Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

<p>Abstract</p> <p>Background</p> <p>Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in <it>Streptococcus pneumoniae</it>. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS) biosynthesis loci of numerous lactic acid bacteria genomes.</p> <p>Results</p> <p>The protein sequence deduced from the <it>wzb </it>gene in <it>Lactobacillus rhamnosus </it>ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP) superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from <it>Escherichia coli </it>cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on <it>p</it>-nitrophenyl-phosphate (<it>p</it>NPP), with an optimal activity in presence of bovine serum albumin (BSA 1%) at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on <it>p</it>-nitrophenyl-phosphate. However, one variant (C6S) showed close to 20% increase in phosphatase activity.</p> <p>Conclusion</p> <p>These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for <it>Streptococcus pneumoniae </it>as well as with the polysaccharide-related phosphatases of Gram negative bacteria.</p

Molecular diversity and high virulence of Legionella pneumophila strains isolated from biofilms developed within a warm spring of a thermal spa.

International audienceABSTRACT: BACKGROUND: Several cases of legionellosis have been diagnosed in the same French thermal spa in 1986, 1994 and 1997. L. pneumophila serogroup 1 (Lp1) strains have been isolated from several patients, but the source of contamination was not identified despite the presence of different Lp1 in water samples of the three natural springs feeding the spa at this period. RESULTS: Our strategy was to investigate L. pneumophila (Lp) strains from natural biofilms developed in a sulphur-rich warm spring of this contaminated site. Biofilm analysis revealed the presence of three Lp serogroups (Lp1, Lp10 and Lp12). Surprisingly, Lp10 and Lp12 were not reported in the previous described studies from water samples. Besides, the new seven Lp1 we isolated exhibit a high molecular diversity and have been differentiated in five classes according to their DNA genome patterns obtained by PFGE and mip sequences. It must be noted that these DNA patterns are original and unknown in databases. Interestingly, the 27 Lp environmental strains we isolated display a higher cytotoxicity and virulence towards the amoeba Acanthamoeba castellanii than those of known Lp1 epidemic strains. CONCLUSION: The characteristics of Legionella pneumophila Lp1 strains isolated from the warm spring are in agreement with their presence in biofilms and their probable long-term persistence in this ecosystem

The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Pneumonia associated with Iegionnaires's disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds

Danièle Atlan : hors de la famille, la précarité

Atlan Danièle. Danièle Atlan : hors de la famille, la précarité. In: Marie Pas Claire, n°10, 1997. Spécial "la famille" et les Assises. pp. 20-23

Spécificité du transport et de l'hydrolyse des peptides chez les bactéries lactiques (aspects nutritionnel et moléculaire)

Pour se développér dans le lait, les bactéries lactiques possèdent un système protéolytique comprenant une protéiase de surface PrtP, un transporteur d'oligopeptides Opp et des peptidases cytoplasmiques. Pour mieux comprendre le contôle de ce système par des peptides du milieu extracellualire, nous avons étudié la spécificité du transport des peptides chez les lactocoques. Cette spécificité est différente selon les souches et ne repose pas exclusivement sur le système Opp. Elle pourrait dépendre d'un second transporteur, Opt, dont la spécificité recouvre partiellement celle d'Opp et dont la biosynthèse est variable selon les souches. La source de carbone joue aussi un rôle sur l'expression de certains composants du système protéolytique. En effet, chez Lactobacillus bulgaricus, l'expression du géne pepQ est soumise à un mécanisme d'activation catabolique, dépendant d'une protéine CcpA-like (PepR1) et de la séquence cre située en amont du promoteur du géne pepQ.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Commission Pauvreté, précarité, exclusion

Atlan Danièle, Moruni , Sandra . Commission Pauvreté, précarité, exclusion. In: Marie Pas Claire, n°10, 1997. Spécial "la famille" et les Assises. p. 34

Analyse physiologique, génétique et moléculaire de lactocoques protéolytiques issus du lait cru de chamelle d'Algérie

Le lait de chamelle est un écosystème bactérien très peu exploré dont nous avons isolé 180 souches protéolytiques, capables d hydrolyser les caséines du lait. Cinq isolats avec une forte activité protéolytique et capables de fermenter le citrate ont été identifiés à Lactococcus lactis subsp. lactis biovar diacetylactis. Ces lactocoques possèdent au moins 3 plasmides (pPRT, pLAC, pCIT) qui portent respectivement les gènes codant pour la protéase de surface PrtP (prtP) responsable de l hydrolyse des caséines du lait, le métabolisme du lactose (lac) et du citrate (cit). Chez 3 isolats, au lieu de l opéron citQRP habituel des biovariants diacetylactis, le cluster citI-citMCDEFGRP a été acquis d autres bactéries lactiques (Leuconostoc). L originalité de l isolat 9B réside aussi dans sa capacité à se développer à 50C et à une spécificité d hydrolyse des caséines différente de celles des PrtPs de type PI et PIII des lactocoques laitiers actuellement connus. Cette spécificité nouvelle (type P IV) pourrait s expliquer par la présence de nombreuses substitutions parmi les 1775 acides aminés de la protease mature. Cette étude a mis en évidence la diversité génétique des lactocoques issus du lait de chamelle qui se traduit par de nouvelles propriétés intéressantes pour l industrie laitièreLittle is known about bacterial ecosystems of dromedary milk. Among 180 proteolytic strains, 5 isolates exhibiting a high proteolytic activity towards milk caseins and a capacity to ferment citrate were identified as Lactococcus lactis subsp. lactis biovar diacetylactis. Analyses revealed at least 3 plasmids (pPRT, pLAC, pCIT) respectively associated with genes coding for a cell-surface protease (prtP), lactose (lac) and citrate (cit) metabolism. Surprisingly, 3 proteolytic isolates exhibited the citI-citMCDEFGRP cluster from Leuconostoc instead of the expected lactococcal citQRP operon. Interestingly, the isolate 9B was also able to grow at 50C and was characterized by an original specificity towards casein compared to those of well-known dairy lactococal PrtPs of PI and PIII types. This new specificity (Type P IV) could be correlated to numerous substitutions revealed in the 1 775 amino acid sequence from PrtP of the strain 9B. This study pointed out the genetic diversity of lactococcal strains present in the dromedary milk which is associated with new properties interesting for the dairy industryLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Élèves en situation de polyhandicap: comportements spontanés en classe

International audienceDespite the law of February 11, 2005, which guarantees the right for schooling for all students with disabilities, only around 13% of young people with profound intellectual and multiple disabilities (PIMD) benefit from part-time schooling in France. The schooling of students with PIMD is particularly challenging because it involves reconsidering curriculum, pedagogy, and assessment. In this observational study, which is part of a larger longitudinal research project, we focused on behaviors students with PIMD spontaneously (i.e., without being explicitly invited to do so) produce in the classroom. The participants were ten students with PIMD, aged 8 to 15, who attended two one-hour classes per week during one school year; in the classroom there were a total of five students with PIMD and two teachers as well as some other professionals (e.g., educator), if necessary. Three randomly selected classes (i.e., one per trimester) were filmed during the school year and analyzed. The results showed that all the students produced spontaneous behaviors. Some of them were exclusively aimed at exploring the material they had at their disposal, but most students also produced spontaneous behaviors addressed to the teachers, the other professionals and/or the other students. The analyses further showed that the teachers didn’t always react to, or take into account the students’ spontaneous behaviors; most of the time, they didn’t seem to notice those behaviors at all