Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1

Abstract

Phage bIL66 is unable to grow on Lactococcus lactis cells harboring the abortive infection gene abiD1. Spontaneous phage mutants able to grow on AbiD1 cells were used to study phage-Abi interaction. A 1.33-kb DNA segment of a mutant phage allowed growth of AbiD1 s phages in AbiD1 cells when present in trans. Sequence analysis of this segment revealed an operon composed of four open reading frames, designated orf1 to orf4. The operon is transcribed 10 min after infection from a promoter presenting an extended ؊10 consensus sequence but no ؊35 sequence. Analysis of four independent AbiD1 r mutants revealed a different point mutation localized in orf1, implying that this open reading frame is needed for sensitivity to AbiD1. However, the sensitivity is partly suppressed when orf3 is expressed in trans on a high-copy-number plasmid, suggesting that AbiD1 acts by decreasing the concentration of an available orf3 product.The growth of a lytic bacteriophage can be arrested by several bacterial phage defense mechanisms (8). One of them is characterized by a normal start of phage development followed by an abrupt interruption, resulting in the release of few or no progeny particles and cell death. Such abortive infections (Abi) were first described for Escherichia coli and Bacillus subtilis and have been identified more recently in Lactococcus lactis. The molecular basis of many of these phage exclusion systems remains unclear. Understanding progressed recently for three of them (28). In every case, phage infection induced deleterious effects for highly conserved cellular components involved either in the translation apparatus or in membrane potential. This results in cell death and phage growth arrest, which blocks the spread of phages to other cells.Four lactococcal genes coding for an Abi phenotype have been cloned and sequenced (2,6,7,9,15). Except for AbiA (16, 23), which has been proposed to interfere with phage DNA replication, the molecular mechanisms of lactococcal phage growth impairment in Abi cells are unknown. AbiD1 is active on two lactococcal phage species, referred to as the 936 and C6A phage groups, respectively (18). The study of phage multiplication in AbiD1 cells could therefore provide insight into both the molecular basis of phage-Abi interactions and the relationship between the two main lactococcal phage species.The spontaneous appearance of mutants of the small-isometric-headed phage bIL66, able to grow on AbiD1 cells, has been observed (11). We report that a 1.33-kb segment from a mutant phage allows the development of sensitive phages when present in AbiD1 cells.Sequence analysis of this segment revealed four putative open reading frames (ORFs) which form an operon. The operon is transcribed 10 min after infection. Two of these ORFs play a role in phage-Abi interaction. Point mutations in orf1 confer AbiD1 r , but the orf3 product is required for the growth of sensitive phages. We propose that the orf1 and abiD1 gene products interact to prevent translation of the orf3 RNA. The amount of orf3 p...

Cloning of a gene encoding nisin resistance from Lactococcus lactis subsp. Iactis M189 which is transcribed from an extended -10 promoter.

“…Recently, a sequence (TtTGt-TATAAT) with no -35 site was identified by primer ex- Liu,HARVEY,and DUNK Vol. 43 tension within the phage bIL66 DNA in L. lactis (Bidnenko et al, 1995). To our knowledge, this study represents the first instance in L. lactis demonstrating the requirement of an extended motif for its promoter function in the absence of a -35 site.…”

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.

“…Lactococcal phages used in this study (bIL170,bIL15,bIL77,bIL120,bIL191) are from our collection and belong to the 936 group as determined by DNA hybridization (B. Cesselin, unpublished results). They were propagated on appropriate Lactococcus lactis strains (IL1403 for bIL170) at 30 mC in M17 broth supplemented with 5 g glucose l − " and 10 mM CaCl # (Bidnenko et al, 1995). Escherichia coli XL1-Blue MRFh (Stratagene), transformed with pBluescript derivatives, was cultivated in Luria-Bertani broth (Maniatis et al, 1982) with 100 µg ampicillin ml − ".…”

“…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).…”

Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain The GenBank accession numbers for the sequences reported in this paper are AF153410–AF153414.

“…The plasmid was subcloned by ligation of a partial Sau3A digest with a BamHI digest of pBluescript SK + . The ligation mixture was used to transform E. coli XL-1 Blue and Lytic phage for IL1403 Bidnenko et al (1995) transformants were selected on plates containing X-Gal. Plasmid DNA from individual white colonies was isolated, checked for the presence of inserts and used for sequence analysis that was performed with an Applied Biosystems 373A automated DNA sequencer.…”