Geobacter sulfurreducens inner membrane cytochrome CbcBA controls electron transfer and growth yield near the energetic limit of respiration

AbstractGeobacter sulfurreducens utilizes extracellular electron acceptors such as Mn(IV), Fe(III), syntrophic partners, and electrodes that vary from +0.4 to −0.3 V vs. Standard Hydrogen Electrode (SHE), representing a potential energy span that should require a highly branched electron transfer chain. Here we describe CbcBA, a bc-type cytochrome essential near the thermodynamic limit of respiration when acetate is the electron donor. Mutants lacking cbcBA ceased Fe(III) reduction at −0.21 V vs. SHE, could not transfer electrons to electrodes between −0.21 and −0.28 V, and could not reduce the final 10% – 35% of Fe(III) minerals. As redox potential decreased during Fe(III) reduction, cbcBA was induced with the aid of the regulator BccR to become one of the most highly expressed genes in G. sulfurreducens. Growth yield (CFU/mM Fe(II)) was 112% of WT in ΔcbcBA, and deletion of cbcL (a different bc-cytochrome essential near −0.15 V) in ΔcbcBA increased yield to 220%. Together with ImcH, which is required at high redox potentials, CbcBA represents a third cytoplasmic membrane oxidoreductase in G. sulfurreducens. This expanding list shows how these important metal-reducing bacteria may constantly sense redox potential to adjust growth efficiency in changing environments.

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Redox potential as a master variable controlling pathways of metal reduction byGeobacter sulfurreducens

10.1101/043059 ◽

2016 ◽

Cited By ~ 1

Author(s):

Caleb E. Levar ◽

Colleen L. Hoffman ◽

Aubrey J. Dunshee ◽

Brandy M. Toner ◽

Daniel R. Bond

Keyword(s):

Redox Potential ◽

Geobacter Sulfurreducens ◽

High Potential ◽

Growth Yields ◽

Redox Potentials ◽

Hydrogen Electrode ◽

Additional Energy ◽

Single Organism ◽

Wide Range ◽

Dependent Pathway

AbstractGeobacter sulfurreducensuses at least two different pathways to transport electrons out of the inner membrane quinone pool before reducing acceptors beyond the outer membrane. When growing on electrodes poised at oxidizing potentials, the CbcL-dependent pathway operates at or below redox potentials of −0.10 V vs. the Standard Hydrogen Electrode (SHE), while the ImcH-dependent pathway operates only above this value. Here, we provide evidence thatG. sulfurreducensalso requires different electron transfer proteins for reduction of a wide range of Fe(III)- and Mn(IV)- (oxyhydr)oxides, and must transition from a high- to low-potential pathway during reduction of commonly studied soluble and insoluble metal electron acceptors. Freshly precipitated Fe(III)-(oxyhydr)oxides could not be reduced by mutants lacking the high potential pathway. Aging these minerals by autoclaving did not change their powder X-ray diffraction pattern, but restored reduction by mutants lacking the high-potential pathway. Mutants lacking the low-potential, CbcL-dependent pathway had higher growth yields with both soluble and insoluble Fe(III). Together, these data suggest that the ImcH-dependent pathway exists to harvest additional energy when conditions permit, and CbcL switches on to allow respiration closer to thermodynamic equilibrium conditions. With evidence of multiple pathways within a single organism, the study of extracellular respiration should consider not only the crystal structure or solubility of a mineral electron acceptor, but rather the redox potential, as this variable determines the energetic reward affecting reduction rates, extents, and final microbial growth yields in the environment.

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An Inner Membrane Cytochrome Required Only for Reduction of High Redox Potential Extracellular Electron Acceptors

mBio ◽

10.1128/mbio.02034-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 79

Author(s):

Caleb E. Levar ◽

Chi Ho Chan ◽

Misha G. Mehta-Kolte ◽

Daniel R. Bond

Keyword(s):

Electron Transfer ◽

Electron Acceptors ◽

Geobacter Sulfurreducens ◽

Extracellular Electron Transfer ◽

Redox Potentials ◽

Inner Membrane ◽

Content Type ◽

Transfer Strategies ◽

Reducing Bacteria ◽

Metal Reducing Bacteria

ABSTRACTDissimilatory metal-reducing bacteria, such asGeobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption ofimcH, encoding an inner membranec-type cytochrome, eliminated the ability ofG. sulfurreducensto reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but theimcHmutant retained the ability to reduce Fe(III) oxides with potentials of ≤−0.1 V versus SHE. TheimcHmutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to −0.1 V versus SHE triggered exponential growth. At potentials of ≤−0.1 V versus SHE, both the wild type and theimcHmutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not triggerimcHmutant growth. These results demonstrate thatG. sulfurreducenspossesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. The redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments whereGeobacteris found.IMPORTANCEInsoluble metal oxides in the environment represent a common and vast reservoir of energy for respiratory microbes capable of transferring electrons across their insulating membranes to external acceptors, a process termed extracellular electron transfer. Despite the global biogeochemical importance of metal cycling and the ability of such organisms to produce electricity at electrodes, fundamental gaps in the understanding of extracellular electron transfer biochemistry exist. Here, we describe a conserved inner membrane redox protein inGeobacter sulfurreducenswhich is required only for electron transfer to high-potential compounds, and we show thatG. sulfurreducenshas the ability to utilize different electron transfer pathways in response to the amount of energy available in a metal or electrode distant from the cell.

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Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III) oxide, Mn(IV) oxide, or electrode reduction by Geobacter sulfurreducens

10.1101/169086 ◽

2017 ◽

Cited By ~ 1

Author(s):

Fernanda Jiménez Otero ◽

Chi Ho Chan ◽

Daniel R. Bond

Keyword(s):

Electron Transfer ◽

Outer Membrane ◽

Electron Acceptor ◽

Single Gene ◽

Gene Clusters ◽

Geobacter Sulfurreducens ◽

Transcriptional Analysis ◽

Growth Defect ◽

Redox Potentials ◽

Wild Type

AbstractAt least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and −0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an ΔomcBC background, such as extEFG, were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens, depending on the available extracellular electron acceptor.

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Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III)-oxide, Mn(IV)-oxide, or electrode reduction by Geobacter sulfurreducens .

10.1101/350553 ◽

2018 ◽

Author(s):

Fernanda Jiménez Otero ◽

Chi Ho Chan ◽

Daniel R Bond

Keyword(s):

Electron Transfer ◽

Outer Membrane ◽

Electron Acceptor ◽

Single Gene ◽

Gene Clusters ◽

Geobacter Sulfurreducens ◽

Transcriptional Analysis ◽

Growth Defect ◽

Redox Potentials ◽

Wild Type

At least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c -type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and -0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an Δ omcBC background, such as extEFG , were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the Δ extABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens , depending on the available extracellular electron acceptor.

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Growth of Geobacter sulfurreducens with Acetate in Syntrophic Cooperation with Hydrogen-Oxidizing Anaerobic Partners

Applied and Environmental Microbiology ◽

10.1128/aem.64.6.2232-2236.1998 ◽

1998 ◽

Vol 64 (6) ◽

pp. 2232-2236 ◽

Cited By ~ 135

Author(s):

Ralf Cord-Ruwisch ◽

Derek R. Lovley ◽

Bernhard Schink

Keyword(s):

Electron Transfer ◽

Electron Acceptor ◽

Hydrogen Transfer ◽

Dry Mass ◽

Geobacter Sulfurreducens ◽

Acetate Oxidation ◽

Yield Data ◽

Partial Pressures ◽

Production Of Hydrogen ◽

Reducing Bacteria

ABSTRACT Pure cultures of Geobacter sulfurreducens and other Fe(III)-reducing bacteria accumulated hydrogen to partial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electron donor if electron release to an acceptor was limiting. G. sulfurreducens coupled acetate oxidation with electron transfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors. Cocultures of G. sulfurreducens and Wolinella succinogenes with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h. The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa. From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to G. sulfurreducens should be about −53 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO2 and H2. However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for G. sulfurreducens) indicated considerably higher energy gains. These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a c-type cytochrome that was found to be excreted byG. sulfurreducens into the culture fluid. Syntrophic acetate degradation was also possible with G. sulfurreducens and Desulfovibrio desulfuricans CSN but only with nitrate as electron acceptor. These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates. These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature.

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The redox potentials of the two-iron plant algal ferredoxins. An electrostatic model

Biochemical Journal ◽

10.1042/bj1330283 ◽

1973 ◽

Vol 133 (2) ◽

pp. 283-287 ◽

Cited By ~ 4

Author(s):

R. J. Kassner ◽

W. Yang

Keyword(s):

Electron Transfer ◽

Free Energy ◽

Redox Potential ◽

Negative Charge ◽

Electrostatic Model ◽

Redox Potentials ◽

Electron Transfer Proteins ◽

Electrostatic Free Energy ◽

The One ◽

Charged Ions

The two-iron–sulphur co-ordination centre in plant and algal ferredoxins is considered as a collection of charged ions whose net negative charge is twice that of the one-iron–sulphur protein rubredoxin. Calculation of the electrostatic free-energy changes for reduction of the two types of proteins indicates that the redox potential of the two-iron–sulphur proteins should be more negative than that of the one-iron–sulphur protein and that in biological systems the ferredoxins should function as one-electron transfer proteins.

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Oxidation of Structural Ferrous Iron in Vermiculites: I. Oxidation by Fe3+

Clay Minerals ◽

10.1180/claymin.1988.023.3.03 ◽

1988 ◽

Vol 23 (3) ◽

pp. 261-270 ◽

Cited By ~ 1

Author(s):

H. Graf ◽

V. Reichenbach ◽

B. Beyme

Keyword(s):

Hydrogen Peroxide ◽

Electron Transfer ◽

Redox Potential ◽

Cation Exchange ◽

Ferrous Iron ◽

Activity Ratio ◽

Redox Potentials ◽

Interlayer Cation ◽

Pt Electrode ◽

Coupled Reactions

AbstractVermiculite prepared from biotite by interlayer cation exchange was reacted with solutions exhibiting redox potentials between 625 and 765 mV. The redox potential was controlled by the Fe2+/Fe3+ activity ratio, measured with a Pt electrode, and kept constant by addition of hydrogen peroxide to balance electron transfer from structural Fe2+ to Fe3+ in solution. Oxidation of structural Fe2+ was followed by Eh-stat titration and the rate of oxidation was shown to depend on the amount of Fe3+ penetrating into interlayer positions. Consequently, it was affected not only by the redox potential, but also by the activity between Fe3+ and all other cations present in solution or in an exchangeable state. Oxidation and cation exchange are coupled reactions. In contrast to the redox potential in solution, the effective redox potential controlling the oxidation of structural Fe2+ was increased by preferential sorption of the Fe3+ ion.

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Electrochemical redox transformations of T1 and T2 copper sites in native Trametes hirsuta laccase at gold electrode

Biochemical Journal ◽

10.1042/bj20041015 ◽

2005 ◽

Vol 385 (3) ◽

pp. 745-754 ◽

Cited By ~ 121

Author(s):

Sergey SHLEEV ◽

Andreas CHRISTENSON ◽

Vladimir SEREZHENKOV ◽

Dosymzhan BURBAEV ◽

Alexander YAROPOLOV ◽

...

Keyword(s):

Electron Transfer ◽

Gold Electrode ◽

Direct Electron Transfer ◽

High Potential ◽

Redox Potentials ◽

Hydrogen Electrode ◽

Trametes Hirsuta ◽

Redox Transformations ◽

Copper Site ◽

Copper Sites

Mediatorless, electrochemically driven, redox transformations of T1 (type 1) and T2 copper sites in Trametes hirsuta laccase were studied by cyclic voltammetry and spectroelectrochemical redox titrations using bare gold electrode. DET (direct electron transfer) between the electrode and the enzyme was observed under anaerobic conditions. From analysis of experimental data it is concluded that the T2 copper site is in DET contact with gold. It was found that electron transfer between the gold surface and the T1 copper site progresses through the T2 copper site. From EPR measurements and electrochemical data it is proposed that the redox potential of the T2 site for high-potential ‘blue’ laccase is equal to about 400 mV versus NHE (normal hydrogen electrode) at pH 6.5. The hypothesis that the redox potentials of the T2 copper sites in low- and high-potential laccases/oxidases from totally different sources might be very similar, i.e. approx. 400 mV, is discussed.

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Geothrix fermentans Secretes Two Different Redox-Active Compounds To Utilize Electron Acceptors across a Wide Range of Redox Potentials

Applied and Environmental Microbiology ◽

10.1128/aem.01460-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 6987-6995 ◽

Cited By ~ 44

Author(s):

Misha G. Mehta-Kolte ◽

Daniel R. Bond

Keyword(s):

Electron Transfer ◽

Redox Potential ◽

Electron Acceptor ◽

Extracellular Electron Transfer ◽

Redox Potentials ◽

Active Compounds ◽

Content Type ◽

Electron Shuttles ◽

Redox Active ◽

Geothrix Fermentans

ABSTRACTThe current understanding of dissimilatory metal reduction is based primarily on isolates from the proteobacterial generaGeobacterandShewanella. However, environments undergoing active Fe(III) reduction often harbor less-well-studied phyla that are equally abundant. In this work, electrochemical techniques were used to analyze respiratory electron transfer by the only known Fe(III)-reducing representative of theAcidobacteria,Geothrix fermentans. In contrast to previously characterized metal-reducing bacteria, which typically reach maximal rates of respiration at electron acceptor potentials of 0 V versus standard hydrogen electrode (SHE),G. fermentansrequired potentials as high as 0.55 V to respire at its maximum rate. In addition,G. fermentanssecreted two different soluble redox-active electron shuttles with separate redox potentials (−0.2 V and 0.3 V). The compound with the lower midpoint potential, responsible for 20 to 30% of electron transfer activity, was riboflavin. The behavior of the higher-potential compound was consistent with hydrophilic UV-fluorescent molecules previously found inG. fermentanssupernatants. Both electron shuttles were also produced when cultures were grown with Fe(III), but not when fumarate was the electron acceptor. This study reveals thatGeothrixis able to take advantage of higher-redox-potential environments, demonstrates that secretion of flavin-based shuttles is not confined toShewanella, and points to the existence of high-potential-redox-active compounds involved in extracellular electron transfer. Based on differences between the respiratory strategies ofGeothrixandGeobacter, these two groups of bacteria could exist in distinctive environmental niches defined by redox potential.

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Redox potentials along the redox-active low-barrier H-bonds in electron transfer pathways

Physical Chemistry Chemical Physics ◽

10.1039/d0cp04265j ◽

2020 ◽

Vol 22 (44) ◽

pp. 25467-25473 ◽

Cited By ~ 1

Author(s):

Keisuke Saito ◽

Manoj Mandal ◽

Hiroshi Ishikita

Keyword(s):

Electron Transfer ◽

Proton Transfer ◽

Redox Potential ◽

Driving Force ◽

Electronic Coupling ◽

Redox Potentials ◽

Redox Active ◽

Electron Transfer Pathways

Local proton transfer along redox-active low-barrier H-bonds can alter the driving force or electronic coupling for electron transfer, as the redox potential values depend on the H+ position in low-barrier H-bonds.

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