Search






Supervisory authorities

Our partners


Home > Departments > Genome Biology > Frédéric BOCCARD : Conformation and segregation of the bacterial chromosome

Frédéric BOCCARD : Group Presentation

Our project aims to reveal the structuring of the chromosome in Escherichia coli and in Pseudomonas species. The objectives are multiple: i) to reveal the principles of chromosome organization, ii) characterize the molecular mechanisms involved, iii) analyze the coordination of chromosome segregation with progression of the cell cycle.

Organization of the bacterial chromosome

The size of DNA molecules is much higher than that of living cells containing them. Condensation of DNA molecules results in the formation of structures called chromosomes. In bacteria, the chromosome is a folded structure called nucleoid. The molecular bases that direct the spatial architecture of the chromosomes have not been identified. Our laboratory is interested in the processes that organize large-scale of two model chromosomes, that of the bacteria Escherichia coli and Pseudomonas aeruginosa.

Organization of the chromosome in E. coli

In 2004, our team has demonstrated that the spatial arrangement of the chromosome in this bacterium was based on the existence of four major structured regions, called macrodomains. In 2008, we identified the nature of the structuring process of one of macrodomains; a new protein, called MatP recognizes specific sites of 13 base pairs (matS sites) present in twenty copies in this 800-kb macrodomain. In recent years, we also identified the nature of the Ori and lateral Left/Right macrodomains. The Ori region of the E. coli chromosome is organized as a MD with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. We have unravelled the structuring nature of the Ori MD and identified the MaoP/maoS system responsible for specifying its properties. A single locus, maoS, acts as a unique organizing center from which MD properties are spread to the entire Macrodomain. 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. We also revealed that the difference between the non-structured regions and the Right and Left lateral MDs relies on their position on the genetic map. A change in the genetic location of oriC generated either by an inversion within the Ori MD or by the insertion of a second oriC modifies the position of Right and Left MDs, 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. 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 lateral MDs in growing cells.
Using 3C-methods combined with genomics together with fluorescence microscopy and genetic approaches, we have disclosed the three-dimensional folding of the E. coli chromosome and characterize the activity of several factors involved in chromatin organization and nucleoid conformation. At short scale, the contact map revealed the presence of domains ranging in size from 50kb to 300kb with long and highly expressed genes frequently found between domains. At large scale, long distance contacts accounted for the presence of macrodomains. By analysing the contact frequency in several mutants, we revealed the modus operandi of the main structural factors as well as their interplay in controlling short- and long-range DNA contacts.

  • The interaction of MatP with the protein ZapB associated to the divisome promotes the anchoring of the ter at midcell. In vitro microscopic observations of MatP-dependant loops of DNA molecules carrying multiple matS suggested that ter organization was mediated by the bridging of distant matS sites. However, 3C analyses did not unveil any discrete in vivo intrachromosomal matS-matS contacts. Furthermore, the chromosome and plasmids carrying matS sites were not brought together in the absence of ZapB, but in the presence of MatP. The same plasmids in the presence of ZapB contacted ter but no discrete matS-matS interactions could be identified. Combined, these results show that MatP does not promote DNA bridges between matS sites neither in cis nor in trans. They also reveal that MatP-ZapB interactions at the divisome are responsible for the clustering of distinct DNA molecules carrying matS sites.
  • Unlike SMC proteins that align chromosome arms in several bacteria, our data support a model where the MukBEF complex promotes long-range contacts along the chromosome arms and where MatP restricts such interactions in the ter region. The interplay of MatP with MukBEF complex cements the role of MatP as an important player promoting the formation of a chromosomal domain by exclusion of the condensin complex.
  • Decades of biochemical and genomic studies have been carried out for the three important NAPs: HU, Fis and H-NS. Yet, the relation between local DNA binding and the in vivo organization of chromosomal DNA over long range scales remained to be tackled. We have identified important insights about the activities of three major NAPs in the control of chromosome conformation: H-NS affects short range contacts while HU and Fis promote long-range contacts in different ways. H-NS effect is in agreement with its modus operandi of silencing extensive regions by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. HU is required along with MukBEF to promote DNA communications in the Mb range. The absence of Fis is less dramatic than HU as the decrease of long-range DNA communications varies along the chromosome. Altogether, by disclosing the different levels of DNA constraints along the chromosome, our results reveal general principles of genome folding in E. coli.
Model for long-range interactions in the E. coli chromosome

Organization of the chromosome in P. aeruginosa

We further highlighted the diversity in chromosome organization by revealing a specific organization for the chromosome of Pseudomonas aeruginosa, an important opportunistic pathogen belonging to the γ-proteobacteria class. We showed that the 6.3 Mb unique circular chromosome of P. aeruginosa is globally oriented from the old pole of the cell to the division plane/new pole according to the oriC-dif axis. The replication machinery is positioned at mid-cell, and chromosomal loci from oriC to dif are moved sequentially to mid-cell prior to replication. The two copies are subsequently segregated to their final subcellular destination in the two halves of the cell. We identified two regions, one surrounding oriC and another surrounding dif, where several chromosomal loci show a biased positioning pattern inside the cell, suggesting that processes responsible for long range chromosome organization exist in P. aeruginosa.

Model of P. aeruginosa chromosome organisation

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 P. aeruginosa, where impairing the ParABS system leads to the formation of anucleate cells. 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 cells 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” (Lagage et al. PLoS Genetics, 2016).

Contact


BOCCARD Frédéric [Senior Researcher - CNRS]
I2BC [Leader]
Conformation and segregation of the bacterial chromosome [Leader]
Fonctions soutien [Leader]
01 69 82 32 17 equipe Gif - Bât 21

published on , updated on