Alain 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.

Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis

Cloning vector plasmids have been constructed on the basis of the broad host range plasmid pAM beta 1 and used for the cloning of a nisin resistance determinant in Streptococcus lactis. They incorporate several desirable features for gene cloning in S. lactis and other transformable Gram-positive bacteria. They carry an easily selectable erythromycin resistance marker, are present at low (6-9) or high (45-85) copy number in S. lactis and possess a convenient polyrestriction site sequence. A significant advantage of these plasmids is their capability to carry and stably maintain very large cloned DNA fragments (up to 30 kilobases).

Molecular diversity and relationship within Lactococcus lactis, as revealed by randomly amplified polymorphic DNA (RAPD)

Lactococcus lactis strains are widely used in industrial dairy fermentations. Conventional phenotypic tests have been used for years to classify members of this species into two subspecies, lactis and cremoris, and play a key role in the choice of strains to be used in particular cheese fermentations. DNA hybridisation techniques have also been used for strain classification, giving rise to two genome homology groups. However, results showed discrepancies between the two methods of classification. We applied the randomly amplified polymorphic DNA fingerprinting (RAPD) technique to resolve previous contradictions in lactococcal classifications. Unlike usual RAPD methods, we use three primers to classify 113 strains and integrate the resulting information by a digitised programme used for this purpose. Our analysis revealed three major RAPD groups, designated G1, G2 and G3. G1 and G3 contain strains of the lactis subspecies, and G2 contains strains of the cremoris subspecies, as previously defined by phenotypic characteristics. Moreover, group G1 corresponds to one genome homology group, and groups G2 and G3 correspond to the second one. The taxonomic structure within L. lactis is therefore unusual: two distinct genetic groups of strains show indistinguishable phenotypes, while conversely, two phenotypically distinct groups are genetically homologous. We hypothesize that a subfamily of the subsp. lactis group gave rise to the cremoris subspecies.

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.

Analysis of the Bacillus subtilis genome sequence reveals nine new T-box leaders

Shiga toxin from Shigella dysenteriae type I and shiga-like toxins produced by several serotypes of Escherichia coli are involved in bacillary dysentery, haemorrhagic colitis and haemolytic uraemic syndrome (O’Brien et al., 1992, Current Topics Microbiol Immunol 180: 65–94), although their exact role in the pathogenesis is not fully understood. These cytotoxins are composed of an enzymatic A subunit linked to a multimeric receptor-binding B subunit. The enzymatic activity of both shiga and shiga-like toxins so far described is surprisingly similar to that identified for ricin and other plant ribosome-inactivating proteins (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237–282): adenine is removed from the major rRNA at a specific adenosine residue thus blocking ribosome function (Endo et al., 1988, Eur J Biochem 171: 45–50). For over a decade it has been common belief that ribosome-inactivating proteins could act only on rRNA within ribosomes. Recently, however, it has been shown that all ribosome-inactivating proteins from plants also depurinate other polyriboand polydeoxyribonucleotides besides rRNA in ribosomes (Barbieri et al., 1994, Nature 372, 624; Barbieri et al., 1997, Nucleic Acids Res 25, 518–522), and the denomination polynucleotide:adenosine glycosidases has been proposed. This prompted us to verify if shiga-like toxin I has polynucleotide:adenosine glycosidase activity. Here we report that indeed activated shiga-like toxin I extensively depurinates DNA from two different sources. The adenine release by activated shiga-like toxin I is reported in Table 1 and Fig. 1. The depurination rate observed with herring-sperm DNA as substrate is similar to the highest reported for plant ribosome-inactivating proteins (Barbieri et al., 1997, ibid ), and in particular is much higher than the rates observed for plant ribosomeinactivating proteins consisting of an A and a B subunit (ricin and related toxins). Activity was detected only with the activated toxin and not with the whole toxin, at odds with the observation made with the above-cited plant toxins, for which removal of the receptor-binding B chain has no effect on the depurination rate on DNA (Barbieri et al., 1997, ibid ). DNA from calf thymus was less sensitive than herring-sperm DNA to enzymatic depurination by shiga-like toxin I, possibly because of the different preparation procedures used, which may yield DNA with nonidentical physicochemical properties. The possibility that the activity observed is due to a contaminant and not to the shiga-like toxin itself is ruled out by (i) the high grade of purification of the toxin prepared by receptor–ligand chromatographic interaction, and (ii) the fact that a significant activity was observed only after activation of the toxin. Shiga-like toxin I also depurinated naked rRNA, tRNA and poly(A), albeit less efficiently. Molecular Microbiology (1998) 29(2), 661–669

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.

tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon.

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon–anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA‐mediated antitermination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.

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.

A Phage Protein Confers Resistance to the Lactococcal Abortive Infection Mechanism AbiP

Phage bIL66M1 is sensitive to the lactococcal abortive infection mechanism AbiP. No spontaneous AbiP-resistant variant could be obtained at a frequency of <10(-10). However, AbiP-resistant variants were readily obtained during infection with both bIL66M1 and the highly homologous AbiP-resistant phage bIL170. Gain of AbiP resistance was due to the acquisition of the e6 gene from bIL170.

Identity elements in trna-mediated transcription antitermination: implication of trna D- and T-arms in mrna recognition

tRNA-mediated transcription antitermination has been shown to control the expression of several amino acid biosynthesis operons and aminoacyl-tRNA-synthetase-encoding genes in Gram-positive bacteria. A model originally put forward by Grundy & Henkin describes the conserved structural features of the leader sequences of these operons and genes. Two sequences of 3 and 4 nt, respectively, take a central position in this model and are thought to be responsible for the binding of the system-specific uncharged tRNA, an interaction which would stabilize the antiterminator conformation of the leader. Here a further evolution of this model is presented based on an analysis of trp regulation in Lactococcus lactis in which a function is assigned to hitherto unexplained conserved structures in the leader sequence. It is postulated that the mRNA-tRNA interaction involves various parts of the tRNA in addition to the anticodon and the acceptor in the original model and that these additional interactions contribute to the recognition of a specific tRNA, and hence to the specificity and efficacy of the regulatory response.