Dissecting the molecular mechanism of directional centromere DNA segregation in Caulobacter Crescentus

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The faithful segregation of chromosomes during cell division is of critical importance for the inheritance of genetic material in all cells. In the bacterium Caulobacter crescentus chromosome replication and segregation occur simultaneously. Initially, the two centromere-like Par proteine complexes near the replicated origin of replication are localized at one cell pole. Then, chromosome segregation is initiated by the rapid movement of one of these complexes from one cell pole to the other. Despite previous identification of key players for Caulobacter chromosome segregation in Caulobacter the generation of force to drive this transport process is not known, nor is the mechanism that orients pole directed transport. Chromosome segregation has been shown to depend on the Par protein system and known polar localized proteins. However, three models have proposed different mechanisms for how subgroups of these proteins interact to mediate chromosome segregation. In the proposed project I will test these models and experimentally determine the mechanism for the transport of the centromere-like complex. By using a combination of cell-free reconstitution techniques, genetic perturbation assays and single-molecule microscopy I will quantitatively analyze the minimal biochemical requirements for centromere segregation in Caulobacter and determine how polar matrix and spatial constrains influence chromosome transport. This projects will provide novel insights into the quantitative dynamics of the centromere transport and yield a deep molecular understanding of the initial steps of bacterial chromosome segregation.