Principal Investigator: Olivier ESPELI, DR2 Cnrs
Genetic and cell biology analysis of chromosome conformation in bacteria
Our laboratory is interested in the analysis of the dynamics chromosome conformation in response to stimuli or stresses coming from the cytoplasm or the environment. We use a combination of genetic, genomic and cell biology tools to monitor chromosome conformation and movements.
Chromosomes are extremely large DNA molecules that are confined in the small space of the nucleus or the nucleoid in bacteria. This strong compaction process relies on a mosaic of structuring factors. An important aspect of the compaction process is that it must be sufficiently plastic to allow large scale deformations that are imposed by the cell cycle during replication, cohesion of sister chromatids and segregation steps (Espeli et al 2008; Possoz et al 2012) and local conformational changes imposed by the expression of genes in response to environmental stimuli or the repair of DNA lesions.
Functions of the E. coli macrodomains
Model bacteria (E. coli, B. subtilis or C. crescentus) are the organisms where chromosome structuring is more extensively described. The DNA is folded into plectonemic supercoiled loops of 10 kb, these loops are assembled in larger domains of 1 MB called macrodomains. We have recently characterized the system that is responsible for the structuring of one macrodomain (the ter Macrodomain) in E. coli. It relies on a protein MatP that can bind to a multiple sites present only in the macrodomain (Mercier et al 2008) (figure 1). MatP provokes the condensation of the Ter macrodomain but also drives its localization to a specific localization in the cell. The Ter macrodomain is anchored at mid-cell to the protein ring in charge of cell division for a long period of the cell cycle (Espeli et al 2012). This specific anchoring of a large region of the chromosome to the machinery responsible of cell division allows a perfect coordination of the chromosome segregation and cell division in the purpose to preserve genomic integrity.
We are now interested in understanding two aspects of macrodomain function: first, their influence on the management of DNA topology during the cell cycle and second, their interplay with the control of gene expression. We are combining transcriptomics and bioinformatics approaches with single cell imaging techniques to monitor chromosome localization variation in response to gene expression and macrodomain structuring.
Sister chromatid cohesion in E. coli
Our work on macrodomains revealed that newly replicated regions remain in close proximity with their sister for 10 to 40 minutes after their replication. This period by homology with the eukaryotic systems has been called sister chromatid cohesion. In eukaryotes a protein complex called cohesins mediates the cohesion. We developed an original assay that measure the proximity of sister loci. We demonstrated that in bacteria the driving force for sister chromatid cohesion is DNA topological links and that topoisomerases are the main regulators of the extent of cohesion (Lesterlin et al 2012). Our working model proposes that topological links, called precatenanes, are formed in between newly replicated chromatids during their replication, these links could be in a sufficient density to bridge the sister chromatid over a 500 to 1000 kb distance (figure 2). At this point a licensing system could allow topoisomerases to remove the topological links. This will trigger chromosome segregation.
We are currently testing this model. We use genomic approaches to map topoisomerases activities on the chromosome, we use molecular biology assay to measure precatenation density and test its relationship with the extent of sister loci cohesion.
In eukaryotes and bacteria the sister chromatid cohesion period represent a significant part of the cell cycle from 20 to 80% according to the loci and organism considered. Alteration of the mechanisms involved in sister chromatid cohesion have severe consequences on chromosome segregation suggesting that cohesion is an essential step of the segregation process, perhaps because it allows the brother chromosomes to accumulate sufficient repulsive forces to provoke a rapid separation when cohesion links will be broken (for review Possoz et al 2012, Le Chat and Espéli, 2012).
Sister cohesion is also important to facilitate DNA repair by homologous recombination. We have observed that some genotoxic molecules provoke an elongation of the sister chromatid cohesion period in E. coli. We are currently trying to understand the mechanisms involved in this process and its usefulness for DNA repair.
Figure 1: Spatial organization of the E. coli chromosome. Three regions of the chromosome were tagged with fluorescent proteins (red, green, blue) the bacteria are represented in black.
Figure 2: Topological constraints around the replication fork. Positive supercoils and precatenanes accumulate respectively ahead and behind the replication fork (grey ball)
Impact of chemotherapeutic drugs on E. coli growth in the absence of RecN
The E. coli chromosome is labelled with the HU-mCherry fluorescent protein, mitomycine C is added for 5 minutes to the growing medium then washed. The total length of the experiment is 4 hours.
- Planchenault, C., Pons, M.C., Schiavon, C., Siguier, P., Rech, J., Guynet, C., Dauverd-Girault, J., Cury, J., Rocha, E.P., Junier, I., Cornet, F., Espéli, O. (2020). Intracellular positioning systems limit the entropic eviction of secondary replicons toward the nucleoid edges in bacterial cells. J. Mol. Biol.
- Demarre, G., Prudent, V., Schenk, H., Rousseau, E., Bringer, M.-A., Barnich, N., Tran Van Nhieu, G., Rimsky, S., De Monte, S., and Espéli, O. (2019). The Crohn’s disease-associated Escherichia coli strain LF82 relies on SOS and stringent responses to survive, multiply and tolerate antibiotics within macrophages. PLoS Pathog. 15, e1008123.
- Lioy, V.S., Cournac, A., Marbouty, M., Duigou, S., Mozziconacci, J., Espéli, O., Boccard, F., and Koszul, R. (2018), Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins. Cell.172, 771-783.e18.
- El Sayyed, H., and Espéli, O. (2018), Mapping E. coli Topoisomerase IV Binding and Activity Sites. Methods Mol. Biol. 1703, 87–94.
- Demarre, G., Prudent, V. & Espéli, O. (2017), Imaging the Cell Cycle of Pathogen E. coli During Growth in Macrophage. Methods Mol. Biol. 1624, 227–236.
- Vickridge, E., Planchenault, C., Cockram, C., Junceda, I.G. & Espéli, O. (2017a), Management of E. coli sister chromatid cohesion in response to genotoxic stress. Nat Commun 8, 14618.
- Vickridge, E., Planchenault, C. & Espéli, O. (2017b), Revealing Sister Chromatid Interactions with the loxP/Cre Recombination Assay. Methods Mol. Biol. 1624, 29–37.
- El Sayyed, H., Le Chat, L., Lebailly, E., Vickridge, E., Pages, C., Cornet, F., Cosentino Lagomarsino, M. & Espéli, O. (2016), Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome. PLoS Genet. 12, e1006025.
- Lagomarsino M.C., Espéli O. & Junier I. (2015), From structure to function of bacterial chromosomes: Evolutionary perspectives and ideas for new experiments. FEBS Lett. 589, 2996–3004.
- Passot, F.M., Nguyen, H.H., Dard-Dascot, C., Thermes, C., Servant, P., Espéli, O. & and Sommer, S. (2015), Nucleoid Organization in the Radioresistant Bacterium Deinococcus radiodurans. Mol. Microbiol. 97, 759–774.
- Junier I., Boccard F. & Espéli 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 Res. Feb 1;42(3):1461-73.
- Le Chat L. & Espéli O. (2012), Let's get 'Fisical' with bacterial nucleoid. Mol Microbiol. Dec;86(6):1285-90.
- Dame R.T., Espéli O., Grainger D.C. & Wiggins P.A. (2012), Multidisciplinary perspectives on bacterial genome organization and dynamics. Mol Microbiol. Dec;86(5):1023-30.
- Lesterlin C., Gigant E., Boccard F. & Espeli O. (2012), Sister chromatid interactions in bacteria revealed by a site specific recombination assay EMBO J, August 15. 31:3468-79.
- Espeli 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, May 11. 31:3198-11.
- Possoz C., Junier I. & Espeli O. (2012), Bacterial chromosome segregation. Front Biosci 17: 1020-1034.
- Rabhi M., Espeli O., Schwartz A., Cayrol B., Rahmouni A.R., Arluison V. & Boudvillain M. (2011), The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators. EMBO J, 30: 2805-2816.
- Mercier R., Petit M.A., Schbath S., Robin S., El Karoui M., Boccard F. & Espeli O. (2008), The MatP/matS site-specific system organizes the terminus region of the E. coli chromosome into a macrodomain. Cell 135(3): 475-485.
- Espeli O., Mercier R. & Boccard F. (2008), DNA dynamics vary according to macrodomain topography in the E. coli chromosome. Mol Microbiol 68(6): 1418-1427.
Espéli Olivier, DR2 CNRS
Rimsky Sylvie, DR2 CNRS
Postdoctoral fellows, PhD Students & Master student:
Singh Parul, Postdoctoral fellow
Camus Adrien, PhD student
Bruder Emma, Master student
Jamai Widjane, Master student
Quenech’du Nicole, IEHC CDF