Molecular mechanisms of microbial iron respiration by Shewanella oneidensis MR-1.

摘要

Microbial metal respiration is central to a variety of biogeochemically important processes including the weathering of clays, biotransformation of Fe- and Mn-bearing minerals, and reductive precipitation of radionuclides (e.g. U(VI) and Tc(VII)); the latter forming the basis for an alternative remediation strategy for radioactive-contaminated field sites. Compared to the wealth of knowledge of other respiratory processes including aerobic respiration and anaerobic respiration using NO3-, SO 42-, fumarate, and other compounds as electron acceptor, the components of the respiratory chain terminating with transition metals have yet to be conclusively identified. Shewanella spp. are present in a variety of freshwater and marine environments and can respire a variety of compounds which span the redox continuum. These include O2, solid and soluble Fe(III), Mn(III), Mn(IV), NO3 -, NO2-, S2O 32-, S0, DMSO, TMAO, fumarate, U(VI) and Tc(VII). Because of this extreme respiratory versatility, Shewanella spp. may predominate in redox-stratified subsurface environments where terminal electron acceptors vary on small temporal and spatial scales.;In the present study, a novel gene deletion system was constructed and used to examine the function of PsrA, the putative thiosulfate reductase in S. oneidensis MR-1. PsrA-null mutants (PSRA1) was unable to grow with S2O32- or S0 as terminal electron acceptor while retaining respiratory ability on all other electron acceptors tested. Additionally, PSRA1 retained mRNA transcripts for downstream genes psrB and psrC, indicating that the psrA mutation was in-frame and did not cause polar mutations. Using the newly constructed suicide vector, genes encoding proteins identified in the peripheral protein fraction, including those present in the Fe(III) terminal reductase complex, were systematically deleted from the S. oneidensis MR-1 genome and the resulting mutant strains were tested for the ability to respire Fe(III). Two mutant strains (DeltaSO3800 and MTRB1) were isolated and further characterized. SO3800 is a putative OM serine protease that displays sequence similarity to known bacterial adhesins. DeltaSO3800 was deficient in adhesion to hematite (Fe2O3), yet retained Fe(III) respiratory ability. DeltaSO3800 also displayed an increase in cell surface exopolymers and decreased electrophoretic mobility. MTRB1 contains a deletion in OM protein MtrB and is abolished in Fe(III) respiratory ability. MtrB contains a conserved N-terminal CXXC motif that may act as electron transfer motif or a structurally important disulfide bond. Both cysteines were independently changed to alanine via site-directed mutagenesis and subsequent "knock-in" complementation using the newly constructed suicide vector. Both MTRB1 and C42A displayed diminished Fe(III) respiratory ability and increased cytochrome content in the peripheral protein fraction while C45A displayed a phenotype similar to wild type. Potential mechanisms of Fe(III) respiration involving C42 and MtrB are proposed.