OUTER MEMBRANE CYTOCHROMES--TOPOLOGY AND FUNCTION

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Anaerobic respiration provides a mechanism to energize the transmembrane proton translocation machinery in the absence of O2. A thorough understanding of anaerobic respiration is essential to understanding bacterial survival in anoxic or hypoxic environments in the human host. Several bacteria, including many human pathogens and components of the normal flora, are capable of anaerobic respiration using soluble compounds (e.g. nitrate, fumarate, etc.) as terminal electron acceptors. Shewanella putrefaciens MR-I can also respire using these soluble compounds but, in addition, it can use solid substrates (e.g. iron(Ill) and manganese(IV)) as electron acceptors. Recent studies in our lab have shown that MR-I localizes a majority of its membrane-bound cytochromes to the outer membrane (OM) when grown under anaerobic conditions. This novel cytochrome distribution could have profound implications for this bacterium's ability to use solid substrates as terminal electron acceptors. We have also shown that (a) the majority of formate-dependent ferric reductase activity is localized in the OM, (b) inhibitors of cytochrome electron transport inhibit the reduction of manganese and iron by MR-I, and (c) formate- dependent ferric reductase of the OM involves c-type cytochromes. Together these data suggest a role for these OM cytochromes in respiratory-linked metal reduction. It seems likely that the terminal components responsible for the transfer of the insoluble electron acceptors must be exposed on the cell surface; the OM cytochromes represent likely candidates for such an electron transfer role. The specific aims of this proposal are: (I) to purify at least two of the three OM cytochromes; and (2) to generate polyclonal antibodies against the purified OM cytochromes, and verify that these antibodies are specific for the respective cytochromes. These studies will provide important tools for future use in the study of the membrane topology and physiologic role of these outer membrane cytochromes.