Biomedical subjects

Yanick Auffray

Publications and source records attributed to Yanick Auffray.

25 records · Page 2Linked to original sources

PubMed2002

Characterization of genes homologous to the general stress-inducible gene gls24 in Enterococcus faecalis and Lactococcus lactis.

Three genes (Ef0604, ymgG, and ytgH) were identified as homologous to the gls24 gene (encoding the general stress protein Gls24) in Enterococcus faecalis V583 and Lactococcus lactis IL1403 genomes sequences, and therefore are part of the 'gls24 family'. The other proteins encoded by the different surrounding genes in each of their respective operons are also highly similar. Our transcriptional analysis showed that Ef0604 and ymgG are not induced under the stress conditions tested. On the other hand, ytgH is induced at the onset of glucose starvation and by heat, ethanolic, osmotic, and CdCl(2) stresses. The transcriptional start site of this operon was identified and the promoter region appears to contain an 'extended -10 Box'. Moreover, the over-expression under several stress conditions of the YtgH protein observed on 2D gel electrophoresis confirms that it corresponds to a general stress protein in L. lactis.

Bacterial Proteins↗

PubMed2002

Isolation and characterization of bile salts-sensitive mutants of Enterococcus faecalis.

A library of insertional mutants of Enterococcus faecalis was constructed; it allowed the isolation and the characterization of 10 mutants affected in resistance to bile salts. Insertion loci of two mutants corresponded to genes of unknown function, while the amino acid sequences deduced from the other loci were homologous to proteins related to DNA repair, oxidative response, transcriptional regulation, dGTP hydrolysis, membrane composition, or cell wall synthesis. Further characterization of one mutant revealed that the insertion within the E. faecalis sagA gene led to a decrease of the resistance towards numerous independent physicochemical stresses, to modifications of the cell wall integrity, and to perturbations of cell division with septation anomalies.

Bacterial Proteins↗

PubMed2002

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.

Chromatography, Gel↗

PubMed2002

The Enterococcus faecalis gene encoding the novel general stress protein Gsp62.

The Enterococcus faecalis general stress protein Gsp62 was purified using two-dimensional gel electrophoresis and its 25 N-terminal amino acid sequence determined. Analysis of the corresponding gene revealed that the gsp62 product is a 172 aa protein. Transcriptional analysis of gsp62 gave evidence for a monocistronic mRNA, the synthesis of which was induced at the onset of stationary phase and in response to heat shock, acid pH, detergents (i.e. SDS or bile salts), ethanol, tert-butyl hydroperoxide, sodium chloride and, to a lesser extent, hydrogen peroxide. 5' rapid amplification of cDNA ends by PCR experiments showed that gsp62 transcription initiates 30 nt upstream of the ATG start codon. Although gsp62 expression was induced in response to various stresses, its disruption had no significant effect on the cell survival after each individual stress. Two-dimensional protein gels from wild-type and mutant cells revealed no pleiotropic effect of the mutation on protein synthesis. Transcriptional fusions with the lacL lacM beta-galactosidase genes showed that an inverted repeat located upstream of the promoter is required for transcriptional induction by environmental stresses but not by entrance into stationary phase. Two distinct mechanisms responding to different signals are thus involved in gsp62 induction.

Amino Acid Sequence↗

PubMed2002

Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production.

Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 microg ml(-1) for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.

Amino Acid Sequence↗

PubMed2002

H(2)O(2), which causes macrophage-related stress, triggers induction of expression of virulence-associated plasmid determinants in Rhodococcus equi.

The response of the intracellular pathogen Rhodococcus equi to H(2)O(2) treatment, a situation potentially encountered after the oxidative burst of alveolar macrophages, was analyzed. Compared to other bacteria, including Deinococcus radiodurans, R. equi showed exceptionally high resistance to this stress. A proteomic approach showed that four polypeptides present in the wild-type strain (85F) are missing in the plasmid-cured strain 85F(P-), and by using a DNA macroarray, we identified two plasmid-encoded vap genes, vapA and vapG, whose expression was highly induced by H(2)O(2) treatment. Whereas the transcript size of vapA was compatible with a monocistronic mRNA, the transcript of vapG was considerably longer. Rapid amplification of cDNA ends PCRs showed that the transcriptional start sites of the two operons were 69 and 269 nucleotides (nt) upstream of the start codon, respectively. Analysis of these leader sequences revealed the presence of a small open reading frame named podG, which encodes a sequence of 55 amino acids preceded by a putative ribosome binding site sequence in the vapG transcript. Taking this result into account, the untranslated leader of the podG/vapG operon is 87 nt. Alignment of this sequence with the leader sequences of vapA and vapD, genes previously shown to be induced by acid, revealed significant homologies. Since our results showed that vapA, vapD, and vapG are genes highly induced by macrophage-related stresses, their gene products may, within the Vap protein family, play a dominant role inside these phagocytic cells and may be the most promising candidates for vaccination strategies.

5' Untranslated Regions↗

PubMed1999

The osmoprotectant glycine betaine inhibits salt-induced cross-tolerance towards lethal treatment in Enterococcus faecalis.

The response of Enterococcus faecalis ATCC 19433 to salt stress has been characterized previously in complex media. In this report, it has been demonstrated that this bacterium actively accumulates the osmoprotectant glycine betaine (GB) from salt-enriched complex medium BHI. To further understand the specific effects of GB and other osmoprotective compounds in salt adaptation and salt-induced cross-tolerance to lethal challenges, a chemically defined medium lacking putative osmoprotectants was used. In this medium, bacterial growth was significantly reduced by increasing concentrations of NaCl. At 0.75 M NaCl, 90% inhibition of the growth rate was observed; GB and its structural analogues restored growth to the non-salt-stressed level. In contrast, proline, pipecolate and ectoine did not allow growth recovery of stressed cells. Kinetic studies showed that the uptake of betaines shows strong structural specificity and occurs through a salt-stress-inducible high-affinity porter [Km = 3.3 microM; Vmax = 130 nmol min(-1) (mg protein)(-1); the uptake activity increased 400-fold in the presence of 0.5 M NaCl]. Moreover, GB and its analogues were accumulated as non-metabolizable cytosolic osmolytes and reached intracellular levels ranging from 1-3 to 1.5 micromol (mg protein)(-1). In contrast to the beneficial effect of GB on the growth of salt-stressed cultures of E. faecalis, its accumulation inhibits the salt-induced cross-tolerance to a heterologous lethal challenge. Indeed, pretreatment of bacterial cells with 0.5 M NaCl induced resistance to 0.3% bile salts (survival of adapted cells increased by a factor of 6800). The presence of GB in the adaptation medium reduced the acquisition of bile salts resistance 680-fold. The synthesis of 11 of the 13 proteins induced during salt adaptation was significantly reduced in the presence of GB. These results raise questions about the actual beneficial effect of GB in natural environments where bacteria are often subjected to various stresses.

Adaptation, Physiological↗