Dokument

Summary:
Accurate positioning of the division site is essential to produce daughter cells with the correct size, and chromosome content. Generally, the first known event of bacterial cell division is the accumulation of FtsZ at the incipient cell division site to form the circumferential, ring-like structure Z-ring. In bacteria, positioning of the division site occurs at the level of formation of the FtsZ-ring. While the cytokinesis machinery is conserved throughout bacteria, the mechanisms to position the Z-ring are diverse. The rod-shaped social bacterium Myxococcus xanthus divides precisely by binary fission at midcell but it lacks all the known systems that regulate Z-ring formation in other bacteria. Instead two novel regulators PomX and PomY together with the ParA ATPase PomZ stimulate formation and positioning of the Z-ring at midcell. PomXYZ interact and form a complex that associates with the nucleoid to translocate towards the mid-nucleoid, which coincides with midcell before nucleoids have segregated, by biased random motion. At the mid-nucleoid, at midcell, the PomXYZ complex undergoes constrained motion, not leaving midcell. Our experimental data show that cluster localization at midcell is independent of FtsZ and that clusters localize at midcell before Z-rings form. By contrast cluster localization at midcell depends on the ATPase PomZ, its associated ability to hydrolyze ATP and its ability to bind non-specifically to DNA. ATP-hydrolysis by PomZ is stimulated by PomX as well as by PomY in the presence of DNA, demonstrating that PomZ is the first ParA ATPase in which ATP-hydrolysis is stimulated by two ATPase activating proteins (AAP’s). We show that interference with ATP-hydrolysis, by mutational analysis, affects cluster translocation towards midcell. PomZ on its own interacts with the nucleoid and recruits a complex of PomX and PomY to the nucleoid. By FRAP experiments we show that PomZ is highly dynamic in the PomXYZ complex, where ATP-hydrolysis takes place and on the nucleoid. Our experimental data support a flux-based mechanism for the positioning of the PomXYZ complex by the ParA ATPase PomZ. In this model, the diffusive random PomZ dynamics on the nucleoid result in diffusive fluxes of PomZ on the nucleoid from either side into the PomXYZ cluster. These fluxes scale with the cellular asymmetry of the cluster within the cell and convert this cellular asymmetry into a PomZ concentration gradient over the PomXYZ complex. This gradient together with the PomZ-associated ATP-hydrolysis translocates the complex towards the higher concentration of PomZ to midcell. At midcell, which coincides with middle of the nucleoid, the diffusive PomZ fluxes equalize, resulting in constrained motion. To understand the molecular details of ATP-hydrolysis, a previously identified pomXK13A,R15A mutant was analyzed for its defect in PomXYZ-dependent positioning of the cell division site. Our in vivo and in vitro data show that the PomXK13A;R15A variant has a defect in stimulating PomZ ATP-hydrolysis, strongly suggesting that the N-terminal part of PomX interacts with PomZ to stimulate ATP-hydrolysis, similar to other ATPase activating proteins. In summary we identified two novel cell division regulators that work in concert with PomZ to position and promote cell division at midcell. Our data provides insights into the function of ParA-ATPases, which in this case is PomZ, and reveal that the PomXYZ system is a novel system that positions cell division at midcell most likely by recruiting the Z-ring to the incipient cell division site.

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