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Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism. doi: 10.1128/JB.00959-09

Guanosine penta- and tetraphosphate [(p)ppGpp] are two unusual nucleotides implied in the bacterial stringent response. In many pathogenic bacteria, mutants unable to synthesize these molecules lose their virulence. In Gram-positive bacteria such as Enterococcus faecalis, the synthesis and degradation of (p)ppGpp mainly depend on the activity of a bifunctional enzyme, encoded by the relA gene. By analysing [DELTA]relA and [DELTA]relQ (which encodes a protein harbouring a ppGpp synthetase activity) deletion mutants, we showed that RelA is by far the main system leading to (p)ppGpp production under our experimental conditions, and during the development of a stringent response induced by mupirocin. We also constructed a mutant ([DELTA]relAsp) in which a small part of the relA gene (about 0.7 kbp) encoding the carboxy-terrninal domain of the RelA protein was deleted. Both relA mutants were more resistant than the wild-type strain to 0.3% bile salts, 25% ethanol and acid (pH 2.3) challenges. Interestingly, the ArelAsp mutant grew better than the two other strains in the presence of 1 mM [H.sub.2][O.sub.2], but did not display increased tolerance when subjected to lethal doses of [H.sub.2][O.sub.2] (45 mM). By contrast, the [DELTA]relA mutant was highly sensitive to 45 mM [H.sub.2][O.sub.2] and displayed reduced growth in a medium containing 1 M NaCl. The two mutants also displayed contrasting virulence phenotypes towards larvae of the Greater Wax Moth infection model Galleria mellonella. Indeed, although the [DELTA]relA mutant did not display any phenotype, the [DELTA]relAsp mutant was more virulent than the wild-type strain. This virulent phenotype should stem from its increased ability to proliferate under oxidative environments. DOI 10.1099/mic.0.026146-0

The gene encoding the manganese-containing superoxide dismutase (MnSOD) of Enterococcus faecalis was characterized. It is transcribed monocistronically from an upstream promoter identified by rapid amplification of cDNA ends (RACE)-PCR. A sodA mutant was constructed and characterized. Growth of the mutant strain was not significantly different from that of its wild-type counterpart in standing and aerated cultures. However, the mutant was more sensitive towards menadione and hydroperoxide stresses. The response to [H.sub.2][O.sub.2] stress was analysed in more detail, and the mode of killing of this oxidant was different under anaerobic and aerobic conditions. Cultures grown and challenged under anaerobic conditions were highly sensitive to treatment with 35 mM [H.sub.2][O.sub.2]. They were largely protected by the iron chelator deferoxamine, which suggested that killing was mainly due to an enhanced Fenton reaction. In contrast, neither strain was protected by the iron chelators deferoxamine and diethylenetriaminepentaacteic acid when grown and challenged under aerobic conditions, which suggested that inactivation of the cells by [H.sub.2][O.sub.2] was due to another killing mode. The sodA mutant was more sensitive under these conditions, showing that MnSOD is also important for protecting the cells from damage under aerobic conditions. Finally, the MnSOD of Ent. faecalis may be considered to be a virulence factor, since survival of the corresponding mutant strain was highly affected inside mouse peritoneal macrophages.

PerR is one of the most important transcriptional regulators involved in the oxidative-stress response in Bacillus subtilis. Here, the homologous gene in Enterococcus faecalis, ranked among the leading causes of nosocomial infection, was characterized and analysed. Phenotype analysis showed that the perR mutant was significantly more resistant to [H.sub.2][O.sub.2] challenge (P< 0.05). Expression of eight genes with potential roles in the oxidative-stress response was determined in the wild-type and perR-mutant strains by real-time quantitative PCR. Surprisingly, low quantitative differences in the transcriptional activity of these genes in the mutant versus wild-type were observed. Likewise, this locus was not involved in survival within murine macrophages, but in the mouse peritonitis model, the perR mutant appeared less lethal than the JH2-2 wild-type strain. The combined results show that PerR affects E. faecalis virulence and that its implication in the transcriptional regulation in this bacterium deviates from the B. subtilis model.

Analysis of the genuine sequence of Enterococcus faecalis allowed the identification of two genes whose protein products showed 33 and 34% identity with those of sigV and yrhM of Bacillus subtilis, respectively. These genes, named sigV and rsiV, are predicted to encode members of the extracytoplasmic function subfamily of eubacterial RNA polymerase sigma and anti-sigma factors, respectively. This group of sigma factors has been shown to regulate gene expression in response to stress conditions, sigV and rsiV were shown to be under the control of the same promoter. The transcriptional start site was determined, and the 1.5-kb mRNA transcript was shown to be overexpressed under glucose and complete starvation, as well as under physicochemical treatments. Three mutants, affected in sigV, rsiV, and both genes, were constructed by double-crossover recombination within the genome of E. faecalis strain JH2-2. Compared with the wild type and the rsiV mutant, the sigV mutants were more susceptible to heat shock, acid, and ethanol treatments and displayed decreased survival during long-term starvation. A nisin-inducible sigV gene construction used in complementation assays restored the wild phenotype of the sigV mutants, confirming the involvement of SigV in the heat shock, ethanol, and acid stress responses. Northern blot analysis carried out with the three mutant strains revealed the inhibition of sigV expression by the related anti-sigma factor gene rsiV. In addition, putative candidates of the sigV regulon determined by computer search for the sigV promoter sequence were analyzed.

A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN ([[sigma].sup.54])-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No '-12, -24' promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by P~His-HPr stimulates its activity, while phosphorylation by P~EII[B.sup.Lev] inhibits it. Disruption of the EII[B.sup.Lev]-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P~EII[B.sup.Lev] and P~His-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P~His-HPr to EII[A.sup.Lev] are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.

Research reveals that gls24 gene encoding a general stress protein is induced at the onset of glucose starvation. Data further indicate that Gls24 alone or in combination with GlsB protein brings about morphological and stress tolerance changes in Enterococcus faecalis.

The enteric microorganism Enterococcus faecalis can survive in an oligotrophic microcosm even after prolonged periods. The ability of the faecalis to survive long periods of complete starvation is due to changes in morphology, development of stress resistance and protein synthesis. The E. faecalis changes its cell surface to irregular shapes, becomes more resistant to environmental changes and synthesize certain proteins to survive in a nutrient-starved environment.

Carbohydrate source depletion decreases growth rate of Lactococcus lactis subsp. lactis IL1403, but increases resistance to heat, ethanol, acid and oxidative and osmotic strains. This cross-protection develops during the transition growth phase and reaches a maximum during entry of cells into the stationary phase. Addition of chloramphenicol or rifamycin does not cancel tolerant cell development, but stimulates resistance in L. lactis subsp. lactis.