Molecular interactions involved in bacterial mitosis – BactMit

Bacterial proliferation relies on a series of essential processes such as genome expression, replication, genome segregation and recombination. By contrast to most of them, the comprehension of genome segregation is still unclear, and the molecular mechanism that drives active separation of DNA molecules is not yet understood at the molecular level, even for well-studied plasmid systems. Genome segregation has been observed in eukaryotic cells by light microscopy for more than hundred years, allowing the precise definition of steps in this process. In bacteria, direct visualization of segregation events is not possible due to their very small size, close to the limit of detection of light microscopy, to the absence of cellular compartments, and to the presence of genomic DNA throughout the cytoplasm.
Low copy number plasmids require a single locus for their faithful maintenance in growing cells. These partition loci, named par, are the archetype of active segregation systems in bacteria. Most bacterial chromosomes also carry homologues of these plasmid partition systems, encoded in the close vicinity of the origin of replication, leading to the hypothesis that these chromosomally-encoded par systems could actively partition the Origin domain and therefore could be responsible for the initial event of the whole chromosome segregation process. However, owing to size, chromosomes need other general processes, such as DNA compaction by condensin-like protein and topoisomerases, chromosome organisation, dimer-resolution and DNA translocase functions, ensuring mid-cell to be cleared of DNA to allow formation of the division septum. Emerging studies also indicated that chromosome partitioning systems have important networking with cell cycle events.
The aim of this programme is to understand at the molecular level the mechanism that actively and faithfully segregates DNA molecules every generation of the bacterial cell cycle; several models are still in competition. We will focus our study on partition complexes, which are key components of partition machineries, equivalent to kinetochore for eukaryotic cells. We will study in detail all interactions involved in this process. In particular, we will analyse interactions that lead to the assembly of partition complex on the centromere, to its ultrastructural organisation, and to concerted displacement of partition complexes towards opposite poles of the cell driven by the motor ATPase.
We will analyse these interactions on two model systems: investigations on the F partition complex will allow deepening our knowledge on this well-studied model and studies on chromosomal replicons of the pathogenic multichromosomal bacteria B. cenocepacia will extend our understanding, which is essentially unknown, of the assembly of partition complexes onto bacterial chromosomes. With these studies, we intend (i) to determine the specificity determinants necessary for partition complex assembly at a genome wide level, (ii) to unravel their ultrastructural organisation and how they interact with the motor ATPase, and (iii) to develop an “ex vivo” partition assay that will allow further definition and characterization of all steps involved in partition. These aspects represent new approaches to unravelling the bacterial partition mechanism.
This fundamental project is upstream of potential applications. However, understanding in details molecular mechanisms underlying bacterial genome segregation, in particular interactions and specificities between each actors, could in the future allow the exploration for new classes of antibiotics directed toward this important and specific step of the bacterial cell cycle. In addition, in the course of the analysis of the centromere specificity, we will participate to the improvement of the detection limit of the SPRi-Plex technology.