Etude du système de résolution des dimères de chromosome chez Vibrio cholerae - Implication dans le contrôle de la lysogénie du phage CTXφ codant pour la toxine cholérique

The majority of the bacteria have a single circular chromosome. At the time of replication, chromosome dimers can be formed by homologous recombination between sister chromatides. Dimerisation of replicating chromosomes prevents the faithful segregation of genetic information between the two daughter cells. To correct this, the tyrosine recombinases, XerC and XerD, resolve dimers by adding an additional crossover at a specific site on the chromosome called dif. In Escherichia coli, the resolution of chromosome dimers is coordinated with cellular division by a septal protein, FtsK. FtsK pumps the DNA of dimers through the division septum until encountering dif, thereby aligning the two sites that the dimer carries. FtsK then activates XerC/D recombination to resolve the dimer into two monomeric chromosomes that can be segregated prior to division.Vibrio cholerae has two circular chromosomes, each one carrying a unique dif site, dif1 for chromosome I and dif2 for chromosome II. The system of resolution of chromosome dimers must therefore manage a higher degree of complexity to ensure the segregation of two chromosomes. Of additional interest, V. cholerae is the agent responsible for the cholera. The choleric toxin, which causes the potentially deadly diarrheas of cholera, is coded by the temperate phage CTXφ. CTXφ is integrated into the genome of its host dif site by hijacking the activity of XerC and XerD. During my thesis, I was interested in the study of the system of resolution of chromosome dimers in V. cholerae. My goal was to not only understand its role in the normal cellular cycle of this multi-chromosomal bacterium, but also to take account of its contribution to the integration of the phage CTXφ.Initially, we sought to understand how the phage CTXφ diverts the recombinases XerC/D to integrate itself in the genome of its host. Our work showed that the single-stranded genome of the phage contains a locus that is able to form a secondary structure which reconstitutes a viable dif site. This site is acted upon by the recombinases XerC/D, recombining it with the bacterial dif site. Through this study, we discovered a novel mode of horizontal transfer of DNA.Secondly, we showed that the resolution of dimers of the two chromosomes of V. cholerae follows the same catalytic pathway, which is controlled by the septal protein FtsK. This is the first example of a common system of maintenance of multiple chromosomes in bacteria. Our results also suggest a synchronization of chromosome dimer resolution in the two replicons and the cellular cycle of the bacterium.Lastly, in E. coli, the role of DNA translocation by FtsK in the segregation of the chromosomes is apparently limited only to the requirement to control the resolution of chromosomes dimers. In V. cholerae, on the contrary, my work shows a more general role of the function of translocation of FtsK in the segregation of the chromosomes.La plupart des bactéries ont un unique chromosome circulaire. Lors de la réplication, des dimères de chromosome peuvent se former par crossover entre chromatides sœurs. La dimérisation des chromosomes empêche la ségrégation de l’information génétique entre les deux cellules filles. Pour prévenir cela, les recombinases à tyrosine, XerC et XerD, résolvent les dimères de chromosomes en ajoutant un crossover au niveau d’un site spécifique du chromosome, appelé dif. Chez Escherichia coli, la résolution des dimères de chromosomes est coordonnée à la division cellulaire par une protéine septale, FtsK. FtsK pompe l’ADN chromosomique du dimère au travers du septum de division jusqu’à mise en contact des deux sites dif que le dimère porte, puis FtsK active la recombinaison XerC/D pour résoudre le dimère en deux chromosomes monomériques ségrégeables.Vibrio cholerae possède deux chromosomes circulaires portant chacun un site dif différent, dif1 pour le chromosome I et dif2 pour le chromosome II. Le système de résolution des dimères de chromosomes doit donc faire face dans cet organisme à un degré de complexité plus élevé pour assurer la ségrégation de deux chromosomes. D’autre part, V. cholerae est l’agent responsable du cholera. La toxine cholérique, qui provoque les diarrhées mortelles du choléra, est codée par le phage tempéré CTXφ. CTXφ s’intègre dans le génome de son hôte au niveau du site dif, en détournant l’action des deux recombinases XerC et XerD de leur fonction de maintenance. Au cours de ma thèse, je me suis intéressée à l’étude du système de résolution des dimères de chromosomes chez V. cholerae à la fois pour comprendre son rôle dans le cycle cellulaire normale de cette bactérie "bi-chromosomique", mais aussi pour comprendre sa contribution à l’intégration du phage CTXφ et donc à l’émergence de nouveaux variants pathogènes.Dans un premier temps, nous avons cherché à savoir comment le phage CTXφ détourne les recombinases XerC/D pour s’intégrer dans le génome de son hôte. Nos travaux montrent que le génome simple brin du phage possède une région capable de former une structure secondaire reconstituant un site dif, qui est pris en charge par les recombinases XerC/D pour être recombinée avec le site dif bactérien. A travers cette étude, nous avons découvert un nouveau mode de transfert horizontal d’ADN.Dans un second temps, nous avons montré que la résolution des dimères des deux chromosomes de V. cholerae suit une même voie recombinationnelle, qui est contrôlée par la protéine septale FtsK. Ceci est le premier exemple d’un système commun de maintenance de multiples chromosomes chez les bactéries. Cela suggère aussi une synchronisation de la résolution des dimères des deux chromosomes au cycle cellulaire de la bactérie.Enfin, chez E. coli, le rôle de translocation de FtsK dans la ségrégation des chromosomes est apparemment limité au seul besoin de contrôler la résolution des dimères de chromosomes. Au contraire, mes travaux montrent un rôle plus général de la fonction de translocation de FtsK dans la ségrégation des chromosomes chez V. cholerae

Replication termination without a replication fork trap

International audienceBacterial chromosomes harbour a unique origin of bidirectional replication, oriC. They are almost always circular, with replication terminating in a region diametrically opposite to oriC, the terminus. The oriC-terminus organisation is reflected by the orientation of the genes and by the disposition of DNA-binding protein motifs implicated in the coordination of chromosome replication and segregation with cell division. Correspondingly, the E. coli and B. subtilis model bacteria possess a replication fork trap system, Tus/ter and RTP/ter, respectively, which enforces replication termination in the terminus region. Here, we show that tus and rtp are restricted to four clades of bacteria, suggesting that tus was recently domesticated from a plasmid gene. We further demonstrate that there is no replication fork system in Vibrio cholerae, a bacterium closely related to E. coli. Marker frequency analysis showed that replication forks originating from ectopic origins were not blocked in the terminus region of either of the two V. cholerae chromosomes, but progressed normally until they encountered an opposite fork. As expected, termination synchrony of the two chromosomes is disrupted by these ectopic origins. Finally, we show that premature completion of the primary chromosome replication did not modify the choreography of segregation of its terminus region

A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae

International audienceBacteria with multiple chromosomes represent up to 10% of all bacterial species. Unlike eukaryotes, these bacteria use chromosome-specific initiators for their replication. In all cases investigated, the machineries for secondary chromosome replication initiation are of plasmid origin. One of the important differences between plasmids and chromosomes is that the latter replicate during a defined period of the cell cycle, ensuring a single round of replication per cell. Vibrio cholerae carries two circular chromosomes, Chr1 and Chr2, which are replicated in a well-orchestrated manner with the cell cycle and coordinated in such a way that replication termination occurs at the same time. However, the mechanism coordinating this synchrony remains speculative. We investigated this mechanism and revealed that initiation of Chr2 replication is triggered by the replication of a 150-bp locus positioned on Chr1, called crtS. This crtS replication-mediated Chr2 replication initiation mechanism explains how the two chromosomes communicate to coordinate their replication. Our study reveals a new checkpoint control mechanism in bacteria, and highlights possible functional interactions mediated by contacts between two chromosomes, an unprecedented observation in bacteria

Macromolecular crowding links ribosomal protein gene dosage to growth rate in Vibrio cholerae

In fast-growing bacteria, the genomic location of ribosomal protein (RP) genes is biased towards the replication origin (oriC). This trait allows optimizing their expression during exponential phase since oriC neighboring regions are in higher dose due to multifork replication. Relocation of s10-spc-α locus (S10), which codes for most of the RP, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction on its dosage, its expression, and bacterial growth rate. However, a mechanism linking S10 dosage to cell physiology has still not been determined.We hypothesized that S10 dosage perturbations impact protein synthesis capacity. Strikingly, we observed that in Vibrio cholerae, protein production capacity was independent of S10 position. Deep sequencing revealed that S10 relocation altered chromosomal replication dynamics and genome-wide transcription. Such changes increased as a function of oriC-S10 distance. Since RP constitutes a large proportion of cell mass, lower S10 dosage could lead to changes in macromolecular crowding, impacting cell physiology. Accordingly, cytoplasm fluidity was higher in mutants where S10 is most distant from oriC. In hyperosmotic conditions, when crowding differences are minimized, the growth rate and replication dynamics were highly alleviated in these strains.The genomic location of RP genes ensures its optimal dosage. However, besides of its essential function in translation, their genomic position sustains an optimal macromolecular crowding essential for maximizing growth. Hence, this could be another mechanism coordinating DNA replication to bacterial growth.Fil: Soler Bistue, Alfonso J. C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Aguilar Pierlé, Sebastián. Institut Pasteur; FranciaFil: Garcia Garcerá, Marc. Institut Pasteur; FranciaFil: Val, Marie Eve. Institut Pasteur; FranciaFil: Sismeiro, Odile. Institut Pasteur; FranciaFil: Varet, Hugo. Institut Pasteur; FranciaFil: Sieira, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Krin, Evelyne. Institut Pasteur; FranciaFil: Skovgaard, Ole. Roskilde Universitet; DinamarcaFil: Comerci, Diego José. Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Investigaciones Biotecnologicas.; ArgentinaFil: Rocha, Eduardo P. C.. Institut Pasteur; FranciaFil: Mazel, Didier. Institut Pasteur; Franci

FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Replicate Once Per Cell Cycle:Replication Control of Secondary Chromosomes

Faithful vertical transmission of genetic information, especially of essential core genes, is a prerequisite for bacterial survival. Hence, replication of all the replicons is tightly controlled to ensure that all daughter cells get the same genome copy as their mother cell. Essential core genes are very often carried by the main chromosome. However they can occasionally be found on secondary chromosomes, recently renamed chromids. Chromids have evolved from non-essential megaplasmids, and further acquired essential core genes and a genomic signature closed to that of the main chromosome. All chromids carry a plasmidic replication origin, belonging so far to either the iterons or repABC type. Based on these differences, two categories of chromids have been distinguished. In this review, we focus on the replication initiation controls of these two types of chromids. We show that the sophisticated mechanisms controlling their replication evolved from their plasmid counterparts to allow a timely controlled replication, occurring once per cell cycle