AEM Accepts, published online ahead of print on 3 August 2007

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Appl. Environ. Microbiol. doi:10.1128/AEM.00146-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of Protein/Protein Interactions Involved in Iron Reduction by Shewanella oneidensis MR-1

Daniel E. Ross, Shane S. Ruebush, Susan L. Brantley, Robert S. Hartshorne, Thomas A. Clarke, David J. Richardson, and Ming Tien^*

Department of Biochemistry and Molecular Biology and Department of Geosciences, The Pennsylvania State University, University Park, PA 16802 and School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK

^* To whom correspondence should be addressed. Email: mxt3{at}psu.edu.

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane (OM): OmcA, MtrB, MtrC; OM-associated: MtrA; periplasm: CctA; cytoplasmic membrane: CymA) were characterized by protein purification, analytical ultracentrifugation and cross-linking methods. Five of these proteins are hemeproteins: OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa) and CymA (21 kDa) and can be visualized after SDS-PAGE by heme staining. We show for the first time that MtrC, MtrA and MtrB form a 198 kDa complex with a 1:1:1 stoichiometry. These proteins co-purify through anion exchange chromatography and the purified complex has the ability to reduce multiple forms of Fe (III) and Mn (IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation and MtrA co-migrates with MtrB in native gels. Protein crosslinking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59kDa. Using antibodies to detect each protein separately, hemeproteins OmcA and MtrC were shown to crosslink yielding the 160 kDa band. Consistent with co-purification results, MtrB cross-links with MtrA, forming high molecular weight bands of approximately 151 and 136 kDa bands.

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