Biosynthesis and regulation of heme C proteins in Shewanella oneidensis strain MR-1

Shewanella oneidensis MR-1 is a facultative anaerobic bacterium that can use a variety of terminal electron acceptors during anaerobic respiration. Since S. oneidensis can use insoluble metal oxides and toxic metals such as iron and uranium, it has become the focus of many researchers as a potential agent in bioremediation. S. oneidensis synthesizes 42 possible c-type cytochromes under several growth conditions. c-type cytochromes are hemeproteins with C-X-X-C-H motifs that play an important role in shuttling electrons between different components of the electron transport chain. Heme synthesis under anaerobic conditions requires hemN gene. In S. oneidensis, hemN is similar to genes encoding anaerobic coproporphyrinogen III oxidases involved in heme biosynthesis in other bacteria. Although, the genome sequence of S. oneidensis contains three genes that are similar to E. coli hemN, our growth and complementation studies of the wild type, hemN mutant and complemented mutant strains of S. oneidensis identified HemN as the major enzyme that is involved in anaerobic heme biosynthesis in S. oneidensis under anaerobic conditions. The other two putative anaerobic coproporphyrinogen III oxidase genes may play a minor role in this process.; A previously identified fccA gene encodes a tetraheme periplasmic soluble flavocytochrome c fumarate reductase. In S. oneidensis the expression of fumarate reductase is induced by fumarate under anaerobic conditions and is regulated by CRP (cAMP Receptor Protein). The differential expression of fccA in the wild type and crp mutants and the presence of several putative CRP binding sites upstream of fccA gene suggest that it is directly regulated by CRP. To confirm this, wild type chromosomal DNA of S. oneidensis was used to amplify and clone a modified crp gene. The His7-tagged CRP protein was expressed, and purified. In vitro electrophoretic mobility gel shift assays were initiated to determine the binding of CRP to the upstream promoter regions of the fccA gene. Our results showed that CRP directly binds and regulates the expression of the fumarate reductase gene in S. oneidensis.