Putative mineral-specific proteins synthesized by a metal reducing bacterium

Abstract

ABSTRACT. Biological force microscopy (BFM) was combined with two-dimensional gel electrophoresis and mass spectrometry to search for evidence of putative mineral-specific outer membrane proteins (OM) synthesized by Shewanella oneidensis for Fe-oxide binding and/or anaerobic Fe(III) reduction. BFM shows that S. oneidensis possess an affinity towards goethite (FeOOH) but not diaspore (AlOOH) under anaerobic conditions, despite the fact that diaspore is isostructural with goethite and has essentially the same surface charge. The worm-like chain model was used to identify force-signatures indicative of putative OM polypeptides that bind to goethite. Two-dimensional protein expression patterns show that over 100 OM proteins are differentially expressed under aerobic versus anaerobic Fe(III) reducing conditions. Peptide mass fingerprinting and tandem mass spectrometry were used to identify several of the protein spots predominately detected when Fe(III) was the terminal electron acceptor. Among those identified were proteins involved in metal reduction, protein transport and secretion, polysaccharide biosynthesis and export, and hypothetical proteins with unknown functions. A comparison of the BFM and proteomic data suggest that a few specific OM proteins are synthesized by S. oneidensis under anaerobic conditions to function in iron oxide binding and/or Fe(III) reduction. This suggests the intriguing possibility that metal reducing bacteria contain the genetic repertoire to make proteins directed at specific inorganic phases.

Specific Bonds between an Iron Oxide Surface and Outer Membrane Cytochromes MtrC and OmcA from Shewanella oneidensis MR-1

“…Perhaps, this longerrange binding feature originates from the unraveling of a dimer of MtrC. In a previous publication, mass peptide analysis identified both MtrC and OmcA in polypeptides having apparent M r s of Ϸ150,000 and Ն210,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (29). These results suggest that MtrC or OmcA can form oligomeric complexes in S. oneidensis MR-1.…”

“…A prerequisite for this type of reaction would be the formation of a stable bond between the cytochrome and an iron oxide surface that would presumably facilitate electronic coupling between the cytochrome and the iron oxide. Evidence supporting this idea has been provided by studies examining the binding forces between living cells of S. oneidensis MR-1 and solid iron oxides (29,32). These studies suggest that S. oneidensis MR-1 actively synthesizes and/or localizes proteins (including MtrC and OmcA) at the mineral interface to function in iron oxide binding and/or reduction.…”

“…Finally, the force spectra for the cytochrome-hematite bonds were compared to previously published spectra collected for a living S. oneidensis MR-1 bacterium and an iron oxide surface (29,32). The force-distance profiles for the pure proteins were remarkably similar to those for the living bacterium, supporting the hypothesis that S. oneidensis MR-1 targets specific c-type cytochromes to the cell surface, where these proteins make direct contact with the Fe(III) oxide.…”

“…In the past, we hypothesized that sawtooth-like signatures for the force between S. oneidensis MR-1 and an Fe(III) oxide indicate that surface-exposed outer membrane proteins form a specific bond with the mineral surface (29,32). Recently, we showed that specific sawtooth force trajectories could be attributed to the formation of a bond between an engineered protein expressed on the surface of a gram-negative bacterium cell and a solid substrate in situ (30).…”

Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1

“…Previous studies suggest that S. oneidensis MR-1 uses outer membrane cytochromes OmcA and MtrC to catalyze the terminal reduction of Fe(III) through direct contact with the extracellular iron oxide mineral (2,8,10,15,16,20,21,23). However, it has yet to be shown whether OmcA or MtrC is actually targeted to the external surface of live S. oneidensis MR-1 cells when Fe(III) serves as the TEA.…”