Mechanism of TLC phage integration into the genome of Vibrio cholerae

La plupart des bactéries ont un unique chromosome circulaire. Lors de la réplication de l’ADN, la circularité lie topologiquement les deux chromatides sœurs résultant de la réplication (caténanes et dimères). Ces liens topologiques doivent être résolus afin de permettre une bonne ségrégation de l’information génétique entre les deux cellules filles au cours de la division cellulaire. Les bactéries possèdent une machinerie très conservée: les recombinases à tyrosines XerC et XerD, capables de résoudre les dimères et une partie des caténanes, en catalysant un crossover au site spécifique dif situé dans la région Ter du chromosome. Lors de ce processus elles réalisent successivement deux échanges de brins. La réaction Xer est spatio-temporellement contrôlée par une protéine du divisome: FtsK. FtsK est une translocase qui pompe l’ADN à travers le septum de division. Lorsqu’elle rencontre une synapse constituée de deux sites dif chargés de XerC et XerD, elle active la catalyse de XerD pour initier le premier échange de brins. Dans un second temps XerC catalyse un second échange de brins indépendamment de FtsK. A ce jour le mécanisme d’activation de XerD n’est pas bien compris. Certains éléments mobiles résolvent leur états multimériques (tels que les plasmides) ou intègrent leur génome dans celui de leur hôte en détournant les recombinases XerCD. On parle d’IMEXs (integrative Mobile Element using Xer). Les éléments mobiles étudiés avant ma thèse utilisaient tous des voies de recombinaison initiées par la catalyse de XerC et ne nécessitant pas l’activation de XerD. Au cours de ma thèse j’ai étudié dans un premier temps le mécanisme d’intégration / excision d’une nouvelle classe d’IMEXs en utilisant comme modèle le phage TLCphi de Vibrio cholerae, la bactérie responsable du choléra. Par des approches de génétique j’ai démontré que TLCphi utilise une voie de recombinaison initiée par la catalyse de XerD et indépendante de FtsK. Mes travaux ont également montré que l’excision du phage participe à l’évolution des souches pandémiques de V.cholerae. Dans une seconde partie, j’ai identifié un facteur phagique qui permet à TLCphi de contourner le contrôle de FtsK sur l’activation de XerD. Ce facteur était une protéine de fonction inconnue présentant un domaine HTH et un domaine DUF3653. Ce dernier est retrouvé dans de nombreux IMEXs. Par des approches de biologie moléculaire j’ai étudié le mécanisme d’action de cette protéine. J’ai reproduit la réaction de recombinaison in vitro et démontré qu’elle active XerD en interagissant directement avec elle. Enfin dans un troisième temps, nous nous sommes intéressés aux disparités observées entre la recombinaison Xer chez E.coli et V.cholerae. En particulier, la recombinaison Xer semble agir seulement sur les dimères chez E.coli alors qu’elle est active également sur les monomères chez V.cholerae. Nous avons démontré que ces divergences de comportement ne viennent pas des Xer elles-mêmes, ni de leurs propriétés d'activations par FtsK. Elles résultent des différentes chorégraphies de ségrégation des chromosomes entre ces deux bactéries et dépendent également des vitesses de croissance.Most of bacteria have a single circular chromosome. During replication of DNA, this circularity can lead to two sister chromatids topologically linked (catenanes and dimers). These topological links have to be solved in order to allow good segregation of genetic information between the two daughter cells during cell division. Bacteria possess a highly conserved machinery: the tyrosine recombinases XerC XerD that are capable to resolve dimers and some catenanes, by catalyzing a crossover at the specific site dif located in the Ter region of the chromosome. During this process they realize two sequentialstrand exchanges.The Xer reaction is spatiotemporally controlled by a protein of the divisome: FtsK. FtsK is a pump that translocates DNA through the septum of division. When FtsK meets a synapse that consists of two dif loaded by XerC and XerD, it activates XerD catalysis that initiates first strand exchange. Secondly XerC catalyzes a second strand exchange independently of FtsK. To date the activation mechanism of XerD is not well understood. Some mobile elements solve their multimeric states (like plasmids) or integrate their genome into the chromosome of their host by using XerCD recombinases. Such integrative elements are named IMEXs (Integrative Mobile Element using Xer). The mobile elements studied before my thesis all used recombination pathways initiated by catalysis of XerC and not requiring activation of XerD .During my PhD I studied at first the integration mechanism / excision of a new class IMEXs using as a model the TLC phage Vibrio cholerae, the bacterium responsible for cholera. By genetic approaches I demonstrated that TLCphi uses a recombination pathway initiated by XerD catalysis and independently of FtsK. My work has also shown that the phage excision participates in the evolution of pandemic strains of V. cholerae. In the second part, I identified a phage factor that allows TLC to bypass the activation of XerD by FtsK. This factor was a protein of unknown function with a HTH domain and a DUF3653 domain. DUF3653 are found in many IMEXs. Using molecular biology approaches, I studied the mechanism of action of this protein. I reproduced the recombination reaction in vitro and demonstrated that this factor activates XerD by directly interacting with it. Finally, we were interested to study disparities between Xer recombination in E.coli and V.cholerae. In particular, the Xer recombination seems to act only on dimers in E.coli while it is also active on monomers in V.cholerae. We have demonstrated that these differences in behaviors do not come from Xer themselves or their activation by FtsK. They result from different choreographies of chromosome segregation between these two bacteria and are also dependent on growth rates

Mécanisme d'intégration du phage TLC dans le génome de Vibrio cholerae

Most of bacteria have a single circular chromosome. During replication of DNA, this circularity can lead to two sister chromatids topologically linked (catenanes and dimers). These topological links have to be solved in order to allow good segregation of genetic information between the two daughter cells during cell division. Bacteria possess a highly conserved machinery: the tyrosine recombinases XerC XerD that are capable to resolve dimers and some catenanes, by catalyzing a crossover at the specific site dif located in the Ter region of the chromosome. During this process they realize two sequentialstrand exchanges.The Xer reaction is spatiotemporally controlled by a protein of the divisome: FtsK. FtsK is a pump that translocates DNA through the septum of division. When FtsK meets a synapse that consists of two dif loaded by XerC and XerD, it activates XerD catalysis that initiates first strand exchange. Secondly XerC catalyzes a second strand exchange independently of FtsK. To date the activation mechanism of XerD is not well understood. Some mobile elements solve their multimeric states (like plasmids) or integrate their genome into the chromosome of their host by using XerCD recombinases. Such integrative elements are named IMEXs (Integrative Mobile Element using Xer). The mobile elements studied before my thesis all used recombination pathways initiated by catalysis of XerC and not requiring activation of XerD .During my PhD I studied at first the integration mechanism / excision of a new class IMEXs using as a model the TLC phage Vibrio cholerae, the bacterium responsible for cholera. By genetic approaches I demonstrated that TLCphi uses a recombination pathway initiated by XerD catalysis and independently of FtsK. My work has also shown that the phage excision participates in the evolution of pandemic strains of V. cholerae. In the second part, I identified a phage factor that allows TLC to bypass the activation of XerD by FtsK. This factor was a protein of unknown function with a HTH domain and a DUF3653 domain. DUF3653 are found in many IMEXs. Using molecular biology approaches, I studied the mechanism of action of this protein. I reproduced the recombination reaction in vitro and demonstrated that this factor activates XerD by directly interacting with it. Finally, we were interested to study disparities between Xer recombination in E.coli and V.cholerae. In particular, the Xer recombination seems to act only on dimers in E.coli while it is also active on monomers in V.cholerae. We have demonstrated that these differences in behaviors do not come from Xer themselves or their activation by FtsK. They result from different choreographies of chromosome segregation between these two bacteria and are also dependent on growth rates.La plupart des bactéries ont un unique chromosome circulaire. Lors de la réplication de l’ADN, la circularité lie topologiquement les deux chromatides sœurs résultant de la réplication (caténanes et dimères). Ces liens topologiques doivent être résolus afin de permettre une bonne ségrégation de l’information génétique entre les deux cellules filles au cours de la division cellulaire. Les bactéries possèdent une machinerie très conservée: les recombinases à tyrosines XerC et XerD, capables de résoudre les dimères et une partie des caténanes, en catalysant un crossover au site spécifique dif situé dans la région Ter du chromosome. Lors de ce processus elles réalisent successivement deux échanges de brins. La réaction Xer est spatio-temporellement contrôlée par une protéine du divisome: FtsK. FtsK est une translocase qui pompe l’ADN à travers le septum de division. Lorsqu’elle rencontre une synapse constituée de deux sites dif chargés de XerC et XerD, elle active la catalyse de XerD pour initier le premier échange de brins. Dans un second temps XerC catalyse un second échange de brins indépendamment de FtsK. A ce jour le mécanisme d’activation de XerD n’est pas bien compris. Certains éléments mobiles résolvent leur états multimériques (tels que les plasmides) ou intègrent leur génome dans celui de leur hôte en détournant les recombinases XerCD. On parle d’IMEXs (integrative Mobile Element using Xer). Les éléments mobiles étudiés avant ma thèse utilisaient tous des voies de recombinaison initiées par la catalyse de XerC et ne nécessitant pas l’activation de XerD. Au cours de ma thèse j’ai étudié dans un premier temps le mécanisme d’intégration / excision d’une nouvelle classe d’IMEXs en utilisant comme modèle le phage TLCphi de Vibrio cholerae, la bactérie responsable du choléra. Par des approches de génétique j’ai démontré que TLCphi utilise une voie de recombinaison initiée par la catalyse de XerD et indépendante de FtsK. Mes travaux ont également montré que l’excision du phage participe à l’évolution des souches pandémiques de V.cholerae. Dans une seconde partie, j’ai identifié un facteur phagique qui permet à TLCphi de contourner le contrôle de FtsK sur l’activation de XerD. Ce facteur était une protéine de fonction inconnue présentant un domaine HTH et un domaine DUF3653. Ce dernier est retrouvé dans de nombreux IMEXs. Par des approches de biologie moléculaire j’ai étudié le mécanisme d’action de cette protéine. J’ai reproduit la réaction de recombinaison in vitro et démontré qu’elle active XerD en interagissant directement avec elle. Enfin dans un troisième temps, nous nous sommes intéressés aux disparités observées entre la recombinaison Xer chez E.coli et V.cholerae. En particulier, la recombinaison Xer semble agir seulement sur les dimères chez E.coli alors qu’elle est active également sur les monomères chez V.cholerae. Nous avons démontré que ces divergences de comportement ne viennent pas des Xer elles-mêmes, ni de leurs propriétés d'activations par FtsK. Elles résultent des différentes chorégraphies de ségrégation des chromosomes entre ces deux bactéries et dépendent également des vitesses de croissance

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

Insights into TLCΦ lysogeny: A twist in the mechanism of IMEX integration

International audienceMany organisms have established symbiotic relationships with acquired mobile genetic elements (MGEs) integrated in their genomes (1). MGEs spread among genomes within and across microbial species through horizontal gene transfer and, once integrated into host chromosome, are disseminated vertically to the progeny, causing rapid evolution of drug resistance, pathogenicity, and virulence traits (2, 3). The MGEs that integrates into the host bacterial chromosomes (IMGEs) either carry their own DNA integration machineries or exploit machineries already existing in the host organisms for integration (4). The latter elements are of interest, in part, because of their contribution to pathogenesis, antimicrobial resistance, and other medically relevant properties (5, 6). One of the host site-specific recombination systems frequently exploited by IMGEs is the widely distributed bacterial chromosome dimer-resolving Xer recombination system, a system that recombines chromosomes at dif site located near where DNA replication terminates (7). As in all organisms, during DNA replication of bacteria, many DNA damages need to be repaired by homologous recombination reaction. For bacteria having circular chromosomes, this often generates circular dimer chromosome, causing problems when the cell divides. Hence, when a pair of unresolved chromosome dimer junctions get trapped at the closing cell division septum, the pair of dif sites with XerC and XerD recombinases bound across the recombination junction encounter FtsK DNA translocation pump, a component of the closing septum complex, whose job is to clear trapped DNA out of the septum. This encounter triggers initiation of recombination by activating XerD to carry out the first strand exchange, generating a Holliday junction recombination intermediate, which is resolved by XerC-mediated second pair of strand exchange (8). XerC is an efficient resolver of the recombination intermediate but a poor recombination initiator. Without FtsK activation, Xer remains essentially silent, avoiding formation of chromosome dimer out of 2 separable replicated … [↵][1]1Email: bhabatosh\at\thsti.res.in. [1]: #xref-corresp-1-

The TLCΦ satellite phage harbors a Xer recombination activation factor

The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site, dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage of Vibrio cholerae, TLCΦ, which integrates into and excises from the dif site of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and beta-proteobacteria, including human, animal, and plant pathogens

Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli

Homologous recombination between the circular chromosomes of bacteria can generate chromosome dimers. They are resolved by a recombination event at a specific site in the replication terminus of chromosomes, dif, by dedicated tyrosine recombinases. The reaction is under the control of a cell division protein, FtsK, which assembles into active DNA pumps at mid-cell during septum formation. Previous studies suggested that activation of Xer recombination at dif was restricted to chromosome dimers in Escherichia coli but not in Vibrio cholerae, suggesting that FtsK mainly acted on chromosome dimers in E. coli but frequently processed monomeric chromosomes in V. cholerae. However, recent microscopic studies suggested that E. coli FtsK served to release the MatP-mediated cohesion and/or cell division apparatus-interaction of sister copies of the dif region independently of chromosome dimer formation. Here, we show that these apparently paradoxical observations are not linked to any difference in the dimer resolution machineries of E. coli and V. cholerae but to differences in the timing of segregation of their chromosomes. V. cholerae harbours two circular chromosomes, chr1 and chr2. We found that whatever the growth conditions, sister copies of the V. cholerae chr1 dif region remain together at mid-cell until the onset of constriction, which permits their processing by FtsK and the activation of dif-recombination. Likewise, sister copies of the dif region of the E. coli chromosome only separate after the onset of constriction in slow growth conditions. However, under fast growth conditions the dif sites separate before constriction, which restricts XerCD-dif activity to resolving chromosome dimers

Initiation of multiple rounds of replication uncouples the final stages of chromosome segregation from cell division.

<p>(A) Rate of <i>dif</i>-cassette (left panel) and <i>recA</i>-dependency (right panel) of <i>dif-</i>cassette excision measured over 16 h in fast (LB) and slow (M9) growing <i>E</i>. <i>coli</i> cells. Mean of 5 independent experiments. <b>*</b>: p <0.01 (t-test with a Two-tailed distribution). (B) Consensus images summarising the cell cycle choreography of the <i>ydeV</i> fluorescence marker (upper panels, GFP) and the cell shape (lower panels, BF) in fast (left panels) and slow (right panels) growing <i>E</i>. <i>coli</i> cells. Only cells that were followed from birth to division were taken into consideration. At each time point, the maximal and minimal intensities of the projections were set to 1 and 0, respectively. We created images describing the evolution of the fluorescence and cell shape in the cell cycle by plotting the different projections of individual cells as a function of time using a jet colour code. In the heat maps, black corresponds to the lowest and dark red to the highest intensities. In the GFP maps the red areas indicate the presence of the <i>ydeV</i> marker, in the BF maps the green line indicates the Septa appearance. Individual fluorescence and cell shape images were compiled into consensus images summarising the results. Y-axis: position along the cell length. X-axis: cell cycle. GT: generation time; n: number of complete cell cycles that were analysed; op: old pole; np: new pole. 0: birth; 1: division. (C) Frequency of cells displaying a single spot (blue line), two spots (red line) and 3 or more spots (black line) as a function of the time before or after septation was detected. The Y-axis serves for both the cell type frequency lines and to the relative spot positions, with 0 indicating 0% frequency or the old pole position and 1 indicating 100% frequency or the new pole position. Left panels: M9-Rich; right panels: M9. The percentage of cells with a single <i>ydeV</i> spot at the time when septation was detected is indicated in blue with the number of observed cells (n) between parentheses. The percentage of new <i>ydeV</i> duplication events 0% per min and 4.3% per min represented at the time when septation was detected is indicated in red. GT: generation time.</p

Fluorescence microscopy snapshot analysis of the position of the <i>dif</i> region in fast growing cells.

<p>(A) Position of the <i>ydeV</i> locus of the <i>E</i>. <i>coli</i> chromosome in cells with no visible indentation. (B) Position of the <i>ydeV</i> locus of the <i>E</i>. <i>coli</i> chromosome in cells with visible indentation. (C) Position of the <i>dif1</i> locus of <i>V</i>. <i>cholerae</i> chr1 in cells with no visible indentation. (D) Position of the <i>dif1</i> locus of <i>V</i>. <i>cholerae</i> chr1 in cells with visible indentation. GT: generation time; n: number of cells analysed; left panels: relative long axis position of foci as a function of cell length; right panels: overall distribution of foci positions; upper panels: cells presenting a single focus; lower panels: cells presenting 2 foci. Cells were arbitrarily oriented.</p