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. 1990 Nov;172(11):6419–6426. doi: 10.1128/jb.172.11.6419-6426.1990

Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis.

C Hill 1, L A Miller 1, T R Klaenhammer 1
PMCID: PMC526828  PMID: 2121714

Abstract

A number of host-encoded phage resistance mechanisms have been described in lactococci. However, the phage genome has not been exploited as a source of additional resistance determinants. A 4.5-kb BamHI-HindIII fragment of phage nck202.50 (phi 50) was subcloned in streptococcus-Escherichia coli shuttle plasmid pSA3 and introduced into Lactococcus lactis NCK203 and MG1363 by protoplast transformation. This cloned phage fragment directed a bacteriophage resistance phenotype designated Per (phage-encoded resistance). Both phi 50 and a distantly related phage, nck202.48 (phi 48), formed small plaques on strain NCK213 at a slightly reduced efficiency of plaquing on the Per+ host. The per locus was further reduced to a 1.4-kb fragment through in vitro deletion analysis. The 1.4-kb fragment was sequenced, and the Per phenotype was found to be associated with a ca. 500-bp region rich in direct and inverted repeats. We present evidence that the Per region contains a phage origin of replication which, in trans, may interfere with phage replication by titration of DNA polymerase or other essential replication factors. It was demonstrated that the Per+ phenotype is not a result of reduced adsorption or action of a restriction and modification system. Per+ activity was not detected against six independent phages which were previously shown to be sensitive to the Hsp+ mechanism. The mutually exclusive resistance mechanisms could be combined to confer resistance to both types of phages (Hsp resistant and Per resistant) in a single host. This is the first description in lactococci of a phage resistance phenotype, other than superinfection immunity, originating from a lactococcal phage genome.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Gasson M. J. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. doi: 10.1128/jb.154.1.1-9.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. Inouye M. Antisense RNA: its functions and applications in gene regulation--a review. Gene. 1988 Dec 10;72(1-2):25–34. doi: 10.1016/0378-1119(88)90124-2. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Kondo J. K., McKay L. L. Transformation of Streptococcus lactis Protoplasts by Plasmid DNA. Appl Environ Microbiol. 1982 May;43(5):1213–1215. doi: 10.1128/aem.43.5.1213-1215.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Matsubara K. M. Interference in phage growth by a resident plasmid lambda dv. I. The mode of interference. Virology. 1972 Dec;50(3):713–726. doi: 10.1016/0042-6822(72)90425-4. [DOI] [PubMed] [Google Scholar]
  11. Matsubara K. Interference in bacteriophage growth by a resident plasmid lambda dv. II. Role of the promoter-operator. J Virol. 1974 Mar;13(3):603–607. doi: 10.1128/jvi.13.3.603-607.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]

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