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Journal of Bacteriology, July 2006, p. 4705-4714, Vol. 188, No. 13
0021-9193/06/$08.00+0     doi:10.1128/JB.01966-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-Type Cytochromes of Shewanella oneidensis MR-1

Liang Shi,* Baowei Chen, Zheming Wang, Dwayne A. Elias, M. Uljana Mayer, Yuri A. Gorby, Shuison Ni, Brian H. Lower, David W. Kennedy, David S. Wunschel, Heather M. Mottaz, Matthew J. Marshall, Eric A. Hill, Alexander S. Beliaev, John M. Zachara, James K. Fredrickson, and Thomas C. Squier

Pacific Northwest National Laboratory, Richland, Washington 99354

Received 22 December 2005/ Accepted 14 April 2006

Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., {Delta}omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the {Delta}omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.

* Corresponding author. Mailing address: Microbiology Group, Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, MSIN P7-50, Richland, WA 99354. Phone: (509) 376-4834. Fax: (509) 372-1632. E-mail: liang.shi{at}pnl.gov.

Journal of Bacteriology, July 2006, p. 4705-4714, Vol. 188, No. 13
0021-9193/06/$08.00+0     doi:10.1128/JB.01966-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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