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International audience; OBJECTIVES: To characterize the naturally occurring beta-lactamase gene identified from a clinical isolate belonging to a novel enterobacterial species that is closely related to Rahnella spp. and Ewingella spp. METHODS: Shotgun cloning and expression in Escherichia coli were performed in order to characterize this resistance determinant. Enzymatic activities were measured by UV spectrophotometry after an ion-exchange chromatography purification procedure. RESULTS: A chromosomal gene coding for the extended-spectrum beta-lactamase (ESBL) SMO-1 was identified from a novel enterobacterial species that is taxonomically related to Rahnella aquatilis and Ewingella americana. The beta-lactamase efficiently hydrolysed penicillins and cefotaxime, and shared 75% amino acid identity with the plasmid-mediated beta-lactamase SFO-1 from Serratia fonticola, 74% amino acid identity with the plasmid-mediated ESBL CTX-M-2 originating from Kluyvera spp. and 72% amino acid identity with the chromosomally encoded and intrinsic RAHN-1 from R. aquatilis. CONCLUSIONS: We have identified a novel enterobacterial species recovered from a clinical specimen, constituting another potential source of acquired ESBL. The ESBL shared significant similarities with the CTX-M-type enzymes.

International audience; OBJECTIVES: To describe a novel extended-spectrum oxacillinase, named OXA-145, differing from narrow-spectrum OXA-35 (from the OXA-10 group) by deletion of residue Leu-165. The genetic environment of bla(OXA-145) and the biochemical properties of OXA-145 are reported. We also assessed the impact of the Leu-165 deletion on the hydrolysis spectrum of the ancestor OXA-10. METHODS: Extended-spectrum β-lactamase OXA-145 was identified in the multidrug-resistant clinical Pseudomonas aeruginosa 08-056, and characterized by isoelectric focusing, PCR and DNA sequencing. Antibiotic susceptibility tests were performed by agar dilution. The resistance profiles conferred by cloned bla(OXA-10), bla(OXA-35), bla(OXA-145) and a bla(OXA-10) derivative obtained by site-directed mutagenesis were determined in Escherichia coli. Kinetic parameters of OXA-35 and OXA-145 were established after purification of His-tagged proteins. RESULTS: The sequence of OXA-145, encoded by a class 1 integron-borne gene in strain 08-056, differed from that of narrow-spectrum penicillinase OXA-35 by a single amino acid deletion (Leu-165) located in the highly conserved omega loop. Deletion of Leu-165 from OXA-35 (yielding OXA-145) or OXA-10 (the progenitor of OXA-35) extended the hydrolysis spectrum to third-generation cephalosporins and to monobactams, while reducing that for penicillins. OXA-145 showed biphasic hydrolysis curves for all the substrates tested. Its activity against nitrocefin was 10-fold higher in the presence of sodium hydrogen carbonate. CONCLUSIONS: OXA-145 is a new extended-spectrum β-lactamase from the OXA-10 group. The deletion of Leu-165 is responsible for a shift in the hydrolysis spectrum from penicillins to third-generation cephalosporins, as well as monobactams. The loss of penicillin hydrolysis was due to a non-carboxylated Lys-73.

International audience; BACKGROUND:Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma, shows intrapatient genetic variability. Although HTLV-1 can replicate via the reverse transcription of virion RNA to a double-stranded DNA provirus (the conventional manner for retroviruses), its predominant mode of replication is via the clonal expansion (mitosis) of the infected cell. This expansion is achieved by the viral oncoprotein Tax, which keeps the infected CD4 T lymphocyte cycling. Because Tax also interferes with cellular DNA repair pathways, we investigated whether somatic mutations of the provirus that occur during the division of infected cells could account for HTLV-1 genetic variability.METHODS:An inverse polymerase chain reaction strategy was designed to distinguish somatic mutations from reverse transcription-associated substitutions. This strategy allows the proviral sequences to be isolated together with flanking cellular sequences. Using this method, we sequenced 208 HTLV-1 provirus 3' segments, together with their integration sites, belonging to 29 distinct circulating cellular clones from infected individuals.RESULTS:For 60% of the clones, 8%-80% of infected cells harbored a mutated HTLV-1 provirus, without evidence of reverse transcription-associated mutations. Mutations within flanking cellular sequences were also identified at a frequency of 2.8 x 10(-4) substitution per base pair. Some of these clones carried multiple discrete substitutions or deletions, indicating progressive accumulation of mutations during clonal expansion. The overall frequency of somatic mutations increased with the degree of proliferation of infected T cells.CONCLUSIONS:These data indicate that, in vivo, HTLV-1 variation results mainly from postintegration events that consist of somatic mutations of the proviral sequence occurring during clonal expansion. The finding of substitutions in flanking sequences suggests that somatic mutations occurring after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which adult T-cell leukemia/lymphoma is the end point.

International audience; A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.

International audience; Vegetative incompatibility is widespread in fungi but its molecular mechanism and biological function are still poorly understood. A way to study vegetative incompatibility is to investigate the function of genes whose mutations suppress this phenomenon. In Podospora anserina, these genes are known as mod genes. In addition to suppressing vegetative incompatibility, mod mutations cause some developmental defects. This suggests that the molecular mechanisms of vegetative incompatibility and development pathway are interconnected. The mod-E1 mutation was isolated as a suppressor of the developmental defects of the mod-D2 strain. We show here that mod-E1 also partially suppresses vegetative incompatibility, strengthening the link between development and vegetative incompatibility. mod-E1 is the first suppressor of vegetative incompatibility characterized at the molecular level. It encodes a member of the Hsp90 family, suggesting that development and vegetative incompatibility use common steps of a signal transduction pathway. The involvement of mod-E in the sexual cycle has also been further investigated.

International audience; We have developed a novel strategy, based on the random insertion by homologous recombination of artificial I-Sce I sites within mammalian repetitive DNA sequences, which should greatly facilitate the high resolution physical mapping of large DNA fragments cloned in YAC. A set of transgenic yeast strains containing appropriately spaced I-Sce I sites within the YAC insert defines a series of nested physical intervals against which new genes, clones or DNA fragments can be mapped by simple hybridisation. Sequential hybridisation using such a series of nested YAC fragments as probes can also allow the rapid sorting of phage or cosmid libraries into contigs. This approach, which has been applied to a YAC containing a 460 kb insert from the mouse X chromosome, may also have applications for the restriction mapping of large genomic segments, mapping of exons and the search for homologous genes.

International audience

Bidirectional communication between cells and their surrounding environment is critical in both normal and pathological settings. Extracellular vesicles (EVs), which facilitate the horizontal transfer of molecules between cells, are recognized as an important constituent of cell-cell communication. In cancer, alterations in EV secretion contribute to the growth and metastasis of tumor cells. However, the mechanisms underlying these changes remain largely unknown. Here, we show that centrosome amplification is associated with and sufficient to promote small extracellular vesicle ( S EV) secretion in pancreatic cancer cells. This is a direct result due of lysosomal dysfunction, caused by increased reactive oxygen species (ROS) downstream of extra centrosomes. Defects in lysosome function promotes multivesicular body fusion with the plasma membrane, thereby enhancing S EV secretion. Furthermore, we find that S EVs secreted in response to amplified centrosomes are functionally distinct and activate pancreatic stellate cells (PSCs). These activated PSCs promote the invasion of pancreatic cancer cells in heterotypic 3-D cultures. We propose that S EVs secreted by cancer cells with amplified centrosomes influence the bidirectional communication between the tumor cells and the surrounding stroma to promote malignancy.