Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis.
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.
Hill
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Department of Food Science, Southeast Center for Dairy Foods Research, North Carolina State University, Raleigh 27695-7624.
Miller
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LA
Klaenhammer
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TR
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D007700:Kinetics; D013294:Lactococcus lactis / Q000235:genetics*; D008242:Lysogeny; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D015183:Restriction Mapping; D013816:Thermodynamics; D014169:Transformation, Bacterial; D014779:Virus Replication