| Cytochrome c is an important component of respiratory chain, Shewanella oneidensis possesses a large number of the c-type cytochromes, give them remarkable ability to respire on a wide range of electron acceptors, including fumarate, TMAO, nitrate, nitrite, and various metallic compounds, thus S. oneidensis becomes a veteran research model for investigating cytochrome c maturation and their function, and molecular mechanism of respiration as well. Based on previous work in the laboratory, in this paper we continued to focus on the function of cytochrome c and its associated biological processes, performed in-depth study in two aspects and achieved some inspiring results:i) tested the physiological roles of all cytochrome c in S. oneidensis and confirmed the essential components of its maturation system through mutagenesis system; ii) analyzed the molecular mechanism underlying nitrite inhibition of fumarate and other EAs respiration.Organizational and functional features of the cytochrome c maturation system in Shewanella oneidensisExisting deletion mutant constructing system has labor-intensive, poor reliability characteristics. In order to improve the efficiency and reliability, we developed a newly att-based mutagenesis approach. First, we obtained the cytochrome c mutants that had not be constructed before and completed the construction of an entire set of c-type cytochrome mutants by virtue of this system. Second, we investigated the cytochrome c maturation (Ccm) system in S. oneidensis. There are two loci predicted to encode components of the Ccm system, SO0259-SO0269 and SO0476-SO0478. The former is proven essential for cytochrome c maturation whereas the latter is dispensable. Unlike the single operon organization observed in other y-proteobacteria, genes at the SO0259-SO0269 locus are uniquely organized into four operons, ccmABCDE, scyA, SO0265, and ccmFGH-SO0269. Functional analysis revealed that the SO0265 gene rather than the scyA and SO0269 genes are relevant to cytochrome c maturation.The effect of reduced cAMP levels caused by nitrite on fumarate respiration in S. oneidensisIn environments where S. oneidensis thrive, multiple EAs are usually present. However, there is surprisingly little knowledge about how multiple EAs are responded and utilized by these bacteria and how their interaction affects ecophysiology of the bacteria. The famous case is that nitrite has been used as preservatives in meat products to inhibit growth of bacterial pathogens. In this study, we demonstrate that nitrite, not through nitric oxide to which it may convert, inhibits respiration of fumarate in S. oneidensis MR-1. This is achieved via the repression of cyclic adenosine monophosphate (cAMP) production, a second messenger required for activation of cAMP-receptor protein (Crp) which plays a primary role in regulation of respiration. If nitrite is not promptly removed, intracellular cAMP levels drop, and this impairs Crp activity. As a result, the production of nitrite reductase NrfA, quinol dehydrogenase CymA, and fumarate reductase FccA is substantially reduced. In contrast, nitrite can be simultaneously respired with trimethylamine N-oxide, resulting in enhanced biomass.Second, we found in S. oneidensis that nitrite inhibits respiration of other periplasmic EAs. This is achieved by inducing production of NapB, the cytochrome c subunit of nitrate reductase. Under this condition, NapB acts as an electron shuttle to transfer electrons from the quinol pool to extracellullar EAs by promiscuously interacting with multiple inner-membrane-bound quinol dehydrogenases. Multiple-component Mtr system, the major electron transport pathway which largely relies on quinol dehydrogenase CymA for electrons and donates them to EAs in extracellular space, is implicated in the process.Briefly, these results reveal a yet unexplored mechanism by which bacteria can regulate multi-branched respiratory networks through an EA that is not only poor for energy generation but toxic to cellular organisms. |
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