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Accueil > Départements > Biologie des Génomes > François-Xavier BARRE : Evolution et Maintenance des Chromosomes Circulaires

Publications

2017


  • P. Brézellec, M. - A. Petit, S. Pasek, I. Vallet-Gely, C. Possoz, et J. - L. Ferat, « Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker », Genome Biology and Evolution, vol. 9, nᵒ 6, p. 1561-1566, 2017.
    Résumé : At the onset of the initiation of chromosome replication, bacterial replicative helicases are recruited and loaded on the DnaA-oriC nucleoprotein platform, assisted by proteins like DnaC/DnaI or DciA. Two orders of bacteria appear, however, to lack either of these factors, raising the question of the essentiality of these factors in bacteria. Through a phylogenomic approach, we identified a pair of genes that could have substituted for dciA. The two domesticated genes are specific of the dnaC/dnaI- and dciA-lacking organisms and apparently domesticated from lambdoid phage genes. They derive from λO and λP and were renamed dopC and dopE, respectively. DopE is expected to bring the replicative helicase to the bacterial origin of replication, while DopC might assist DopE in this function. The confirmation of the implication of DopCE in the handling of the replicative helicase at the onset of replication in these organisms would generalize to all bacteria and therefore to all living organisms the need for specific factors dedicated to this function.
    Mots-clés : DBG, dciA, dnaC, EMC2, lambda phage, OCB, replication initiation, replicative helicase, viral gene domestication.

  • T. N. Dalia, S. H. Yoon, E. Galli, F. - X. Barre, C. M. Waters, et A. B. Dalia, « Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases », Nucleic Acids Research, 2017.
    Résumé : Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
    Mots-clés : DBG, EMC2.


  • E. Galli, C. Midonet, E. Paly, et F. - X. Barre, « Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli », PLOS Genetics, vol. 13, nᵒ 3, p. e1006702, mars 2017.


  • E. Galli, E. Paly, et F. - X. Barre, « Late assembly of the Vibrio cholerae cell division machinery postpones septation to the last 10% of the cell cycle », Scientific Reports, vol. 7, p. 44505, mars 2017.

  • M. Poidevin, E. Galli, Y. Yamaichi, et F. - X. Barre, « WGADseq: Whole Genome Affinity Determination of Protein-DNA Binding Sites », Methods in Molecular Biology (Clifton, N.J.), vol. 1624, p. 53-60, 2017.
    Résumé : We present a method through which one may monitor the relative binding affinity of a given protein to DNA motifs on the scale of a whole genome. Briefly, the protein of interest is incubated with fragmented genomic DNA and then affixed to a column. Washes with buffers containing low salt concentrations will remove nonbound DNA fragments, while stepwise washes with increasing salt concentrations will elute more specifically bound fragments. Massive sequencing is used to identify eluted DNA fragments and map them on the genome, which permits us to classify the different binding sites according to their affinity and determine corresponding consensus motifs (if any).
    Mots-clés : DBG, EMC2, EQYY, Genomics, High-throughput sequencing, Next-generation sequencing (NGS), Site-specific DNA binding protein.

  • A. K. Sinha, A. Durand, J. - M. Desfontaines, I. Iurchenko, H. Auger, D. R. F. Leach, F. - X. Barre, et B. Michel, « Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme », PLoS genetics, vol. 13, nᵒ 10, p. e1006895, 2017.
    Résumé : 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.
    Mots-clés : DBG, EMC2, NGS, PF, STABAC.

2016


  • P. Brézellec, I. Vallet-Gely, C. Possoz, S. Quevillon-Cheruel, et J. - L. Ferat, « DciA is an ancestral replicative helicase operator essential for bacterial replication initiation », Nature Communications, vol. 7, p. 13271, 2016.
    Résumé : Delivery of the replicative helicase onto DNA is an essential step in the initiation of replication. In bacteria, DnaC (in Escherichia coli) and DnaI (in Bacillus subtilis) are representative of the two known mechanisms that assist the replicative helicase at this stage. Here, we establish that these two strategies cannot be regarded as prototypical of the bacterial domain since dnaC and dnaI (dna[CI]) are present in only a few bacterial phyla. We show that dna[CI] was domesticated at least seven times through evolution in bacteria and at the expense of one gene, which we rename dciA (dna[CI] antecedent), suggesting that DciA and Dna[CI] share a common function. We validate this hypothesis by establishing in Pseudomonas aeruginosa that DciA possesses the attributes of the replicative helicase-operating proteins associated with replication initiation.
    Mots-clés : B3S, DBG, EMC2, FAAM, OCB.


  • E. Galli, M. Poidevin, R. Le Bars, J. - M. Desfontaines, L. Muresan, E. Paly, Y. Yamaichi, et F. - X. Barre, « Cell division licensing in the multi-chromosomal Vibrio cholerae bacterium », Nature Microbiology, vol. 1, nᵒ 9, p. 16094, juin 2016.
    Mots-clés : DBG, EMC2, EQYY, IMAGIF, PF, PHOT.

  • E. Martínez, J. Campos-Gómez, et F. - X. Barre, « CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications », Bacteriophage, vol. 6, nᵒ 2, p. e1128512, 2016.
    Résumé : For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages.
    Mots-clés : Cholera, DBG, EMC2, filamentous phage, integrative mobile element, rolling circle replication, Xer recombination.

  • C. Midonet et F. - X. Barre, « How Xer-exploiting mobile elements overcome cellular control », Proceedings of the National Academy of Sciences of the United States of America, vol. 113, nᵒ 30, p. 8343-8345, 2016.

  • M. - E. Val, M. Marbouty, F. de Lemos Martins, S. P. Kennedy, H. Kemble, M. J. Bland, C. Possoz, R. Koszul, O. Skovgaard, et D. Mazel, « A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae », Science Advances, vol. 2, nᵒ 4, p. e1501914, 2016.
    Résumé : Bacteria 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.
    Mots-clés : Bacterial Proteins, cell cycle, Cell Cycle Checkpoints, Cholera, chromosome, Chromosome Segregation, Chromosomes, Bacterial, DBG, DNA Replication, EMC2, Gene Expression Regulation, Bacterial, Genome, Bacterial, multipartite genome, pathogens, Plasmids, replication, replication initiation, Replication Origin, secondary chromosome, Vibrio, Vibrio cholerae.

2015


  • E. Martínez, E. Paly, et F. - X. Barre, « CTXφ Replication Depends on the Histone-Like HU Protein and the UvrD Helicase », PLoS genetics, vol. 11, nᵒ 5, p. e1005256, 2015.
    Résumé : The Vibrio cholerae bacterium is the agent of cholera. The capacity to produce the cholera toxin, which is responsible for the deadly diarrhea associated with cholera epidemics, is encoded in the genome of a filamentous phage, CTXφ. Rolling-circle replication (RCR) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles. A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome. The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome, such as the Rep SF1 helicase. In contrast, we show here that the histone-like HU protein of V. cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ. We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair, UvrD, rather than Rep. In addition to CTXφ, we show that VGJφ, a representative member of a second family of vibrio integrative filamentous phages, requires UvrD and HU for RCR while TLCφ, a satellite phage, depends on Rep and is independent from HU.
    Mots-clés : Bacterial Proteins, DBG, DNA Helicases, DNA-Binding Proteins, EMC2, Gene Deletion, Genome, Viral, Inovirus, Vibrio cholerae, Virus Replication.
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Publications Principales avant 2015

- Midonet, C., Das, B., Paly, E., and Barre, F.-X. (2014). XerD-mediated FtsK-independent integration of TLCφ into the Vibrio cholerae genome. Proc. Natl. Acad. Sci. U. S. A. 2014 Nov 25 ;111(47):16848-53.

- Demarre, G., Galli, E., Muresan, L., Paly, E., David, A., Possoz, C., and Barre, F.-X. (2014). Differential Management of the Replication Terminus Regions of the Two Vibrio cholerae Chromosomes during Cell Division. PLoS Genet. 10, e1004557.

- David, A., Demarre, G., Muresan, L., Paly, E., Barre, F.-X., and Possoz, C. (2014). The two Cis-acting sites, parS1 and oriC1, contribute to the longitudinal organisation of Vibrio cholerae chromosome I. PLoS Genet. 10, e1004448.

- Bischerour, J., Spangenberg, C., and Barre, F.-X. (2012). Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration. EMBO J. 31, 3757–3767.

- Das, B., Bischerour, J., and Barre, F.-X. (2011). VGJphi integration and excision mechanisms contribute to the genetic diversity of Vibrio cholerae epidemic strains. Proc. Natl. Acad. Sci. U. S. A. 108, 2516–2521.

- Dubarry, N., Possoz, C., and Barre, F.X. (2010). Multiple regions along the Escherichia coli FtsK protein are implicated in cell division. Mol Mic 78, 1088–1100.

- Dubarry, N., and Barre, F.X. (2010). Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK. EMBO J 29, 597–605.

- Das, B., Bischerour, J., Val, M.-E., and Barre, F.-X. (2010). Molecular keys of the tropism of integration of the cholera toxin phage. Proc. Natl. Acad. Sci. U. S. A. 107, 4377–4382.

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