ruvA Mutants that resolve Holliday junctions but do not reverse replication forks

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination

Multiple Pathways of Genome Plasticity Leading to Development of Antibiotic Resistance

The emergence of multi-resistant bacterial strains is a major source of concern and has been correlated with the widespread use of antibiotics. The origins of resistance are intensively studied and many mechanisms involved in resistance have been identified, such as exogenous gene acquisition by horizontal gene transfer (HGT), mutations in the targeted functions, and more recently, antibiotic tolerance through persistence. In this review, we focus on factors leading to integron rearrangements and gene capture facilitating antibiotic resistance acquisition, maintenance and spread. The role of stress responses, such as the SOS response, is discussed

Influence of very short patch mismatch repair on SOS inducing lesions after aminoglycoside treatment in Escherichia coli.

International audienceLow concentrations of aminoglycosides induce the SOS response in Vibrio cholerae but not in Escherichia coli. In order to determine whether a specific factor present in E. coli prevents this induction, we developed a genetic screen where only SOS inducing mutants are viable. We identified the vsr gene coding for the Vsr protein of the very short patch mismatch repair (VSPR) pathway. The effect of mismatch repair (MMR) mutants was also studied. We propose that lesions formed upon aminoglycoside treatment are preferentially repaired by VSPR without SOS induction in E. coli and by MMR when VSPR is impaired

Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance▿†

Antibiotic resistance development has been linked to the bacterial SOS stress response. In Escherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS in Vibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation with E. coli, all these antibiotics induce SOS in V. cholerae

SOS, the formidable strategy of bacteria against aggressions

International audienceThe presence of an abnormal amount of single-stranded DNA in the bacterial cell constitutes a genotoxic alarm signal that induces the SOS response, a broad regulatory network found in most bacterial species to address DNA damage. The aim of this review was to point out that beyond being a repair process, SOS induction leads to a very strong but transient response to genotoxic stress, during which bacteria can rearrange and mutate their genome, induce several phenotypic changes through differential regulation of genes, and sometimes acquire characteristics that potentiate bacterial survival and adaptation to changing environments. We review here the causes and consequences of SOS induction, but also how this response can be modulated under various circumstances and how it is connected to the network of other important stress responses. In the first section, we review articles describing the induction of the SOS response at the molecular level. The second section discusses consequences of this induction in terms of DNA repair, changes in the genome and gene expression, and sharing of genomic information, with their effects on the bacteria's life and evolution. The third section is about the fine tuning of this response to fit with the bacteria's 'needs'. Finally, we discuss recent findings linking the SOS response to other stress responses. Under these perspectives, SOS can be perceived as a powerful bacterial strategy against aggressions

Rôle de l hélicase RuvAB lors du retournement de fourche de réplication chez la bactérie Escherichia coli

Les fourches de réplication arrêtées peuvent être retournées lors de la réaction de retournement des fourches de réplication (RFR) chez la bactérie Escherichia coli. Cette réaction consiste en l appariement des brins nouvellement synthétisés de la fourche pour former une structure à quatre branches, la Jonction de Holliday (JH). RuvAB est une hélicase induite par la présence de lésions de l ADN. Elle se fixe spécifiquement sur les JH formées lors de la recombinaison homologue (RH) et permet leur résolution par la résolvase RuvC dans le complexe RuvABC. Nous avons montré que RuvAB retourne les fourches de réplication dans les mutants dnaEts, holD, et partiellement rep mais pas dans les mutants priA et dnaNts. Nous proposons donc que RuvAB possède deux fonctions distinctes : sa fonction dans la RH qui est bien décrite et un nouveau rôle dans la réplication. Une mutagénèse aléatoire dans ruvA nous a permis d isoler et caractériser des mutants de dissociation de fonction, où le RFR est totalement ou partiellement aboli, tandis que divers tests de recombinaison (recombinaison Hfr, réparation par RH des lésions après irradiation UV ou traitement à la mitomycine C) montrent que la RH n est pas affectée in vivo. Les mutants sont affectés pour la fixation à l'ADN, l octamérisation et la stimulation de l'activité hélicase de RuvB in vitro. Les protéines RuvA affectées sont donc incapables de convertir des fourches de réplication en jonctions de Holliday tandis qu elles restent capables de faire de la migration de branche et de résoudre les intermédiaires de recombinaison in vivo. Les données indiquent que les deux fonctions de RuvAB peuvent être séparées et que la réaction de RFR est plus exigeante que la RH.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation

International audienceConjugation is one mechanism for intra-and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria

Multiple Pathways of Genome Plasticity Leading to Development of Antibiotic Resistance

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Connecting Environment and Genome Plasticity in the Characterization of Transformation-Induced SOS Regulation and Carbon Catabolite Control of the Vibrio cholerae Integron Integrase

International audienceThe human pathogen Vibrio cholerae carries a chromosomal superintegron (SI). The SI contains an array of hundreds of gene cassettes organized in tandem which are stable under conditions when no particular stress is applied to bacteria (such as during laboratory growth). Rearrangements of these cassettes are catalyzed by the activity of the associated integron integrase. Understanding the regulation of integrase expression is pivotal to fully comprehending the role played by this genetic reservoir for bacterial adaptation and its connection with the development of antibiotic resistance. Our previous work established that the integrase is regulated by the bacterial SOS response and that it is induced during bacterial conjugation. Here, we show that transformation, another horizontal gene transfer (HGT) mechanism, also triggers integrase expression through SOS induction, underlining the importance of HGT in genome plasticity. Moreover, we report a new cyclic AMP (cAMP)-cAMP receptor protein (CRP)-dependent regulation mechanism of the integrase, highlighting the influence of the extracellular environment on chromosomal gene content. Altogether, our data suggest an interplay between different stress responses and regulatory pathways for the modulation of the recombinase expression, thus showing how the SI remodeling mechanism is merged into bacterial physiology