Genesis and maintenance of bacterial secondary chromosomes – MAGISBAC

The project is divided in five tasks:
I) In silico analysis of bacterial secondary replicons with emphasis on enterobacteria. In particular, analysis of housekeeping functions involved in replication, conjugation and segregation. Identifying key evolutions associated with plasmid domestication and finally modeling the process of domestication.
II) In silico building of a predictive model for secondary replicon folding and positioning using a polymer thermodynamics framework that successfully predicted the fate of chromosomes.
III) Establishing the mechanisms of two key events distinguishing plasmids from secondary chromosomes - the control of replication initiation and the pattern of segregation - in enterobacteria and vibrionaceae.
IV) Generalise these findings to other bacteria of biomedical and industrial interest using genome-wide approaches.
V) Integrating the results obtained to design de novo bacterial replicon with predicted properties.

We have analysed the dynamics of secondary replicons, the mechanisms used and their evolution, focussing on two major processes for mechanistic studies: the control of replication and sister replicons unlinking by TopoIV (resolution of intercatenated forms) and XerCD/dif recombination (resolution of dimeric forms).
We have shown that:
- Replication of Vibrio cholera Chr2 is triggered by a 150-bp locus positioned on Chr1, called crtS, explaining how the two chromosomes coordinate their replication (Val et al, 2016; Bland et al, 2017).
- The location of genes is correlated with fitness in V. cholerae (Soler-Bistué et al, 2017).
- Xer recognition sequences (xrs) are a hotspot for decatenation by TopoIV (El Sayyed et al, 2016).
- The FtsK control protein can differentiate between xrs devoted to dimer resolution from those involved in mobile elements integration (Fournes et al, 2016).
- Integrative and extra-chromosomal elements have specific traits that can be advantageous in different situations and may explain the continual existence of domesticated plasmids (in progress).
- A third of a collection of 1000 enterobacterial secondary replicon carries xrs. Both the presence of xrs and the way recombination is controlled depends on the size of the replicon. Clearly larger replicons (above 200 kb) carry xrs with very few exceptions and use a chromosome-like mechanism involving the FtsK protein (in progress).
- In a Worm Like Chain polymer models, replicon behave differently depending on their size (in progress).
- Synthetic replicons carrying the V. cholerae chr2 replication origin. replicate in E. coli in a controlled manner depending on the presence and positioning of the crtS site (in progress).

Our project aims at providing both a global and detailed view of how large mobile genetic elements with capacities to profoundly change the physiology of bacteria adapt to their host and become domesticated. This involves thorough in silico analysis to establish both global rules for the entire bacterial world and the specificities of different taxa. Importantly, this global view integrates a detailed description of key mechanisms that evolve in their way to domestication. These mechanisms are investigated in model organisms but we will also extend functional approaches to other bacteria of biomedical interest. Tackling these important questions by a multidisciplinary approach is leading to several publications in both specialised and top ranking general journals. In addition, mathematical modelling produces a general predictive model for replicons dynamics in bacteria valuable for future studies and bioinformatics produce original programs and databases.
The mechanisms involved in maintenance of large plasmids and secondary chromosomes are poorly understood. These replicons however carry complex traits involved in interaction of bacteria with eukaryotes and therefore merit particular attention. In particular, they are frequently involved in the development of pathogenesis. These replicons are thus important targets to consider when designing new antibacterial strategies. Understanding how they evolve and are maintained in bacteria is of primary importance in this respect.
Understanding the fate of large bacterial replicons is also of crucial importance for synthetic biology. It is now possible to synthetize a whole bacterial genome. Constructing new organisms with complex new traits requires designing large DNA constructs along with the associated replicons. Our project provides a blueprint for such replicons and should change the way we anticipate the construction of synthetic organisms with new traits and properties.

- Bland, M.J., Ducos-Galand, M., Val, M.-E., and Mazel, D. (2017). An att site-based recombination reporter system for genome engineering and synthetic DNA assembly. BMC Biotechnol. 17.
- Fournes, F., Crozat, E., Pages, C., Tardin, C., Salomé, L., Cornet, F., and Rousseau, P. (2016). FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex. Proc. Natl. Acad. Sci. 201523178.
- Sayyed, H.E., Chat, L.L., Lebailly, E., Vickridge, E., Pages, C., Cornet, F., Lagomarsino, M.C., and Espéli, O. (2016). Mapping Topoisomerase Iv Binding and Activity Sites on the E . Coli Genome. PLOS Genet 12, e1006025.
- Soler-Bistué, A., Timmermans, M., and Mazel, D. (2017). The Proximity of Ribosomal Protein Genes to oriC Enhances Vibrio cholerae Fitness in the Absence of Multifork Replication. mBio 8.
- Val, M.-E., Marbouty, M., Martins, F. de L., Kennedy, S.P., Kemble, H., Bland, M.J., Possoz, C., Koszul, R., Skovgaard, O., and Mazel, D. (2016). A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae. Sci. Adv. 2, e1501914.
- Vickridge, E., Planchenault, C., Cockram, C., Junceda, I.G., and Espéli, O. (2017). Management of E. coli sister chromatid cohesion in response to genotoxic stress. Nat. Commun. 8, 14618.
- Logiciel ConjScan, Cury J and Rocha EPC, article submitted, available at github.com/gem-