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Applied and Environmental Microbiology, April 2006, p. 2925-2935, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2925-2935.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Reduction of Soluble and Insoluble Iron Forms by Membrane Fractions of Shewanella oneidensis Grown under Aerobic and Anaerobic Conditions

Shane S. Ruebush,1 Susan L. Brantley,2 and Ming Tien1*

Department of Biochemistry and Molecular Biology,1 Department of Geosciences, Pennsylvania State University, University Park, Pennsylvania 168022

Received 20 November 2005/ Accepted 17 January 2006

The effect of iron substrates and growth conditions on in vitro dissimilatory iron reduction by membrane fractions of Shewanella oneidensis MR-1 was characterized. Membrane fractions were separated by sucrose density gradients from cultures grown with O2, fumarate, and aqueous ferric citrate as the terminal electron acceptor. Marker enzyme assays and two-dimensional gel electrophoresis demonstrated the high degree of separation between the outer and cytosolic membrane. Protein expression pattern was similar between chelated iron- and fumarate-grown cultures, but dissimilar for oxygen-grown cultures. Formate-dependent ferric reductase activity was assayed with citrate-Fe3+, ferrozine-Fe3+, and insoluble goethite as electron acceptors. No activity was detected in aerobic cultures. For fumarate and chelated iron-grown cells, the specific activity for the reduction of soluble iron was highest in the cytosolic membrane. The reduction of ferrozine-Fe3+ was greater than the reduction of citrate-Fe3+. With goethite, the specific activity was highest in the total membrane fraction (containing both cytosolic and outer membrane), indicating participation of the outer membrane components in electron flow. Heme protein content and specific activity for iron reduction was highest with chelated iron-grown cultures with no heme proteins in aerobically grown membrane fractions. Western blots showed that CymA, a heme protein involved in iron reduction, expression was also higher in iron-grown cultures compared to fumarate- or aerobic-grown cultures. To study these processes, it is important to use cultures grown with chelated Fe3+ as the electron acceptor and to assay ferric reductase activity using goethite as the substrate.

* Corresponding author. Mailing address: 408 Althouse Laboratory, Department of Biochemistry and Molecular Biology, University Park, PA 16802. Phone: (814) 863-1165. Fax: (814) 863-7024. E-mail: mxt3{at}psu.edu.

Applied and Environmental Microbiology, April 2006, p. 2925-2935, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2925-2935.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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