1,721,035 research outputs found
The pearling transition provides evidence of force-driven endosomal tubulation during Salmonella infection
No full textBacterial pathogens exploit eukaryotic pathways for their own end. Upon ingestion, Salmonella enterica serovar Typhimurium passes through the stomach and then catalyzes its uptake across the intestinal epithelium. It survives and replicates in an acidic vacuole through the action of virulence factors secreted by a type three secretion system located on Salmonella pathogenicity island 2 (SPI-2). Two secreted effectors, SifA and SseJ, are sufficient for endosomal tubule formation, which modifies the vacuole and enables Salmonella to replicate within it. Two-color, superresolution imaging of the secreted virulence factor SseJ and tubulin revealed that SseJ formed clusters of conserved size at regular, periodic intervals in the host cytoplasm. Analysis of SseJ clustering indicated the presence of a pearling effect, which is a force-driven, osmotically sensitive process. The pearling transition is an instability driven by membranes under tension; it is induced by hypotonic or hypertonic buffer exchange and leads to the formation of beadlike structures of similar size and regular spacing. Reducing the osmolality of the fixation conditions using glutaraldehyde enabled visualization of continuous and intact tubules. Correlation analysis revealed that SseJ was colocalized with the motor protein kinesin. Tubulation of the endoplasmic reticulum is driven by microtubule motors, and in the present work, we describe how Salmonella has coopted the microtubule motor kinesin to drive the force-dependent process of endosomal tubulation. Thus, endosomal tubule formation is a force-driven process catalyzed by Salmonella virulence factors secreted into the host cytoplasm during infection
High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms
Full text linkWe have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure
Study of molecular mechanisms of stress response in Oenococcus oeni and implementation of tools for the functional exploration of enological genes
No full textO. oeni est responsable de la fermentation malolactique des vins. Elle doit en permanence s’adapter aux fluctuations physico-chimiques de son environnement. La production de protéines Hsp constitue un mécanisme majeur d’adaptation de la bactérie à son environnement. Chez O. oeni, la protéine CtsR est l’unique régulateur identifié à ce jour des gènes hsp. Ce manuscrit aborde la caractérisation des mécanismes de régulation de la réponse au stress chez O. oeni. Une partie de ce travail a consisté à développer un nouvel outil d’expression de gènes chez O. oeni. Cet outil a permis l’étude de la fonction in vivo du gène hsp18 par une technique de modulation de l’expression de gènes par synthèse d’ARN antisens (ARNas). La production d’ARNas ciblant l’ARN messager du gène hsp18 entraîne une diminution du taux protéique de Lo18 et induit une perte de cultivabilité en conditions de stress. Ces résultats montrent, pour la 1ère fois in vivo, l’implication de Lo18 dans la thermotolérance et l’acidotolérance de O. oeni. Cette même approche appliquée au gène ctsR a induit une perte de cultivabilité en conditions de stress confirmant le rôle clef du locus ctsR dans la réponse au stress de O. oeni. Les mécanismes de régulation de l’activité de CtsR ont été appréhendés par complémentation d’un mutant ctsR déficient de B. subtilis via l’expression de ctsR de O. oeni. Des tests de thermoinduction mettent en évidence la thermosensibilité du CtsR de O. oeni dont l’activité est levée à une température inférieure à 33°C. Le pSIPSYN est un outil prometteur valorisé au cours de ce travail par une étude évaluant l’impact de deux estérases de O. oeni, EstA2 et EstA7 sur le profil aromatique du vin.O. oeni is responsible for wine malolactic fermentation. As any organism, O. oeni tries to adapt its physiology to environmental fluctuations by producing Hsp proteins encoded by the hsp genes. In O. oeni, CtsR is currently the only regulator of hsp genes. As an alternative to the lack of genetic tool, with the goal of understanding the mechanisms of O. oeni stress response, we developed a new expression vector, the pSIPSYN, to produce antisense RNA targeting of hsp18 mRNA. The synthesis of hsp18 asRNA leads to the decrease in the protein level of Lo18 and induced a loss of cultivability after heat or acid shock showing for the first time in vivo involvement of Lo18 in thermotolerance and acidotolerance in O. oeni. The O. oeni ability of the membrane fluidity restoration of after ethanol stress was strongly affected in the presence of asRNAof hsp18 gene. Then, the ctsR function in O. oeni was investigated with this new genetic tool. Inhibition of the ctsR expression by asRNA approach induced a loss of cultivability after heat or acid shock confirming the key role of ctsR locus in the O. oeni stress response. B. subtilis was used to characterize the regulation of CtsR activity. The ctsR gene of O. oeni was expressed to complement a B. subtilis ctsR-deficient strain and restore the wild-type phenotype. Thermoinduction tests performed to understand the thermosensibility of CtsR showed that O. oeni CtsR is a specific thermosensor inactivated at a temperature threshold below 33°C. The pSIPSYN is a promising tool valorized in this work through an oenological study by evaluating of the impact of O. oeni two esterases, and EstA2 EstA7 on wine ester profile
Nonselective bottlenecks control the divergence and diversification of phase-variable bacterial populations
Full text linkPhase variation occurs in many pathogenic and commensal bacteria and is a major generator of genetic variability. A putative advantage of phase variation is to counter reductions in variability imposed by nonselective bottlenecks during transmission. Genomes of Campylobacter jejuni, a widespread food-borne pathogen, contain multiple phase-variable loci whose rapid, stochastic variation is generated by hypermutable simple sequence repeat tracts. These loci can occupy a vast number of combinatorial expression states (phasotypes) enabling populations to rapidly access phenotypic diversity. The imposition of nonselective bottlenecks can perturb the relative frequencies of phasotypes, changing both within-population diversity and divergence from the initial population. Using both in vitro testing of C. jejuni populations and a simple stochastic simulation of phasotype change, we observed that single-cell bottlenecks produce output populations of low diversity but with bimodal patterns of either high or low divergence. Conversely, large bottlenecks allow divergence only by accumulation of diversity, while interpolation between these extremes is observed in intermediary bottlenecks. These patterns are sensitive to the genetic diversity of initial populations but stable over a range of mutation rates and number of loci. The qualitative similarities of experimental and in silico modeling indicate that the observed patterns are robust and applicable to other systems where localized hypermutation is a defining feature. We conclude that while phase variation will maintain bacterial population diversity in the face of intermediate bottlenecks, narrow transmission-associated bottlenecks could produce host-to-host variation in bacterial phenotypes and hence stochastic variation in colonization and disease outcomes
When the going gets tough: survival strategies and environmental signaling networks in Bacillus subtilis
No full textInternational audienceRegulatory pathways involving two-component histidine kinase/response regulator proteins of Bacillus subtilis are highly interconnected and form a signal transduction network controlling stationary-phase adaptive responses. These include chemotaxis and motility, degradative enzyme synthesis, antibiotic production, natural competence for DNA uptake, and sporulation. Many of these responses are mutually exclusive, with different control levels involving protein-environment, protein-protein and protein-DNA interactions, allowing the bacteria to adapt rapidly to environmental changes
Grasping at Shadows: Revealing the Elusive Nature of Essential Genes
No full textInternational audienceThe conundrum of studying a gene that is essential for bacterial viability poses a nontrivial genetic challenge that has been met with mixed success using multiple strategies. Most of the time, cursory analyses merely report the inability to inactivate a gene of interest, hence suggesting it is vital for the cell [...
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Caracterisation du couple modulateur/effecteur DegS/DegU controlant la synthese d'une classe d'enzymes degradatifs et l'expression des genes de competence chez Bacillus subtilis
No full textSIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 84824 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc
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