The long-range goal of this project is to determine the mechanism used by bacteria to select the proper division site at midcell in preference to other potential division sites that are located elsewhere within the cell. Since the products of the three min genes, minC, minD and minE, are responsible for this site-selection process, we will attempt to define the mechanism of action of each of the gene products in the site-selection process. Previous work has shown that MinD localizes to potential division sites at poles and midcell in the absence of the other Min proteins, and that coexpression of MinE or domains of MinE causes redistribution of membrane-associated Gfp-MinD into structures whose position depends on the presence of the MinE topological specificity domain. The major immediate aims of the proposal will be: i. To define the determinants in MinD that are responsible for its localization to poles and midcell, its role in formation of the MinE ring, and its ability to activate the MinC division inhibitor; ii. To determine the molecular basis of the different membrane-associated MinD structures that are induced by coexpression of Gfp-MinD with MinE and with the N-terminal region of MinE, using a combination of fluorescence microscopy, molecular genetics, and membrane-biochemistry; iii. To determine the localization pattern of MinC and the effects of MinC on localization of MinD and MinE; iv. To complete the high-resolution three-dimensional structure of the MinE topological specificity domain and to map topological specificity mutations of MinE to the three-dimensional structure; v. To characterize new minicell mutants that map outside of the minCDE locus and determine whether any of them code for the topological targets for MinD and MinE localization.
