AU722216B2 - Nucleic acid sequences and plasmids comprising at least one phage resistance mechanism, bacteria containing them and their use - Google Patents
Nucleic acid sequences and plasmids comprising at least one phage resistance mechanism, bacteria containing them and their use Download PDFInfo
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- AU722216B2 AU722216B2 AU64217/96A AU6421796A AU722216B2 AU 722216 B2 AU722216 B2 AU 722216B2 AU 64217/96 A AU64217/96 A AU 64217/96A AU 6421796 A AU6421796 A AU 6421796A AU 722216 B2 AU722216 B2 AU 722216B2
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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Description
AUSTRALIA
PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): -SYSTEMS DIO-T IDUSTRIES SK ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.
INVENTION TITLE: Nucleic acid sequences and plasmids comprising mechanism, bacteria containing them and their use at least one phage resistance •ooo o •eoe oe ••go •ooo The following statement is a full description of this invention, including the best method of performing it known to me/us:- The present invention relates to novel nucleic acid sequences and plasmids capable of hybridizing therewith and carrying at least one phage resistance mechanism, to the lactic acid bacteria containing these sequences or these plasmids, in particular the lactococci belonging to the species Lactococcus lactis to the use of certain strains of these lactococci for transferring a phage resistance mechanism, especially by conjugation, to strains of industrial interest, particularly in the dairy industry, and to the use of certain strains of Lactococcus lactis for obtaining these plasmids.
Lactic acid bacteria are involved in the production and preservation of a large number of foodstuffs such as cheeses, butter, yogurts, sausage or sauerkraut.
Among these, dairy products occupy a particularly important position. The industrial conversion of milk is carried out in ever larger fermentation vats, in which the appearance of phages of lactic acid bacteria can have serious or even catastrophic consequences, namely a variation in the characteristics, especially organoleptic characteristics, of the final product, a loss of product present in the vat, and the need to decontaminate the latter as well as the surrounding installations.
dairy industry is therefore in urgent need of novel means and novel methods of rendering bacteria more resistant to phages.
The phages of lactic acid bacteria belong to three major homology groups S 20 (II) and (III) defined by DNA/DNA hybridization studies according to RELANO P. staL, (1987), J. Gen. Microbiol. 133, 3053-3063. Groups and (III) comprise only virulent phages. Group (II) comprises virulent phages and temperate phages. The homologies are strong within one and the same group and very weak between groups. The phages of group have an oblong nucleocapsid while the phages of groups (II) and (III) have an isometric nucleocapsid.
Several natural phage resistance mechanisms are known to exist, the three main ones being: inhibition of phage adsorption; in this mechanism, the adsorption of the phage by the bacterium is inhibited or delayed.
30 -the restriction/modification system; this system involves a restriction enzyme, which degrades the DNA of the phage as soon as it enters the bacterium.
abortive infection; according to this third mechanism, phage adsorption is normal but phage multiplication does not take place.
These mechanisms are described in detail by SANDERS M. in Biochimie (1988), 411-421.
The development of phage resistant lactic acid bacteria has already been the subject of numerous studies.
In this connection, reference may be made to the following articles in particular: VLEGELS et a; Neth. Milk and Dairy J. 43, (1989), 245-259; SANDERS and KLAENHAMMER; Applied and Environ. Microbiol. (1983), vol. 46, 1125-1133; these articles relate to plasmids which inhibit phage adsorption; Audrey W. JARVIS; Applied and Environ. Microbiol.; March 1988, p. 777-783; EP-A3-0 208 468; COFFEY eLaL; Neth. Milk and Dairy J. 43, (1989), 229-244; KLAENHAMMER and SANOZKY; Journal of General Microbiology (1985), 131,1531-1541; DURMAZ et a; J. Bacteriol. (1992), 7463-7469; these articles describe plasmids which confer phage resistance by the abortive infection mechanism; JOSEPHSEN and KLAENHAMMER, Plasmid 23,71-75, (1990); 20 MOINEAU et a; Applied and Environ. Microbiol. (1995), 2193-2202; US patent 4 883 756; GAUTIER and CHOPIN; Applied and Environ. Microbiol. (1987), 53, p. 923- 927; McLANDSBOROUGH et al.; Applied and Environ. Microbiol. (1995), 2023- 2026; these last articles describe especially plasmids which confer phage resistance by the restriction/modification mechanism.
Patent application EP-A1-452224 also describes a DNA sequence comprising at least one phage resistance mechanism; this DNA sequence contains a 30 functional portion of the HindIII-HindII fragment of about 3.3 kb of plasmid pPF144-1 present in the Escherichia coli strain deposited on 9th April 1991 in the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, France) under no. 1-1070.
This HindIII-HindIII fragment of about 3.3 kb was isolated from plasmid pPF144 contained in the Lactococcus lactis ssp lactis strain deposited in the CNCM on 12th April 1990 under no. 1-945, which is a transconjugant resulting from the crossing of the donor strain Lactococcus lactis ssp lactis S91, deposited in the CNCM on 12th April 1990 under no. 1-940, and the recipient strain Lactococcus lactis ssp lactis S45, derived from the strain Lactococcus lactis ssp lactis C2-LL described by McKay Let 1977, in J. Bacteriol. 257-265. This fragment carries one or more phage resistance mechanisms.
Following this work, a DNA sequence of 1.9 kb which by itself confers phage resistance was isolated from this HindIII-HindIII DNA sequence of 3.3 kb.
This novel DNA sequence of about 1.9 kb was described in EP-A1-643 134.
The Applicant has isolated a fragment of 3704 base pairs (bp) from the total DNA of the strain Lactococcus lactis ssp diacetylactis S94, deposited in the CNCM on 12th April 1990 under no. 1-941, by partial digestion with the restriction enzyme Sau3A.
This fragment carries one or more phage resistance mechanisms and by itself confers phage resistance.
From this DNA sequence of 3704 bp, the Applicant subsequently isolated a DNA sequence of 1345 bp which by itself confers phage resistance.
20 The present invention therefore relates to two novel DNA sequences comprising at least one phage resistance mechanism, said sequences respectively containing 3704 bp and 1345 bp and consisting of: a) the DNA sequences having the nucleic acid series of [SEQ ID No 1] or [SEQ ID No 3]; b) the DNA sequences hybridizing with the above sequences or a fragment thereof; and c) the corresponding mRNA and cDNA sequences.
The sequences [SEQ ID No. 2] and [SEQ ID No. 4] are the amino acid sequences deduced from the sequences [SEQ ID No. 1] and [SEQ ID No. 3] respectively.
The sequence [SEQ ID No. 3] is a DNA fragment of 1345 bp contained within the sequence [SEQ ID No. 1].
This fragment of 1345 bp can be obtained by the PCR method using the following two oligonucleotides: oligonucleotide no. 1 [SEQ No. TCATAGGATCCAATAATCAACTAGCAATTCG 3' fBamHI oligonucleotide no. 2 [SEQ No. 6]: CATTAGTCGACAAATACAGGCTCTATAAAGC 3' SaI The invention particularly relates to the DNA sequences respectively containing: 3704 bp and having the nucleic acid sequence [SEQ ID No. 1] below; and 1345 bp and having the nucleic acid sequence [SEQ ID No. 3] below.
The invention further relates to the DNA sequences which have a high degree of homology with the DNA sequences [SEQ ID No. 1] and [SEQ ID No. 3] above. A high degree of homology means a homology (ratio of the identical nucleotides to the total number of nucleotides) of at least 70%, preferably at least of the nucleotide sequences when they are aligned according to the maximum homology by the optimal sequence alignment method of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443-453. This method is used especially in the UWGCG software of the University of Wisconsin: Devereux t al.. 1984, Nucl. Ac. Res. 12, 8711-8721 option GAP.
The present invention particularly relates to the DNA sequences which hybridize with the DNA sequence [SEQ ID Nol] and [SEQ ID No 3] or a fragment thereof. In the present specification the term "hybridization" designated the 25 conventional hybridization conditions and more particularly the stringent hybridization conditions.
The invention further relates to the plasmids transformed with one of the nucleic acid sequences according to the invention. A possible example of these plasmids is plasmid pLDP1 into which one of the DNA sequences according to the invention has been cloned by the customary techniques known to those skilled in the art. Plasmid pLDP1 is derived from plasmid pVA838 (Macrina F.L. Let a Gene 19, 345-353) by deletion of the 1523 bp fragment between the HindIII site and the EcoRI site (1523) and replacement thereof with 54 base pairs corresponding to the multiple cloning sites of plasmid pUC18 (Yanisch-Perron C. et al.: 1985 Gene 33, 103-119) which are flanked by the EcoRI and HindIII sites.
The invention further relates to the phage resistant lactic acid bacteria, preferably belonging to the species Lactococcus lactis, which contain at least one of the nucleic acid sequences or one plasmid as defined above.
These nucleic acid sequences or these plasmids may have been introduced into the lactic acid bacteria by conjugation, transformation, protoplast fusion or any other gene transfer method well known to those skilled in the art.
The lactic acid bacteria which can advantageously be transformed with the DNA sequences according to the invention, or a plasmid containing them, are for example the strains of Lactococcus lactis ssp cremoris, Lactococcus lactis ssp lactis and Lactococcus lactis ssp lactis var, diacetylactis.
These strains transformed in this way can be used for transmitting a phage resistance mechanism to a strain of industrial interest by conjugation, transformation, transduction, protoplast fusion or any other gene transfer method well known to those skilled in the art. This mechanism can be carried by a plasmid or by another part of the bacterial genome. If it is carried by a plasmid, it is e* advantageously transferred by conjugation.
2The invention further relates to the phage resistant strains of industrial S* 20 interest obtained in this way.
The invention will be understood more clearly with the aid of the following Examples, which comprise experimental results and a discussion thereof. Some of these Examples relate to experiments performed for the purpose of carrying out the invention; others relate to embodiments of the invention, which are of course given 25 purely by way of illustration.
The majority of the techniques described in these Examples, which are well known to those skilled in the art, are explained in detail in the work by Sambrook, Fritsch and Maniatis entitled "Molecular cloning; a Laboratory Manual", published in 1989 by Cold Spring Harbor Press, New York (2nd edition).
S* 30 The following description will be understood more clearly with the aid of Figures 1 and 2 below, which respectively show: FIG. 1: Organization of the ORF deduced from the sequence of the fragment of 3704 base pairs.
FIG. 2: Amplification, by PCR, of 2 internal fragments of the fragment of 3704 bp conferring phage resistance.
Example 1: Sequence of the fragment of 3704 bp The strain Lactococcus lactis ssp diacetylactis S94, deposited in the CNCM on 12thApril 1990 under no. 1-941, contains one or more phage resistance mechanisms. In particular, it has transferred plasmid pPF72 by conjugation into the recipient strain Lactococcus lactis S45, derived from the strain Lactococcus lactis C2-LL described by McKay et al., 1977, J. Bacteriol. 257-265, said plasmid conferring resistance to phages 0 53 (group I) and 0 59 (group III). Other phage resistance mechanisms may be present in the strain 1-941. It is for this reason that a total DNA library was constructed from the strain 1-941. The restriction fragment of 3704 bp conferring phage resistance, obtained by a partial digestion of the total DNA of the strain S94 (1-941) with the restriction enzyme Sa3A, was cloned into vector pLDP1 at the BamHI site. The recombinant plasmid pLAB206 obtained confers a total resistance to phage 0 59 to the strain Lactococcus lactis ssp lactis MG 1363 disclosed by GASSON M.J. (1983) in J. Bacteriol. 154 1-9, hereinafter named strain L. lactis S56 or S56.
20 The nucleic acid sequence of this fragment of 3704 bp, determined by the method of Sanger taL. (PNAS USA, 14, 5463, 1977), is the sequence [SEQ ID No. 1] below.
Analysis of the sequence showed that the fragment of 3704 bp possesses 2 complete open reading frames (ORF1 and ORF2) and two truncated open reading frames (ORF3 and ORF4) containing only the N-terminal parts (Figure 1).
Example 2: Amplification of an internal fragment of the 3704 bp fragment by PCR The PCR (Polymerase Chain Reaction) technique, described for example in 30 the work by Maniatis cited above, makes it possible to amplify a DNA fragment contained between two appropriately chosen oligonucleotides. This amplified DNA can easily be cloned if restriction sites are provided by the oligonucleotides.
In fact, the sequences of these oligonucleotides can contain, at their 5' end, a heterologous part of the DNA to be amplified, consisting of 10 to 12 base pairs, for example, 6 of which constitute a restriction site.
This technique was applied in order to determine whether one or other of the two open reading frames (ORF1, ORF2) identified in the nucleotide sequence of the 3704 bp fragment corresponds to a phage resistance gene, but also in order to form a probe specific to this gene.
This was done by synthesizing 4 oligonucleotides of 31 and 32 bases (6 of which constitute a restriction site).
These 4 oligonucleotides have the following sequences: oligonucleotide no. 1 [SEQ No. TCATAGGATCCAATAATCAACTAGCAATTCG 3' BamHI oligonucleotide no. 2 [SEQ No. 6]: CATIAGTCGACAAATACAGGCTCTATAAAGC 3' Sall oligonucleotide no. 3 [SEQ No. 7]: TCATAGGATCCTTAACAATAAAATTACTCTGC 3' BamHI **oligonucleotide no. 4 [SEQ No. 8]: 20 5' CATTAGTCGACITAAATITGATATTGATTGCG 3' Sal Their locations on the 3704 bp fragment are indicated in Figure 2.
The oligonucleotides no. 1 and 2 made it possible to amplify a DNA fragment of 1345 bp containing ORF1 of 1038 bp in the form of a BamHI-alI 25 fragment, by virtue of the restriction sites provided by the oligonucleotides, enabling directional cloning into shuttle plasmid pLDP1.
Likewise, the oligonucleotides no. 3 and 4 made it possible to amplify a DNA fragment of 1152 bp containing ORF2 of 573 bp in the form of a BamHI- Sail fragment, as above.
30 The DNA fragments were amplified by PCR, starting from a total DNA preparation of the strain S94 (1-941), with conventional Taq polymerase.
8 The PCR products were purified by phenol/chloroform extraction, precipitated with ethanol, digested with the restriction enzymes BamHI and SaI and cloned into shuttle vector pLDP1 open at the BamHI and SaI sites.
Cloning of these fragments into vector pLDP1 produced recombinant plasmids pLAB207 and pLAB 208. These plasmids were introduced into the strain E. coli TG1 and into the strain L. lactis S56.
The introduction of plasmids pLAB207 and pLAB 208 into the strain L lactis S56 made it possible to determine whether they confer phage resistance.
The results pertaining to the cloning of the various amplified DNA fragments are summarized in Table 1 below: TABLE 1 Pair of oligo- Size of the Sites added Cloned into Phenotype* nucleotides fragment pLDP1 1-2 1345 bp BamHI-Sal pLAB207 pr 3-4 1152 bp BamHI-SalI pLAB208 pr 15 phenotypes: pr phage resistance pr- no phage resistance Example 3: Phage resistance conferred by plasmid pLAB207 Plasmid pLAB207 was introduced into the strain L. lactis S56.
The phage resistance of the clones obtained was tested by performing a titration (PFU/ml) with phages 0 53 and 0 59.
The results are given below: Strain Phage 0 53 Phage 59 (III) Titer Size of the Titer Size of the (PFU/ml) plates (PFU/ml) plates (mm) (mm) S56 1010 3 3.109 2 S56 (pLDP1) 6.109 3 2.109 2 S56 106 0.5 0 (pLAB207) PFU/ml plate forming units per ml Plasmid pLAB207 containing the PCR-amplified fragment of 1345 bp corresponding to ORF1 confers phage resistance.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
*S S SEQUENCE LISTING GENERAL INFORMATION:
APPLICANT:
NAME: SYSTEMS BIO INDUSTRIES STREET: 4, Place des Ailes CITY: Boulogne Billancourt COUNTRY: France POSTAL CODE (ZIP): 92641 Cedex TELEPHONE: 47 12 25 TELEFAX: 47 12 26 56 (ii) TITLE OF INVENTION: Nucleic acid sequences and plasmids comprising at least one phage resistance mechanism, bacteria containing them, and their use (iii) NUMBER OF SEQUENCES: 8 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 S"(vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:
CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: FR 95 09980 S(B) FILING DATE: 22-AUG-1995 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 3704 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Lactococcus lactis (ix) FEATURE: NAME/KEY: CDS LOCATION:1095. .2132 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GATCAAAACT
TTTGrrGAA
AATTAAAGCA
AAGACTTCCA
rITC'ITGCCT
TAAAAGACGT
GAAAAGTGGT
TCAG TTGCA
ACTCAAAATC
TCATTATAGT
GATGTTAMAT
GATAGTGTAC
AAGATACATC
'FTGTATATCA
TTCAAAGTTA
AAGTGAAAAA
AACTAAAAAA
TTCTG'TITG
TAGTGGGAGA
GATGCAAATG
ACAGAGGCTC
AGCGCAGAAA
ATTACACCTA
ATAG ITGGTG
AAAATGTAAA
AGTGAC ITAA GTA 1TCCAAT
ATGTAAATAT
ITGCAAAATG
GAGTTAGAAA
TACTMAGTTA
AATGCTAATA
ACGATACAAG
AATACAGGCT
TAAAATGGGG
AA ITATTI= ATTA FTATAT GAGTAATrrAA CAGCAGG ITA
ACAAACCACA
AAAAGCCAGA
TTGGACTAT
ATTATATAAA
TACAAAAATA
AAGTAAAAAA
TAAGCTTGGA
GTATTCCAAA
TTGAGCCAAT
AGGTATCGAT
AAAATATAAT
G TT'rrATC
CTATAAAGCT
CAAATTGGGG
TTGAATTAGT
AATTATATT
GGCAAATCAT
TCAAT'rAAGT AG'PrGTTAAT A FTACCTAAG
AATGGTGGGT
AACATGGCTT
=1IAGTIG
GATTTGGCTA
TGGCCCAAT
TGTAGTCACA
AATTACAAGT
AATAAAGTAG
TAAAAGAATA
TATGCCGAGT
CTCAAAGTGT
GCAMr'CAAG
OFITITTAT
ATAT'ITATAG
CTAAAACCAG
CAAGAAACTA
ACTGGAAAGT
ACAGGAGAAG
GCTACTAT
TAGCCATGTT
TAATACCTAA
TATTAGAACC
AGAGAAAATT
GTAAATACAC
GAGC 1T1'TA
TCGTCAATAA
AACGAAATAC
ATGTA TI TTTGATfGG
AAATAATACT
ACCCAAAAAA
TITTCT'ITCA
GTAAATAAAA CAATGAAAAA 120 18o 240 300 360 420 48o 540 600 660 720 780 840 900 960 1020 1080 1130 -too 09 *000 a*9 0 0* S *000
GAGGOTT~AG
TGGCAAAGTT
TGATAAAATA
GCAGGA ITGT
TCACTATAGA
TTAAGAGTAT
TTGCATA'TTT
'rrATAGTTAT
ATAAACTTGT
AATGGGCATA
ATAAAAAACC
TTAGATAGTT
AAAGCTCTTA
TCAAATATGT
TTTCGAGATT
TGAGTTAAGT
TACA ATG AAA T1TA TAT ATA GT T GGT Met Lys Leu Tyr Ile Val Giy TCA CAC MAT CTG AMA ACA TCA TAT ACA GAG 1IT Ser His Asn Leu Lys Thr Ser Tyr Thr Clu Phe 20 GMA CAT AGG GAT GMA OTA TAT ACA GAT GMA GAT Giu His Arg Asp Glu Val Tyr Thr Asp Glu Asp AAT GGC 'Tr GAT ITA Asn Gly Phe Asp Leu AGA 1TA TAT TT'A 'FrG Arg Leu Tyr Leu Leu TTA ATA ATA TCA AMA Leu Ile le Ser Lys 1178 1226 GGA GAT ATT TTA CGA MAT TIT GAA AGA TAT TGT CAA OCA MAT GAT TTA Gly Asp Ile Leu Arg Asn Phe Giu Arg Tyr Cys Gin Pro Asn Asp Leu 4550 55 1274 TGG AGO GAT 'TTT GAA GAA CAG ACT GAG AAA ATA ATA TOA GAA Ile Ile Ser Giu Trp Ser Asp Phe Giu Glu Gin Thr Giu ATA ATC TTT GAA GAA Ile Ile Phe Giu Giu 85 Lys 70 ATA TCA Ile Ser TTA GAT Leu Asp GAG GGG AAA Giu Gly Lys Afl GGA GGT Ile Giy Giy
ATTL
Ile GAA TGG AGO TGC Giu Trp Ser Cys
A
Lys 1322 1370 14118 GAG AGG CAT AAA Glu Arg His Lys TPT A FI GAT TGT TT AAA MAG TTA ATA Phe Ile Asp Cys Phe Lys Lys Leu Ile 100 105 GAT ACT GAT ATA TAT CAT MAT GAA ATT Asp Thr Asp Ile Tyr His Asn Giu Ile 120 MAA A Lys Lys 110 CMA GAT AGT ACA Gin Asp Ser Thr rrc Phe 115 1466
CAA
Gin 125 GAG TAT GCC TTA AAG Giu Tyr Aia Leu Lys 130 TAT IT AAO AAT AAG Tyr Phe Asn Asn Lys 135 GTA AGG GAA OTA Vai Arg Giu Leu 1514 a a a. ATA TGG GTT' CCA Ile Trp Val Pro TAT 1TA TAT AlT AGT Tyr Leu Tyr Ile Ser 145 MAT ACG AAA AAT ATO Asn Thr Lys Asn Ile 165 Phe 150 CAA GAA TOG ATA Gin Giu Trp Ile CAA AOG Gin Thr 155 1562 1610 ATT ATT TTA Ie Ile Leu TCT GTA AT Ser Vai Ile 175
AAA
Lys 160 TAT GAA ATA GAT GMA GAT TOG Tyr Giu Ile Asp Giu Asp Ser 170 TOO 'TTT MAC TAT Ser Phe Asn Tyr AAT ACT ATG GMA Asn Thr Met Giu
GAT
Asp 185 OTT TAT CMA Val Tyr Gin 1658 1706 CMA A Gin Lys 190 GAT GTA OTA OAT ITA Asp Val Leu His Leu 195 CAT GGT TOT GTA AAA AAT OTA GAA GAT His Giy Ser Val Lys Asn Leu Giu Asp 200
UTA
Vai 205 Leu GTA 1TA GGA TIT Vai Leu Giy Phe GAA ACA AGT 'Tr Giu Thr Ser Phe 225
CAT
His 210 TOG OCA GAA ATA GAO Ser Pro Giu Ile Asp 215 GAO AAA ITA OCA OGG Asp Lys Leu Pro Gly 220 1754 1802 ACG AAG GAA MIT AAG GAA Thr Lys Giu Phe Lys Giu 230 ACA CAA AGA Thr Gin Arg ATG MIT Met Phe 235 GCA OAA Ala Glu CAG GOT Gin Gly 240 TOT AGA AAG O'ir AAT Ser Arg Lys Leu Asn 245 TOT AAT AAG TFIT TAT OAT GAA 1850 Ser Asn Lys Phe Tyr Asp Glu 250 AAT ATA OGA AGA M~I TAT AAA OCA Asn Ile Gly Arg Phe Tyr Lys Pro 255 260 GTA MAT CTT FPA A GAO TCA AT Asp Ser Ile 1898 Val Asn Leu Leu Lys 265 GAG TOT T'IT O'1T AAA MAT Glu Ser Phe Val Lys Asn 270 AMAMAT A FT OAT GMA OTA ATA ATO ETA GOT Ile Ile Leu Gly Lys Asn Ile His Glu 275 GAO TOG GTT TAT 'TT Asp Trp Val Tyr Phe 295 Val 280 1946 1994
OAT
His 285 TOA TAT MAT MAG Ser Tyr Asn Lys
ATA
le 290 AAA GMA OTA GTC AGA Lys Oiu Leu Val Arg 300 TOO 000 OOT GAA OCA AAA Cys Ala Pro Giu Ala Lys 305 TAT 'ITA MI~ AGO TAT TAT TOO CAA Tyr Leu Phe Ser 310 Tyr Tyr Ser Gin AAT OAT Asn Asp 315 2042 AAA GAA Lys Giu MOC ATA MAT MAA Afl Asn Ile Asn Lys Ile 320 ATA TTO Ile Leu 325 GAO AAT AAA TT Oiu Asn Lys Phe OAT ATT OAT Asp Ile Asp 330 2090 TOO GMA AMA Oys Glu Lys 335 ATA OAT GTA OAT AAC AAA AIT AAA AAA OAT le His Vai Asp Asn Phe Lys Ile Lys Lys Asp 340 345 2132
TAGOAAAAAT
TTAGCCATG
TGATTTTMAC
AGGOGMOCGA
MATCACTTr
CCTOACAGGT
TAAATATCO
CATTGTAOTG
ACTGACTAAA
MOCATTTGCA
AATGGGMOA
TTGGCOAMTG
TOCTAGGAMA
AAAATGCAGT
GAOGTTC TT
GTOAGTAMAT
C'TTMATG
TTAACTAGCA
TTAGGAGT'IT
ACTTOATACO
AGTMAAGMAT
C1TITACAAO OTACGT'rITG
GTCATTAGAT
ATATOTOOMA
TCAGOTATTG
GCATGACA T
TMATOTOOAA
OTGTOMTMA
AO ITGGATAC
TTAGMAATA
AOGOOOOATA
COOTTITOM
AAACCAAAMA
O!ITAGTrGGGT
TATATGATTA
OGATAGAATA
AOOOTAAAAO
GOTOAGOTCO
MAAAATTAOG
OAATTOCTT
GGACTOTTOG
MAGCAGGAGT
TACATOOATA
GAAOTTOT
TOGATACACT
TOGGTAAGAG
AACTrrGAGO
GGOTMOAAGO
COTCAAGGG
OTT'IGGAAG
TAAGA77AO
TATATAGA
TOTTAAAGTO
TATCATCTCT
TAGATTTGTC
GAAMAGGATT
TAATTCCAGO
TOTOOOAAAT
MITTGTGTAG
GMAGTACTGT
TCTGAAATTIA
CATTOCCTTCA
GGCATTACAA
OAATTGGTGO
OATAACTAAO
ACACTOOTT
TGTGCTA FTA
ATAGCAOGAO
TTTATAAAA
CCGOAITGOC TAO'ITGATTA
TOTGGTOCA
AAITI'OM TT
AAAAAAGAGA
ITCT'ITCGCT
OTATOATGOA
TGAOTOAAOA
TOTATOAOAT
OOOTTTGTMA
CGAATOOAOT
AOOTOTTTAA
CGOOOGAOMA
TTAOO'PITAA
OAATCTGATO
GATAAATOOT
TAAGTOATAT
TTA FrrAT AAA FTACTCC GTGA TTAGO
GAAMOO'ITO
MAAOTOGOTO
TCATG'TOTT
GTGAOTGAOO
OCrrCCTCGGC AGCCTGA TOC CA TOGTAGOO
GACAOMAAOG
AOAOOAOTOO
AGOGAOAATA
GTO'IMCAOT
TTGCGAGATG
CTCAATOGT
AAGTOGTGOG
TOOMATGAM
TAAAGCAGAG
2192 2252 2312 2372 2432 2492 2552 2612 2672 2732 2792 2852 2912 2972 3032 3092 3152 3212 ATTOGGG TT TCTTAATT
TAATTTI'ATT
TATOACTOCT
TGGAATAAAG
AGCAAAGAAA
CGCAGTCCAA
ATATACCOCA
TAAAATACCA
ATGAAATTTC
G'PrAA FITrr
ACCGAAAATC
ATAATCAGGG
ATGATAAAAA
T'ITAAATA
ATAACATTCC
ATCACTAAAG
ACTCTGAGAT
ATCAGAGATI'
CTAAAAGGAG
CAATAATTGC
CCCGAAATCC
AGAGATGCCA
CAA ITAACAG
ATAAGATTAA
AGAAATTCAA
A'1PCCAAAAA
GACTAGGOCA
TCCAATGAGT
CCCAGAACCT
ATCTCTGACA
CCCAAAGAGC
GTCAAGAACT
C ITCATGGGT TTTCTGAT AAAAATGAAG AGG FTAACAA
GCTCCTGCTC
C'1rGTAAAAA
AAATAATGAA
AAGGTAACAG
AGTTGAATAA
AAGCTTAA
CTAAAGGATA
CCAAAAGCGT
G'ITGAGTCAA
GTGAAATCAG
GGACTTTCAG
AAAAGGCAAA
AATAATTAAA
3272 3332 3392 3452 3512 3572 3632 3692 3704 A ITTTCCCGA TC INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 346 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met 1 Lys Lys Leu Tyr Ile Val Gly Asn Gly 5 Phe 10 Asp Leu Ser His Asn Leu Thr Ser Tyr Thr Giu Phe Arg Thr Leu 25 Ile Tyr Leu Leu Giu Giu Val Tyr Arg Asn Phe Asp Giu Asp Leu 40 Ile Ser Lys Gly Trp His Arg Asp Asp Ile Leu Ser Asp Phe Giu Arg Tyr Gin Pro Asn Asp Giu Giu Leu Gin Thr Giu Ile Ile Phe Giu Giu 85 Ile Lys Ile Ile Ser Giu 70 Giu Trp Ser Cys Lys 90 Asp Cys Phe Lys Lys Ser Giu Gly Lys Ile Asp Ile Giy Giy Giu Arg His Lys Ser Thr Phe 115 Leu Lys Tyr 130 Tyr Leu Tyr Phe 100 Asp Leu Ile Lys Thr Asp Ile Tyr His Asn Giu Ile Gin Lys Gin Asp 110 Giu Tyr Aia Trp Vai Pro 120 Val Phe Asn Asn Lys Arg Giu Leu Tyr 140 Thr Ile Ser Phe Gin Giu Trp Ile Gin Ile Ile Leu 145 Asn Lys 160 Ser Thr Lys Asn Ile 165 Giu Ile Asp Giu Ser Ser Val 170 Val Phe Asn Tyr Thr 180 Asn Thr Met Giu Asp 185 Tyr Gin Gin Lys Asp Vai 190 Leu His Leu His Gly Ser Val Lys Asn Leu Glu Asp Val Val Leu Gly 195 200 205 Phe His Ser Pro Glu Ile Asp Asp Lys Leu Pro Gly Leu Glu Thr Ser 210 215 220 Phe Thr Lys Glu Phe Lys Glu Thr Gin Arg Met Phe Ala Glu Gin Gly 225 230 235 240 Ser Arg Lys Leu Asn Ser Asn Lys Phe Tyr Asp Glu Asn Ile Gly Arg 245 250 255 Phe Tyr Lys Pro Val Asn Leu Leu Lys Asp Ser Ile Glu Ser Phe Val 260 265 270 Lys Asn Lys Asn Ile His Glu Val Ile Ile Leu Gly His Ser Tyr Asn 275 280 285 Lys Ile Asp Trp Val Tyr Phe Lys Glu Leu Val Arg Cys Ala Pro Glu 290 295 300 Ala Lys Tyr Leu Phe Ser Tyr Tyr Ser Gin Asn Asp Lys Glu Asn Ile 305 310 315 320 Asn Lys Ile Ile Leu Glu Asn Lys Phe Asp Ile Asp Cys Glu Lys Ile 325 330 335 His Val Asp Asn Phe Lys Ile Lys Lys Asp 340 345 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1345 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Lactococcus lactis (ix) FEATURE: NAME/KEY: CDS LOCATION:246..1283 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAATACAGGC TCTATAAAGC TCTCAAAGTG TTCACTATAG AAAAGCTCTT AAAGTGAAAA ATAAAATGGG GCAAATTGGG GGCATTTCAA GTTAAGAGTA TTCAAATATG TAACTAAAAA 120 AAATTATTTT TlTGAATAG TGTTITTCTA r1TGOATATT TITOTGAGAT TTTCTGTTII GATATTATA TAATTATATT TATATTTATA GTTATAGTTA TTGAGTAAG TTAGTGGGAG ATACA ATG AAA TTA TAT ATA GTT GGT AAT GGC TTT GAT TTA TCA CAC Met Lys Leu Tyr Ile Val Gly Asn Gly Phe Asp Leu Ser His 180 240 287
AAT
Asn CTG AAA ACA TCA Leu Lys Thr Ser ACA GAG TT Thr Olu Phe AGA TTA Arg Leu 25 TAT TTA TTG GAA CAT Tyr Leu Leu Glu His AGG GAT GAA GTA TAT Arg Asp Giu Val Tyr ATT TrA CGA AAT Trr Ile Leu Arg Asn Phe ACA OAT GAA GAT TTA Thr Asp Glu Asp Leu 40 ATA ATA TCA AAA GGA GAT Ile Ile Ser Lys Gly Asp CCA AAT GAT TTA TGG AGO Pro Asn Asp Leu Trp Ser GAA AGA TAT Glu Arg Tyr OAT T GAA Asp Phe Glu GAA CAG ACT GAG Glu Gin Thr Glu
AAA
Lys 70 ATA ATA TCA GAA ATA TCA GAG GGG Ile Ile Ser Giu Ile Ser Giu Gly
S
AAA
Lys
GGT
Gly
CAA
Gin
TAT
Tyr
GTT
Vai Leu Leu ATT ATA ATC TIT GAA GAA lie Ile lie Phe Giu Glu GAG AGG CAT AAA TTT ATT Glu Arg His Lys Phe Ile 100 GAA TOG AGO TGC Glu Trp Ser Cys TTA GAT ATT GGA Leu Asp lie Gly GAT TGT T Asp Cys Phe AAA AAG ITA Lys Lys Leu 105 ATA AAA AAA Ile Lys Lys 110 575 GAT AOT ACA Asp Ser Thr GCC TTA AAG Ala Leu Lys 130 TTC GAT ACT GAT ATA TAT Phe Asp Thr Asp Ile Tyr 115 120 TAT TTI AAC AAT AAG OTA Tyr Phe Asn Asn Lys Val 135 CAT AAT GAA ATT CAA GAG His Asn Giu Ile Gin Glu 125 623 AGO GAA OTA Arg Giu Leu
TAT
Tyr 140 ATA TGG lie Trp CCA TAT Pro Vyr 145 TTA TAT A T AOT TTI Leu Tyr Ile Ser Phe 150 CAA OAA TGO ATA Gin Glu Trp Ile
CAA
Gin 155 ACG ATT ATT Thr lie Ile AAA AAT ACG Lys Asn Thr 160 AAA AAT ATO Lys Asn Ile 165 TAT OAA ATA GAT GAA GAT TOG TOT OTA 767 Tyr Glu Ile Asp Giu Asp Ser Ser Vai 170 Ile 175 TOO TTr AAC TAT Ser Phe Asn Tyr
ACA
Thr 180 AAT ACT ATG GAA Asn Thr Met Glu GAT G'IT TAT CAA CAA AAA Asp 185 Val Tyr Gin Gin GAT GTA OTA OAT 'ITA OAT GGT TOT OTA Asp Vai Leu His Leu His Giy Ser Vai 195
AAA
Lys 200 AAT OTA GAA CAT CTA CTA Asn Leu Giu Asp Vai Vai 205 LTA GGA T Leu Gly Phe ACA ACT Trrr Thr Ser Phe 225
OAT
His 210 TCGCOCA GMA ATA GAO Ser Pro Ciu Ile Asp 215 GAO AAA TrA OCA OGG TTA GAA Asp Lys Leu Pro Giy Leu Giu 220 ACO AAG GMA 'TTT MC Thr Lys Giu Phe Lys 230 GMA ACA CAA AGA Giu Thr Gin Arg
ATG
Met 235 T'IT GOA GAA Phe Aia Giu OAG COT Gin Ciy 240 TOT AGA AAG OTT Ser Arg Lys Leu
AAT
Asn 245 TOT AAT AAG 'ITT TAT CAT GAA AAT ATA Ser Asn Lys Phe Tyr Asp Giu Asn Ile 250 1007
GGA
Ciy 255 AGA TTT TAT AMA OOA Arg Phe Tyr Lys Pro 260 CTA AAT CIT TTA AAA Vai Asn Leu Leu Lys 265 GAO TOA ATT GAG Asp Ser Ile Giu
TOT
Ser 270 TT OTT AMA MT Phe Vai Lys Asn
AMA
Lys 275 AAT ATr OAT GAA Asn Ile His Glu
GTA
Val 280 ATA ATO TI'A GOT OAT TOA Ile Ile Leu Cly His Ser 285 TAT MAT MCG Tyr Asn Lys
ATA
Ile 290 GAO TOG GO[T TAT TT Asp Trp Vai Tyr Phe 295 AAA GAA OTA OTC AGA TGO CO Lys Ciu Leu Vai Arg Cys Aia 300 1055 1103 1151 1199 1247 1293 COT CAA OA AMA TAT TTA Pro Ciu Aia Lys Tyr Leu 305 rrr AGO Phe Ser 310 TAT TAT TOO Tyr Tyr Ser MOC ATA Asn Ile 320 AAT AAA ATT ATA ITG Asn Lys Ile Ile Leu 325 GAG AAT AAA TT Ciu Asn Lys Phe AAA AIT AAA AAA Lys Ile Lys Lys 341 CAA MAT CAT AMA CM Gin Asn Asp Lys Ciu 315 CAT ATT1 CAT TGO CAA Asp Ile Asp Oys Ciu 330 CAT TAGOAAAAAT Asp
AAA
Lys 335 ATA OAT GTA CAT Ile His Vai Asp TTAGCAG ITT TATATGA [TA TATPTATACA OOOCAA TGC TAG ITGATTA 'TT14 1345 18 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 346 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Lys Leu Tyr Ile Val Gly Asn Gly Phe Asp Leu Ser His Asn Leu 1 5 10 Lys Thr Ser Tyr Thr Glu Phe Arg Leu Tyr Leu Leu Glu His Arg Asp 25 Glu Val Tyr Thr Asp Glu Asp Leu Ile Ile Ser Lys Gly Asp Ile Leu 40 Arg Asn Phe Glu Arg Tyr Cys Gin Pro Asn Asp Leu Trp Ser Asp Phe 55 Glu Glu Gin Thr Glu Lys Ile Ile Ser Glu Ile Ser Glu Gly Lys Ile 70 75 Ile Ile Phe Glu Glu Glu Trp Ser Cys Lys Leu Asp Ile Gly Gly Glu A85 90 Arg His Lys Phe Ile Asp Cys Phe Lys Lys Leu Ile Lys Lys Gin Asp 100 105 110 Ser Thr Phe Asp Thr Asp Ile Tyr His Asn Glu Ile Gin Glu Tyr Ala 115 120 125 Leu Lys Tyr Phe Asn Asn Lys Val Arg Glu Leu Tyr Ile Trp Val Pro 130 135 140 Tyr Leu Tyr Ile Ser Phe Gln Glu Trp Ile Gin Thr Ile Ile Leu Lys 145 150 155 160 Asn Thr Lys Asn Ile Tyr Glu Ile Asp Glu Asp Ser Ser Val Ile Ser 165 170 175 Phe Asn Tyr Thr Asn Thr Met Glu Asp Val Tyr Gin Gin Lys Asp Val 180 185 190 Leu His Leu His Gly Ser Val Lys Asn Leu Glu Asp Val Val Leu Gly 195 200 205 Phe His Ser Pro Glu Ile Asp Asp Lys Leu Pro Gly Leu Glu Thr Ser 210 215 220 Phe Thr Lys Glu Phe Lys Glu Thr Gln Arg Met Phe Ala Glu Gin Gly 225 230 235 240 Ser Arg Lys Leu Asn Ser Asn Lys Phe Tyr Asp Glu Asn Ile Gly Arg 245 250 255 Phe Tyr Lys Pro Val Asn Leu Leu Lys Asp Ser Ile Glu Ser Phe Val 260 265 270 a a Lys Asn Lys Asn Ile His Glu Val Ile Ile Leu Gly His Ser Tyr Asn 275 280 285 Lys Ile Asp Trp Val Tyr Phe Lys Glu Leu Val Arg Cys Ala Pro Glu 290 295 300 Ala Lys Tyr Leu Phe Ser Tyr Tyr Ser Gin Asn Asp Lys Glu Asn Ile 305 310 315 320 Asn Lys Ile Ile Leu Glu Asn Lys Phe Asp Ile Asp Cys Glu Lys Ile 325 330 335 His Val Asp Asn Phe Lys Ile Lys Lys Asp 340 345 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc_signal LOCATION:6..11 OTHER INFORMATION:/function= "BamHI restriction site" (ix) FEATURE: NAME/KEY: misc structure LOCATION:12..31 OTHER INFORMATION:/function= "sequence homologous to the cDNA corresponding to nucleotides 1326-1345 of SEQ ID NO: 3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TCATAGGATC CAATAATCAA CTAGCAATTC G INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc_signal LOCATION:6..11 OTHER INFORMATION:/function= "SalI restriction site" (ix) FEATURE: NAME/KEY: miscstructure LOCATION:12..31 OTHER INFORMATION:/function= "sequence homologous to to nucleotides 1-20 of SEQ ID NO:3" SEQUENCE DESCRIPTION: SEQ ID NO: 6: CATTAGTCGA CAAATACAGG CTCTATAAAG C 31 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:
NO
(iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc_signal LOCATION:6..11 OTHER INFORMATION:/function= "BamHI restriction site" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TCATAGGATC CTTAACAATA AAATTACTCT GC 32 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc_signal LOCATION:6..11 S* OTHER INFORMATION:/function= "SalI restriction site" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CATTAGTCGA CTTAAATTTG ATATTGATTG CG 32
C.
Claims (6)
1. A nucleic acid sequence comprising at least one phase resistance mechanism, said sequence consisting of: the DNA sequences having the nucleic acid series of [SEQ ID NO:1] or [SEQ ID NO:3]; the DNA sequences hybridizing with the above sequences; and the corresponding mRNA and cDNA sequences.
2. A DNA sequence comprising at least one phage resistance mechanism, selected from the sequences [SEQ ID NO:1] and [SEQ ID NO:3] or a sequence having at least 70% homology with said sequences [SEQ ID NO:1] and [SEQ ID NO:3].
3. A plasmid comprising at least one phage resistance mechanism and containing a nucleic acid sequence according to claim 1.
4. A plasmid comprising at least one phage resistance mechanism and containing a DNA sequence according to claim 2. 20
5. A phage resistant lactic acid bacterium which contains at least one plasmid according to claim 3 or claim 4. a
6. Use of the lactic acid bacteria according to claim 5 for conferring a phage resistance mechanism to strains of industrial interest. S7. A nucleic acid sequence according to claim 1, a plasmid according to claim 3 or a bacterium according to claim 5 substantially as hereinbefore described with reference to the Examples. Dated this 2 3 rd day of May 2000. SKW Biosystems By its Patent Attorneys /I Davies Collison Cave
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9509980 | 1995-08-22 | ||
FR9509980A FR2738015B1 (en) | 1995-08-22 | 1995-08-22 | DNA SEQUENCES AND PLASMIDS COMPRISING AT LEAST ONE PHASE RESISTANCE MECHANISM, BACTERIA CONTAINING THEM AND THEIR USE |
Publications (2)
Publication Number | Publication Date |
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AU6421796A AU6421796A (en) | 1997-02-27 |
AU722216B2 true AU722216B2 (en) | 2000-07-27 |
Family
ID=9482030
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU64217/96A Ceased AU722216B2 (en) | 1995-08-22 | 1996-08-22 | Nucleic acid sequences and plasmids comprising at least one phage resistance mechanism, bacteria containing them and their use |
Country Status (1)
Country | Link |
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AU (1) | AU722216B2 (en) |
-
1996
- 1996-08-22 AU AU64217/96A patent/AU722216B2/en not_active Ceased
Non-Patent Citations (1)
Title |
---|
J. BACTERIAL, 174(22), PP 7463-9 (1992) * |
Also Published As
Publication number | Publication date |
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AU6421796A (en) | 1997-02-27 |
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