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. 1992 Nov;174(22):7463–7469. doi: 10.1128/jb.174.22.7463-7469.1992

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

E Durmaz 1, D L Higgins 1, T R Klaenhammer 1
PMCID: PMC207445  PMID: 1429469

Abstract

The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.

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Selected References

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  1. Alatossava T., Klaenhammer T. R. Molecular Characterization of Three Small Isometric-Headed Bacteriophages Which Vary in Their Sensitivity to the Lactococcal Phage Resistance Plasmid pTR2030. Appl Environ Microbiol. 1991 May;57(5):1346–1353. doi: 10.1128/aem.57.5.1346-1353.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson D. G., McKay L. L. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 1983 Sep;46(3):549–552. doi: 10.1128/aem.46.3.549-552.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behnke D., Malke H. Bacteriophage interference in Streptococcus pyogenes. I. Characterization of prophage--host systems interfering with the virulent phage A25. Virology. 1978 Mar;85(1):118–128. doi: 10.1016/0042-6822(78)90416-6. [DOI] [PubMed] [Google Scholar]
  4. 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 Dec;57(12):3547–3551. doi: 10.1128/aem.57.12.3547-3551.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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 Jan;49(1):115–119. doi: 10.1128/aem.49.1.115-119.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Froseth B. R., Harlander S. K., McKay L. L. Plasmid-mediated reduced phage sensitivity in Streptococcus lactis KR5. J Dairy Sci. 1988 Feb;71(2):275–284. doi: 10.3168/jds.S0022-0302(88)79555-7. [DOI] [PubMed] [Google Scholar]
  8. Gautier M., Chopin M. C. Plasmid-Determined Systems for Restriction and Modification Activity and Abortive Infection in Streptococcus cremoris. Appl Environ Microbiol. 1987 May;53(5):923–927. doi: 10.1128/aem.53.5.923-927.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Higgins D. L., Sanozky-Dawes R. B., Klaenhammer T. R. Restriction and modification activities from Streptococcus lactis ME2 are encoded by a self-transmissible plasmid, pTN20, that forms cointegrates during mobilization of lactose-fermenting ability. J Bacteriol. 1988 Aug;170(8):3435–3442. doi: 10.1128/jb.170.8.3435-3442.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hill C., Massey I. J., Klaenhammer T. R. Rapid method to characterize lactococcal bacteriophage genomes. Appl Environ Microbiol. 1991 Jan;57(1):283–288. doi: 10.1128/aem.57.1.283-288.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hill C., Miller L. A., Klaenhammer T. R. In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage. J Bacteriol. 1991 Jul;173(14):4363–4370. doi: 10.1128/jb.173.14.4363-4370.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hill C., Miller L. A., Klaenhammer T. R. Nucleotide sequence and distribution of the pTR2030 resistance determinant (hsp) which aborts bacteriophage infection in lactococci. Appl Environ Microbiol. 1990 Jul;56(7):2255–2258. doi: 10.1128/aem.56.7.2255-2258.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hill C., Pierce K., Klaenhammer T. R. The conjugative plasmid pTR2030 encodes two bacteriophage defense mechanisms in lactococci, restriction modification (R+/M+) and abortive infection (Hsp+). Appl Environ Microbiol. 1989 Sep;55(9):2416–2419. doi: 10.1128/aem.55.9.2416-2419.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hill C., Romero D. A., McKenney D. S., Finer K. R., Klaenhammer T. R. Localization, cloning, and expression of genetic determinants for bacteriophage resistance (Hsp) from the conjugative plasmid pTR2030. Appl Environ Microbiol. 1989 Jul;55(7):1684–1689. doi: 10.1128/aem.55.7.1684-1689.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jarvis A. W., Fitzgerald G. F., Mata M., Mercenier A., Neve H., Powell I. B., Ronda C., Saxelin M., Teuber M. Species and type phages of lactococcal bacteriophages. Intervirology. 1991;32(1):2–9. doi: 10.1159/000150179. [DOI] [PubMed] [Google Scholar]
  16. Jarvis A. W., Klaenhammer T. R. Bacteriophage Resistance Conferred on Lactic Streptococci by the Conjugative Plasmid pTR2030: Effects on Small Isometric-, Large Isometric-, and Prolate-Headed Phages. Appl Environ Microbiol. 1986 Jun;51(6):1272–1277. doi: 10.1128/aem.51.6.1272-1277.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klaenhammer T. R. Development of bacteriophage-resistant strains of lactic acid bacteria. Biochem Soc Trans. 1991 Aug;19(3):675–681. doi: 10.1042/bst0190675. [DOI] [PubMed] [Google Scholar]
  18. Klaenhammer T. R., Sanozky R. B. Conjugal transfer from Streptococcus lactis ME2 of plasmids encoding phage resistance, nisin resistance and lactose-fermenting ability: evidence for a high-frequency conjugative plasmid responsible for abortive infection of virulent bacteriophage. J Gen Microbiol. 1985 Jun;131(6):1531–1541. doi: 10.1099/00221287-131-6-1531. [DOI] [PubMed] [Google Scholar]
  19. Kondo J. K., McKay L. L. Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning. Appl Environ Microbiol. 1984 Aug;48(2):252–259. doi: 10.1128/aem.48.2.252-259.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krüger D. H., Bickle T. A. Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts. Microbiol Rev. 1983 Sep;47(3):345–360. doi: 10.1128/mr.47.3.345-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McKay L. L., Baldwin K. A., Walsh P. M. Conjugal transfer of genetic information in group N streptococci. Appl Environ Microbiol. 1980 Jul;40(1):84–89. doi: 10.1128/aem.40.1.84-91.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McKay L. L., Baldwin K. A., Zottola E. A. Loss of lactose metabolism in lactic streptococci. Appl Microbiol. 1972 Jun;23(6):1090–1096. doi: 10.1128/am.23.6.1090-1096.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McKay L. L., Bohanon M. J., Polzin K. M., Rule P. L., Baldwin K. A. Localization of Separate Genetic Loci for Reduced Sensitivity towards Small Isometric-Headed Bacteriophage sk1 and Prolate-Headed Bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2. Appl Environ Microbiol. 1989 Oct;55(10):2702–2709. doi: 10.1128/aem.55.10.2702-2709.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Powell I. B., Ward A. C., Hillier A. J., Davidson B. E. Simultaneous conjugal transfer in Lactococcus to genes involved in bacteriocin production and reduced susceptibility to bacteriophages. FEMS Microbiol Lett. 1990 Oct;60(1-2):209–213. doi: 10.1016/0378-1097(90)90373-x. [DOI] [PubMed] [Google Scholar]
  25. Sanders M. E., Klaenhammer T. R. Characterization of Phage-Sensitive Mutants from a Phage-Insensitive Strain of Streptococcus lactis: Evidence for a Plasmid Determinant that Prevents Phage Adsorption. Appl Environ Microbiol. 1983 Nov;46(5):1125–1133. doi: 10.1128/aem.46.5.1125-1133.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanders M. E., Klaenhammer T. R. Restriction and modification in group N streptococci: effect of heat on development of modified lytic bacteriophage. Appl Environ Microbiol. 1980 Sep;40(3):500–506. doi: 10.1128/aem.40.3.500-506.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sanders M. E. Phage resistance in lactic acid bacteria. Biochimie. 1988 Mar;70(3):411–422. doi: 10.1016/0300-9084(88)90215-5. [DOI] [PubMed] [Google Scholar]
  28. Steenson L. R., Klaenhammer T. R. Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages. Appl Environ Microbiol. 1985 Oct;50(4):851–858. doi: 10.1128/aem.50.4.851-858.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Terzaghi B. E., Sandine W. E. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975 Jun;29(6):807–813. doi: 10.1128/am.29.6.807-813.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tsao S. G., Brunk C. F., Pearlman R. E. Hybridization of nucleic acids directly in agarose gels. Anal Biochem. 1983 Jun;131(2):365–372. doi: 10.1016/0003-2697(83)90185-9. [DOI] [PubMed] [Google Scholar]
  31. de Bruijn F. J., Lupski J. R. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids--a review. Gene. 1984 Feb;27(2):131–149. doi: 10.1016/0378-1119(84)90135-5. [DOI] [PubMed] [Google Scholar]
  32. van de Guchte M., van der Vossen J. M., Kok J., Venema G. Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1989 Jan;55(1):224–228. doi: 10.1128/aem.55.1.224-228.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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