Bacterial cytokinesis is mediated by the septal ring; a cytoskeletal-like organelle that is associated with the cytoplasmic membrane at the site of division. The long-term goals of the proposed research are to understand the molecular structure, assembly, and function of the septal ring in E. coli. The FtsZ ring is a major component of the septal ring, and its formation is an early step in the assembly of the organelle. FtsZ is a soluble, tubulin -like, GTPase which forms homopolymers in vitro. this self assembly reaction is likely to drive formation of the FtsZ ring in vivo, but how this process is initiated at specific sites in the cell is not known. The recently discovered integral inner-membrane protein ZipA is also an early component of the septal ring, binds FtsZ directly, and is essential for cell division. The primary aims of this proposal are to understand the importance of the ZipA-FtsZ interaction in the assembly/function of the septal ring, as well as to identify and characterize interactions between ZipA and/or FtsZ and other cell components. Specifically: i) to test the idea that ZipA triggers FtsZ polymerization at potential division sites, it will be determined whether ZipA is required for FtsZ ring formation in vivo, and whether purified ZipA affects the GTPase and polymerization activities of FtsZ in vitro; ii) Two proteins, P75 and P41, have already been shown to specifically bind to the FtsZ-ZipA complex, and their identification and characterization will be vigorously pursued; iii) Possible interactions between FtsZ/ZipA and specific candidates (FtsA, PBP3, and MinC) will be tested by measurement of several biochemical parameters in purified systems; iv) Affinity blotting and expression cloning techniques will be used to search for additional interacting factors in an unbiased approach. In addition to FtsZ and ZipA, FtsA is also part of the septal ring, and this is likely to be true for each of the other essential division proteins (FtsI, K, L, N, Q, W) as well. It is also likely that many specific interactions occur among all (putative) septal ring components. Taking advantage of powerful methods that have recently become available, it is proposed to complement and extend the studies above; fluorescence microscopy will be used to localize putative septal ring components (v) as well as to determine the relative order of recruitment of each component into the organelle (vi), and rapid affinity blotting protocols will be applied to efficiently search for possible interactions among all division factors (vii). The results of this project can be expected to provide valuable new insights into the molecular mechanisms that underlie the cell division process.
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