Analysis of three phage resistance mechanisms and a recA homologue encoded by the lactococal plasmid pNP40

Abstract

Phage represent a major problem in dairy fermentations Investigation of the phage resistance mechanisms employed by naturally insensitive lactococcal strains should aid m the development of rational strategies to help combat the problem. The lactococcal plasmid pNP40 from Lactococcus lactis ssp lactis biovar diacetylactis confers complete resistance to the prolate-headed 0c2 and the small isometnc-headed 0712 in L lactis ssp lactis MG1614, and has been used successfully to construct commercially valuable starter strains. In this study, the determinants for two independent abortive infection phage resistance systems (AbiE and AbiF) were cloned AbiF was shown to act at the level of phage DNA replication while AbiE operates post-replication, possibly at the level of transcription/translation or phage packaging/release pNP40 was also found to mediate resistance to 0c2 by a novel earlyacting phage resistance mechanism which we propose prevents phage DNA penetration into the host. Sequence analysis of the detemmants for AbiE and AbiF demonstrated that two overlapping ORF's, of 861 bp and 894 bp respectively, are required for expression of AbiE while a single 1026 bp ORF encodes AbiF Two ORF's are located between the abiE and abiF determinants, one of which codes for a RecA homologue. This represents, to our knowledge, the first recA gene located on a plasmid. Using a RecA deficient lactococcal strain, it was established that while the chromosomally-encoded RecA is required for full phenotypic expression of AbiF, the pNP40-encoded RecA has no discernible role in phage resistance. Thus, pNP40-directed phage insensitivity is mediated by three independent phage resistance systems AbiE, AbiF and a novel phage DNA penetration blocking mechanism