OUTER MEMBRANE CYTOCHROMES--TOPOLOGY AND FUNCTION

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DESCRIPTION: A thorough understanding of anaerobic respiraiton is essential to understanding bacterial energy generation and survival and growth in anoxic or hyosix environments in humans and environmental reservoirs. While many bacteria can use soluble electron acceptors, Shewanella putrefaciens MR-1 can also use highly insoluble substrates (e.g. oxidized manganese or iron) for anaerobic respiration. This bacterium has a novel cytochrome distribution, localizing most of its membrane-bound cytochromes to its outer membrane (OM) under anaerobic growth conditions, where they could potentially make direct contact with extracellular insoluble electron acceptors. The long-term goals of our research are to use this bacterium as a model to understand electron transport systems involved with the use of insoluble electron acceptors, with particular emphasis here on the role of OM cytochromes. We propose that one or more of the OM cytochromes are required for the use of insoluble electron acceptors, and that one or more of these cytochromes is exposed on the cell surface (where they could directly contact the insoluble electron acceptors). The specific aims of this proposal are: To determine if the OM cytochromes are required for the use of insoluble electron acceptors, or other anaerobic respiratory processes. Mutants that are deficient in one or more OM cytochromes will be generated and studied for their anaerobic respiratory capabilities. We will restore these mutants to wild-type by complementation with wild-type DNA and then re-examine their electron transport capabilities. To determine the cell surface exposure of the OM cytochromes in anaerobically grown cells. A multifaceted approach will be used, including immunoadsorption, radioiodination of cell surface components, and protease susceptibility studies. These studies will provide essential information on a novel energy-generating electron transport chain that appears to involve components of both the cytoplasmic and outer membrane; they will broaden the current vision of anaerobic respiration as a process not only confined to the cytoplasmic membrane and periplasm, but one which can include electron transport links to the extracellular environment through components in the OM. This will greatly enhance our understanding of bacterial survival strategies under diverse environments and could eventually lead to new strategies for controlling or inhibiting bacterial growth. They will also contribute to environmental health by expanding our knowledge on the mechanisms by which manganese, iron, and various heavy metals are mobilized into water supplies.