The molecular mechanisms that serve to couple DNA replication, chromosome segregation and cell division are largely unknown in bacteria. This led a considerable interest to the study of Escherichia coli FtsK, an essential cell division protein that assembles into DNA-pumps to transfer chromosomal DNA between the two daughter cell compartments during septation. Indeed, our recent work suggests that FtsK might regulate the late stages of septation to ensure DNA is fully cleared from the septum before it is allowed to close. This would be the first example of a cell cycle checkpoint in bacteria.FtsK-mediated DNA transfer is required in 15% of the cells at each generation in E. coli, in which it serves to promote the resolution of topological problems arising from the circularity of the chromosome by Xer recombination. However, the FtsK checkpoint could be a more general feature of the bacterial cell cycle since FtsK is highly conserved among eubacteria, including species that do not possess a Xer system. Indeed, preliminary results from the lab indicate that DNA transfer by FtsK is required independently of Xer recombination in Vibrio cholerae.To confirm the existence and the generality of the FtsK checkpoint in bacteria, we will determine the different situations that lead to a requirement for FtsK-mediated DNA transfer by studying chromosome segregation and cell division in V. cholerae. In parallel, we will apply new fluorescent microscopy tools to follow the progression of cell division and chromosome segregation in single live bacterial cells. PALM will notably serve to probe the structure of the FtsK DNA-pumps at a high spatial resolution, FRET will be used to determine their timing of assembly and their interactions with the other cell division proteins, and TIRF will serve to follow in real time their activity with respect to the progression of chromosome dimer resolution, chromosome segregation, and septum closure.