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Applied and Environmental Microbiology, September 2007, p. 5797-5808, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00146-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Daniel E. Ross,¹ Shane S. Ruebush,^1, Susan L. Brantley,² Robert S. Hartshorne,³ Thomas A. Clarke,³ David J. Richardson,³ and Ming Tien^1*

Department of Biochemistry and Molecular Biology,¹ Department of Geosciences, The Pennsylvania State University, University Park, Pennsylvania 16802,² School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom³

Received 19 January 2007/ Accepted 20 July 2007

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis 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 copurify 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 comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.

Published ahead of print on 3 August 2007.

Present address: Department of Chemistry, Montana State University, Bozeman, MT.

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