Marie-Christine Chopin

Two plasmid-determined restriction and modification systems in Streptococcus lactis

Two restriction and modification systems were found in Streptococcus lactis strain IL594 which was found to contain 9 plasmids designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments, we showed that a restriction and modification system was related to the presence of pIL6 or pIL7. The pIL6-determined restriction and modification system was confirmed by cotransfer of the plasmid and of the restriction and modification system to a plasmid-free, nonrestricting, and nonmodifying derivative of S. lactis IL594.

Combinational variation of restriction modification specificities in Lactococcus lactis

Three genes coding for a type I R‐M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403. In addition, plasmids were found that encode only the HsdS subunit that directs R‐M specificity. The presence of these plasmids in IL1403 conferred a new R‐M phenotype on the host, indicating that the plasmid‐encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits. Such combinational variation of type I R‐M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.

Dramatic decay of phage transcripts in lactococcal cells carrying the abortive infection determinant AbiB

The abortive infection determinant AbiB prevents growth of the sensitive phage bIL170, but not of the resistant phage bIL41, on Lactococcus lactis strain IL1403. Here we show that AbiB promotes a dramatic degradation of sensitive phage transcripts, starting 10–15 min after infection. The decay of the transcripts is the probable cause of the arrest of the sensitive phage development. Mapping of the 5′ end of degradation products established that they result from endonucleolytic cleavage preferentially at U/U, A/U and U/A sites. We propose that an early product of the sensitive phage either induces the synthesis or stimulates the activity of an RNase in an AbiB+ cell.

Resistance of 17 mesophilic lactic Streptococcus bacteriophages to pasteurization and spray-drying.

For 17 phages active against Streptococcus cremoris, Str. lactis and Str. lactis subsp. diacetylactis, the killing efficiency of pasteurization (log No/N) at 72 degrees C for 15 s in skim-milk showed large variations from greater than 6 to 0; the efficienty of killing during spray-drying ranged from 3.7 to 0.2 and phages survived well storage of milk powder at room temperature. Destruction in a heat exchanger was found to be greater than that calculated from biphasic curves obtained by heating phages in sealed ampoules. No relationship was established between lytic classification of phages and their heat resistance.

Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC

The 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.

Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii

We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria.

CHARACTERIZATION OF IS1201, AN INSERTION SEQUENCE ISOLATED FROM LACTOBACILLUS HELVETICUS

IS1201, a 1387-bp insertion sequence isolated from Lactobacillus helveticus, was identified by its nucleotide (nt) sequence. It carries a single open reading frame encoding a 369-amino-acid protein, which shares homology with transposases found in a class of related IS, including ISRm3 from Rhizobium meliloti, IS256 from Staphylococcus aureus, IS6120 from Mycobacterium smegmatis, IS1081 from M. bovis, IST2 from Thiobacillus ferroxidans and IS406 from Pseudomonas cepacia. IS1201 has terminal inverted repeats of 24 bp in length and a target site duplication of 8 bp. Its copy number ranges from 3 to about 16 per L. helveticus genome. No homology was found between the nt sequence of IS1201 and those of the other bacterial IS from the same class. These results, together with previous observations [de los Reyes-Gavilán et al., Appl. Environ. Microbiol., 58 (1992) 3429-3432], confirm that IS1201 can be used as a specific DNA probe for the identification of L. helveticus strains.

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

We describe here a new lactococcal abortive phage infection system, designated AbiP. AbiP is effective against some lactococcal phages of one prevalent group, 936, but not against phages from the other two groups (c6A and P335). It was identified in the Lactococcus lactis subsp. cremoris strain IL420, on the native plasmid pIL2614. AbiP is encoded by a single gene, expressed in an operon with a second gene. In this work, abiP is shown to affect both the replication and transcription of phage DNA. In AbiP(+) cells, phage DNA replication is arrested approximately 10 min after infection. Levels of middle and late phage transcripts are lower in AbiP(+) than in AbiP(-) cells, probably due to the smaller amount of phage DNA. By contrast, early phage transcripts are more abundant in AbiP(+) than in AbiP(-) cells, suggesting that the switch-off, which occurs 15 min after infection in AbiP(-) cells, is prevented in AbiP(+) cells.

P087, a lactococcal phage with a morphogenesis module similar to an Enterococcus faecalis prophage.

The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.

Activation of mRNA translation by phage protein and low temperature: the case of Lactococcus lactis abortive infection system AbiD1

BackgroundAbortive infection (Abi) mechanisms comprise numerous strategies developed by bacteria to avoid being killed by bacteriophage (phage). Escherichia coli Abis are considered as mediators of programmed cell death, which is induced by infecting phage. Abis were also proposed to be stress response elements, but no environmental activation signals have yet been identified. Abis are widespread in Lactococcus lactis, but regulation of their expression remains an open question. We previously showed that development of AbiD1 abortive infection against phage bIL66 depends on orf1, which is expressed in mid-infection. However, molecular basis for this activation remains unclear.ResultsIn non-infected AbiD1+ cells, specific abiD1 mRNA is unstable and present in low amounts. It does not increase during abortive infection of sensitive phage. Protein synthesis directed by the abiD1 translation initiation region is also inefficient. The presence of the phage orf1 gene, but not its mutant AbiD1R allele, strongly increases abiD1 translation efficiency. Interestingly, cell growth at low temperature also activates translation of abiD1 mRNA and consequently the AbiD1 phenotype, and occurs independently of phage infection. There is no synergism between the two abiD1 inducers. Purified Orf1 protein binds mRNAs containing a secondary structure motif, identified within the translation initiation regions of abiD1, the mid-infection phage bIL66 M-operon, and the L. lactis osmC gene.ConclusionExpression of the abiD1 gene and consequently AbiD1 phenotype is specifically translationally activated by the phage Orf1 protein. The loss of ability to activate translation of abiD1 mRNA determines the molecular basis for phage resistance to AbiD1. We show for the first time that temperature downshift also activates abortive infection by activation of abiD1 mRNA translation.