Spatial regulation of cell division by the nucleoid occlusion protein SlmA in Escherichia coli
University of Kansas
Microbiology, Molecular Genetics & Immunology
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Spatial regulation of cell division in bacteria occurs at the stage of Z ring formation, a cytoskeletal element that bacterial cells employ for assembly of the cell division machinery. In the model organism Escherichia coli, spatial regulation of Z ring formation is dependent on two partially redundant negative regulatory systems, the Min system, which prevents Z ring formation near the cell poles, and Nucleoid Occlusion (NO), which prevents Z ring formation over the nucleoid. The effector of the Min system, MinC, prevents assembly of the Z ring in its vicinity by antagonizing FtsZ polymerization and membrane attachment, the two essential activities required for FtsZ to assemble into the Z ring. Previous studies have shown that the effector of the NO system, SlmA, is a DNA associated FtsZ inhibitor that is activated by binding to a SlmA binding sequence (SBS). The SlmA binding site on FtsZ has not been identified and how SBS bound SlmA prevents FtsZ assembly into the Z ring in its vicinity is controversial. In this study, we show that SBS bound SlmA acts in a similar manner to MinC, antagonizing FtsZ polymerization and membrane attachment. In the first part of this thesis, two FtsZ mutants were isolated that are resistant to de-localized SBS bound SlmA, which has been shown to block Z ring formation throughout the cell and cause cell death. By characterizing these two FtsZ mutants, we found that SBS activated SlmA antagonizes FtsZ polymerization and the efficacy of SlmA to antagonize FtsZ polymerization depends upon the length of the DNA molecule containing the SBS. The longer the bound SBS DNA molecule (14-30 bp), the more efficiently SlmA disassembles FtsZ polymers; SlmA bound to the shorter SBS DNA molecule is missing several DNA contacts likely explaining the weaker impact on FtsZ polymerization. Even though the isolated ftsZ mutations conferred resistance to the action of SlmA in vivo and in vitro, they did not disrupt FtsZ-SlmA binding. One of the ftsZ mutations increased the bundling of FtsZ polymers in vitro, indicating that it provides resistance to SlmA by increasing FtsZ lateral interactions. The other ftsZ mutation alters a residue in the H7 helix of FtsZ. This helix mediates the conformational change between the two sub-domains of FtsZ during assembly suggesting that SBS bound SlmA antagonizes FtsZ polymerization by reversing this conformational change and that the mutation is resistant to this affect. In the second part of the project, we found that SlmA binds to FtsZ largely through the conserved C-terminal tail of FtsZ, a region critical for FtsZ-ZipA and FtsZ-FtsA interactions and therefore attachment of FtsZ filaments to the membrane. More importantly, we found that SlmA requires the presence of the conserved C-terminal tail of FtsZ to disassemble FtsZ polymers. As the conserved C-terminal tail of FtsZ is not required for FtsZ polymerization, this unexpected finding suggests that SlmA binding to the FtsZ tail allows it to bind to a secondary site in the globular domain of FtsZ to antagonize FtsZ polymerization. This two binding site model is consistent with the observation that SlmA forms a sandwich like complex with FtsZ truncations lacking the conserved C-terminal tail and our finding that ftsZ mutations in the globular domain of FtsZ confer resistance to the action of SlmA. Collectively, our results suggest that SlmA antagonizes Z ring formation in its vicinity in at least two ways: first, SBS bound SlmA competes with ZipA and FtsA for the conserved C-terminal tail of FtsZ preventing membrane attachment of FtsZ filaments; and, second, the binding to the conserved C-terminal tail of FtsZ brings the SBS-SlmA complexes close to FtsZ filaments such that SlmA can actively disassemble FtsZ polymers by reversing the conformational change occurring upon FtsZ assembly. ZipA and FtsA promote Z ring assembly by tethering FtsZ filaments to the membrane through the conserved FtsZ tail. In contrast, MinC and SlmA promote Z ring disassembly by binding the tail because they also have an antagonistic effect on FtsZ polymers. Competition for the FtsZ tail between Z ring promoting factors and Z ring disassembling factors may be an important way to regulate Z ring formation. The remarkable similarity between MinC and SlmA also indicates that antagonizing FtsZ polymerization and FtsZ filaments membrane attachment simultaneously may be a universal mechanism for FtsZ spatial regulators to antagonize Z ring formation in their vicinity.
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