Chorégraphie de ségrégation des deux chromosomes de Vibrio cholerae

L objectif de cette thèse est de définir la chorégraphie de ségrégation des deux chromosomes circulaires de Vibrio cholerae, c est à dire le positionnement de l information génétique au cours de la croissance de la cellule, ainsi que les mécanismes dirigeant ces ségrégations. Il a longtemps été supposé que les bactéries étaient trop petites pour avoir une organisation intra-cellulaire, et le manque de techniques appropriées ne permettait pas d infirmer cette hypothèse. Or la taille des chromosomes comparée à celle de la bactérie impose une compaction et aujourd hui, de nouvelles techniques de microscopie et d analyse génétique permettent d affirmer que les chromosomes bactériens étudiés jusqu à maintenant ont tous une organisation et une chorégraphie de ségrégation précises et différentes selon les espèces. Toutes les espèces étudiées à ce jour ont un chromosome circulaire unique : la réplication du chromosome commence à une origine unique bidirectionnelle, les deux fourches de réplication se déplacent le long des deux bras de réplication (ou réplichores) et finissent la réplication au terminus, diamétralement à l opposée de l origine de réplication sur la carte du chromosome. Peu d espèces ont été étudiées, et Vibrio cholerae émerge progressivement comme un nouveau modèle : son génome est réparti sur deux chromosomes, et la chorégraphie de plusieurs chromosomes dans une cellule n a jamais été décrite. De plus, cette espèce semble être au croisement évolutif entre Caulobacter crescentus et Escherichia coli : Vibrio cholerae a d une part une morphologie en croissant, des systèmes de partition aux origines et un positionnement de l origine du chromosome I, semblables à C. crescentus, et d autre part un système de compaction du terminus et un set de gènes impliqués dans la maintenance du chromosome ayant co-évolué, qu on ne retrouve que dans peu d espèces proches d E. coli. Une autre caractéristique intéressante de V. cholerae est que le chromosome II semble avoir été acquis récemment et n est donc peut être pas gouverné par les mêmes mécanismes que le chromosome I, comme en témoignent le positionnement de son origine et son terminus, inédits pour des chromosomes bactériens. Parmi les Vibrios (environ 60 espèces principalement retrouvées dans les environnements aquatiques), certaines espèces sont des pathogènes dévastateurs pour les poissons, le corail, les crustacés ou les fruits de mer. Mais la plus documentée est Vibrio cholerae, car elle provoque chez l Humain une maladie provoquée par l ingestion d eau contaminée qui peut être mortelle si le patient n est pas réhydraté à temps. Bien que facilement traitable, le choléra fait encore de nombreuses victimes dans les pays en développement où les structures de santé et les règles d hygiène font parfois défaut. Ainsi l étude de Vibrio cholerae présente un intérêt médical, mais également par extension aux autres Vibrios, un intérêt environnemental non négligeable.The aim of this thesis is to define the segregation choreography of the two circular chromosomes of Vibrio cholerae, which is the positionning of the genetic information during cell growth, as well as the mecanisms directing those segregations. It was supposed for a long time that bacteria were too small to have a intra-cellular organization and the lack of appropriate tools could not prove this hypothesis wrong. The size of the chromosomes compared to the size of the cell means there has to be a compaction and today, new tools for microscopy and genetic analysis allow us to affirm that all bacterial chromosomes studied so far have an organization and a segregation choreography which are precise and different between specie. Most bacterial specie studied to this day have a unique circular chromosome : the replication of the chromosome starts at a unique and bidirectionnal origin, both replication forks move along the two replication arms (or replichores) and end the replication at the terminus which is diametrically to the opposite of the origin on the chromosome map. A few specie have been studied, and Vibrio cholerae progressively emerges as a new model : its genome is divided between two chromosomes, and the choreography of several chromosomes in a cell has never been described. Moreover, this species seems to be at the crossover between Caulobacter crescentus and Escherichia coli : Vibrio cholerae as on one hand, a crescent shape, partition systems positionned at both origins and a positionning of the chromosome I origin similar to C. crescentus, and on the other hand a compaction system of the terminus and a set of genes involved on the maintenance of chromosomes that one only finds in very few specie closely related to E. coli. An other interesting characteristic of V. cholerae is that the chromosome II seems to have been acquired recently and thus might not be governed by the same mecanisms as the chromosome I, as shown by the positionning of its origin and terminus which are completely new to bacterial chromosomes. Among Vibrios (about 60 species mostly found in aquatic environments), some species are devastating pathogens for fish, coral, crustacean and shellfish. But the most documented one is Vibrio cholerae, because it induces a disease in humans caused by the ingestion of contaminated water, which can be deadly if the patient is not rehydrated on time. Although easily treatable, cholera still makes a lot of victims in developing countries where health structures and basic hygiene sometimes lack dramatically. The study of Vibrio cholerae has a medical interest, but also by extention to other Vibrios, a non-negligible environmental interest.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Asymmetric DNA requirements in Xer recombination activation by FtsK

In bacteria with circular chromosomes, homologous recombination events can lead to the formation of chromosome dimers. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover by two tyrosine recombinases, XerC and XerD, at a specific site on the chromosome, dif. Recombination depends on a direct contact between XerD and a cell division protein, FtsK, which functions as a hexameric double stranded DNA translocase. Here, we have investigated how the structure and composition of DNA interferes with Xer recombination activation by FtsK. XerC and XerD each cleave a specific strand on dif, the top and bottom strand, respectively. We found that the integrity and nature of eight bottom-strand nucleotides and three top-strand nucleotides immediately adjacent to the XerD-binding site of dif are crucial for recombination. These nucleotides are probably not implicated in FtsK translocation since FtsK could translocate on single stranded DNA in both the 5′–3′ and 3′–5′ orientation along a few nucleotides. We propose that they are required to stabilize FtsK in the vicinity of dif for recombination to occur because the FtsK–XerD interaction is too transient or too weak in itself to allow for XerD catalysis

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant

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

DNA mechanics as a tool to probe helicase and translocase activity

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

<p><u>(A) Circular map of the <i>E</i>. <i>coli</i> chromosome</u>: <i>oriC</i>, <i>dif</i> and <i>terD</i> to <i>terB</i> sites are indicated. Numbers refer to the chromosome coordinates (in kb) of MG1655. (<u>B) Linear map of the terminus region:</u> chromosome coordinates are shown increasing from left to right, as in the marker frequency panels (see Figure 1C for example), therefore in the opposite direction to the circular map. In addition to <i>dif</i> and <i>ter</i> sites, the positions of the <i>parS</i>_pMT1 sites used for microscopy experiments are indicated. (<u>C) MFA analysis of terminus DNA loss in the <i>recB</i> mutant</u>: sequence read frequencies of exponential phase cells normalized to the total number of reads were calculated for each strain. Ratios of normalized reads in isogenic wild-type and <i>recB</i> mutant are plotted against chromosomal coordinates (in kb). The profile ratio of the terminus region is enlarged and the profile of the corresponding entire chromosomes is shown in inset. Original normalized profiles used to calculate ratios are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.s005" target="_blank">S1 Fig</a>. The position of <i>dif</i> is indicated by a red arrow. The <i>ter</i> sites that arrest clockwise forks (<i>terC</i>, <i>terB</i>, green arrow) and counter-clockwise forks (<i>terA</i>, <i>terD</i>, blue arrow) are shown. <u>(D) Schematic representation of focus loss in the <i>recB</i> mutant:</u> Time-lapse microscopy experiments showed that loss of a focus in the <i>recB</i> mutant occurs concomitantly with cell division in one of two daughter cells, and that the cell that keeps the focus then generates a focus-less cell at each generation. The percentage of initial events was calculated as the percentage of cell divisions that generate a focus-less cell, not counting the following generations. In this schematic representation, two initial events occurred (generations #2 and #7) out of 9 generations, and focus loss at generation #2 is heritable. Panels shown in this figure were previously published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.ref019" target="_blank">19</a>] and are reproduced here to introduce the phenomenon.</p

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

ABSTRACT Many mobile elements take advantage of the highly-conserved chromosome dimer resolution system of bacteria, Xer. They participate in the transmission of antibiotic resistance and pathogenicity determinants. In particular, the toxin-linked cryptic satellite phage (TLCΦ) plays an essential role in the continuous emergence of new toxigenic clones of the Vibrio cholerae strain at the origin of the ongoing 7 th cholera pandemic. The Xer machinery is composed of two chromosomally-encoded tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific 28 base pair site of bacterial chromosomes, dif . The activity of XerD depends on a direct contact with a cell division protein, FtsK, which spatially and temporally constrains the process. TLCΦ encodes for a XerD-activation factor (XafT), which drives the integration of the phage into the dif site of the primary chromosome of V. cholerae independently of FtsK. However, XerD does not bind to the attachment site ( attP ) of TLCΦ, which raised questions on the integration process. Here, we compared the integration efficiency of thousands of synthetic mini-TLCΦ plasmids harbouring different attP sites and assessed their stability in vivo . In addition, we compared the efficiency with which XafT and the XerD activation domain of FtsK drive recombination reactions in vitro . Taken together, our results suggest that XafT promotes the formation of synaptic complexes between canonical Xer recombination sites and imperfect sites

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

ABSTRACT Many mobile elements take advantage of the highly-conserved chromosome dimer resolution system of bacteria, Xer. They participate in the transmission of antibiotic resistance and pathogenicity determinants. In particular, the toxin-linked cryptic satellite phage (TLCΦ) plays an essential role in the continuous emergence of new toxigenic clones of the Vibrio cholerae strain at the origin of the ongoing 7 th cholera pandemic. The Xer machinery is composed of two chromosomally-encoded tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific 28 base pair site of bacterial chromosomes, dif . The activity of XerD depends on a direct contact with a cell division protein, FtsK, which spatially and temporally constrains the process. TLCΦ encodes for a XerD-activation factor (XafT), which drives the integration of the phage into the dif site of the primary chromosome of V. cholerae independently of FtsK. However, XerD does not bind to the attachment site ( attP ) of TLCΦ, which raised questions on the integration process. Here, we compared the integration efficiency of thousands of synthetic mini-TLCΦ plasmids harbouring different attP sites and assessed their stability in vivo . In addition, we compared the efficiency with which XafT and the XerD activation domain of FtsK drive recombination reactions in vitro . Taken together, our results suggest that XafT promotes the formation of synaptic complexes between canonical Xer recombination sites and imperfect sites

How Xer-exploiting mobile elements overcome cellular control

International audienceMost strains of Neisseria gonorrheae (Ng), the causative agent of the sexually transmitted disease gonorrheae, and a few strains of Neisseria meningitidis (Nm), which is responsible for a large number of meningitides, harbor a 57-kb horizontally acquired genetic element, the gonococcal genomic island (GGI) (1⇓–3). Certain versions of the GGI are associated with disseminated gonococcal infection (1, 4). In addition, the GGI encodes numerous homologs of type IV secretion system genes, which are necessary for DNA secretion and facilitate natural transformation of the Neisseria (1, 2, 4). GGI are found integrated at the chromosomal dimer resolution site of their host chromosome, dif, and are flanked by a partial repeat of it, difGGI (Fig. 1A) (1, 5). The dif site is the target of two highly conserved chromosomally encoded tyrosine recombinases, XerC and XerD, which normally serve to resolve dimers of circular chromosomes through the addition of a crossover between directly repeated dif sites (6). This reaction raises questions on how GGI could be stably maintained (5). The results presented by Fournes et al. (7) in PNAS shed a new light on this apparent paradox