Outer membrane cytochromes of Shewanella putrefaciens MR-1: spectral analysis, and purification of the 83-kDa c-type cytochrome

Abstract

The metal-reducing bacterium Shewanella putrefaciens MR-1 is known to localize a majority of its membrane-bound cytochromes to its outer membrane when grown under anaerobic conditions. In this study, pyridine hemochrome spectra confirmed that these outer membrane cytochromes are c-type, and electrophoretic data demonstrated the presence of four distinct outer membrane cytochromes, with apparent molecular masses of 150, 83, 65, and 53 kDa. Fourth-order derivative analysis of 77 K spectra of the outer membrane revealed four spectrally distinct c-type hemes, with peaks at 545.4, 548.0, 550.6, and 552.6 nm. Outer membrane cytochromes in the reduced state were rapidly re-oxidized by oxidized iron and manganese, which have previously been shown to serve as electron acceptors for anaerobic respiration in this bacterium. The 83-kDa outer membrane cytochrome was purified and a specific polyclonal antibody was generated against this protein. Western blot analysis demonstrated that the vast majority of this protein was localized to the outer membrane and an intermediate density membrane fraction of similar composition. Its levels, but not its subcellular distribution, were somewhat influenced by the electron acceptor used to support anaerobic growth, with levels higher in fumarate-grown cells relative to iron(III)- or trimethylamine N-oxide-grown cells. Its specific content in cells grown under aerobic conditions was only 14% of that of fumarate-grown cells, suggesting that a switch to anaerobic conditions significantly increases the de novo synthesis of this outer membrane cytochrome.

Reduction of Fe(III) colloids by Shewanella putrefaciens: A kinetic model

“…Several outer-membrane c-type cytochromes capable of mediating electron transfer to Fe(III) and Mn(IV) oxides have been isolated from Shewanella cultures (Arnold et al, 1988;Myers and Myers, 1997;Beliaev and Saffarini, 1998;Caccavo, 1999;Blakeney et al, 2000). Their abundance depends on the growth conditions of the bacteria, with values of 0.45 · 10 À2 and 1.96 · 10 À2 lmol c-type cytochrome per mg of protein reported for cultures of S. putrefaciens 200 grown under aerobic and microaerobic conditions, respectively (Picardal et al, 1993).…”

Putative mineral-specific proteins synthesized by a metal reducing bacterium

“…Since this protein is so prominent in 2D gels and observed only under anaerobic Fe(III) reducing conditions, efforts are currently underway to identify the protein(s) contained within this chain. discussion A great deal is known about the transfer of electrons from S. oneidensis to Fe(III) (Arnold and others, 1990;Myers and Nealson, 1990;Myers and Myers, 1992;DiChristina and Delong, 1994;Roden and Zachara, 1996;Caccavo and others, 1997;Fredrickson and others, 1998;Zachara and others, 1998;Caccavo, 1999;Newman and Kolter, 2000;Roden and others, 2000;Hernandez and Newman, 2001;Myers, 2001, 2002). However, there are at least two fundamental questions that have not been adequately addressed in the literature with respect to electron transfer to solid Fe(III) phases.…”

“…Unlike oxygen, Fe(III) in a solid form cannot diffuse across the OM to the cytosolic membrane, which in most organisms, is the location of the enzymes responsible for electron transfer, proton translocation, and production of adenosine triphosphate. Certain Gram-negative, dissimilatory ironreducing bacteria (for example, S. oneidensis), which are common in soils, surface waters, and subsurface environments, have developed a resourceful solution to this problem by using a unique system of proteins that shuttle electrons from an energy source in the cytoplasm, across the cytosolic membrane and periplasmic space to the bacterium's OM (Myers and Myers, 1992;Myers and Myers, 2000). Once there, putative iron reductases transfer the electrons directly to Fe(III) in the crystal structure of Fe(III)-bearing minerals (Arnold and others, 1988;Myers and Myers, 1992;Myers and Myers, 1998).…”

“…Certain Gram-negative, dissimilatory ironreducing bacteria (for example, S. oneidensis), which are common in soils, surface waters, and subsurface environments, have developed a resourceful solution to this problem by using a unique system of proteins that shuttle electrons from an energy source in the cytoplasm, across the cytosolic membrane and periplasmic space to the bacterium's OM (Myers and Myers, 1992;Myers and Myers, 2000). Once there, putative iron reductases transfer the electrons directly to Fe(III) in the crystal structure of Fe(III)-bearing minerals (Arnold and others, 1988;Myers and Myers, 1992;Myers and Myers, 1998). It is important to note that others have recently shown that iron-reducing bacteria may possess a richer repertoire of mechanisms to reduce Fe(III) oxyhydroxides that also includes extracellular quinones (Newman and Kolter, 2000;Hernandez and Newman, 2001).…”

Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

“…The metal-reducing bacterium Shewanella oneidensis MR-1 is one of the most extensively studied organisms for understanding such EET processes (4)(5)(6). MR-1 expresses a significant quantity of cell-surface redox-active proteins, namely the c-type cytochromes (c-Cyts), which form a hypothetical protein complex "OmcAMtrCAB" (6,7) in which these proteins work together to transport electrons generated by the intracellular metabolic oxidation of organic matter to solid extracellular electron acceptors as a terminal process in microbial respiration (8)(9)(10). The electron transfer (ET) chain from the inner to outer membrane (OM) is called the metal reduction (Mtr) pathway and is responsible for both metal-oxide reduction reactions and anodic current production in microbial fuel cells (MFCs) (5,9).…”