Cysteine-mediated electron transfer in syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes

Syntrophic cocultures of Geobacter sulfurreducens and Wolinella succinogenes oxidize acetate with nitrate as terminal electron acceptor. It has been postulated earlier that electrons are transferred in these cocultures not via hydrogen, but via a different carrier, e.g., a small c-type cytochrome that is detected in the supernatant of growing cultures. In the present study, L -cysteine, which was provided as a reducing agent, was found to mediate the electron transfer between the two partners. Low concentrations of L -cysteine or L -cystine (10-100 microM) supported syntrophic growth, and no acetate oxidation was observed in the absence of cysteine or cystine. Cell suspensions of G. sulfurreducens or coculture cell suspensions reduced cystine to cysteine, and suspensions of W. succinogenes or coculture suspensions oxidized cysteine with nitrate, as measured by the formation or depletion of free thiol groups. Added cysteine was rapidly oxidized by the coculture during growth, but the formed cystine was not entirely rereduced even under acceptor-limited conditions. The redox potential prevailing in acetate-oxidizing cocultures was -160 to -230 mV. Sulfide at low concentrations supported syntrophic growth as well and could replace cysteine. Neither growth nor acetate degradation was found with D-cysteine, homocysteine, cysteamine, 3-mercaptopropionate, dithiothreithol, thioglycolate, glutathione, coenzyme M, dimethylsulfoxide, trimethylamine- N-oxide, anthraquinone-2,6-disulfonate, or ascorbate.     

Selective enrichment of Geobacter sulfurreducens from anaerobic granular sludge with quinones as terminal electron acceptors

A quinone-respiring, enrichment culture derived from methanogenic granular sludge was phylogenetically characterized by using a combined cloning-denaturing gradient gel electrophoresis (DGGE) method, which revealed that the consortium developed was dominated by a single microorganism: 97% related, in a sequence of 1520 base pairs, to Geobacter sulfurreducens. The enrichment culture could grow with acetate, formate or H₂ when humic acids, the humic model compound, anthraquinone-2,6-disulfonate (AQDS), or chelated Fe(III) was provided as a terminal electron acceptor. The occurrence of a humic acid- or quinone-respiring microorganism in the microbial community of a wastewater treatment system suggests that this type of microorganisms may play a potential role in anaerobic bioreactors treating humus-containing wastewaters.     

Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens

A novel fluorescence technique for monitoring the redox status of c-type cytochromes in Geobacter sulfurreducens was developed in order to evaluate the capacity of these extracytoplasmic cytochromes to store electrons during periods in which an external electron acceptor is not available. When intact cells in which the cytochromes were in a reduced state were excited at a wavelength of 350 nm, they fluoresced with maxima at 402 and 437 nm. Oxidation of the cytochromes resulted in a loss of fluorescence. This method was much more sensitive than the traditional approach of detecting c-type cytochromes via visible light absorbance. Furthermore, fluorescence of reduced cytochromes in individual cells could be detected via fluorescence microscopy, and the cytochromes in a G. sulfurreducens biofilm, remotely excited with an optical fibre, could be detected at distances as far as 5 cm. Fluorescence analysis of cytochrome oxidation and reduction of the external electron acceptor, anthraquinone-2,6-disulfonate, suggested that the extracytoplasmic cytochromes of G. sulfurreducens could store approximately 10(7) electrons per cell. Independent analysis of the haem content of the cells determined from analysis of incorporation of (55)Fe into cytochromes provided a similar estimate of cytochrome electron-storage capacity. This electron-storage capacity could, in the absence of an external electron acceptor, permit continued electron transfer across the inner membrane sufficient to supply the maintenance energy requirements for G. sulfurreducens for up to 8 min or enough proton motive force to power flagella motors for G. sulfurreducens motility. The fluorescence approach described here provides a sensitive method for evaluating the redox status of Geobacter species in culture and/or its environments. Furthermore, these results suggest that the periplasmic and outer-membrane cytochromes of Geobacter species act as capacitors, allowing continued electron transport, and thus viability and motility, for Geobacter species as they move between heterogeneously dispersed Fe(III) oxides during growth in the subsurface.     

Comparative genomic analysis of Geobacter sulfurreducens KN400, a strain with enhanced capacity for extracellular electron transfer and electricity production

ABSTRACT: BACKGROUND: A new strain of Geobacter sulfurreducens, strain KN400, produces more electrical current in microbial fuel cells and reduces insoluble Fe(III) oxides much faster than the wildtype strain, PCA. The genome of KN400 was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved. RESULTS: Both genomes were re-annotated, resulting in 14 fewer genes (net) in the PCA genome; 28 fewer (net) in the KN400 genome; and ca. 400 gene start and stop sites moved. 96% of genes in KN400 had clear orthologs with conserved synteny in PCA. Most of the remaining genes were in regions of genomic mobility and were strain-specific or conserved in other Geobacteraceae, indicating that the changes occurred post-divergence. There were 27,270 single nucleotide polymorphisms (SNP) between the genomes. There was significant enrichment for SNP locations in non-coding or synonymous amino acid sites, indicating significant selective pressure since the divergence. 25% of orthologs had sequence differences, and this set was enriched in phosphorylation and ATP-dependent enzymes. Substantial sequence differences (at least 12 non-synonymous SNP/kb) were found in 3.6% of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. Genes known to be involved in electron transport, those used in the metabolic cell model, and those that exhibit changes in expression during growth in microbial fuel cells were examined in detail. CONCLUSIONS: The improvement in external electron transfer in the KN400 strain does not appear to be due to novel gene acquisition, but rather to changes in the common metabolic network. The increase in electron transfer rate and yield in KN400 may be due to changes in carbon flux towards oxidation pathways and to changes in ATP metabolism, both of which indicate that the overall energy state of the cell may be different. The electrically conductive pili appear to be unchanged, but cytochrome folding, localization, and redox potentials may all be affected, which would alter the electrical connection between the cell and the substrate.     

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.     

Multiple roles of released c-type cytochromes in tuning electron transport and physiological status of Geobacter sulfurreducens

Dissimilatory metal-reducing bacteria (DMRB) can transfer electrons to extracellular insoluble electron acceptors and play important roles in geochemical cycling, biocorrosion, environmental remediation, and bioenergy generation. c-type cytochromes (c-Cyts) are synthesized by DMRB and usually transported to the cell surface to form modularized electron transport conduits through protein assembly, while some of them are released as extracellularly free-moving electron carriers in growth to promote electron transport. However, the type of these released c-Cyts, the timing of their release, and the functions they perform have not been unrevealed yet. In this work, after characterizing the types of c-Cyts released by Geobacter sulfurreducens under a variety of cultivation conditions, we found that these c-Cyts accumulated up to micromolar concentrations in the surrounding medium and conserved their chemical activities. Further studies demonstrated that the presence of c-Cyts accelerated the process of microbial extracellular electron transfer and mediated long-distance electron transfer. In particular, the presence of c-Cyts promoted the microbial respiration and affected the physiological state of the microbial community. In addition, c-Cyts were observed to be adsorbed on the surface of insoluble electron acceptors and modify electron acceptors. These results reveal the overlooked multiple roles of the released c-Cyts in acting as public goods, delivering electrons, modifying electron acceptors, and even regulating bacterial community structure in natural and artificial environments.     

pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer

The limitation of pH inside electrode‐respiring biofilms is a well‐known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode‐respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm. Biotechnol. Bioeng. 2012; 109: 2651–2662. © 2012 Wiley Periodicals, Inc.     

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

Background  

The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.     

Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e⁻/H⁺ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met⁵⁸, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e⁻/H⁺ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.     

Graphite electrode as a sole electron donor for reductive dechlorination of tetrachlorethene by Geobacter lovleyi

The possibility that graphite electrodes can serve as the direct electron donor for microbially catalyzed reductive dechlorination was investigated with Geobacter lovleyi. In an initial evaluation of whether G. lovleyi could interact electronically with graphite electrodes, cells were provided with acetate as the electron donor and an electrode as the sole electron acceptor. Current was produced at levels that were ca. 10-fold lower than those previously reported for Geobacter sulfurreducens under similar conditions, and G. lovleyi anode biofilms were correspondingly thinner. When an electrode poised at -300 mV (versus a standard hydrogen electrode) was provided as the electron donor, G. lovleyi effectively reduced fumarate to succinate. The stoichiometry of electrons consumed to succinate produced was 2:1, the ratio expected if the electrode served as the sole electron donor for fumarate reduction. G. lovleyi effectively reduced tetrachloroethene (PCE) to cis-dichloroethene with a poised electrode as the sole electron donor at rates comparable to those obtained when acetate serves as the electron donor. Cells were less abundant on the electrodes when the electrodes served as an electron donor than when they served as an electron acceptor. PCE was not reduced in controls without cells or when the current supply to cells was interrupted. These results demonstrate that G. lovleyi can use a poised electrode as a direct electron donor for reductive dechlorination of PCE. The ability to colocalize dechlorinating microorganisms with electrodes has several potential advantages for bioremediation of subsurface chlorinated contaminants, especially in source zones where electron donor delivery is challenging and often limits dechlorination.     

Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells

Geobacter sulfurreducens developed highly structured, multilayer biofilms on the anode surface of a microbial fuel cell converting acetate to electricity. Cells at a distance from the anode remained viable, and there was no decrease in the efficiency of current production as the thickness of the biofilm increased. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili. These pili may represent an electronic network permeating the biofilm that can promote long-range electrical transfer in an energy-efficient manner, increasing electricity production more than 10-fold.     

Comparative proteomics analysis of the root apoplasts of rice seedlings in response to hydrogen peroxide

Background

Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H₂O₂) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H₂O₂-responsive proteins in the apoplast of rice seedling roots.

Methodology/Principal Findings

14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H₂O₂ for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H₂O₂ were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H₂O₂ conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H₂O₂-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H₂O₂ stress.

Conclusions/Significance

The present study is the first apoplast proteome investigation of plant seedlings in response to H₂O₂ and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H₂O₂ response.     

A Periplasmic and Extracellular c-Type Cytochrome of Geobacter sulfurreducens Acts as a Ferric Iron Reductase and as an Electron Carrier to Other Acceptors or to Partner Bacteria

An extracellular electron carrier excreted into the growth medium by cells of Geobacter sulfurreducens was identified as a c-type cytochrome. The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate. It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm. The midpoint redox potential determined by redox titration was −0.167 V. With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes c of Desulfovibrio salexigens and Desulfuromonas acetoxidans. The G. sulfurreducens cytochrome c reduced ferrihydrite (Fe(OH)₃), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates. Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly. G. sulfurreducens reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate. Wolinella succinogenes was able to reduce externally provided cytochrome c of G. sulfurreducens with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor. A coculture could be established in which G. sulfurreducens reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by W. succinogenes in the presence of nitrate. We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.     

Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture

Geobacter sulfurreducens strain PCA oxidized acetate to CO2 via citric acid cycle reactions during growth with acetate plus fumarate in pure culture, and with acetate plus nitrate in coculture with Wolinella succinogenes. Acetate was activated by succinyl-CoA:acetate CoA-transferase and also via acetate kinase plus phosphotransacetylase. Citrate was formed by citrate synthase. Soluble isocitrate and malate dehydrogenases NADP+ and NAD+, respectively. Oxidation of 2-oxoglutarate was measured as benzyl viologen reduction and strictly CoA-dependent; a low activity was also observed with NADP+. Succinate dehydrogenase and fumarate ductase both were membrane-bound. Succinate oxidation was coupled to NADP+ reduction whereas fumarate reduction was coupled to NADPH and NADH Coupling of succinate oxidation to NADP+ or cytochrome(s) reduction required an ATP-dependent reversed electron transport. Net ATP synthesis proceeded exclusively through electron transport phosphorylation. During fumarate reduction, both NADPH and NADH delivered reducing equivalents into the electron transport chain, which contained a menaquinone. Overall, acetate oxidation with fumarate proceeded through an open loop of citric acid cycle reactions, excluding succinate dehydrogenase, with fumarate reductase as the key reaction for electron delivery, whereas acetate oxidation in the syntrophic coculture required the complete citric acid cycle.     

Biofilm and Nanowire Production Leads to Increased Current in Geobacter sulfurreducens Fuel Cells

Geobacter sulfurreducens developed highly structured, multilayer biofilms on the anode surface of a microbial fuel cell converting acetate to electricity. Cells at a distance from the anode remained viable, and there was no decrease in the efficiency of current production as the thickness of the biofilm increased. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili. These pili may represent an electronic network permeating the biofilm that can promote long-range electrical transfer in an energy-efficient manner, increasing electricity production more than 10-fold.