Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC

Elena Bidnenko,* S. Dusko Ehrlich and Marie-Christine ChopinINRA, Laboratoire de Ge´ne´tique Microbienne, 78352Jouy-en-Josas Cedex, France.SummaryThe function of the Lactococcus lactis bacterio-phagebIL66middletime-expressedoperon(M-operon),involvedin sensitivity totheabortiveinfection mechan-ism AbiD1, was examined. Expression of the M-operonis detrimental to Escherichia coli cells, induces theSOS response and is lethal to recA and recBC E.coli mutants, which are both deficient in recombina-tional repair of chromosomal double-stranded breaks(DSBs). The use of an inducible expression systemallowedustodemonstratethattheM-operon-encodedproteins generate a limited number of randomly dis-tributed chromosomal DSBs that are substrates forExoV-mediated DNA degradation. DSBs were alsoshown to occur upstream of the replication initiationpoint of unidirectionally theta-replicating plasmids.The characteristics of the DSBs lead us to proposethat the endonucleolytic activity of the M-operon isnot specific to DNA sequence, but rather to branchedDNA structures. Genetic and physical analysis per-formed with different derivatives of the M-operonindicated that two orfs(orf2 and orf3) are neededfor nucleolytic activity. The orf3 product has aminoacid homology with the E. coliRuvC Holliday junctionresolvase. By site-specific mutagenesis, we haveshown that one of the amino acid residues constitut-ing the active centre of RuvC enzyme (Glu-66) andconserved in ORF3 (Glu-67) is essential for thenucleolytic activity of the M-operon gene product(s).We therefore propose that orf2 and orf3 of theM-operon code for a structure-specific endonuclease(M-nuclease), which might be essential for phagemultiplication.IntroductionIn response to a phage threat, bacteria evolve differentdefence strategies, one of which is abortive phage infec-tion (Abi). Abi is characterized by a normal initial phaseof infection (i.e. phage adsorption and DNA injection), fol-lowed by an interruption of phage development, leading tothe release of few or no phage progeny particles, and tothe death of the infected cell (for a review, see Molineux,1991; Snyder, 1995). Lactococcus lactis species are indus-triallyimportantmicroorganismsthatareunderstrongselec-tive pressure from the bacteriophages present in the dairyenvironment. These organisms were found to possess vari-ousAbimechanisms,andthegenesinvolvedin13of themhave been cloned and sequenced (Hill, 1993; Daly et al.,1996; Prevots et al., 1996; Deng et al., 1997; Emond etal., 1997; Su et al., 1997; Y.-M. Deng and N. W. Dunn,unpublished). However, the primary effects on phage deve-lopment are presently known only for AbiA, AbiB and AbiD1(Hill et al., 1991; Anba et al., 1995; Parreira et al., 1996;Dinsmore and Klaenhammer, 1997). We have proposedpreviously that AbiD1 acts by decreasing the concentra-tion of an essential middle-expressed phage gene product(Bidnenko et al., 1995). The target for the AbiD1 mechan-ism was identified on the phage bIL66 genome, using theability of the phage to form spontaneous AbiD1-resistantmutants (Bidnenko et al., 1995). Phage bIL66 contains amiddle-expressed operon (M-operon) composed of foursmall open reading frames, designated orf1 to orf4 (42,43, 160 and 43 codons respectively). Mutations conferringresistance to the AbiD1 mechanism were mapped to orf1of the M-operon, indicating that this ORF is needed forsensitivity to AbiD1. Moreover, the sensitivity is partlysuppressed when the M-operon from phage AbiD1