All cells exhibit biochemical reactions which occur exclusively at specific locations within their membranes. The fundamental question is why the reactions occur at one site instead of a seemingly equivalent location elsewhere. The two most significant of these processes in the bacterium Escherichia coli are related to cell division: formation of the septal division plane, and chromosomal segregation to daughter cells. The long-range objective is to understand the molecular mechanism by which the cell controls the site-specificity of these events. This proposal is designed to determine how the membrane of E. coli contributes to this mechanism. Improvements will be made in methods which previously demonstrated that membrane vesicles can be separated into classes more extensive than the classical inner/outer membrane definition. These methods include separation by charge (electrophoresis of vesicles through dilute agarose) and by size (chromatography through Sephacryl- SlOOO. Additional techniques will be developed, including rapid assays and fractionation based on lectin affinity. These methods will help answer the following questions: do proteins have specific sequences which direct them to different vesicle sub-populations; do major cell division proteins interact with one another in """"""""septation subdomains;"""""""" and does the chromosome interact with a particular membrane structure? The first question will be addressed by surveying the location of proteins fused to the reporter enzyme, alkaline phosphatase. The remaining questions will be addressed by: immunoblot localization of proteins in vesicles of wild-type and mutant bacteria; localization of penicillin-binding proteins; genetic characterization using the X174 lysis protein E; and nucleic acid hybridization to detect DNA- vesicle associations, using oriC and mini-F plasmids as experimental probes. In the long term, knowing how E. coli creates and maintains organization within its membrane and how that structure contributes to cell division may form the basis for understanding how biochemical processes are spatially restricted, how this arrangement regulates cell growth and division, and how these processes may be inhibited to control bacterial infections.
