Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC

Abstract

SummaryThe function of the Lactococcus lactis bacteriophage bIL66 middle time-expressed operon (M-operon), involved in sensitivity to the abortive infection mechanism AbiD1, was examined. Expression of the M-operon is detrimental to Escherichia coli cells, induces the SOS response and is lethal to recA and recBC E. coli mutants, which are both deficient in recombinational repair of chromosomal double-stranded breaks (DSBs). The use of an inducible expression system allowed us to demonstrate that the M-operon-encoded proteins generate a limited number of randomly distributed chromosomal DSBs that are substrates for ExoV-mediated DNA degradation. DSBs were also shown to occur upstream of the replication initiation point of unidirectionally theta-replicating plasmids. The characteristics of the DSBs lead us to propose that the endonucleolytic activity of the M-operon is not specific to DNA sequence, but rather to branched DNA structures. Genetic and physical analysis performed with different derivatives of the M-operon indicated that two orfs (orf2 and orf3 ) are needed for nucleolytic activity. The orf3 product has amino acid homology with the E. coli RuvC Holliday junction resolvase. By site-specific mutagenesis, we have shown that one of the amino acid residues constituting the active centre of RuvC enzyme (Glu-66) and conserved in ORF3 (Glu-67) is essential for the nucleolytic activity of the M-operon gene product(s). We therefore propose that orf2 and orf3 of the M-operon code for a structure-specific endonuclease (M-nuclease), which might be essential for phage multiplication.

Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are bIL170, AF009630; bIL120, AY054975; bIL15, AY054976; bIL191, AY054977; bIL77, AY054978.

“…The c2 group has the same features, whereas the P335 group A.-M. Crutz-Le Coq and others contains mainly temperate phages that are not necessarily closely related at the DNA sequence level (Chopin et al, 2001). In the 936 group, only a few genomic segments (essentially encoding the holin, lysin, major structural protein, structure-specific endonuclease and ori fragment) have been studied experimentally (Bidnenko et al, 1998 ;Chandry et al, 1997 ;Chung et al, 1991 ;Kim & Batt, 1991a). In the c2 and P335 groups, a number of structural proteins have been experimentally characterized as well as endolysins, origins of replication and transcriptional regulators, or the lysogeny module in the case of temperate phages (see Table 2 for selected references).…”

“…gp3 of phage bIL66 has been shown to express a structure-specific endonuclease activity (Bidnenko et al, 1998). Structure-specific endonucleases cleave branched DNA structures generated during replication or recombination.…”

“…They belong to different structural families and could have diverse physiological roles in replication, recombination, repair, maturation or packaging of phage DNA. gp3 of bIL66 is homologous to the E. coli RuvC Holliday-junction resolvase (Bidnenko et al, 1998) and has been shown to be involved in phage Sequence analysis of lactococcal phage bIL170 sensitivity to the AbiD1 abortive infection mechanism (Bidnenko et al, 1995). Its exact role in phage development is still unknown, but it is most probably essential for phage multiplication under certain conditions (Bidnenko et al, 1995).…”

The Lactococcal Abortive Phage Infection System AbiP Prevents both Phage DNA Replication and Temporal Transcription Switch

“…This is indeed the case for AbiD1. This mechanism, effective on all 936 and c6A phages tested (references 5 and 19, respectively; M. C. Chopin, unpublished data), interferes with a gene coding for an essential endonuclease homologous to RuvC (4,5), which is highly conserved in the two phage groups. Finally, AbiA (24) is the only lactococcal system that is effective on all three groups.…”

“…AbiT, which has no effect on either DNA replication or transcription, has been proposed to be activated by one of the late phage mRNAs or proteins and to rapidly cause premature cell death (7). AbiD1 has been proposed to decrease an essential RuvClike endonucleolytic activity (4,5), resolving branched DNA structures, and thus conceivably to be involved in phage DNA replication and/or packaging.…”