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Title: Identification and Characterization of UndA-HRCR-6, an Outer Membrane Endecaheme c-Type Cytochrome of Shewanella sp. Strain HRCR-6

Abstract

The outer membrane decaheme c-type cytochromes (c-Cyt) MtrC and OmcA of Shewanella oneidensis MR-1(MR-1) play critical roles in extracellular reduction of iron [Fe(III)] oxides and uranium [ U(VI)]. To identify and characterize the outer membrane c-Cyts found in the metal-reducing Shewanella strains isolated from the Hanford Reach of the Columbia River (HRCR), 7 HRCR isolates were tested for the presence of mtrC, omcA and undA1 (a gene encoding a putative 11-heme c-Cyt) homologues in their genomes. All 7 tested strains possessed an mtrC homologue, while 3 strains had an omcA homologue and the remaining 4 strains contained an undA1 homologue. The coding region of an undA1 homologue from HRCR isolate 6 was cloned and sequenced. Because it was 93% identical to the UndA of S. baltica OS223, the protein product encoded by this sequenced gene was named as UndA-HRCR6. In MR-1, UndA-HRCR6 (i) restored an MR-1 mutant’s ability to reduce solid phase ferrihydrite at 40% of that for MR-1 wild type, (ii) increased extracellular formation of UO2 associated with the outer membrane and extracellular polymeric substances in a U(VI) reduction assay and (iii) was secreted to the extracellular environment by bacterial type II secretion system. UndA-HRCR6 was purified from themore » membrane fraction following its overexpression in MR-1 cells. Purified UndA-HRCR6 possessed 11 heme-Fe and reduced ferric complexes. Collectively, these results show that UndA-HRCR6 is an outer membrane endecaheme c-Cyt and can serve an extracellular metal reductase with functions similar to that of MR-1 MtrC and OmcA.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1023724
Report Number(s):
PNNL-SA-77918
Journal ID: ISSN 0099-2240; AEMIDF; 13994; KP1702030; TRN: US201120%%1060
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology, 77(15):5521-5523; Journal Volume: 77; Journal Issue: 15
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; BACTERIA; COLUMBIA RIVER; COMPLEXES; CYTOCHROMES; FUNCTIONS; GENES; IRON OXIDES; MEMBRANES; METALS; OXIDOREDUCTASES; PROTEINS; REDUCTION; SECRETION; SOLIDS; URANIUM; Environmental Molecular Sciences Laboratory

Citation Formats

Shi, Liang, Belchik, Sara M., Wang, Zheming, Kennedy, David W., Dohnalkova, Alice, Marshall, Matthew J., Zachara, John M., and Fredrickson, Jim K. Identification and Characterization of UndA-HRCR-6, an Outer Membrane Endecaheme c-Type Cytochrome of Shewanella sp. Strain HRCR-6. United States: N. p., 2011. Web. doi:10.1128/AEM.00614-11.
Shi, Liang, Belchik, Sara M., Wang, Zheming, Kennedy, David W., Dohnalkova, Alice, Marshall, Matthew J., Zachara, John M., & Fredrickson, Jim K. Identification and Characterization of UndA-HRCR-6, an Outer Membrane Endecaheme c-Type Cytochrome of Shewanella sp. Strain HRCR-6. United States. doi:10.1128/AEM.00614-11.
Shi, Liang, Belchik, Sara M., Wang, Zheming, Kennedy, David W., Dohnalkova, Alice, Marshall, Matthew J., Zachara, John M., and Fredrickson, Jim K. 2011. "Identification and Characterization of UndA-HRCR-6, an Outer Membrane Endecaheme c-Type Cytochrome of Shewanella sp. Strain HRCR-6". United States. doi:10.1128/AEM.00614-11.
@article{osti_1023724,
title = {Identification and Characterization of UndA-HRCR-6, an Outer Membrane Endecaheme c-Type Cytochrome of Shewanella sp. Strain HRCR-6},
author = {Shi, Liang and Belchik, Sara M. and Wang, Zheming and Kennedy, David W. and Dohnalkova, Alice and Marshall, Matthew J. and Zachara, John M. and Fredrickson, Jim K.},
abstractNote = {The outer membrane decaheme c-type cytochromes (c-Cyt) MtrC and OmcA of Shewanella oneidensis MR-1(MR-1) play critical roles in extracellular reduction of iron [Fe(III)] oxides and uranium [ U(VI)]. To identify and characterize the outer membrane c-Cyts found in the metal-reducing Shewanella strains isolated from the Hanford Reach of the Columbia River (HRCR), 7 HRCR isolates were tested for the presence of mtrC, omcA and undA1 (a gene encoding a putative 11-heme c-Cyt) homologues in their genomes. All 7 tested strains possessed an mtrC homologue, while 3 strains had an omcA homologue and the remaining 4 strains contained an undA1 homologue. The coding region of an undA1 homologue from HRCR isolate 6 was cloned and sequenced. Because it was 93% identical to the UndA of S. baltica OS223, the protein product encoded by this sequenced gene was named as UndA-HRCR6. In MR-1, UndA-HRCR6 (i) restored an MR-1 mutant’s ability to reduce solid phase ferrihydrite at 40% of that for MR-1 wild type, (ii) increased extracellular formation of UO2 associated with the outer membrane and extracellular polymeric substances in a U(VI) reduction assay and (iii) was secreted to the extracellular environment by bacterial type II secretion system. UndA-HRCR6 was purified from the membrane fraction following its overexpression in MR-1 cells. Purified UndA-HRCR6 possessed 11 heme-Fe and reduced ferric complexes. Collectively, these results show that UndA-HRCR6 is an outer membrane endecaheme c-Cyt and can serve an extracellular metal reductase with functions similar to that of MR-1 MtrC and OmcA.},
doi = {10.1128/AEM.00614-11},
journal = {Applied and Environmental Microbiology, 77(15):5521-5523},
number = 15,
volume = 77,
place = {United States},
year = 2011,
month = 8
}
  • Pertechnetate, 99Tc(VII)O4-, is a highly mobile radionuclide contaminant at U.S. Department of Energy sites that can be enzymatically reduced by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble Tc(IV)O2(s). In other microorganisms, Tc(VII)O4- reduction is generally considered to be catalyzed by hydrogenase. Here, we provide evidence that although the NiFe hydrogenase of MR-1 was involved in the H2-driven reduction of Tc(VII)O4- (presumably through a direct coupling of H2 oxidation and Tc(VII) reduction), the deletion of both hydrogenase genes did not completely eliminate the ability of MR-1 to reduce Tc(VII). With lactate as themore » electron donor, mutants lacking the outer membrane c-type cytochromes MtrC and OmcA or the proteins required for the maturation of c-type cytochromes were defective in reducing Tc(VII) to nanoparticulate TcO2·nH2O(s) relative to MR-1 or a NiFe hydrogenase mutant. In addition, reduced MtrC and OmcA were oxidized by Tc(VII)O4-, confirming the capacity for direct electron transfer from these OMCs to TcO4-. c-Type cytochrome-catalyzed Tc(VII) reduction could be a potentially important mechanism in environments where organic electron donor concentrations are sufficient to allow this reaction to dominate.« less
  • The identification of electron transfer proteins that couple soluble redox carriers to electrode surfaces has great potential in permitting the development of scalable bioreactors. In this respect, the 85 kDa outer membrane decaheme cytochrome OmcA (SO1779) from Shewanella oneidensis MR-1 can reduce soluble Fe(III) chelates, and has previously been suggested to function in concert with other membrane proteins as one of the terminal electron donors in the metal reductase protein complex of Shewanella oneidensis MR-1.1 Shewanella is a facultative anaerobe that can reduce a range of different metal oxides, including iron [Fe(III)], manganese [Mn(III/IV)], chromium [Cr(IV)] and uranium [U(VI)]2, andmore » whose metabolic diversity has considerable promise for both the bioremediation of organic and metal contaminants as well as in the design of microbial fuel cells3. Biofuel cells offer a potential means to couple the breakdown of bio-wastes to generate power4. Miniaturization of these fuel cells is dependent on the elimination of the currently necessary membrane between cathode and anode compartments.5 It has been demonstrated that the immobilization of redox active proteins, such as glucose oxidase, on electrodes with redox active polymer coatings renders the membrane unnecessary.6 The identification of a purified metal-reducing enzyme able to densely bind and directly donate electrons to iron-oxide coated electrodes (commonly used to increase electron transfer efficiencies7) has great potential to contribute to fuel cell design. To identify the terminal electron donors in the S. oneidensis metal reductase system, and to explore whether isolated proteins can directly bind and mediate electron transfer reactions to reduce solid metals, we have purified OmcA and measured its ability to bind and transfer electrons to solid Fe2O3 in the mineral hematite.« less
  • Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) atmore » its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are consistent with the inherent complexity associated with correct heme insertion and acylation of MtrC that occurs in the periplasm prior to its targeting to the outer membrane. These latter results suggest that MtrC protein trafficking to the outer membrane and its subsequent degradation are tightly regulated, which is consistent with cellular processing pathways that target MtrC to extracellular structures and their possible role in promoting electron transfer from Shewanella to extracellular acceptors.« less
  • The outer membrane cytochrome OmcA functions as a terminal metal reductase in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. The ten-heme centers shuttle electrons from the transmembrane donor complex to extracellular electron acceptors. Here, the crystallization and preliminary crystallographic analysis of OmcA are reported. Crystals of OmcA were grown by the sitting-drop vapor-diffusion method using PEG 20 000 as a precipitant. The OmcA crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 93.0, b = 246.0, c = 136.6 {angstrom}, = 90, {beta} = 97.8, {gamma} = 90{sup o}. X-ray diffraction data were collected to a maximummore » resolution of 3.25 {angstrom}.« less
  • The outer membrane cytochrome OmcA functions as a terminal metal reductase in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. The ten-heme centers shuttle electrons from the transmembrane donor complex to extracellular electron acceptors. Here, the crystallization and preliminary crystallographic analysis of OmcA are reported. Crystals of OmcA were grown by the sitting-drop vapor-diffusion method using PEG 20 000 as a precipitant. The OmcA crystals belonged to space group P21, with unit-cell parameters a = 93.0, b = 246.0, c = 136.6 A ° , * = 90, * = 97.8, * = 90*. X-ray diffraction data were collected to amore » maximum resolution of 3.25 A ° .« less