Lactococcus lactis Lytic Bacteriophages of the P335 Group Are Inhibited by Overexpression of a Truncated CI Repressor

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Abstract
Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage φ31 encodes a genetic switch region of c I and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative c I repressor gene of phage φ31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length c I gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the c I gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage φ31 was inhibited to an efficiency of plaquing (EOP) of 10 −6 in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the φ31 EOP to −7 . Phage φ31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the φ31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.