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Accueil > Départements > Biologie des Génomes > Frédéric BOCCARD : Conformation et Ségrégation du chromosome bactérien

Publications de l’équipe


  • A. M. Kretsch, G. L. Morgan, J. Tyne, E. Meyers, I. Vallet-Gely, et B. Li, « Discovery of (Dihydro)pyrazine N-Oxides via Genome Mining in Pseudomonas », Organic Letters, vol. 20, nᵒ 16, p. 4791-4795, août 2018.
    Résumé : Overexpression of the Pseudomonas virulence factor (pvf) biosynthetic operon led to the identification of a family of pyrazine N-oxides (PNOs), including a novel dihydropyrazine N,N'-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)PNOs was characterized, and a biosynthetic pathway for (d)PNOs was proposed. This work highlights the unique chemistry catalyzed by pvf-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway.
    Mots-clés : bacteria, biofilm formation, biosynthesis, DBG, escherichia-coli, OCB, oxygenase, peptide, pyrazines, synthetase, syringae, virulence.

  • V. S. Lioy et F. Boccard, « Conformational Studies of Bacterial Chromosomes by High-Throughput Sequencing Methods », Methods in Enzymology, vol. 612, p. 25-45, 2018.
    Résumé : The development of next-generation sequencing technologies has allowed the application of different methods dedicated to the study of DNA-protein interactions and chromosome conformation to entire bacterial genome. By combining these approaches, the role of various parameters and factors involved in gene expression and chromosome organization can be disclosed at the molecular level over the full genome. Here we describe two methods that profoundly revolutionized our vision of DNA-protein interactions and spatial organization of chromosomes. Chromosome conformation capture (3C) coupled to deep sequencing (3C-seq) enables studies of the genome-wide chromosome folding and its control by different parameters and structural factors. Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) revealed the extent and regulation of DNA-protein interactions in vivo and highlight the role of structural factors in the control of chromosome organization. In this chapter, we describe a detailed protocol of 3C-seq and ChIP-seq experiments that, when combined, allows the spatial study of the chromosome and the factors that promote specific folding. Data processing and analysis for both experiments are also discussed.
    Mots-clés : 3C-seq, ChIP-seq, Chromosome conformation, Condensin, DBG, Nucleoid-associated proteins, OCB.

  • V. S. Lioy, A. Cournac, M. Marbouty, S. Duigou, J. Mozziconacci, O. Espéli, F. Boccard, et R. Koszul, « Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins », Cell, vol. 172, nᵒ 4, p. 771-783.e18, févr. 2018.
    Résumé : Summary As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.
    Mots-clés : bacteria, chromatin, DBG, genome organization, Hi-C, HU, MatP, OCB, SMC.


  • 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, juin 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.

  • S. Duigou et F. Boccard, « Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains », PLoS genetics, vol. 13, nᵒ 5, p. e1006758, mai 2017.
    Résumé : The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell.
    Mots-clés : DBG, OCB.


  • 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, nov. 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.

  • V. Lagage, F. Boccard, et I. Vallet-Gely, « Regional Control of Chromosome Segregation in Pseudomonas aeruginosa », PLoS genetics, vol. 12, nᵒ 11, p. e1006428, nov. 2016.
    Résumé : Chromosome segregation in bacteria occurs concomitantly with DNA replication, and the duplicated regions containing the replication origin oriC are generally the first to separate and migrate to their final specific location inside the cell. In numerous bacterial species, a three-component partition machinery called the ParABS system is crucial for chromosome segregation. This is the case in the gammaproteobacterium Pseudomonas aeruginosa, where impairing the ParABS system is very detrimental for growth, as it increases the generation time and leads to the formation of anucleate cells and to oriC mispositioning inside the cell. In this study, we investigate in vivo the ParABS system in P. aeruginosa. Using chromatin immuno-precipitation coupled with high throughput sequencing, we show that ParB binds to four parS site located within 15 kb of oriC in vivo, and that this binding promotes the formation of a high order nucleoprotein complex. We show that one parS site is enough to prevent anucleate cell formation, therefore for correct chromosome segregation. By displacing the parS site from its native position on the chromosome, we demonstrate that parS is the first chromosomal locus to be separated upon DNA replication, which indicates that it is the site of force exertion of the segregation process. We identify a region of approximatively 650 kb surrounding oriC in which the parS site must be positioned for chromosome segregation to proceed correctly, and we called it "competence zone" of the parS site. Mutant strains that have undergone specific genetic rearrangements allow us to propose that the distance between oriC and parS defines this "competence zone". Implications for the control of chromosome segregation in P. aeruginosa are discussed.
    Mots-clés : DBG, OCB.

  • M. Valens, A. Thiel, et F. Boccard, « The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain », PLoS genetics, vol. 12, nᵒ 9, p. e1006309, sept. 2016.
    Résumé : The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼-¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed.
    Mots-clés : DBG, OCB.
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Une liste de publication « majeures »

  • Lioy. V. S., Cournac, A., Marbouty, M., Duigou, S., Mozziconacci J., Espéli, O., Boccard, F.*, Koszul, R.* (2018) Multiscale structuring of the E. coli chromosome by nucleoid-associated and condensin proteins. Cell 172 : 771–783.
  • Duigou, S., Boccard, F. (2017) Long range chromosome organization in E. coli : the position of the replication origin defines the Non-Structured regions and the Right and Left macrodomains. PLoS Genetics 13(5) : e1006758.
  • Lagage, V., Boccard, F.*, Vallet-Gely, I*. (2016) Regional control of chromosome segregation in Pseudomonas aeruginosa. PLoS Genetics 12 (11) : e1006428.
  • Valens, M., Thiel, A., Boccard, F. (2016) The MaoP/maoS site specific system organizes the Ori region of the E. coli chromosome into a Macrodomain. PLoS Genetics 12(9):e1006309.
  • Boccard, F. (2014) An emerging integrated view of the bacterial chromosome, from the genome to the nucleoid. Current Opinion in Microbiology, 22, vii-x.
  • Junier, I., Boccard, F., Espeli, O. (2014) Polymer modeling of the E. coli genome reveals the involvement of locus positioning and macrodomain structuring for the control of chromosome conformation and segregation. Nucleic Acids Research. 42(3):1461-1473.
  • Vallet-Gely, I. Boccard, F. (2013) Chromosomal organization and segregation in Pseudomonas aeruginosa. PLoS Genetics 9(5) : e1003492.
  • Dupaigne, P., Tonthat, N.K., Espéli, O., Whitfill, T., Boccard, F.,* Schumacher M.A.* (2012) Molecular basis for a protein-mediated DNA bridging mechanism that functions in long-range condensation of the E. coli chromosome. Molecular Cell, 48 : 560-571.
  • Lesterlin, C., Gigant, E., Boccard, F., Espéli, O. (2012) Sister-chromatids interaction in bacteria revealed by a site-specific recombination assay. EMBO J., 31 : 3468-3479.
  • Espéli, O., Borne, R., Dupaigne, P., Thiel, A., Gigant, E., Mercier, R., Boccard, F. (2012) A MatP divisome interaction coordinates chromosome segregation with cell division in E. coli. EMBO J. 31 : 3198-3211.
  • Thiel, A., Valens, M., Vallet-Gely, I., Espéli, O., Boccard, F. (2012) Long range chromosome organization in E. coli : A site-specific system isolates the Ter macrodomain. PLoS Genetics, 8 (4) : e1002672.
  • Mercier, R., Petit, M.-A., Schbath, S., Robin, S., El Karoui, M., Boccard, F.*, Espéli, O.* (2008) The MatP/matS site specific system organizes the Terminus region of the E. coli chromosome into a Macrodomain. Cell, 135 : 475-485.
  • Espéli, O., Mercier, R., Boccard, F. (2008) DNA dynamics vary according to macrodomain topography in the E. coli chromosome. Molecular Microbiology, 68 : 1418–1427.
  • Esnault, E., Valens, M., Espéli, O., Boccard, F. (2007) Chromosome structuring limits genome plasticity in E. coli. PLoS Genetics, 3 : e226.
  • Vodovar, N., Vallenet, D., Cruveiller, S., Rouy, Z., Barbe, V., Acosta, C., Cattolico, L., Jubin, C., Lajus, A., Segurens, B., Vacherie, B., Wincker, P., Weissenbach, J., Lemaitre, B., Médigue, C., and Boccard, F. (2006). Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila. Nature Biotechnology, 24, 673 – 679.
  • Vodovar, N., Vinals, M., Liehl, P., Basset, A., Degrouard, J., Spellman, P., Boccard, F., and Lemaitre, B. (2005) Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species. Proceedings of the National Academy of Sciences USA 102 : 11414-11419
  • Boccard, F., Esnault, E., Valens, M. (2005) Spatial arrangement and macrodomain organization of bacterial chromosomes. Molecular Microbiology 57 :9-16.
  • Moulin, L., Rahmouni, A.R., Boccard, F. (2005) Topological insulators inhibit diffusion of transcription-induced positive supercoils in the chromosome of E. coli. Molecular Microbiology, 55 : 601-610.
  • Valens, M., Penaud, S., Rossignol, M., Cornet, F., Boccard, F. (2004) Macrodomain organization of the E. coli chromosome. EMBO J., 23 : 4330-4341.

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