In:
Journal of Bacteriology, American Society for Microbiology, Vol. 188, No. 13 ( 2006-07), p. 4705-4714
Abstract:
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., Δ 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 Δ 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 ( K d 〈 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.
Type of Medium:
Online Resource
ISSN:
0021-9193
,
1098-5530
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2006
detail.hit.zdb_id:
1481988-0
SSG:
12
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