The long term objective of this proposal is to determine the molecular mechanisms involved in the process of cell division in bacteria. Our efforts have focused on the ftsZ gene, which is an essential cell division gene in both E. coli and B. subtilis. Previously, we have shown that FtsZ is localized at the leading edge of the septum in these bacteria in a pattern designated the FtsZ ring. A variety of evidence indicates that this ring is a key feature of bacterial cell division. A model was proposed that suggested the FtsZ ring was a cytoskeletal element that mediated septation. More recent findings show that FtsZ is a GTPase and self assembles into filaments in vitro which is consistent with the model. The present proposal will use a more sensitive immunofluorescent technique to further explore FtsZ localization to try and determine the requirements for FtsZ localization. In addition, the sensitivity of this technique should allow the localization of other division proteins to be addressed. Combining this technique with genetics should allow a dependency pattern for localization of proteins to the division site to be established. In order to further characterize protein interactions occurring among division proteins and to identify potential novel proteins, especially the hypothetical protein responsible for targeting FtsZ to the division site, we will use the yeast 2-hybrid system as well as biochemical approaches. We have already used the yeast 2-hybrid system to document interaction between FtsZ and both FtsA and the division inhibitor SulA. In addition, interaction among the Min proteins, involved in division site selection, has been characterized and will be further studied as these proteins must also contact topological markers. Further study will be conducted on FtsZ assembly. Using the mutants we have available and we will examine the effects of SulA and FtsA on the assembly process.
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