Outer membrane cytochromes of Shewanella putrefaciens MR-1: spectral analysis, and purification of the 83-kDa c-type cytochrome.
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.
Myers
C R
CR
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA. cmyers@post.its.mcw.edu
Myers
J M
JM
D000332:Aerobiosis; D000693:Anaerobiosis; D000906:Antibodies; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005290:Ferric Compounds / Q000378:metabolism; D006092:Gram-Negative Facultatively Anaerobic Rods / Q000737:chemistry*; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D000906:Antibodies; D003574:Cytochrome c Group; D005290:Ferric Compounds; D008345:Manganese