Conductive Pili Research Articles

Protein Nanowires: the Electrification of the Microbial World and Maybe Our Own.

Electrically conductive protein nanowires appear to be widespread in the microbial world and are a revolutionary "green" material for the fabrication of electronic devices. Electrically conductive pili (e-pili) assembled from type IV pilin monomers have independently evolved multiple times in microbial history as have electrically conductive archaella (e-archaella) assembled from homologous archaellin monomers. A role for e-pili in long-range (micrometer) extracellular electron transport has been demonstrated in some microbes. The surprising finding of e-pili in syntrophic bacteria and the role of e-pili as conduits for direct interspecies electron transfer have necessitated a reassessment of routes for electron flux in important methanogenic environments, such as anaerobic digesters and terrestrial wetlands. Pilin monomers similar to those found in e-pili may also be a major building block of the conductive "cables" that transport electrons over centimeter distances through continuous filaments of cable bacteria consisting of a thousand cells or more. Protein nanowires harvested from microbes have many functional and sustainability advantages over traditional nanowire materials and have already yielded novel electronic devices for sustainable electricity production, neuromorphic memory, and sensing. e-pili can be mass produced with an Escherichia coli chassis, providing a ready source of material for electronics as well as for studies on the basic mechanisms for long-range electron transport along protein nanowires. Continued exploration is required to better understand the electrification of microbial communities with microbial nanowires and to expand the "green toolbox" of sustainable materials for wiring and powering the emerging "Internet of things."

High-efficiency methanogenesis via kitchen wastes served as ethanol source to establish direct interspecies electron transfer during anaerobic Co-digestion with waste activated sludge

Kitchen wastes (KW) have been widely investigated for bio-ethanol production, while no study utilizes KW as ethanol source to stimulate the methanogenic communities to perform direct interspecies electron transfer (DIET), since the excess acidity contained after the biological ethanol-type fermentation pretreatment (BEFP) can seriously inhibit the DIET-based syntrophic metabolism. In this study, a strategy that utilized waste activated sludge (WAS) as co-substrate to relieve the excess acidity after BEFP during anaerobic co-digestion (AcoD) was proposed. The results showed that, under the mixed ratio of 1:2 and 1:5 (KW:WAS, volume ratio), both methane production and organic compound removal evidently increased, compared with that treating the sole WAS. Conversely, under the other mixed ratios (sole KW, 5:1, 2:1 and 1:1), no methane but the evident hydrogen production was detected, and syntrophic metabolism of organic acids and alcohols was prevented. Three-dimensional excitation emission matrix (3D-EEM) analysis showed that the protein-like organic compounds contained in both KW and WAS were effectively degraded. Furthermore, the maximum methane production potential from WAS during AcoD (260.5 ± 4.1 and 264.3 ± 2.7 mL/g-COD) was higher than that treating sole WAS (250.8 ± 0.1 mL/g-COD). Microbial community analysis showed that, some genera capable of metabolizing the complex organic compounds with the reduction of the elemental sulfur or equipped with the electrically conductive pili were specially enriched during AcoD under the mixed ratio of 1:2 and 1:5. They might proceed DIET with methanogens, such as Methanosarcina and Methanospirillum species, to maintain the syntrophic metabolism effective and stable, since the abundance of both Methanosarcina and Methanospirillum species evidently increased.

Global transcriptional analysis of Geobacter sulfurreducens under palladium reducing conditions reveals new key cytochromes involved.

Geobacter sulfurreducens is capable of reducing Pd(II) to Pd(0) using acetate as electron donor; however, the biochemical and genetic mechanisms involved in this process have not been described. In this work, we carried out transcriptome profiling analysis to identify the genes involved in Pd(II) reduction in this bacterium. Our results showed that 252 genes were upregulated while 141 were downregulated during Pd(II) reduction. Among the upregulated genes, 12 were related to energy metabolism and electron transport, 50 were classified as involved in protein synthesis, 42 were associated to regulatory functions and transcription, and 47 have no homologs with known function. RT-qPCR data confirmed upregulation of genes encoding PilA, the structural protein for electrically conductive pili, as well as c-type cytochromes GSU1062, GSU2513, GSU2808, GSU2934, GSU3107, OmcH, OmcM, PpcA, and PpcD under Pd(II)-reducing conditions. ΔpilA and ΔpilR mutant strains showed 20% and 40% decrease in the Pd(II)-reducing capacity, respectively, as compared to the wild type strain, indicating the central role of pili in this process. RT-qPCR data collected during Pd(II) reduction also confirmed downregulation of omcB, omcC, omcZ, and omcS genes, which have been shown to be involved in the reduction of Fe(III) and electrodes. The present study contributes to elucidate the mechanisms involved in Pd(II) reduction by G. sulfurreducens. Graphical Abstract KEY POINTS: • Transcriptome analysis provided evidence on Pd(II) reduction by G. sulfurreducens. • Results indicate that electrically conductive pili is involved in Pd(II) reduction. • G. sulfurreducens was not able to grow under Pd(II)-reducing conditions. • The study contributes to a better understanding of the mechanisms in Pd(II) reduction.

Potential interactions between syntrophic bacteria and methanogens via type IV pili and quorum-sensing systems

Interspecies electron transfer plays an important role in syntrophic methanogenesis. Direct interspecies electron transfer (DIET) between syntrophic oxidizers and methanogens via conductive pili has been only confirmed in some specific co-cultures. This study examined potential syntrophic cooperation via type IV pili and quorum sensing between widespread syntrophic bacteria and methanogens through a metagenomic analysis of 12 anaerobic sludge samples. We found that Methanosaeta and Methanosarcina, which are reported to have DIET ability, were dominant in most methanogenic samples. Putative conductive pili genes were found in some typical syntrophic bacteria, which has rarely been reported previously. The existence of diverse quorum-sensing genes suggested that various quorum-sensing systems might participate in the communication of anaerobic microorganisms. Specifically, the diffusible signal factor and 3′-5′ cyclic diguanosine monophosphate related genes were mainly assigned to syntrophic bacteria. These results suggest that the combined regulation of these signals might be responsible for the biosynthesis of type IV pili and affect syntrophic interaction during methanogenesis. These novel results provide fresh evidence to support the widespread existence of DIET in anaerobic methanogenic systems; therefore, regulating the quorum-sensing system may promote syntrophic interaction.

How does electron transfer occur in microbial fuel cells?

Microbial fuel cells (MFCs) have emerged as a promising technology for sustainable wastewater treatment coupled with electricity generation. A MFC is a device that uses microbes as catalysts to convert chemical energy present in biomass into electrical energy. Among the various mechanisms that drive the operation of a MFC, extracellular electron transfer (EET) to the anode is one of the most important. Exoelectrogenic bacteria can natively transfer electrons to a conducting surface like the anode. The mechanisms employed for electron transfer can either be direct transfer via conductive pili or nanowires, or mediated transfer that involves either naturally secreted redox mediators like flavins and pyocyanins or artificially added mediators like methylene blue and neutral red. EET is a mechanism wherein microorganisms extract energy for growth and maintenance from their surroundings and transfer the resulting electrons to the anode to generate current. The efficiency of these electron transfer mechanisms is dependent not only on the redox potentials of the species involved, but also on microbial oxidative metabolism that liberates electrons. Attempts at understanding the electron transfer mechanisms will boost efforts in giving rise to practical applications. This article covers the various electron transfer mechanisms involved between microbes and electrodes in microbial fuel cells and their applications.

Syntrophus conductive pili demonstrate that common hydrogen-donating syntrophs can have a direct electron transfer option.

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron transfer because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Heterologous expression of the putative S. aciditrophicus type IV pilin gene in Geobacter sulfurreducens yielded conductive pili of the same diameter (4 nm) and conductance of the native S. aciditrophicus pili and enabled long-range electron transport in G. sulfurreducens. S. aciditrophicus lacked abundant c-type cytochromes often associated with DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.

Molecular evidence for the adaptive evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in natural environments.

The electrically conductive pili (e-pili) of Geobacter species enable extracellular electron transfer to insoluble metallic minerals, electrodes and other microbial species, which confers biogeochemical significance and global prevalence on Geobacter in diverse anaerobic environments. E-pili are constructed by truncated PilA which is considered to have evolved from full-length pilin by gene fission under positive evolutionary selection. However, this hypothesis is based on phylogenetic analysis and has not yet been experimentally confirmed. Here, we reconstructed an ancestral strain of G. sulfurreducens (designated COMB) carrying full-length PilA by combining genes GSU1496 and GSU1497. The results demonstrated that strain COMB expressed and assembled the full-length fused PilA and exhibited an outer membrane c-type cytochrome profile similar to the wild-type strain. Surprisingly, the generated COMB-pili were also conductive, indicating the evolution of truncated PilA did not occur for conductivity. Moreover, strain COMB minimally reduced Fe(III) oxides but maintained its ability to respire electrodes, demonstrating the truncation of pilin enables iron respiration. This study provides the first experimentalevidence that the truncation of pilin in Geobacter species confers adaption to Fe(III)-mineral-mediated selective pressures, and suggests an evolutionary event during which the separation of the GSU1497 gene helped Geobacter survive and thrive in natural environments.

Direct Extracellular Electron Transfer of the Geobacter sulfurreducens Pili Relevant to Interaromatic Distances.

Microorganisms can transfer electrons directly to extracellular acceptors, during which organic compounds are oxidized to carbon dioxide. One of these microbes, Geobacter sulfurreducens, is well known for the “metallic-like” conductivity of its type IV pili. However, there is no consensus on what the mechanism for electron transfer along these conductive pili is. Based on the aromatic distances and orientations of our predicted models, the mechanism of electron transfer in the Geobacter sulfurreducens (GS) pili was explored by quantum chemical calculations with Marcus theory of electron transfer reactions. Three aromatic residues from the N-terminal α-helix of the GS pilin subunit are packed together, resulting in a continuous pi-pi interaction chain. The theoretical conductance (4.69 μS/3.85 μS) of the predicted models is very similar to that in the experiments reported recently (3.40 μS). These findings offer a new concept that the GS pili belongs to a new class of proteins that can transport electrons through pi-pi interaction between aromatic residues and also provide a valuable tool for guiding further researches of these conductive pili, to investigate their roles in biogeochemical cycling, and potential applications in biomaterials, bioelectronics, and bioenergy.

Voltammetric study of conductive planar assemblies of Geobacter nanowire pilins unmasks their ability to bind and mineralize divalent cobalt.

Geobacter bacteria assemble a helical peptide of the Type IVa pilin subclass as conductive pili decorated with metal binding and reduction sites. We used recombinant techniques to synthesize thiolated pilin derivatives and self-assembled them on gold electrodes as a monolayer thatconcentrated the metal traps at the liquid interface. Cyclic and step potential voltammetry demonstrated the conductivity of the pilin films and their ability to bind and reductively precipitate divalent cobalt (Co2+) in a diffusion-controlled reaction characterized by fast binding kinetics, efficient charge transfer, and three-dimensional nanoparticle growth at discreet sites. Furthermore, cobalt oxidation at the pilin film was slower than on bare gold, consistent with a peptide optimized for metal immobilization. These properties make recombinant pilins attractive building blocks for the synthesis of novel biomaterials for the immobilization of toxic cationic metals that, like Co2+, are sparingly soluble and, thus, less mobile and bioavailable as reduced species.

Flagella act as Geobacter biofilm scaffolds to stabilize biofilm and facilitate extracellular electron transfer

Flagella are widely expressed in electroactive biofilms; however, their actual role is unknown. To understand the role of flagella, two Geobacter sulfurreducens strains (KN400 and PCA, with and without flagella, respectively) were selected. We restored flagellum expression in trans in strain PCA and prevented flagellum expression in strain KN400. Electrochemical results showed that flagellum restoration in strain PCA promoted current generation, while flagellum deletion in strain KN400 impaired current production. However, the expression of conductive pili and outer surface c-type cytochromes was not affected. Further microscopic analyses demonstrated that flagella promoted the formation of thicker biofilms and served as biofilm matrix scaffolds to accommodate more extracellular cytochromes with an orderly arrangement, which increased the electron diffusion rate within the biofilm. Our findings reveal an unprecedented structural role for flagella in stabilizing electroactive biofilms and highlight the importance of cytochromes in electron transfer across biofilms, which will deepen our understanding of biofilm conductivity.

Geobacter Protein Nanowires.

The study of electrically conductive protein nanowires in Geobacter sulfurreducens has led to new concepts for long-range extracellular electron transport, as well as for the development of sustainable conductive materials and electronic devices with novel functions. Until recently, electrically conductive pili (e-pili), assembled from the PilA pilin monomer, were the only known Geobacter protein nanowires. However, filaments comprised of the multi-heme c-type cytochrome, OmcS, are present in some preparations of G. sulfurreducens outer-surface proteins. The purpose of this review is to evaluate the available evidence on the in vivo expression of e-pili and OmcS filaments and their biological function. Abundant literature demonstrates that G. sulfurreducens expresses e-pili, which are required for long-range electron transport to Fe (III) oxides and through conductive biofilms. In contrast, there is no definitive evidence yet that wild-type G. sulfurreducens express long filaments of OmcS extending from the cells, and deleting the gene for OmcS actually increases biofilm conductivity. The literature does not support the concern that many previous studies on e-pili were mistakenly studying OmcS filaments. For example, heterologous expression of the aromatic-rich pilin monomer of Geobacter metallireducens in G. sulfurreducens increases the conductivity of individual nanowires more than 5,000-fold, whereas expression of an aromatic-poor pilin reduced conductivity more than 1,000-fold. This more than million-fold range in nanowire conductivity was achieved while maintaining the 3-nm diameter characteristic of e-pili. Purification methods that eliminate all traces of OmcS yield highly conductive e-pili, as does heterologous expression of the e-pilin monomer in microbes that do not produce OmcS or any other outer-surface cytochromes. Future studies of G. sulfurreducens expression of protein nanowires need to be cognizant of the importance of maintaining environmentally relevant growth conditions because artificial laboratory culture conditions can rapidly select against e-pili expression. Principles derived from the study of e-pili have enabled identification of non-cytochrome protein nanowires in diverse bacteria and archaea. A similar search for cytochrome appendages is warranted. Both e-pili and OmcS filaments offer design options for the synthesis of protein-based “green” electronics, which may be the primary driving force for the study of these structures in the near future.

Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire

Electrically conductive pili from Geobacter species, termed bacterial nanowires, are intensely studied for their biological significance and potential in the development of new materials. Using cryo-electron microscopy, we have characterized nanowires from conductive G. sulfurreducens pili preparations that are composed solely of head-to-tail stacked monomers of the six-heme C-type cytochrome OmcS. The unique fold of OmcS — closely wrapped around a continuous stack of hemes that can serve as an uninterrupted path for electron transport — generates a scaffold that supports the unbranched chain of hemes along the central axis of the filament. We present here, at 3.4 Å resolution, the structure of this cytochrome-based filament and discuss its possible role in long-range biological electron transport.

Metagenomic analysis reveals that activated carbon aids anaerobic digestion of raw incineration leachate by promoting direct interspecies electron transfer

The raw fresh leachate from municipal solid waste (MSW) incineration plants contains high concentrations of volatile fatty acids (VFAs), ammonia and metals, all compounds that severely limit anaerobic digestion treatment efficiencies. These inhibitory compounds make reactor systems unstable, causing reactor start-up periods to take more than 100 days, even when the leachate is diluted significantly. In this study, granular activated carbon (GAC) was incorporated into a bioreactor fed with raw incineration leachate. Addition of GAC allowed direct treatment of raw incineration leachate without any start-up acclimation period, while the non-amended control reactor soured immediately and collapsed within 17 days. When hydraulic retention time (HRT) of the GAC-amended reactor was stepwise decreased to increase organic loading rates (OLR) to 25.0 kgCOD/(m3·d), COD removal efficiencies remained stable at >90%. Metagenomic analysis of the GAC-amended reactor revealed that Geobacter and Methanosarcina, species known to participate in direct interspecies electron transfer (DIET), were more abundant in the GAC-amended reactor than the seed sludge. In addition, the abundance of genes coding for proteins thought to be involved in DIET such as electrically conductive pili and the outer membrane c-type cytochrome, OmcS, increased significantly, while genes involved in fermentation, and nitrate (narG) and sulfate (dsrA) reduction dropped significantly as the experiment progressed. These results are significant because this is the first detailed investigation into the metabolic capabilities of microbial communities involved in efficient treatment of raw incineration leachate within biomethanogenic reactors that did not require a long start-up period.

Assessing Possible Mechanisms of Micrometer-Scale Electron Transfer in Heme-Free Geobacter sulfurreducens Pili.

The electrically conductive pili of Geobacter sulfurreducens are of both fundamental and practical interest. They facilitate extracellular and interspecies electron transfer (ET) and also provide an electrical interface between living and nonliving systems. We examine the possible mechanisms of G. sulfurreducens electron transfer in regimes ranging from incoherent to coherent transport. For plausible ET parameters, electron transfer in G. sulfurreducens bacterial nanowires mediated only by the protein is predicted to be dominated by incoherent hopping between phenylalanine (Phe) and tyrosine (Tyr) residues that are 3 to 4 Å apart, where Phe residues in the hopping pathways may create delocalized "islands." This mechanism could be accessible in the presence of strong oxidants that are capable of oxidizing Phe and Tyr residues. We also examine the physical requirements needed to sustain biological respiration via nanowires. We find that the hopping regimes with ET rates on the order of 108 s-1 between Phe islands and Tyr residues, and conductivities on the order of mS/cm, can support ET fluxes that are compatible with cellular respiration rates, although sustaining this delocalization in the heterogeneous protein environment may be challenging. Computed values of fully coherent electron fluxes through the pili are orders of magnitude too low to support microbial respiration. We suggest experimental probes of the transport mechanism based on mutant studies to examine the roles of aromatic amino acids and yet to be identified redox cofactors.

A pilin chaperone required for the expression of electrically conductive Geobacter sulfurreducens pili.

Mechanisms controlling the expression of the electrically conductive pili (e-pili) of Geobacter species are of interest because of the important role of e-pili in diverse biogeochemical processes, anaerobic digestion and electromicrobiological applications. We investigated the function of the protein, designated Spc (short pilin chaperone), encoded by the gene immediately downstream from the gene for PilA, the monomer that assembles into e-pili. Multiple lines of evidence suggest that Spc forms an oligomer that is associated with the inner membrane. Mutating the start codon of spc to prevent translation increased the transcript abundance of pilA but greatly diminished the abundance of PilA, and e-pili could no longer be detected. Cross-linking, protein capture and two-hybrid studies demonstrated that Spc and PilA interacted. Two sites in PilA for electrostatic interaction with Spc were identified. The results demonstrate that Spc is required for PilA stability prior to incorporation into e-pili, suggesting that Spc has a chaperone function that may be specific to the relatively short PilA monomers that assemble into e-pili. These results are important for identifying microorganisms likely to express e-pili from (meta)genomic data and for the construction of microbial strains expressing e-pili.

Potential of direct interspecies electron transfer in synergetic enhancement of methanogenesis and sulfate removal in an up-flow anaerobic sludge blanket reactor with magnetite

Anaerobic digestion (AD) has been widely applied in the treatment of industrial wastewater containing oxidized sulfur compounds. However, the production of hydrogen sulfide usually limits the syntrophic metabolism proceeded by interspecies hydrogen transfer (IHT), due to its corrosive and toxic properties. The current study was in an attempt to establish direct interspecies electron transfer (DIET) to resist the toxic inhibition from hydrogen sulfide and keep syntrophic metabolism stable. The results showed that, in the presence of magnetite, the methane production was improved about 3–10 folds at each ratio of COD/SO42−, while the enhancement of methanogenesis had almost no negative effect on sulfate reduction. With magnetite, the sludge conductance increased about 3 folds, but the concentration of c-type cytochromes decreased, suggesting that the potential DIET via both electrically conductive pili and outer surface c-type cytochromes was established. Microbial community revealed that, Veillonella species, the Fe(III)-reducing genus capable of reducing sulfate to hydrogen sulfide, were specially enriched with magnetite. Together with the relatively higher abundance of Methanothrix and Methanosarcina species, the novel DIET between Fe(III)/sulfate-reducing genus and methanogens was inferred to be responsible for the synergetic enhancement of methanogenesis and sulfate removal.

The electrifying physiology of Geobacter bacteria, 30 years on.

The family Geobacteraceae, with its only valid genus Geobacter, comprises deltaproteobacteria ubiquitous in soil, sediments, and subsurface environments where metal reduction is an active process. Research for almost three decades has provided novel insights into environmental processes and biogeochemical reactions not previously known to be carried out by microorganisms. At the heart of the environmental roles played by Geobacter bacteria is their ability to integrate redox pathways and regulatory checkpoints that maximize growth efficiency with electron donors derived from the decomposition of organic matter while respiring metal oxides, particularly the often abundant oxides of ferric iron. This metabolic specialization is complemented by versatile metabolic reactions, respiratory chains, and sensory networks that allow specific members to adaptively respond to environmental cues to integrate organic and inorganic contaminants in their oxidative and reductive metabolism, respectively. Thus, Geobacteraceae are important members of the microbial communities that degrade hydrocarbon contaminants under iron-reducing conditions and that contribute, directly or indirectly, to the reduction of radionuclides, toxic metals, and oxidized species of nitrogen. Their ability to produce conductive pili as nanowires for discharging respiratory electrons to solid-phase electron acceptors and radionuclides, or for wiring cells in current-harvesting biofilms highlights the unique physiological traits that make these organisms attractive biological platforms for bioremediation, bioenergy, and bioelectronics application. Here we review some of the most notable physiological features described in Geobacter species since the first model representatives were recovered in pure culture. We provide a historical account of the environmental research that has set the foundation for numerous physiological studies and the laboratory tools that had provided novel insights into the role of Geobacter in the functioning of microbial communities from pristine and contaminated environments. We pay particular attention to latest research, both basic and applied, that has served to expand the field into new directions and to advance interdisciplinary knowledge. The electrifying physiology of Geobacter, it seems, is alive and well 30 years on.

Maghemite (γ-Fe2O3) nanoparticles enhance dissimilatory ferrihydrite reduction by Geobacter sulfurreducens: Impacts on iron mineralogical change and bacterial interactions

Microbially mediated bioreduction of iron oxyhydroxide plays an important role in the biogeochemical cycle of iron. Geobacter sulfurreducens is a representative dissimilatory iron-reducing bacterium that assembles electrically conductive pili and cytochromes. The impact of supplementation with γ-Fe2O3 nanoparticles (NPs) (0.2 and 0.6 g) on the G. sulfurreducens-mediated reduction of ferrihydrite was investigated. In the overall performance of microbial ferrihydrite reduction mediated by γ-Fe2O3 NPs, stronger reduction was observed in the presence of direct contact with γ-Fe2O3 NPs than with indirect contact. Compared to the production of Fe(II) derived from biotic modification with ferrihydrite alone, increases greater than 1.6- and 1.4-fold in the production of Fe(II) were detected in the biotic modifications in which direct contact with 0.2 g and 0.6 g γ-Fe2O3 NPs, respectively, occurred. X-ray diffraction analysis indicated that magnetite was a unique representative iron mineral in ferrihydrite when active G. sulfurreducens cells were in direct contact with γ-Fe2O3 NPs. Because of the sorption of biogenic Fe(II) onto γ-Fe2O3 NPs instead of ferrihydrite, the addition of γ-Fe2O3 NPs could also contribute to increased duration of ferrihydrite reduction by preventing ferrihydrite surface passivation. Additionally, electron microscopy analysis confirmed that the direct addition of γ-Fe2O3 NPs stimulated the electrically conductive pili and cytochromes to stretch, facilitating long-range electron transfer between the cells and ferrihydrite. The obtained findings provide a more comprehensive understanding of the effects of iron oxide NPs on soil biogeochemistry.

Geobacter Strains Expressing Poorly Conductive Pili Reveal Constraints on Direct Interspecies Electron Transfer Mechanisms

ABSTRACTCytochrome-to-cytochrome electron transfer and electron transfer along conduits of multiple extracellular magnetite grains are often proposed as strategies for direct interspecies electron transfer (DIET) that do not require electrically conductive pili (e-pili). However, physical evidence for these proposed DIET mechanisms has been lacking. To investigate these possibilities further, we constructed Geobacter metallireducens strain Aro-5, in which the wild-type pilin gene was replaced with the aro-5 pilin gene that was previously shown to yield poorly conductive pili in Geobacter sulfurreducens strain Aro-5. G. metallireducens strain Aro-5 did not reduce Fe(III) oxide and produced only low current densities, phenotypes consistent with expression of poorly conductive pili. Like G. sulfurreducens strain Aro-5, G. metallireducens strain Aro-5 displayed abundant outer surface cytochromes. Cocultures initiated with wild-type G. metallireducens as the electron-donating strain and G. sulfurreducens strain Aro-5 as the electron-accepting strain grew via DIET. However, G. metallireducens Aro-5/G. sulfurreducens wild-type cocultures did not. Cocultures initiated with the Aro-5 strains of both species grew only when amended with granular activated carbon (GAC), a conductive material known to be a conduit for DIET. Magnetite could not substitute for GAC. The inability of the two Aro-5 strains to adapt for DIET in the absence of GAC suggests that there are physical constraints on establishing DIET solely through cytochrome-to-cytochrome electron transfer or along chains of magnetite. The finding that DIET is possible with electron-accepting partners that lack highly conductive pili greatly expands the range of potential electron-accepting partners that might participate in DIET.

Syntrophic growth with direct interspecies electron transfer between pili-free Geobacter species.

Direct interspecies electron transfer (DIET) may prevail in microbial communities that show methanogenesis and anaerobic methane oxidation and can be an electron source to support anaerobic photosynthesis. Previous mutagenic studies on cocultures of defined Geobacter species indicate that both conductive pili and extracellular cytochromes are essential for DIET. However, the actual functional role of the pili in DIET is uncertain, as the pilus mutation strategy used in these studies affected the extracellular cytochrome profile. Here we repressed the function of pili by deleting the pilus polymerization motor PilB in both Geobacter species. The PilB mutation inhibited the pilus assembly but did not alter the pattern of extracellular cytochromes. We report that the two pilus-free Geobacter species can form aggregates and grow syntrophically with DIET. The results demonstrate that the Gmet_2896 cytochrome of Geobacter metallireducens plays a key role in DIET and that conductive pili are not necessary to facilitate DIET in cocultures of Geobacter species, and they suggest cytochromes by themselves can meditate DIET, deepening the understanding of DIET.