTTTACCTTATTCCTCTTCCAATCCAGCAGTCAGTCAAGAAAAACAAGAA7020GAGTGTGATGACATTGGTTTAGCCATCAAAAATTTATTAGAAAAAGATATTAAACCAAGT7080GATATCATGACCAAAGAAGCTTTTGAAAATGCCATAACAATTGTCATGGTCCTTGGAGGC7140TCAACCAATGCTGTGCTTCATATCATTGCAATGGCAAATGCCATTGGTGTAGAAATTACG7200CAAGATGATTTCCAACGTATTTCAGATATTATCCCTGTTCTTGGCGATTTCAAACCGAGC7260GGAAAATATATGATGGAAGATCTGCACAAAATTGGTGGCCTTCCTGCTGTTTTGAAATAC7320CTACTTAAAGAAGGAAAACTTCACGGTGATTGTTTGACCGTCACAGGTAAAACTTTGGCT7380GAAAATGTTGAAACAGCATTAGATTTGGACTTTGACAGTCAAGATATTATGCGACCACTA7440AAAAATCCAATTAAAGCTACTGGACATTTACAAATTTTGTACGGTAATCTTGCCCAAGGG7500GGTTCTGTTGCAAAAATTTCTGGTAAAGAAGGCGAATTTTTCAAAGGAACAGCTCGTGTT7560TTTGACGGAGAACAACACTTTATCGATGGCATTGAGTCTGGCCGATTGCATGCCGGTGAT7620GTTGCGGTCATTAGAAATATTGGCCCAGTCGGAGGTCCGGGAATGCCAGAGATGTTAAAA7680CCAACCTCAGCATTAATTGGAGCAGGACTTGGAAAATCTTGTGCCCTAATTACTGACGGA7740AGATTTTCTGGTGGCACACACGGCTTTGTTGTGGGTCATATCGTCCCTGAAGCAGTTGAA7800GGTGGGTTGATTGGTTTAGTTGAAGATGATGATATTATCGAAATTGATGCGGTGAATAAT7860AGTATTAGTTTAAAAGTTTCTAATGAAGAAATTGCTAAACGACGTGCCAATTATCAAAAA7920CCAACCCCTAAAGCAACGCGTGGTGTTCTTGCAAAATTTGCCAAACTTACGCGCCCCGCT7980AGTGAAGGTTGCGTTACAGATTTATAGAAAGGTTTGAATGAAAAAAATAAAGTTAGAAAA8040ACCTACTTCCGGTTCCCAACTTGTTCTCCAAACCTTAAAAGAACTTGGAGTAGAAATTAT8100TTTTGGTTATCCTGGTGGGGCCATGCTCCCCTTGTATGATGCGATTCATAATTTTGAAGG8160AATTCAACATATTTTAGCCCGTCATGAGCAAGGAGCAACGCATGAAGCCGAAGGTTACGC8220TAAATCGTCTGGTAAAGTTGGTGTCGTCGTTGTTACGTCAGGACCAGGAGCGACTAATGC8280AGTAACCGGAATTGCTGACGCTTATCTTGATTCAGTCCCATTGTTAGTTTTCACAGGTCA8340AGTTGGCCGTCAGTCAATTGGTAAAGATGCTTTTCAAGAAGCAGATACTGTTGGAATTAC8400AGCCCCAATTACAAAATATAATTATCAAATTAGGGAAACCGCAGATATTCCAAGAATTGT8460TACAGAAGCCTATTATTTGGCAAGGACAGGACGTCCTGGACCAGTAGAAATTGATTTACC8520AAAAGATGTTTCCACCCTTGAAGTCACTGAAATTAATGACCCAAGCTTGAATCTTCCTCA8580TTATCACGAAAGTGAAAAAGCGACTGATGAACAATTGCAAGAATTACTGACAGAACTTTC8640TGTCAGTAAAAAACCAGTCATTATTGCTGGCGGAGGAATTAATTATTCTGGCTCAGTTGA8700TATTTTCAGAGCATTTGTCGAAAAATATCAAATTCCAGTTGTTTCTACATTGCTTGGCTT8760AGGAACATTACCAATCAGCCACGAATTGCAACTAGGAATGGCAGGAATGCACGGTTCATA8820CGCTGCAAATATGGCTTTAGTTGAAGCTGACTATATTATTAATTTGGGATCACGTTTTGA8880CGATAGAGTTGTATCCAATCCTGCAAAATTTGCTAAAAATGCTGTCGTTGCTCATATTGA8940TATTGACGCTGCTGAACTTGGCAAAATTGTAAAAACCGATATTCCAATCCTTTCTGATTT9000GAAAGCGGCTTTAAGCAGACTTTTGCAATTAAATAAGGTCAGGACTGACTTTAATGATTG9060GATTAAAACTGTCATTGAAAATAAAGAGAAAGCACCATTTACTTATGAGCCCCAAAACCA9120TGATATCCGTCCACAGGAAACAATTAAATTAATTGGAGAATACACTCAAGGAGATGCAAT9180CATTGTAACTGACGTTGGGCAACATCAAATGTGGGTGGCGCAATATTATCCTTATAAAAA9240TGCAAGGCAACTTATTACTTCTGGGGGAATGGGAACGATGGGCTTTGGCATTCCTGCAGC9300AATCGGTGCAAAGCTGGCACAGCCAAATAAAAATGTCATTGTTTTTGTTGGCGATGGTGG9360CTTTCAAATGACTAATCAAGAATTAGCATTACTTAATGGCTACGGTATTGCAATCAAAGT9420TGTGCTGATTAATAATCATTCATTGGGAATGGTACGTCAATGGCAAGAATCATTCTATGA9480AGAGCGACGTTCACAATCGGTTTTTGATGTTGAACCCAATTTTCAATTGTTAGCCGAAGC9540TTATGGCATCAAACATGTTAAGTTAGATAATCCAAAAACTTTGGCTGATGATTTAAAAAT9600TATTACAGAAGATGAGCCAATGCTTATTGAAGTTCTAATTTCAAAATCTGAGCATGTTTT9660ACCAATGATACCAGCTGGATTACACAATGACGAAATGATTGGACTTCATTTTACTGATAA9720GAATGAGGAGATAGATAATGCGTAGAATGATTATCGCAAAACTTCATAACGTGACAGGAA9780TTATGAATCGATTTACCGCCGTTCTCAATCGAAGGCAAGTGAACATTCTCTCAATTACCG9840CTGGAGTTACAGAAAGTCAAGACTTAACTCATACCACTTTTGTTATTGAAGTTGATCATC9900TTGATGAAGTAGAACAAATCATTAAACAATTAAATCGCTTAATAGATGTAATTGAAGTAG9960CTGATATTACTGATTTTCCTCATGTAGAACGTGAAGTCGTCTTGATTAAAGTATCAGCTC10020CACCGACCATTAGGGCAGAAATTTTTACAATGATTGAACCTTTTAGAGTAAATGTAGTTG10080ATGTCAATCTGGAAAATGTCACCATTCAATTAACGGGTGATTCAGCAAAAATCGAAGCAC10140TTATTGAGGTTGTTAGTCCTTATGGCATTCTAAATATGGCTCGGACAGGTAGTGCAGGTT10200TTGAGCGTGGCTAAATTTAAATAAGTTAACAAATAAATAGAAAAATAGAGGAAACAAAAA10260TGGCAGTTACAATGTATTATGAAGATGATGTAGAAGTATCAGCACTTGCTGGAAAGCAAA10320TTGCAGTAATCGGTTATGGTTCACAAGGACATGCTCACGCACAGAATTTGCGTGATTCTG10380GTCACAACGTTATCATTGGTGTGCGCCACGGAAAATCTTTTGATAAAGCAAAAGAAGATG10440GCTTTGAAACATTTGAAGTAGGAGAAGCAGTAGCTAAAGCTGATGTTATTATGGTTTTGG10500CACCAGATGAACTTCAACAATCCATTTATGAAGAGGACATCAAACCAAACTTGAAAGCAG10560GTTCAGCACTTGGTTTTGCTCACGGATTTAATATCCATTTTGGCTATATTAAAGTACCAG10620AAGACGTTGACGTCTTTATGGTTGCGCCTAAGGCTCCAGGTCACCTTGTCCGTCGGACTT10680ATACTGAAGGTTTTGGTACACCAGCTTTGTTTGTTTCACACCAAAATGCAAGTGGTCATG10740CGCGTGAAATCGCAATGGATTGGGCCAAAGGAATTGGTTGTGCTCGAGTGGGAATTATTG10800AAACAACTTTTAAAGAAGAAACAGAAGAAGATTTGTTTGGAGAACAAGCTGTTCTATGTG10860GAGGTTTGACAGCACTTGTTGAAGCCGGTTTTGAAACACTGACAGAAGCTGGATACGCTG10920GCGAATTGGCTTACTTTGAAGTTTTGCACGAAATGAAATTGATTGTTGACCTCATGTATG10980AAGGTGGTTTTACTAAAATGCGTCAATCCATCTCAAATACTGCTGAGTTTGGCGATTATG11040TGACTGGTCCACGGATTATTACTGACGAAGTTAAAAAGAATATGAAGCTTGTTTTGGCTG11100ATATTCAATCTGGAAAATTTGCTCAAGATTTCGTTGATGACTTCAAAGCGGGGCGTCCAA11160AATTAATAGCCTATCGCGAAGCTGCAAAAAATCTTGAAATTGAAAAAATTGGGGCAGAGC11220ACGTCAAGCAATGCCATTCACACAATCTGGTGATGACGATGCCTTTAAAATCTATCAGTA11280ATTTCTCTTATTGATTGAACAAAAACATAAAAGCATTTTATGGAGGAATGACATAAATGA11340TAAGTGCCAAAGAGGTTGAAGATGCCTATGATTTGTTAAAAGCAGTTGTCACTAAAACAC11400CTTTACAATTAGACCCTTACCTTTCCAATAAATATCAAGCAAATATTTACTTAAAAGAAG11460TTGTCACTAAAACACCTTTACAATTAGACCCTTACCTTTCCAATAAATATCAAGCAAATA11520TTTACTTAAAAGAAGAAAACTTACAGAAAGTTCGTTCTTTTAAATTACGAGGAGCTTATT11580ATTCTATCAGTAAATTATCTGATGAGCAACGCTCTAAAGGAGTGGTTTGTGCCTCAGCAG11640GAAATCATGCACAAGGGGTTGCTTTTGCTGCAAATCAATTAAATATTTCTGCGACAATTT11700TTATGCCCGTTACCACACCTAACCAAAAAATTTCACAAGTTAAATTTTTTGGCGAAAGTC11760ACGTAACAATTCGTTTAATTGGTGATACTTTTGATGAATCAGCCAGAGCAGCAAAAGCTT11820TTTCTCAAGATAATGACAAACCATTTATAGACCCTTTTGATGATGAAAATGTAATTGCTG11880GTCAAGGGACAGTGGCTTTAGAAATTTTTGCGCAAGCTAAAAAACAAGGAATAAGTTTAG11940ATAAGATTTTTGTACAGATTGGTGGAGGTGGTTTAATTGCAGGAATTACTGCCTACAGTA12000AGGAGCGCTATCCCCAAACTGAAATTATCGGAGTTGAAGCAAAAGGGGCAACAAGTATGA12060AAGCTGCCTACTCTGCTGGTCAGCCCGTCACCTTGGAACACATTGATAAATTTGCTGACG12120GAATTGCGGTTGCGACTGTCGGTCAGAAAACTTACCAACTTATTAATGACAAAGTGAAAC12180AATTGCTTGCGGTTGATGAAGGTTTAATTTCTCAAACCATACTCGAATTGTATTCAAAAT12240TAGGAATTGTCGCCGAGCCAGCAGGTGCAACATCTGTTGCCGCACTTGAACTTATTAAAG12300ATGAAATCAAGGGTAAAAATATCGTCTGTATCATCAGCGGCGGAAATAATGATATTAGTC12360GAATGCAAGAAATTGAAGAAAGAGCTTTGGTTTATGAAGGTCTAAAACATTATTTTGTCA12420TTAACTTTCCTCAAAGACCAGGATCCTTACGAACTTTTGTCAGTGATATTTTAGGGCCAA12480ATGATGATATCACCCGATTTGAGTACATCAAAAGGGCTGATAAAGGTAAAGGACCTTGTC12540TTGTTGGGATTTTACTTTCAGATGCTAGTGATTATGATTCATTGATTAATCGGATTGAAA12600GATTTGATAATCGTTATGTTAACTTACGTGGAAATGATAGTTTATACGAACTTTTGGTCT12660AACTAACCAATTGGTTTGAGCCATTTTCTAGTTTCAATTCTCTTTAAATCACTAGAAATT12720(2) INFORMATION FOR SEQ ID NO: 12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1728 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ilvB(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:ATGAAAAAAATAAAGTTAGAAAAACCTACTTCCGGTTCCCAACTTGTTCTCCAAACCTTA60AAAGAACTTGGAGTAGAAATTATTTTTGGTTATCCTGGTGGGGCCATGCTCCCCTTGTAT120GATGCGATTCATAATTTTGAAGGAATTCAACATATTTTAGCCCGTCATGAGCAAGGAGCA180ACGCATGAAGCCGAAGGTTACGCTAAATCGTCTGGTAAAGTTGGTGTCGTCGTTGTTACG240TCAGGACCAGGAGCGACTAATGCAGTAACCGGAATTGCTGACGCTTATCTTGATTCAGTC300CCATTGTTAGTTTTCACAGGTCAAGTTGGCCGTCAGTCAATTGGTAAAGATGCTTTTCAA360GAAGCAGATACTGTTGGAATTACAGCCCCAATTACAAAATATAATTATCAAATTAGGGAA420ACCGCAGATATTCCAAGAATTGTTACAGAAGCCTATTATTTGGCAAGGACAGGACGTCCT480GGACCAGTAGAAATTGATTTACCAAAAGATGTTTCCACCCTTGAAGTCACTGAAATTAAT540GACCCAAGCTTGAATCTTCCTCATTATCACGAAAGTGAAAAAGCGACTGATGAACAATTG600CAAGAATTACTGACAGAACTTTCTGTCAGTAAAAAACCAGTCATTATTGCTGGCGGAGGA660ATTAATTATTCTGGCTCAGTTGATATTTTCAGAGCATTTGTCGAAAAATATCAAATTCCA720GTTGTTTCTACATTGCTTGGCTTAGGAACATTACCAATCAGCCACGAATTGCAACTAGGA780ATGGCAGGAATGCACGGTTCATACGCTGCAAATATGGCTTTAGTTGAAGCTGACTATATT840ATTAATTTGGGATCACGTTTTGACGATAGAGTTGTATCCAATCCTGCAAAATTTGCTAAA900AATGCTGTCGTTGCTCATATTGATATTGACGCTGCTGAACTTGGCAAAATTGTAAAAACC960GATATTCCAATCCTTTCTGATTTGAAAGCGGCTTTAAGCAGACTTTTGCAATTAAATAAG1020GTCAGGACTGACTTTAATGATTGGATTAAAACTGTCATTGAAAATAAAGAGAAAGCACCA1080TTTACTTATGAGCCCCAAAACCATGATATCCGTCCACAGGAAACAATTAAATTAATTGGA1140GAATACACTCAAGGAGATGCAATCATTGTAACTGACGTTGGGCAACATCAAATGTGGGTG1200GCGCAATATTATCCTTATAAAAATGCAAGGCAACTTATTACTTCTGGGGGAATGGGAACG1260ATGGGCTTTGGCATTCCTGCAGCAATCGGTGCAAAGCTGGCACAGCCAAATAAAAATGTC1320ATTGTTTTTGTTGGCGATGGTGGCTTTCAAATGACTAATCAAGAATTAGCATTACTTAAT1380GGCTACGGTATTGCAATCAAAGTTGTGCTGATTAATAATCATTCATTGGGAATGGTACGT1440CAATGGCAAGAATCATTCTATGAAGAGCGACGTTCACAATCGGTTTTTGATGTTGAACCC1500AATTTTCAATTGTTAGCCGAAGCTTATGGCATCAAACATGTTAAGTTAGATAATCCAAAA1560ACTTTGGCTGATGATTTAAAAATTATTACAGAAGATGAGCCAATGCTTATTGAAGTTCTA1620ATTTCAAAATCTGAGCATGTTTTACCAATGATACCAGCTGGATTACACAATGACGAAATG1680ATTGGACTTCATTTTACTGATAAGAATGAGGAGATAGATAATGCGTAG1728(2) INFORMATION FOR SEQ ID NO: 13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 477 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ilvN(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:ATGCGTAGAATGATTATCGCAAAACTTCATAACGTGACAGGAATTATGAATCGATTTACC60GCCGTTCTCAATCGAAGGCAAGTGAACATTCTCTCAATTACCGCTGGAGTTACAGAAAGT120CAAGACTTAACTCATACCACTTTTGTTATTGAAGTTGATCATCTTGATGAAGTAGAACAA180ATCATTAAACAATTAAATCGCTTAATAGATGTAATTGAAGTAGCTGATATTACTGATTTT240CCTCATGTAGAACGTGAAGTCGTCTTGATTAAAGTATCAGCTCCACCGACCATTAGGGCA300GAAATTTTTACAATGATTGAACCTTTTAGAGTAAATGTAGTTGATGTCAATCTGGAAAAT360GTCACCATTCAATTAACGGGTGATTCAGCAAAAATCGAAGCACTTATTGAGGTTGTTAGT420CCTTATGGCATTCTAAATATGGCTCGGACAGGTAGTGCAGGTTTTGAGCGTGGCTAA477(2) INFORMATION FOR SEQ ID NO: 14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2231 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(vi) ORIGINAL SOURCE:(A) ORGANISM: Lactococcus lactis subsp. lactis(C) INDIVIDUAL ISOLATE: ilvBN(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:ATTTATAGAAAGGTTTGAATGAAAAAAATAAAGTTAGAAAAACCTACTTCCGGTTCCCAA60CTTGTTCTCCAAACCTTAAAAGAACTTGGAGTAGAAATTATTTTTGGTTATCCTGGTGGG120GCCATGCTCCCCTTGTATGATGCGATTCATAATTTTGAAGGAATTCAACATATTTTAGCC180CGTCATGAGCAAGGAGCAACGCATGAAGCCGAAGGTTACGCTAAATCGTCTGGTAAAGTT240GGTGTCGTCGTTGTTACGTCAGGACCAGGAGCGACTAATGCAGTAACCGGAATTGCTGAC300GCTTATCTTGATTCAGTCCCATTGTTAGTTTTCACAGGTCAAGTTGGCCGTCAGTCAATT360GGTAAAGATGCTTTTCAAGAAGCAGATACTGTTGGAATTACAGCCCCAATTACAAAATAT420AATTATCAAATTAGGGAAACCGCAGATATTCCAAGAATTGTTACAGAAGCCTATTATTTG480GCAAGGACAGGACGTCCTGGACCAGTAGAAATTGATTTACCAAAAGATGTTTCCACCCTT540GAAGTCACTGAAATTAATGACCCAAGCTTGAATCTTCCTCATTATCACGAAAGTGAAAAA600GCGACTGATGAACAATTGCAAGAATTACTGACAGAACTTTCTGTCAGTAAAAAACCAGTC660ATTATTGCTGGCGGAGGAATTAATTATTCTGGCTCAGTTGATATTTTCAGAGCATTTGTC720GAAAAATATCAAATTCCAGTTGTTTCTACATTGCTTGGCTTAGGAACATTACCAATCAGC780CACGAATTGCAACTAGGAATGGCAGGAATGCACGGTTCATACGCTGCAAATATGGCTTTA840GTTGAAGCTGACTATATTATTAATTTGGGATCACGTTTTGACGATAGAGTTGTATCCAAT900CCTGCAAAATTTGCTAAAAATGCTGTCGTTGCTCATATTGATATTGACGCTGCTGAACTT960GGCAAAATTGTAAAAACCGATATTCCAATCCTTTCTGATTTGAAAGCGGCTTTAAGCAGA1020CTTTTGCAATTAAATAAGGTCAGGACTGACTTTAATGATTGGATTAAAACTGTCATTGAA1080AATAAAGAGAAAGCACCATTTACTTATGAGCCCCAAAACCATGATATCCGTCCACAGGAA1140ACAATTAAATTAATTGGAGAATACACTCAAGGAGATGCAATCATTGTAACTGACGTTGGG1200CAACATCAAATGTGGGTGGCGCAATATTATCCTTATAAAAATGCAAGGCAACTTATTACT1260TCTGGGGGAATGGGAACGATGGGCTTTGGCATTCCTGCAGCAATCGGTGCAAAGCTGGCA1320CAGCCAAATAAAAATGTCATTGTTTTTGTTGGCGATGGTGGCTTTCAAATGACTAATCAA1380GAATTAGCATTACTTAATGGCTACGGTATTGCAATCAAAGTTGTGCTGATTAATAATCAT1440TCATTGGGAATGGTACGTCAATGGCAAGAATCATTCTATGAAGAGCGACGTTCACAATCG1500GTTTTTGATGTTGAACCCAATTTTCAATTGTTAGCCGAAGCTTATGGCATCAAACATGTT1560AAGTTAGATAATCCAAAAACTTTGGCTGATGATTTAAAAATTATTACAGAAGATGAGCCA1620ATGCTTATTGAAGTTCTAATTTCAAAATCTGAGCATGTTTTACCAATGATACCAGCTGGA1680TTACACAATGACGAAATGATTGGACTTCATTTTACTGATAAGAATGAGGAGATAGATAAT1740GCGTAGAATGATTATCGCAAAACTTCATAACGTGACAGGAATTATGAATCGATTTACCGC1800CGTTCTCAATCGAAGGCAAGTGAACATTCTCTCAATTACCGCTGGAGTTACAGAAAGTCA1860AGACTTAACTCATACCACTTTTGTTATTGAAGTTGATCATCTTGATGAAGTAGAACAAAT1920CATTAAACAATTAAATCGCTTAATAGATGTAATTGAAGTAGCTGATATTACTGATTTTCC1980TCATGTAGAACGTGAAGTCGTCTTGATTAAAGTATCAGCTCCACCGACCATTAGGGCAGA2040AATTTTTACAATGATTGAACCTTTTAGAGTAAATGTAGTTGATGTCAATCTGGAAAATGT2100CACCATTCAATTAACGGGTGATTCAGCAAAAATCGAAGCACTTATTGAGGTTGTTAGTCC2160TTATGGCATTCTAAATATGGCTCGGACAGGTAGTGCAGGTTTTGAGCGTGGCTAAATTTA2220AATAAGTTAAC2231(2) INFORMATION FOR SEQ ID NO: 15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iii) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:CTAGTGAAGGTTGCGTTACA20(2) INFORMATION FOR SEQ ID NO: 16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iii) ANTI-SENSE: YES(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:TGCCATTTTTGTTTCCTCTA20__________________________________________________________________________

Number	Date	Country
0 436 886 A1	Dec 1990	EPX
0 500 188 A2	Feb 1992	EPX

Nucleic acid coding for an .alpha.-acetolactate synthase from lactococcus and its applications

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Starrenburg et al., "Citrate Fermentation by Lactococcus and Leuconostoc spp.," Applied and Environmental Microbiology, 57(12):3535-3540 (Dec. 1991).
Snoep et al., "Isolation, Characterization, and Physiological Role of the Pyruvate Dehydrogenase Complex and .alpha.-Acetolactate Synthase of Lactococcus lactis subsp. lactis bv. diacetylactis," Journal of Bacteriology, 174(14):4838-4841 (Jul. 1992).
Zahler et al., ".alpha.-Acetolactate Synthesis by Bacillus subtilis," Biosynth. Branched Chain Amino Acids, Proc. Workshop 1988, pp. 25-32 (1990).
Godon et al. "Branched-Chain Amino Acid Biosynth. Gene in . . . " J. Bacteriol. 174, 6580-6589. Oct. 1992.
Hugenholtz et al. "Diacetyl production by different . . . " Appl. Microbiol. Biotechnol. 38, 17-22. 1992.