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Microbial consortia that cooperatively exchange electrons play a key role in the anaerobic processing of organic matter. Interspecies hydrogen transfer is a well-documented strategy for electron exchange in dispersed laboratory cultures, but cooperative partners in natural environments often form multispecies aggregates. We found that laboratory evolution of a coculture of Geobacter metallireducens and Geobacter sulfurreducens metabolizing ethanol favored the formation of aggregates that were electrically conductive. Sequencing aggregate DNA revealed selection for a mutation that enhances the production of a c-type cytochrome involved in extracellular electron transfer and accelerates the formation of aggregates. Aggregate formation was also much faster in mutants that were deficient in interspecies hydrogen transfer, further suggesting direct interspecies electron transfer.

Methanothrix is widely distributed in natural and artificial anoxic environments and plays a major role in global methane emissions. It is one of only two genera that can form methane from acetate dismutation and through participation in direct interspecies electron transfer (DIET) with exoelectrogens. Although Methanothrix is a significant member of many methanogenic communities, little is known about its physiology. In this study, transcriptomics helped to identify potential routes of electron transfer during DIET between Geobacter metallireducens and Methanothrix thermoacetophila . Additions of magnetite to cultures significantly enhanced growth by acetoclastic methanogenesis and by DIET, while granular activated carbon (GAC) amendments impaired growth. Transcriptomics suggested that the OmaF-OmbF-OmcF porin complex and the octaheme outer membrane c -type cytochrome encoded by Gmet_0930, were important for electron transport across the outer membrane of G. metallireducens during DIET with Mx. thermoacetophila . Clear differences in the metabolism of Mx. thermoacetophila when grown via DIET or acetate dismutation were not apparent. However, genes coding for proteins involved in carbon fixation, the sheath fiber protein MspA, and a surface-associated quinoprotein, SqpA, were highly expressed in all conditions. Expression of gas vesicle genes was significantly lower in DIET- than acetate-grown cells, possibly to facilitate better contact between membrane-associated redox proteins during DIET. These studies reveal potential electron transfer mechanisms utilized by both Geobacter and Methanothrix during DIET and provide important insights into the physiology of Methanothrix in anoxic environments. Methanothrix is a significant methane producer in a variety of methanogenic environments including soils and sediments as well as anaerobic digesters. Its abundance in these anoxic environments has mostly been attributed to its high affinity for acetate and its ability to grow by acetoclastic methanogenesis. However, Methanothrix species can also generate methane by directly accepting electrons from exoelectrogenic bacteria through direct interspecies electron transfer (DIET). Methane production through DIET is likely to further increase their contribution to methane production in natural and artificial environments. Therefore, acquiring a better understanding of DIET with Methanothrix will help shed light on ways to (i) minimize microbial methane production in natural terrestrial environments and (ii) maximize biogas formation by anaerobic digesters treating waste.

Extracellular electron transfer by Geobacter species through surface appendages known as microbial nanowires 1 is important in a range of globally important environmental phenomena 2 , as well as for applications in bio-remediation, bioenergy, biofuels and bioelectronics. Since 2005, these nanowires have been thought to be type 4 pili composed solely of the PilA-N protein 1 . However, previous structural analyses have demonstrated that, during extracellular electron transfer, cells do not produce pili but rather nanowires made up of the cytochromes OmcS 2 , 3 and OmcZ 4 . Here we show that Geobacter sulfurreducens binds PilA-N to PilA-C to assemble heterodimeric pili, which remain periplasmic under nanowire-producing conditions that require extracellular electron transfer 5 . Cryo-electron microscopy revealed that C-terminal residues of PilA-N stabilize its copolymerization with PilA-C (to form PilA-N–C) through electrostatic and hydrophobic interactions that position PilA-C along the outer surface of the filament. PilA-N–C filaments lack π-stacking of aromatic side chains and show a conductivity that is 20,000-fold lower than that of OmcZ nanowires. In contrast with surface-displayed type 4 pili, PilA-N–C filaments show structure, function and localization akin to those of type 2 secretion pseudopili 6 . The secretion of OmcS and OmcZ nanowires is lost when pilA-N is deleted and restored when PilA-N–C filaments are reconstituted. The substitution of pilA-N with the type 4 pili of other microorganisms also causes a loss of secretion of OmcZ nanowires. As all major phyla of prokaryotes use systems similar to type 4 pili, this nanowire translocation machinery may have a widespread effect in identifying the evolution and prevalence of diverse electron-transferring microorganisms and in determining nanowire assembly architecture for designing synthetic protein nanowires. Structural, functional and localization studies reveal that Geobacter sulfurreducens pili cannot behave as microbial nanowires, instead functioning in a similar way to secretion pseudopili to export cytochrome nanowires that are essential for extracellular electron transfer.

Microorganisms are a primary control on the redox-induced cycling of iron in the environment. Despite the ability of bacteria to grow using both Fe(II) and Fe(III) bound in solid-phase iron minerals, it is currently unknown whether changing environmental conditions enable the sharing of electrons in mixed-valent iron oxides between bacteria with different metabolisms. We show through magnetic and spectroscopic measurements that the phototrophic Fe(II)-oxidizing bacterium Rhodopseudomonas palustris TIE-1 oxidizes magnetite (Fe3O4) nanoparticles using light energy. This process is reversible in co-cultures by the anaerobic Fe(III)-reducing bacterium Geobacter sulfurreducens. These results demonstrate that Fe ions bound in the highly crystalline mineral magnetite are bioavailable as electron sinks and electron sources under varying environmental conditions, effectively rendering magnetite a naturally occurring battery.

Methylmercury is a potent neurotoxin produced in natural environments from inorganic mercury by anaerobic bacteria. However, until now the genes and proteins involved have remained unidentified. Here, we report a two-gene cluster, hgcA and hgcB, required for mercury methylation by Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. In either bacterium, deletion of hgcA, hgcB, or both genes abolishes mercury methylation. The genes encode a putative corrinoid protein, HgcA, and a 2[4Fe-4S] ferredoxin, HgcB, consistent with roles as a methyl carrier and an electron donor required for corrinoid cofactor reduction, respectively. Among bacteria and archaea with sequenced genomes, gene orthologs are present in confirmed methylators but absent in nonmethylators, suggesting a common mercury methylation pathway in all methylating bacteria and archaea sequenced to date.

The in situ stimulation of Fe(III) oxide reduction by Geobacter bacteria leads to the concomitant precipitation of hexavalent uranium [U(VI)] from groundwater. Despite its promise for the bioremediation of uranium contaminants, the biological mechanism behind this reaction remains elusive. Because Fe(III) oxide reduction requires the expression of Geobacter's conductive pili, we evaluated their contribution to uranium reduction in Geobacter sulfurreducens grown under pili-inducing or noninducing conditions. A pilin-deficient mutant and a genetically complemented strain with reduced outer membrane c-cytochrome content were used as controls. Pili expression significantly enhanced the rate and extent of uranium immobilization per cell and prevented periplasmic mineralization. As a result, pili expression also preserved the vital respiratory activities of the cell envelope and the cell's viability. Uranium preferentially precipitated along the pili and, to a lesser extent, on outer membrane redox-active foci. In contrast, the pilus-defective strains had different degrees of periplasmic mineralization matching well with their outer membrane c-cytochrome content. X-ray absorption spectroscopy analyses demonstrated the extracellular reduction of U(VI) by the pili to mononuclear tetravalent uranium U(IV) complexed by carbon-containing ligands, consistent with a biological reduction. In contrast, the U(IV) in the pilin-deficient mutant cells also required an additional phosphorous ligand, in agreement with the predominantly periplasmic mineralization of uranium observed in this strain. These findings demonstrate a previously unrecognized role for Geobacter conductive pili in the extracellular reduction of uranium, and highlight its essential function as a catalytic and protective cellular mechanism that is of interest for the bioremediation of uranium-contaminated groundwater.

In anaerobic biota, reducing equivalents (electrons) are transferred between different species of microbes [interspecies electron transfer (IET)], establishing the basis of cooperative behaviors and community functions. IET mechanisms described so far are based on diffusion of redox chemical species and/or direct contact in cell aggregates. Here, we show another possibility that IET also occurs via electric currents through natural conductive minerals. Our investigation revealed that electrically conductive magnetite nanoparticles facilitated IET from Geobacter sulfurreducens to Thiobacillus denitrificans , accomplishing acetate oxidation coupled to nitrate reduction. This two-species cooperative catabolism also occurred, albeit one order of magnitude slower, in the presence of Fe ions that worked as diffusive redox species. Semiconductive and insulating iron-oxide nanoparticles did not accelerate the cooperative catabolism. Our results suggest that microbes use conductive mineral particles as conduits of electrons, resulting in efficient IET and cooperative catabolism. Furthermore, such natural mineral conduits are considered to provide ecological advantages for users, because their investments in IET can be reduced. Given that conductive minerals are ubiquitously and abundantly present in nature, electric interactions between microbes and conductive minerals may contribute greatly to the coupling of biogeochemical reactions.

Electroactive microbes have significant environmental impacts, as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal cofactors common in much of biological electron transport. Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens . However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c -type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90% of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in Escherichia coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c -type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90% filaments with a 3-nm diameter, but greatly reduced conductance, further indicating that the 3-nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c -type cytochromes, including OmcS, were deleted yielded only 3-nm-diameter filaments with the same conductance as in the wild type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens , consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer. IMPORTANCE Electroactive microbes have significant environmental impacts, as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal cofactors common in much of biological electron transport. This study demonstrates that e-pili are abundant filaments emanating from Geobacter sulfurreducens , which serves as a model for long-range extracellular electron transfer in direct interspecies electron transfer, dissimilatory metal reduction, microbe-electrode exchange, and corrosion caused by direct electron uptake from Fe(0). The methods described in this study provide a simple strategy for evaluating the distribution of conductive filaments throughout the microbial world with an approach that avoids artifactual production and/or enrichment of filaments that may not be physiologically relevant.

The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G. uraniireducens , were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G. metallireducens were investigated. Heterologously expressing the PilA gene of G. metallireducens in G. sulfurreducens yielded a G. sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH 7 had conductivities of 277 ± 18.9 S/cm (mean ± standard deviation), which is 5,000-fold higher than the conductivity of G. sulfurreducens pili at pH 7 and nearly 1 million-fold higher than the conductivity of G. uraniireducens pili at the same pH. A potential explanation for the higher conductivity of the G. metallireducens pili is their greater density of aromatic amino acids, which are known to be important components in electron transport along the length of the pilus. The G. metallireducens pili represent the most highly conductive pili found to date and suggest strategies for designing synthetic pili with even higher conductivities. IMPORTANCE e-pili are a remarkable electrically conductive material that can be sustainably produced without harsh chemical processes from renewable feedstocks and that contain no toxic components in the final product. Thus, e-pili offer an unprecedented potential for developing novel materials, electronic devices, and sensors for diverse applications with a new “green” technology. Increasing e-pili conductivity will even further expand their potential applications. A proven strategy is to design synthetic e-pili that contain tryptophan, an aromatic amino acid not found in previously studied e-pili. The studies reported here demonstrate that a productive alternative approach is to search more broadly in the microbial world. Surprisingly, even though G. metallireducens and G. sulfurreducens are closely related, the conductivities of their e-pili differ by more than 3 orders of magnitude. The ability to produce e-pili with high conductivity without generating a genetically modified product enhances the attractiveness of this novel electronic material. e-pili are a remarkable electrically conductive material that can be sustainably produced without harsh chemical processes from renewable feedstocks and that contain no toxic components in the final product. Thus, e-pili offer an unprecedented potential for developing novel materials, electronic devices, and sensors for diverse applications with a new “green” technology. Increasing e-pili conductivity will even further expand their potential applications. A proven strategy is to design synthetic e-pili that contain tryptophan, an aromatic amino acid not found in previously studied e-pili. The studies reported here demonstrate that a productive alternative approach is to search more broadly in the microbial world. Surprisingly, even though G. metallireducens and G. sulfurreducens are closely related, the conductivities of their e-pili differ by more than 3 orders of magnitude. The ability to produce e-pili with high conductivity without generating a genetically modified product enhances the attractiveness of this novel electronic material.

The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of direct interspecies electron transfer (DIET) in the environment. Here we tested eleven new co-culture combinations between methanogens and electrogens. Previously, only the most electrogenic Geobacter paired by DIET with Methanosarcinales methanogens, namely G . metallireducens and G . hydrogenophilus . Here we provide additional support, and show that five additional Methanosarcinales paired with G . metallireducens , while a strict hydrogenotroph could not. We also show that G . hydrogenophilus , which is incapable to grow with a strict hydrogenotrophic methanogen, could pair with a strict non-hydrogenotrophic Methanosarcinales . Likewise, an electrogen outside the Geobacter cluster ( Rhodoferrax ferrireducens ) paired with Methanosarcinales but not with strict hydrogenotrophic methanogens. The ability to interact with electrogens appears to be conserved among Methanosarcinales , the only methanogens with c -type cytochromes, including multihemes (MHC). Nonetheless, MHC, which are often linked to extracellular electron transfer, were neither unique nor universal to Methanosarcinales and only two of seven Methanosarcinales tested had MHC. Of these two, one strain had an MHC-deletion knockout available, which we hereby show is still capable to retrieve extracellular electrons from G . metallireducens or an electrode suggesting an MHC-independent strategy for extracellular electron uptake.