Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells.
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omcZ is among the most up-regulated genes in current-harvesting biofilms, and of the outer-surface genes tested only pilA and omcZ deletions severely inhibit current production and current-harvesting biofilm formation.
"only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode"
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The omcZ deletion has no effect on biofilm formation when fumarate is the electron acceptor.
"In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor."
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Plasmid-borne omcZ restores wild-type current to the deletion mutant.
"When these mutations were complemented by expressing pilA or omcZ on a plasmid, wild type levels of current production were restored (Table 4)."
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Deleting omcZ does not affect growth with fumarate, Fe(III) citrate or Fe(III) oxide as electron acceptor.
"Deletion of omcZ had no impact on growth with any of these electron acceptors (data not shown)."
Purification and characterization of OmcZ, an outer-surface, octaheme c-type cytochrome essential for optimal current production by Geobacter sulfurreducens.
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The 30 kDa OmcZS is a cleavage product of the 50 kDa OmcZL that retains all eight hemes, one of them on a CX14CH motif.
"OmcZ(S) is a cleaved product of OmcZ(L) retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX(14)CH"
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OmcZS hemes span -420 to -60 mV, with a midpoint near -220 mV versus SHE.
"the midpoint reduction potential of OmcZ(S) is approximately -220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (-420 to -60 mV)"
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Purified OmcZS transfers electrons to soluble metals, Mn(IV) oxide and AQDS in vitro, but not to Fe(III) oxide.
"OmcZ(S) transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide."
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OmcZS is the predominant extracellular form.
"Subcellular-localization studies suggested that OmcZ(S) was the predominant extracellular form of OmcZ."
Specific localization of the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter sulfurreducens.
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Immunogold labelling places OmcZ in the extracellular matrix of fumarate-grown cell aggregates.
"OmcZ was dispersed throughout the extracellular matrix surrounding the cells that accumulated at the bottom of the culture tubes of cells grown under standard conditions with fumarate as the electron acceptor."
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In anode-grown biofilms, OmcZ is concentrated at the biofilm-electrode interface; fumarate-grown biofilms on the same graphite do not show this accumulation.
"When G. sulfurreducens grew as a biofilm on a graphite electrode that served as an anode and the sole electron acceptor for growth, OmcZ was highly concentrated at the biofilm-electrode interface."
Identification of an extracellular polysaccharide network essential for cytochrome anchoring and biofilm formation in Geobacter sulfurreducens.
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Extracellular c-type cytochromes, including OmcZ, are held in a polysaccharide-bound matrix that is largely lost from the xap (GSU1501) mutant.
"The matrix from the mutant contained 60% less sugar and was nearly devoid of c-type cytochromes such as OmcZ."
Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens.
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OzpA (GSU2075) is required to convert OmcZL to OmcZS, and an ozpA mutant produces as little current as an omcZ mutant.
"the ozpA mutant accumulated OmcZL and did not produce OmcZS, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode"
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OzpA expressed in E. coli cleaves OmcZL to OmcZS.
"A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZL and generated OmcZS as the proteolytic product."
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OmcZL cleavage activity is found only in the culture supernatant fraction.
"only the culture supernatant protein fraction showed OmcZL cleavage activity, indicating that the mature form of OmcZ, OmcZS, can be produced outside the cells"
Structure of Geobacter cytochrome OmcZ identifies mechanism of nanowire assembly and conductivity.
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A 3.5 Å cryo-EM structure of native OmcZ filaments, purified from an omcS-disrupted strain, resolves all eight hemes per subunit.
"At this resolution, the backbone density along with many bulky sidechain densities, such as aromatic residues and all eight haem densities, are visible"
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Seven hemes form a linear chain and heme VI is surface exposed.
"In OmcZ, haems I–V and VII form a linear, central, electron transport chain whereas haem VI is surface exposed"
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Deleting the heme II CX14CH loop slows electron transfer from reconstituted OmcZ filaments to extracellular acceptors.
"Using reconstituted OmcZ, we find that deletion of the haem II loop reduces the rate of electron transfer by OmcZ nanowires to extracellular electron acceptors compared with the WT"
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Purified OzpA cleaves OmcZ50 into OmcZ30, which self-assembles into filaments indistinguishable from native ones.
"We found that purified OzpA cleaves both native and recombinant OmcZ50 into OmcZ30 which then self-assembled into nanowires"
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OmcZ is not essential for Fe(III) oxide reduction by cells.
"Despite higher conductivity and solvent exposure of haems, OmcZ is not essential for bacterial reduction of Fe(III) oxide, an extracellular electron acceptor abundant in soil"
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Purified OmcZ filaments do not reduce riboflavin.
"We found that purified OmcZ nanowires do not reduce riboflavin"
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omcZ is co-transcribed with ozpA in an operon that also includes a peptidyl-prolyl cis-trans isomerase gene (GSU2074).
"we confirmed co-transcription of omcZ and ozpA in G. sulfurreducens"
Structure of Geobacter OmcZ filaments suggests extracellular cytochrome polymers evolved independently multiple times.
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An independent cryo-EM structure of OmcZ filaments shows a branched heme arrangement with one highly exposed heme per subunit and a heme packing different from OmcS and OmcE.
"A branched heme arrangement within OmcZ leads to a highly surface exposed heme in every subunit, which may account for the formation of conductive biofilm networks, and explain the higher measured conductivity of OmcZ filaments."
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Cytochrome filaments appear to have evolved more than once.
"This new structural evidence suggests that conductive cytochrome polymers arose independently on more than one occasion from different ancestral multiheme proteins."
Electric field stimulates production of highly conductive microbial OmcZ nanowires.
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An applied electric field stimulates OmcZ filament production in G. sulfurreducens biofilms; the filaments were reported to be about 30 S/cm conductive and stiffer than OmcS filaments.
"stimulates production of cytochrome OmcZ nanowires with 1,000-fold higher conductivity (30 S cm-1) and threefold higher stiffness (1.5 GPa) than the cytochrome OmcS nanowires"
Structure of Geobacter pili reveals secretory rather than nanowire behaviour.
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OmcZ reaches the extracellular fraction of wild-type cells but stays in the periplasm without PilA-N, and in trans PilA-N/PilA-C restores secretion.
"Our subcellular localization experiments revealed that both OmcS and OmcZ are present in the extracellular fraction of wild-type cells but remain in the periplasm for ΔpilA-N cells"
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The PilA-N–C filament is needed to export OmcZ.
"Our studies thus show that the unique PilA-N–C filaments are essential for the translocation of OmcZ nanowires"
The varied roles of pilA-N, omcE, omcS, omcT, and omcZ in extracellular electron transfer by Geobacter sulfurreducens.
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ΔomcZ was impaired in ferrihydrite reduction, anode reduction and co-culture with G. metallireducens.
"Deletion of omcT, omcZ or pilA-N alone impaired bacterial ability to reduce ferrihydrite and anodes and to form the co-culture."
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In a hybL (uptake hydrogenase) mutant background, omcZ deletion alone abolished growth in co-culture with G. metallireducens.
"However, deletion of only omcZ or pilA-N or both omcS and omcT abolished the ability of G. sulfurreducens without hydrogenase gene hybL to co-culture with G. metallireducens"
Iron Corrosion via Direct Metal-Microbe Electron Transfer.
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In strain ACLHF, which cannot use H2, omcZ deletion prevents growth on Fe(0), and complementation restores it.
"Deletion of the gene for omcZ prevented growth on Fe(0)"
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The authors interpret this as OmcZ taking part in electron uptake from Fe(0).
"These results suggest that OmcZ is also involved in electron transfer from Fe(0) into the cell."
Dissecting the Structural and Conductive Functions of Nanowires in Geobacter sulfurreducens Electroactive Biofilms.
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Of the three filament types in G. sulfurreducens, OmcZ contributes to current both structurally and conductively.
"The OmcZ nanowire played both a structural and a conductive function to contribute to current generation."
Real-time spatial gene expression analysis within current-producing biofilms.
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The omcZ promoter is active uniformly throughout anode biofilms, so expression differences do not explain why OmcZ accumulates at the anode.
"there are factors other than gene expression differences influencing the distribution of OmcZ"
Structural Determinants of Redox Conduction Favor Robustness over Tunability in Microbial Cytochrome Nanowires.
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Structure-based calculations predict that redox conductivity of OmcE, OmcS and OmcZ filaments differs by less than 10^3-fold, with small redox currents at physiological bias.
"Given the conserved energetic parameters, redox conductivity differs by < 103-fold among the cytochrome "nanowires"."
To be or not to be a cytochrome: electrical characterizations are inconsistent with Geobacter cytochrome 'nanowires'.
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This perspective argues that reported electrical characterizations of cytochrome filaments, including OmcZ, are artefactual or physically implausible, while accepting the filaments as redox-active cytochromes.
"prior electrical and spectroscopic structural characterizations are argued to be physiologically irrelevant or physically implausible for the known cytochrome filaments because of experimental artifacts and sample impurities"
Electron transport across the cell envelope via multiheme c-type cytochromes in Geobacter sulfurreducens.
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No environmentally relevant condition has been found in which OmcZ is as strongly and specifically up-regulated as on poised anodes.
"no environmentally relevant growth conditions have been identified to date in which OmcZ is as strongly and uniquely upregulated as on poised anodes"
Homologous overexpression of omcZ, a gene for an outer surface c-type cytochrome of Geobacter sulfurreducens by single-step gene replacement.
Electrode-associated growth reveals structure of OmcZ-type cytochrome nanowires in anaerobic methanotrophic archaea
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The filament-forming part of G. sulfurreducens OmcZ is 258 residues long and carries one heme on a CX14CH motif; an archaeal OmcZ-type cytochrome (OmcZ2 of 'Ca. Methanoperedens vercellensis') has eight canonical hemes on a 330-residue filament-forming part.
"In contrast, the nanowire-forming part of OmcZGs comprises 258 residues and includes one heme bound by a non-canonical motif (CX14CH)."
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Bacterial and archaeal OmcZ share a conserved core architecture, including three His-His motifs that link adjacent heme pairs.
"Central to this organization are three conserved histidine-histidine (HH) motifs that link adjacent heme pairs in both OmcZ2Mv and OmcZGs together."
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The C-terminal region removed during maturation is predicted to form two Ig-like beta-sandwich domains in G. sulfurreducens OmcZ.
"AlphaFold predictions of full-length OmcZ proteins indicate that the cleaved C-terminal regions adopt β-sandwich folds resembling immunoglobulin-like domains, with three such domains in OmcZ2Mv and two in OmcZGs"
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G. sulfurreducens OmcZ has only three N-glycosylation motifs and no detectable glycan density.
"whereas OmcZGs contains only three such motifs and shows no detectable extra densities"
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Bacterial OmcZ homologues form their own phylogenetic group (Group VI), separate from archaeal groups I-V.
"Group VI contains bacterial OmcZ homologs"
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G. sulfurreducens OmcZ filaments aggregate into dense networks between cells and electrodes, unlike the straighter archaeal filaments.
"Whereas OmcZGs nanowires aggregate extensively and densely fill the extracellular space between bacterial cells and electrodes"
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Archaea assemble OmcZ filaments with no prolyl isomerase in the gene cluster, which calls into question the proposed role of the Geobacter isomerase in OmcZ folding.
"Surprisingly, these archaea assemble OmcZ into nanowires even in the apparent absence of prolyl isomerase involved in folding bacterial OmcZ."