DNA Sequence Analysis of Three Lactococcus lactis Plasmids Encoding Phage Resistance Mechanisms

  • I. Boucher
    Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Canada, G1K 7P4
    Search for articles by this author
  • Author Footnotes
    1 Current address: Chr. Hansen, Inc. 9015 West Maple Street, Milwaukee, WI 53214-4298.
    É. Émond
    1 Current address: Chr. Hansen, Inc. 9015 West Maple Street, Milwaukee, WI 53214-4298.
    Department of Food Science and Nutrition, Centre de Recherche en Science et Technologie du Lait (STELA), Faculté des Sciences de l’Agriculture et de l’Alimentation, Université Laval, Québec, Canada, G1K 7P4
    Search for articles by this author
  • M. Parrot
    Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Canada, G1K 7P4
    Search for articles by this author
  • S. Moineau
    Corresponding author.
    Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Canada, G1K 7P4
    Search for articles by this author
  • Author Footnotes
    1 Current address: Chr. Hansen, Inc. 9015 West Maple Street, Milwaukee, WI 53214-4298.
      This paper is only available as a PDF. To read, Please Download here.


      The three Lactococcus lactis plasmids pSRQ700, pSRQ800, and pSRQ900 encode the previously described anti-phage resistance mechanisms LlaDCHI, AbiK, and AbiQ, respectively. Since these plasmids are likely to be introduced into industrial Lactococcus lactis strains used to manufacture commercial fermented dairy products, their complete DNA sequences were determined and analyzed. The plasmids pSRQ700 (7784 bp), pSRQ800 (7858 bp), and pSRQ900 (10,836 bp) showed a similar genetic organization including a common lactococcal theta-type replicon. A second replication module showing features of the pMV158 family of rolling circle replicons was also found on pSRQ700. The theta replication regions of the three plasmids were associated with two additional coding regions, one of which encodes for HsdS, the specificity subunit of the type I restriction/modification system. When introduced into L. lactis IL1403, the HsdS of pSRQ800 and pSRQ900 conferred a weak resistance against phage P008 (936 species). These results indicated that both HsdS subunits can complement the chromosomally encoded type I restriction/modification system in IL1403. The genes involved in the phage resistance systems LlaDCHI, AbiK, and AbiQ were found in close proximity to and downstream of the replication modules. In pSRQ800 and pSRQ900, transfer origins and putative tyrosine recombinases were found upstream of the theta replicons. Genes encoding recombination proteins were also found on pSRQ700. Finally, open reading frames associated with bacteriocin production were found on pSRQ900, but no anti-lactococcal activity was detected. Based on our current knowledge, these three plasmids are safe and suitable for food-grade applications.

      Abbreviation key:

      EOP (efficiency of plaquing), IR (inverted repeats), IRR (right inverted repeat), IS (insertion sequence), ORF (open reading frame), RBS (ribosome binding site), RC (rolling circle), R/M (restriction/modification), SD (Shine-Dalgarno)


        • Abremski K.E.
        • Hoess R.H.
        Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein.
        J. Biol. Chem. 1984; 259: 1509-1514
        • Abremski K.E.
        • Hoess R.H.
        Evidence for a second conserved arginine residue in the integrase family of recombination proteins.
        Protein Eng. 1992; 5: 87-91
        • Allison G.E.
        • Fremaux C.
        • Klaenhammer T.R.
        Expansion of bacteriocin activity and host range upon complementation of two peptides encoded within the lactacin F operon.
        J. Bacteriol. 1994; 176: 2235-2241
        • Argos P.
        • Landy A.
        • Abremski K.
        • Egan J.B.
        • Haggard-Ljunquist E.
        • Hoess R.H.
        • Kahn M.L.
        • Kalionis B.
        • Narayana S.V.L.
        • Pierson III, L.S.
        • Sternberg N.
        • Leong J.M.
        The integrase family of site-specific recombinases: regional similarities and global diversity.
        EMBO J. 1986; 5: 433-440
        • Benachour A.
        • Frère J.
        • Flahaut S.
        • Novel G.
        • Auffray Y.
        Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus.
        Mol. Gen. Genet. 1997; 255: 504-513
        • Cluzel P.-J.
        • Chopin A.
        • Ehrlich S.D.
        • Chopin M.-C.
        Phage abortive infection mechanism from Lactococcus lactis subsp. lactis, expression of which is mediated by an Iso-ISS1 element.
        Appl. Environ. Microbiol. 1991; 57: 3547-3551
        • Cooper L.P.
        • Dryden D.T.F.
        The domains of a type I DNA methyltransferase: interactions and role in recognition of DNA methylation.
        J. Mol. Biol. 1994; 236: 1011-1021
        • Dao M.L.
        • Ferretti J.J.
        Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptococcal genes.
        Appl. Environ. Microbiol. 1985; 49: 115-119
        • Del Solar G.
        • Giraldo R.
        • Ruiz-Echevarría M.J.
        • Espinosa M.
        • Díaz-Orejas R.
        Replication and control of circular bacterial plasmids.
        Microbiol. Mol. Biol. Rev. 1998; 62: 434-464
        • Del Solar G.
        • Puyet A.
        • Espinosa M.
        Initiation signals for the conversion of single stranded to double strandedDNAforms in the streptococcal plasmid pLS1.
        Nucleic Acids Res. 1987; 15: 5561-5580
        • Devereux J.
        • Haeberli P.
        • Smithies O.
        A comprehensive set of sequence analysis programs for the VAX.
        Nucleic Acids Res. 1984; 12: 387-395
        • Diep D.B.
        • Havarstein L.S.
        • Nes I.F.
        Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11.
        J. Bacteriol. 1996; 178: 4472-4483
        • Duan K.
        • Chun-Quiang L.
        • Supple S.
        • Dunn N.W.
        Involvement of antisense RNA in replication control of the lactococcal plasmid pND324.
        FEMS. Microbiol. Lett. 1998; 164: 419-426
        • Durrell S.R.
        • Raghunathan G.
        • Guy H.R.
        Modeling the ion channel structure of cecropin.
        Biophys. J. 1992; 63: 1623-1631
        • Ehrlich S.D.
        • Bruand C.
        • Sozhamannan S.
        • Dabert P.
        • Gros M.-F.
        • Jannière L.
        • Gruss A.
        Plasmid replication and structural stability in Bacillus subtilis.
        Res. Microbiol. 1991; 142: 869-873
        • Emond E.
        • Dion E.
        • Walker S.A.
        • Vedamuthu E.R.
        • Kondo J.K.
        • Moineau S.
        AbiQ, an abortive infection mechanism from Lactococcus lactis.
        Appl. Environ. Microbiol. 1998; 64: 4748-4756
        • Emond E.
        • Holler B.J.
        • Boucher I.
        • Vandenbergh P.A.
        • Vedamuthu E.R.
        • Kondo J.K.
        • Moineau S.
        Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis.
        Appl. Environ. Microbiol. 1997; 63: 1274-1283
        • Esposito D.
        • Scocca J.J.
        The integrase family of tyrosine recombinases: evolution of a conserved active site domain.
        Nucleic Acids Res. 1997; 25: 3605-3614
        • Forde A.
        • Fitzgerald G.F.
        Bacteriophage defence systems in lactic acid bacteria.
        Antonie van Leeuvenhoek. 1999; 76: 89-113
        • Furuya N.
        • Komano T.
        Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence.
        J. Bacteriol. 1997; 179: 7291-7297
        • Garnier T.
        • Saurin W.
        • Cole S.T.
        Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for procaryotic site-specific recombinases.
        Mol. Microbiol. 1987; 1: 371-376
        • Gravesen A.
        • Josephsen J.
        • Von Wright A.
        • Vogensen F.K.
        Characterization of the replicon from the lactococcal thetareplicating plasmid pJW563.
        Plasmid. 1995; 34: 105-118
        • Gravesen A.
        • Von Wright A.
        • Josephsen J.
        • Vogensen F.K.
        Replication regions of two pairs of incompatible lactococcal thetareplicating plasmids.
        Plasmid. 1997; 38: 115-127
        • Higgins C.F.
        • Hyde S.C.
        • Mimmack M.M.
        • Gileadi U.
        • Gill D.R.
        • Gallagher M.P.
        Binding protein-dependent transport systems.
        J. Bioenerg. Biomembr. 1990; 22: 571-592
        • Jannière L.
        • Gruss A.
        • Ehrlich S.D.
        Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and other gram-positive bacteria. ASM Press, Washington, DC1993: 625-644
        • Kiewiet R.
        • Bron S.
        • Venema G.
        • Seegers J.F.M.L.
        Theta replication of the lactococcal plasmid pWV02.
        Mol. Microbiol. 1993; 10: 319-327
        • Kiewiet R.
        • Kok J.
        • Seegers J.F.M.L.
        • Venema G.
        • Bron S.
        The mode of replication is amajor factor in segregational plasmid instability in Lactococcus lactis.
        Appl. Environ. Microbiol. 1993; 59: 358-364
        • Klaenhammer T.R.
        Genetics of bacteriocins produced by lactic acid bacteria.
        FEMS. Microbiol. Rev. 1993; 12: 39-86
        • Lanka E.
        • Wilkins B.M.
        DNA processing in bacterial conjugation.
        Annu. Rev. Biochem. 1995; 64: 141-169
        • Lucey M.
        • Daly C.
        • Fitzgerald G.
        Identification and sequence analysis of the replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503.
        FEMS. Microbiol. Lett. 1993; 110: 249-256
        • Ludwig W.
        • Seewaldt E.
        • Klipper-Balz R.
        • Schleifer K.H.
        • Magrum L.
        • Woese C.R.
        • Fox G.E.
        • Steckebrandt E.
        The phylogenetic position of Streptococcus and Enterococcus.
        J. Gen. Microbiol. 1985; 131: 543-551
        • Mahillon J.
        • Chandler M.
        Insertion sequences.
        Microbiol. Mol. Biol. Rev. 1998; 62: 725-774
        • McKay L.L.
        Functional properties of plasmids in lactic streptococci.
        Antonie van Leeuwenhoek. 1983; 49: 259-274
        • Mills D.A.
        • Choi C.K.
        • Dunny G.M.
        • McKay L.L.
        Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer.
        Appl. Environ. Microbiol. 1994; 60: 4413-4420
        • Moineau S.
        Application of phage resistance in lactic acid bacteria.
        Antonie van Leeuvenhoek. 1999; 76: 377-382
        • Moineau S.
        • Walker S.A.
        • Vedamuthu E.R.
        • Vandenbergh P.A.
        Cloning and sequencing of LlaDCHI restriction and modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system.
        Appl. Environ. Microbiol. 1995; 61: 2193-2202
        • Nordstrom K.
        • Austin S.J.
        Mechanisms that contribute to the stable segregation of plasmids.
        Annu. Rev. Genet. 1989; 23: 37-70
        • Novick R.P.
        Plasmid incompatibility.
        Microbiol. Rev. 1987; 51: 381-395
        • O'Sullivan D.J.
        • Klaenhammer T.R.
        Rapid mini-prep isolation of high quality plasmid DNA from Lactococcus lactis and Lactobacillus spp.
        Appl. Environ. Microbiol. 1993; 59: 2730-2733
        • O'Sullivan D.
        • Twomey D.P.
        • Coffey A.
        • Hill C.
        • Fitzgerald G.F.
        • Ross R.P.
        Novel type IRestriction Specificities through Domain Shuffling of HSDS Subunits in Lactococcus Lactis.
        Mol. MicroBiol. 2000; 36: 866-875
        • Perreten V.
        • Schwarz F.
        • Cresta L.
        • Boeglin M.
        • Dasen G.
        • Teuber M.
        Antibiotic resistance spread in food.
        Nature. 1997; 389: 801-802
        • Polzin K.M.
        • Shimizu-Kadota M.
        Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.
        J. Bacteriol. 1987; 169: 5481-5488
        • Sadowski P.D.
        • Beatty L.G.
        • Clary D.
        • Ollerhead S.
        McMacken R. Kelly T. DNA Replication and Recombination. Alan R. Liss Inc., New York, NY1987: 691-701
        • Sambrook J.
        • Fritsh E.E.
        • Maniatis T.
        Molecular Cloning: A Laboratory Manual.
        2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989
        • Sanders M.E.
        • Klaenhammer T.R.
        Restriction/modification in group N streptococci: effect of heat on development of modified lytic bacteriophage.
        Appl. Environ. Microbiol. 1980; 40: 500-506
        • Schouler C.
        • Gautier M.
        • Ehrlich S.D.
        • Chopin M.-C.
        Combinational variation of restriction modification specificities in Lactococcus lactis.
        Mol. Microbiol. 1998; 28: 169-178
        • Seegers J.F.M.L.
        • Bron S.
        • Francke C.M.
        • Venema G.
        • Kiewiet R.
        Themajority of lactococcal plasmids carries a highly related replicon.
        Microbiology. 1994; 140: 1291-1300
        • Seegers J.F.M.
        • van Sinderen D.
        • Fitzgerald G.F.
        Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain.
        Microbiology. 2000; 146: 435-443
        • Swinfield T.J.
        • Jannière L.
        • Ehrlich S.D.
        • Minton N.P.
        Characterization of a region of the Enterococcus faecalis plasmid pAMβ1 which enhances the segregational stability of pAMβ1-derived cloning vectors in Bacillus subtilis.
        Plasmid. 1991; 26: 209-221
        • Terzaghi B.E.
        • Sandine W.E.
        Improved medium for lactic streptococci and their bacteriophages.
        Appl. Microbiol. 1975; 29: 807-813
        • van Kranenberg R.
        • de Vos W.M.
        Characterization of multiple regions involved in replication and mobilization of plasmid pNZ4000 coding for exopolysaccharide production in Lactococcus lactis.
        J. Bacteriol. 1998; 180: 5285-5290
        • Venema K.
        • Dost M.H.R.
        • Beun P.A.H.
        • Haandrikman A.J.
        • Venema G.
        • Kok J.
        The genes for secretion and maturation of lactococcins are located on the chromosome of Lactococcus lactis IL1403.
        Appl. Environ. Microbiol. 1996; 62: 1689-1692
        • von Wright A.
        • Sibakov M.
        Genetic modification of lactic acid bacteria.
        in: von Wright A. Salminen S. Lactic Acid Bacteria. Marcel Dekker, New York, NY1993: 161-198
        • von Wright A.
        • Wessels S.
        • Tynkkynen S.
        • Saarela M.
        Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant.
        Appl. Environ. Microbiol. 1990; 56: 2029-2035
        • Walker J.E.
        • Saraste M.
        • Runswick M.J.
        • Gay N.J.
        Distantly related sequences in the α-and β-subunits of ATPsynthetase, myosin, kinases and other ATP requiring enzymes and a common nucleotide binding fold.
        EMBO. J. 1982; 1: 945-951
        • Wells J.M.
        • Wilson P.W.
        • LePage R.W.F.
        Improved cloning vectors and transformation procedure for Lactococcus lactis.
        J. Appl. Bacteriol. 1993; 74: 629-636
        • Wilson G.W.
        Restriction and modification systems.
        Annu. Rev. Genet. 1993; 25: 585-627