OUTER MEMBRANE CYTOCHROMES--TOPOLOGY AND FUNCTION

无氧呼吸提供了一种机制，使 无氧条件下的跨膜质子转运机。一个 对无氧呼吸的透彻了解对于 了解细菌在缺氧或低氧环境中的生存情况 人类的主人。几种细菌，包括许多人类病原体和 正常菌群的组成部分，能够进行厌氧呼吸 使用可溶化合物(如硝酸盐、富马酸等)AS终端 电子受体。腐败希瓦氏杆菌MR-I也能呼吸 使用这些可溶化合物，但除此之外，它还可以使用固体 作为电子受体的底物(如铁(11)和锰(4))。 我们实验室最近的研究表明，MR-I定位了大部分 生长时与外膜(OM)结合的细胞色素 在厌氧条件下。这种新的细胞色素分布可以 对这种细菌使用固体的能力有深远的影响 作为终端电子受体的底物。我们还表明：(A) 依赖于甲酸盐的铁还原酶的大部分活性是 (B)细胞色素电子传递的抑制剂 抑制MR-I对锰和铁的还原，以及(C)甲酸盐- OM的依赖铁还原酶涉及c型细胞色素。 综上所述，这些数据表明这些OM细胞色素在 与呼吸相关的金属还原。 似乎很可能是负责终端的组件 不溶性电子受体的转移必须暴露在 细胞表面；OM细胞色素代表了可能的候选者 一种电子转移作用。这项建议的具体目的是：(一) 提纯三种OM细胞色素中的至少两种；和(2) 制备针对纯化的OM细胞色素的多克隆抗体， 并验证这些抗体是否针对各自的 细胞色素。 这些研究将为今后在研究中使用提供重要的工具 膜的拓扑结构和这些外膜的生理作用 细胞色素。

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.