Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.     

Metabolic Efficiency of Geobacter sulfurreducens Growing on Anodes with Different Redox Potentials

Microorganisms respiring Fe(III) in the environment face a range of redox potentials of the prospective terminal ferric electron acceptors, because Fe(III) can be present in different minerals or organic complexes. We investigated the adaptation of Geobacter sulfurreducens to this range by exposing the bacteria to different redox potentials between the electron donor acetate and solid, extracellular anodes in a microbial fuel-cell set-up. Over a range of anode potentials from ?0.105 to +0.645 V versus standard hydrogen electrode, G. sulfurreducens produced identical amounts of biomass per electron respired. This indicated that the organism cannot utilize higher available energies for energy conservation to ATP, and confirmed recent studies. Either the high potentials cannot be used due to physiological limitations, or G. sulfurreducens decreased its metabolic efficiency, and less biomass per unit of energy was produced. In this case, G. sulfurreducens “wasted” energy at high-potential differences, most likely as heat to fuel growth kinetics.     

Identification of an uptake hydrogenase required for hydrogen-dependent reduction of Fe(III) and other electron acceptors by Geobacter sulfurreducens

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens.     

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

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 by G. 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.     

Metabolic response of <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> towards electron donor/acceptor variation

Background  

Geobacter sulfurreducens is capable of coupling the complete oxidation of organic compounds to iron reduction. The metabolic response of G. sulfurreducens towards variations in electron donors (acetate, hydrogen) and acceptors (Fe(III), fumarate) was investigated via ¹³C-based metabolic flux analysis. We examined the ¹³C-labeling patterns of proteinogenic amino acids obtained from G. sulfurreducens cultured with ¹³C-acetate.     

Lack of Electricity Production by Pelobacter carbinolicus Indicates that the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability to Fuel Cell Anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

Going Wireless: Fe(III) Oxide Reduction without Pili by Geobacter sulfurreducens Strain JS-1

Previous studies have suggested that the conductive pili of Geobacter sulfurreducens are essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport through G. sulfurreducens biofilms is more effective via pili.     

Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.     

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.     

Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate,formate and/or hydrogen as electron donor

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO₂ fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO₂, and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.     

Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile

Background

In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated.

Results

The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III).

Conclusion

This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.     

A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production

The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity.     

Microbially Mediated Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5- Triazine by Extracellular Electron Shuttling Compounds

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.     

Localization and Solubilization of the Iron(III) Reductase of Geobacter sulfurreducens

The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain a c-type cytochrome.     

Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c 7 family

Cytochromes c ₇ are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins—phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c ₇ family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of ¹H–¹⁵N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.     

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH₂QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH₂QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for ¹³C,¹⁵N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and ¹H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.     

Thermodynamic properties of triheme cytochrome PpcF from Geobacter metallireducens reveal unprecedented functional mechanism

The bacterium Geobacter metallireducens is highly efficient in long-range extracellular electron transfer, a process that relies on an efficient bridging between the cytoplasmic electron donors and the extracellular acceptors. The periplasmic triheme cytochromes are crucial players in these processes and thus the understanding of their functional mechanism is crucial to elucidate the extracellular electron transfer processes in this microorganism. The triheme cytochrome PpcF from G. metallireducens has the lowest amino acid sequence identity with the remaining cytochromes from the PpcA-family of G. sulfurreducens and G. metallireducens, making it an interesting target for structural and functional studies. In this work, we performed a detailed functional and thermodynamic characterization of cytochrome PpcF by the complementary usage of NMR and visible spectroscopic techniques. The results obtained show that the heme reduction potentials are negative, different from each other and are also modulated by the redox and redox-Bohr interactions that assure unprecedented mechanistic features to the protein. The results showed that the order of oxidation of the hemes in cytochrome PpcF is maintained in the entire physiological pH range. The considerable separation of the hemes' redox potential values facilitates a sequential transfer within the chain of redox centers in PpcF, thus assuring electron transfer directionality to the electron acceptors.     

U(VI) Reduction by Diverse Outer Surface c-Type Cytochromes of Geobacter sulfurreducens

Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.     

Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp.Geobacter sulfurreducens is a member of the metal-reducing Geobacteraceae family and was originally isolated based on its ability to transfer electrons from internal oxidative reactions to extracellular electron acceptors such as insoluble Fe(III) or Mn(IV) oxides (5). G. sulfurreducens is also able to use an electrode as its sole electron acceptor for respiration, a phenotype which has many possible biotechnological applications (28, 29), and serves as a useful tool for direct measurement of electron transfer rates (2, 31). As G. sulfurreducens was the first Geobacteraceae genome sequence available (34) and the only member of this family with a robust genetic system (7), it serves as a model organism for extracellular electron transfer studies.The proteins facilitating electron transfer to insoluble Fe(III) oxides by individual Geobacter cells and how these cells interact in multicellular biofilms are not fully understood. Many genes implicated in Fe(III) and electrode reduction were identified based on proteomic and microarray analysis of cultures grown with fumarate versus Fe(III) citrate as a terminal electron acceptor (9, 15, 35). More recently, similar expression data from Fe(III) oxide and electrode-grown cultures have also become available (8, 12, 16). In most extracellular electron transfer studies, outer membrane proteins (such as c-type cytochromes) have been the focus (4, 23, 27, 32), leading to targeted knockout studies of at least 14 cytochromes to date.To reduce an insoluble electron acceptor, Geobacter spp. must achieve direct contact with the substrate (36). While contact with small Fe(III) oxide particles may be transient, growth on Fe(III)-coated surfaces or electron-accepting electrodes requires biofilm formation (31, 39). For example, when G. sulfurreducens produces an exponentially increasing rate of electron transfer at an electrode, this demonstrates that all newly divided cells remain embedded in the growing, conductive biofilm (2, 31). Thus, in addition to the need for an array of outer membrane cytochromes, there is also a need for control of both cell-cell contact and cell-surface contact.While a genetic system for G. sulfurreducens has been developed, conjugal transfer of a plasmid or a transposon has not been reported (7). The broad-host-range cloning vector pBBR1MCS-2 has previously been electroporated into G. sulfurreducens, but its mobilization capabilities were not utilized (7). Similarly, a number of suicide vectors have been identified for G. sulfurreducens, but none have been used to deliver transposons for mutagenesis. mariner-based transposon mutagenesis systems have been successful in a variety of Bacteria and Archaea, producing random insertions (20, 25, 40, 41, 43, 46, 48, 49). For example, genes involved in Shewanella oneidensis cytochrome maturation were discovered using the modified transposon mini-Himar RB1 (3).In this work, we describe a system for the conjugal transfer of the pBBR1MCS family of plasmids from Escherichia coli to G. sulfurreducens, which allowed transposon mutagenesis based on pMiniHimar RB1. Under strictly anaerobic conditions, a library of insertion mutants was constructed and screened to identify genes putatively involved in attachment and Fe(III) citrate reduction. Approximately 8,000 insertion mutants were isolated, with insertions distributed throughout the G. sulfurreducens chromosome. Subsequent characterization revealed mutants defective in metal reduction but unaffected in all aspects of electrode reduction, as well as mutants able to reduce metals but incapable of electrode reduction. These observations greatly expand the list of Geobacter mutants with defects in respiration or biofilm formation, and this library serves as a resource for further screening of extracellular electron transfer phenotypes.