SummaryThe only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication fork helicase. In contrast, in Bacillus subtilis, in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus. This gene is located at 55Њ on the chromosome and belongs to a putative operon together with a ligase gene (lig ) and two unknown genes named pcrB and yerH. As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli. PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep ¹ phenotype on E. coli. Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.

SummaryThree 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.

blL67 is a broad-host-range prolate-headed phage that is active against Lactococcus cells. The complete phage genome sequence of 22 195 bp was established. Thirty-seven open reading frames (ORFs) organized in two clusters were identified. Functions were assigned to the putative products of six of the ORFs on the basis of comparison of the deduced amino acid sequences to known proteins, analysis of structural features of the proteins and search for conserved motifs. These were a DNA polymerase, a protein involved in recombination, a lysin, a terminase subunit, a structural protein and a holin.

SummaryStudies of cellular responses to stress conditions such as heat, oxygen or starvation have revealed the existence of numerous specific or interactive response pathways. We previously observed in Lactococcus lactis that inactivation of the recA gene renders the lactococcal strain sensitive not only to DNA-damaging agents but also to oxygen and heat. To further examine the stress response pathways in L. lactis, we isolated thermoresistant insertional mutants (Trm) of the recA strain. Eighteen independent trm mutations were identified and characterized. We found that mutations map in only seven genes, implicated in purine metabolism (deoB, guaA and tktA), phosphate uptake (pstB and pstS ), mRNA stability (pnpA) and in one uncharacterized gene (trmA). All the trm mutations, with the exception of trmA, confer multiple stress resistance to the cell. Some of the mutations confer improved heat stress resistance not only in the recA but also in the wild-type context. Our results reveal that cellular metabolic pathways are intimately related to stress response and that the flux of particular metabolites, notably guanine and phosphate, may be implicated in stress response in lactococci.

SummaryIt has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli. Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD ::Tn5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD-mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA, recF, recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD þ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.

The plasmid-encoded RepE protein is absolutely essential and rate-limiting for replication of the promiscuous plasmid pAM beta 1 originating from Enterococcus faecalis. We previously showed that the rep gene is transcribed from a promoter that is negatively regulated (approximately 10-fold reduction) by the CopF repressor. In this report, we show that this transcription is decreased a further approximately 10-times by a countertranscript-driven transcriptional attenuation system. Extensive mutagenesis revealed that this system operates by a mechanism similar to that previously described for the unrelated repC gene of plasmid pT181.

The dnaD gene of Bacillus subtilis was identified within a 104 kb DNA segment cloned into a yeast artificial chromosome. The nucleotide sequence of the wild type and dnaD23 mutant genes were determined. dnaD is predicted to encode a protein of 232 amino acids with no similarity to proteins in the data banks.

The 200 kb region of the Bacillus subtilis chromosome spanning from 255 to 275O on the genetic map was sequenced. The strategy applied, based on use of yeast artificial chromosomes and multiplex Long Accurate PCR, proved to be very efficient for sequencing a large bacterial chromosome area. A total of 193 genes of this part of the chromosome was classified by level of knowledge and biological category of their functions. Five levels of gene function understanding are defined. These are: (i) experimental evidence is available of gene product or biological function; (ii) strong homology exists for the putative gene product with proteins from other organisms; (iii) some indication of the function can be derived from homologies with known proteins; (iv) the gene product can be clustered with hypothetical proteins; (v) no indication on the gene function exists. The percentage of detected genes in each category was: 20,28,20,15 and 17, respectively. In the sequenced region, a high percentage of genes are implicated in transport and metabolic linking of glycolysis and the citric acid cycle. A functional connection of several genes from this region and the genes close to 140" in the chromosome was also observed.