Implication of terminal segregation processes in the Evolution and Maintenance of bacterial Circular Chromosomes – EMC2

The genome of several members of many different bacterial genera is divided on multiple non-homologous chromosomes. For instance, the genome of Vibrio cholerae, which lives as a saprophyte in briny waters but also causes cholera in humans, consists of two circular DNA molecules of 2.96 Mbp and 1.07 Mbp. Multipartite genomes in bacteria seem to offer a selective advantage for the adaptation to very different environmental conditions. Nevertheless, most bacteria harbour a single chromosome, which suggests that the machineries responsible for the coordinated replication and segregation of the bacterial genome with cell division are not easily adapted to the management of multiple chromosomes and/or that this adaptation imposes a heavy burden on the metabolism of the cell. In contrast to bacteria, there is no apparent limit to the size and numbers of chromosomes in eukaryotic cells. A major difference between bacteria and eukaryotes is intrinsic to the structure of chromosomes: in bacteria, chromosomes are generally covalently closed circular DNA molecules while they are linear in eukaryotes. DNA circularity can result in the formation of two major topological threats for the segregation of genetic information at the time of cell division, chromosome dimers and catenated chromosomes: catenation links result from the replication of circular chromosomes because of the helical nature of DNA; chromosome dimers are created by odd numbers of crossovers due to homologous recombination between circular chromosomes. We refer to the resolution of these topological problems as terminal segregation. Our past research efforts have been devoted to the understanding of the mechanisms of terminal segregation in Escherichia coli, which harbours a single chromosome. We reasoned that in bacteria with multiple circular non-homologous chromosomes, more numerous and/or novel kinds of topological problems could arise. For instance, catenation links could be trapped between non-homologous chromosomes when a double strand break is repaired by homologous recombination. Thus, DNA circularity could be one of the major limits to the number of chromosomes in bacteria. This prompted us to explore the importance of terminal segregation in the Evolution and Maintenance of multiple non-homologous Circular Chromosomes. We chose to work with V. cholerae as a model organism because of its importance in health and economical problems, the availability of a complete genome sequence, its easy handling in the laboratory, the existence of methods to generate gene knockouts or replacements, large enough cells for microscopic observation of the cellular localisation of proteins or chromosome regions, and because of its relatively close relationship to E. coli, our previous model organism. Finally, animal models exist to study gut infection by V. cholerae, giving the opportunity to perform some of the studies in a more 'natural' environment than petri dishes. In this project, we want to determine the different kinds of topological problems that the presence of the two circular non-homologous chromosomes generates in V. cholerae, how frequently these events occur and what are the molecular mechanisms that allow their resolution. To this aim, we propose to use a combination of classical molecular biology and genetics to dissect the different functions performed by FtsK in V. choleare, a protein that plays a central role in terminal segregation. We will also use fluorescence microscopy to directly compare the timing of activity of FtsK with the timing of segregation of the chromosomes in live cells. Chromosomal regions and cell division proteins will be simultaneously visualized using available fluorescent proteins of different colours. The observation of the formation of active FtsK complexes will be based on FRET. Finally, we will develop bacterial chromosome engineering techniques to modify the architecture of the V. cholerae genome, and thereby directly test how the presence of a single circular chromosome, of two non-homologous circular chromosomes or of combinations of circular and linear chromosomes affect the frequency and kind of topological problems that can be created. In addition to their importance for our understanding of the life cycle of bacteria with multipartite genomes, this exploration should also shade a new light on the mechanisms controlling terminal segregation in bacteria in general.