Regulation of Chlamydial Divisome Assembly - The obligate intracellular pathogen Chlamydia trachomatis is the leading bacterial cause of sexually transmitted infections. This coccoid, gram-negative bacterium does not encode several proteins essential for binary fission including FtsZ, the major regulator of binary fission in most bacteria, and we previously showed that Chlamydia divides by a polarized budding process. Our data indicate that chlamydial budding is driven by a novel hybrid division machinery that contains elements of the divisome (FtsK, PBP3) and elongasome (MreB, MreC, and PBP2) machinery defined in other bacterial systems. However, many questions regarding the mechanisms that regulate this polarized division process remain unanswered. Two of the most interesting questions are how the site of polarized budding is defined in this coccoid organism, and how do MreB, MreC, and PBP2 function with elements of the divisome to drive polarized budding. Although Chlamydia does not encode several proteins essential for binary fission, it expresses FtsK, a DNA translocase that accumulates at the septum of bacteria where it couples the segregation of replicated chromosomes to the septation of daughter cells during division. The translocase activity of FtsK is dependent upon the recognition of specific DNA sequences (KOPS sites) by the γ domain at the C-terminus of the protein. The γ domain of FtsK also interacts with XerD and activates XerCD-dependent recombination at dif sites near the chromosomal terminus, which is required for the decatenation of chromosomal dimers that arise during replication to monomers to enable their segregation. We show here that FtsK in Chlamydia accumulates in two foci in budding cells, one at the septum of the dividing cell and one at the base of the progenitor mother cell. This pattern of localization was also observed for PBP2, PBP3, MreB, and MreC suggesting these proteins function as part of a divisome complex that coordinates chromosomal segregation with septation during division. Our data suggest that the proteins that accumulate at the base of the mother cell correspond to an immature divisome complex where the bud will emerge in the next round of division, and FtsK initiates the assembly of immature divisomes by associating with XerD bound to the chromosomal dif site. The studies described in this proposal will test the novel hypothesis that the site of divisome assembly in Chlamydia is specified by the binding of FtsK to XerD that is associated with the chromosomal dif site. In addition, we will test the hypothesis that interactions between elongasome and divisome components of the hybrid division machinery of Chlamydia are critical for divisome assembly and the recruitment of MreB to the complex activates peptidoglycan synthesis required for polarized budding. Our proposed studies will be significant as they will be the first to define the molecular mechanisms that regulate divisome function in an organism that lacks FtsZ. In addition, defining the mechanisms associated with the novel polarized division process of Chlamydia may lead to the development of specific therapeutics for eliminating chlamydial genital tract infections in humans.
