@article {3055, title = {Extracellular Electron Exchange Capabilities of and .}, journal = {Environ Sci Technol}, volume = {55}, year = {2021}, month = {2021 Dec 07}, pages = {16195-16203}, abstract = {

Microbial extracellular electron transfer plays an important role in diverse biogeochemical cycles, metal corrosion, bioelectrochemical technologies, and anaerobic digestion. Evaluation of electron uptake from pure Fe(0) and stainless steel indicated that, in contrast to previous speculation in the literature, and are not able to directly extract electrons from solid-phase electron-donating surfaces. grew with Fe(III) as the electron acceptor, but did not. reduced Fe(III) oxide occluded within porous alginate beads, suggesting that it released a soluble electron shuttle to promote Fe(III) oxide reduction. Conductive atomic force microscopy revealed that the pili are electrically conductive and the expression of a gene encoding an aromatics-rich putative pilin was upregulated during growth on Fe(III) oxide. The expression of genes for multi-heme -type cytochromes was not upregulated during growth with Fe(III) as the electron acceptor, and genes for a porin-cytochrome conduit across the outer membrane were not apparent in the genome. The results suggest that has adopted a novel combination of strategies to enable extracellular electron transport, which may be of biogeochemical and technological significance.

}, keywords = {Desulfovibrio, Electron Transport, Electrons, Ferric Compounds, Geobacter, Oxidation-Reduction}, issn = {1520-5851}, doi = {10.1021/acs.est.1c04071}, author = {Liang, Dandan and Liu, Xinying and Woodard, Trevor L and Holmes, Dawn E and Smith, Jessica A and Nevin, Kelly P and Feng, Yujie and Lovley, Derek R} } @article {3057, title = {Generation of High Current Densities in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor.}, journal = {Microbiol Spectr}, volume = {9}, year = {2021}, month = {2021 Oct 31}, pages = {e0087721}, abstract = {

Geobacter sulfurreducens is commonly employed as a model for the study of extracellular electron transport mechanisms in the species. Deletion of , which is known to encode the pilus assembly motor protein for type IV pili in other bacteria, has been proposed as an effective strategy for evaluating the role of electrically conductive pili (e-pili) in G. sulfurreducens extracellular electron transfer. In those studies, the inhibition of e-pili expression associated with deletion was not demonstrated directly but was inferred from the observation that deletion mutants produced lower current densities than wild-type cells. Here, we report that deleting did not diminish current production. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-type G. sulfurreducens or when e-pili are expressed heterologously from the G. sulfurreducens pilin gene in Escherichia coli. Immunogold labeling demonstrated that a G. sulfurreducens strain expressing a pilin monomer with a His tag continued to express His tag-labeled filaments when was deleted. These results suggest that a reinterpretation of the results of previous studies on G. sulfurreducens deletion strains may be necessary. Geobacter sulfurreducens is a model microbe for the study of biogeochemically and technologically significant processes, such as the reduction of Fe(III) oxides in soils and sediments, bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity, direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments, and metal corrosion. Elucidating the phenotypes associated with gene deletions is an important strategy for determining the mechanisms for extracellular electron transfer in G. sulfurreducens. The results reported here demonstrate that we cannot replicate the key phenotype reported for a gene deletion that has been central to the development of models for long-range electron transport in G. sulfurreducens.

}, keywords = {Bacterial Proteins, Electric Conductivity, Electron Transport, Fimbriae Proteins, Fimbriae, Bacterial, Gene Deletion, Geobacter, Geologic Sediments, Microscopy, Atomic Force, Oxidoreductases}, issn = {2165-0497}, doi = {10.1128/Spectrum.00877-21}, author = {Ueki, Toshiyuki and Walker, David J F and Nevin, Kelly P and Ward, Joy E and Woodard, Trevor L and Nonnenmann, Stephen S and Lovley, Derek R} } @article {3064, title = {Solvent-Induced Assembly of Microbial Protein Nanowires into Superstructured Bundles.}, journal = {Biomacromolecules}, volume = {22}, year = {2021}, month = {2021 Mar 08}, pages = {1305-1311}, abstract = {

Protein-based electronic biomaterials represent an attractive alternative to traditional metallic and semiconductor materials due to their environmentally benign production and purification. However, major challenges hindering further development of these materials include (1) limitations associated with processing proteins in organic solvents and (2) difficulties in forming higher-order structures or scaffolds with multilength scale control. This paper addresses both challenges, resulting in the formation of one-dimensional bundles composed of electrically conductive protein nanowires harvested from the microbes and . Processing these bionanowires from common organic solvents, such as hexane, cyclohexane, and DMF, enabled the production of multilength scale structures composed of distinctly visible pili. Transmission electron microscopy revealed striking images of bundled protein nanowires up to 10 μm in length and with widths ranging from 50-500 nm (representing assembly of tens to hundreds of nanowires). Conductive atomic force microscopy confirmed the presence of an appreciable nanowire conductivity in their bundled state. These results greatly expand the possibilities for fabricating a diverse array of protein nanowire-based electronic device architectures.

}, keywords = {Electric Conductivity, Electron Transport, Geobacter, Nanowires, Solvents}, issn = {1526-4602}, doi = {10.1021/acs.biomac.0c01790}, author = {Sun, Yun-Lu and Montz, Brian J and Selhorst, Ryan and Tang, Hai-Yan and Zhu, Jiaxin and Nevin, Kelly P and Woodard, Trevor L and Ribbe, Alexander E and Russell, Thomas P and Nonnenmann, Stephen S and Lovley, Derek R and Emrick, Todd} } @article {3072, title = {Syntrophus conductive pili demonstrate that common hydrogen-donating syntrophs can have a direct electron transfer option.}, journal = {ISME J}, volume = {14}, year = {2020}, month = {2020 Mar}, pages = {837-846}, abstract = {

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H 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.

}, keywords = {Deltaproteobacteria, Electric Conductivity, Electron Transport, Electrons, Fimbriae Proteins, Fimbriae, Bacterial, Formates, Geobacter, Hydrogen}, issn = {1751-7370}, doi = {10.1038/s41396-019-0575-9}, author = {Walker, David J F and Nevin, Kelly P and Holmes, Dawn E and Rotaru, Amelia-Elena and Ward, Joy E and Woodard, Trevor L and Zhu, Jiaxin and Ueki, Toshiyuki and Nonnenmann, Stephen S and McInerney, Michael J and Lovley, Derek R} } @article {3084, title = { Strains Expressing Poorly Conductive Pili Reveal Constraints on Direct Interspecies Electron Transfer Mechanisms.}, journal = {mBio}, volume = {9}, year = {2018}, month = {2018 Jul 10}, abstract = {

Cytochrome-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 strain Aro-5, in which the wild-type pilin gene was replaced with the pilin gene that was previously shown to yield poorly conductive pili in strain Aro-5. strain Aro-5 did not reduce Fe(III) oxide and produced only low current densities, phenotypes consistent with expression of poorly conductive pili. Like strain Aro-5, strain Aro-5 displayed abundant outer surface cytochromes. Cocultures initiated with wild-type as the electron-donating strain and strain Aro-5 as the electron-accepting strain grew via DIET. However, Aro-5/ 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. DIET is thought to be an important mechanism for interspecies electron exchange in natural anaerobic soils and sediments in which methane is either produced or consumed, as well as in some photosynthetic mats and anaerobic digesters converting organic wastes to methane. Understanding the potential mechanisms for DIET will not only aid in modeling carbon and electron flow in these geochemically significant environments but will also be helpful for interpreting meta-omic data from as-yet-uncultured microbes in DIET-based communities and for designing strategies to promote DIET in anaerobic digesters. The results demonstrate the need to develop a better understanding of the diversity of types of e-pili in the microbial world to identify potential electron-donating partners for DIET. Novel methods for recovering as-yet-uncultivated microorganisms capable of DIET in culture will be needed to further evaluate whether DIET is possible without e-pili in the absence of conductive materials such as GAC.

}, keywords = {Cytochromes, Electron Transport, Ferric Compounds, Fimbriae, Bacterial, Geobacter, Microbial Interactions, Oxidation-Reduction}, issn = {2150-7511}, doi = {10.1128/mBio.01273-18}, author = {Ueki, Toshiyuki and Nevin, Kelly P and Rotaru, Amelia-Elena and Wang, Li-Ying and Ward, Joy E and Woodard, Trevor L and Lovley, Derek R} } @article {3094, title = {Expressing the Geobacter metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity.}, journal = {mBio}, volume = {8}, year = {2017}, month = {2017 Jan 17}, abstract = {

UNLABELLED: 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 {\textpm} 18.9 S/cm (mean {\textpm} 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.

}, keywords = {Electric Conductivity, Electrodes, Electron Transport, Ferric Compounds, Fimbriae Proteins, Gene Expression, Geobacter, Oxidation-Reduction, Recombinant Proteins}, issn = {2150-7511}, doi = {10.1128/mBio.02203-16}, author = {Tan, Yang and Adhikari, Ramesh Y and Malvankar, Nikhil S and Ward, Joy E and Woodard, Trevor L and Nevin, Kelly P and Lovley, Derek R} } @article {3092, title = {Metatranscriptomic Evidence for Direct Interspecies Electron Transfer between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils.}, journal = {Appl Environ Microbiol}, volume = {83}, year = {2017}, month = {2017 May 01}, abstract = {

The possibility that (formerly ) and species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of accounted for over half of the PilA gene sequences in metagenomic libraries and 42\% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with species, but sequences most closely related to putative e-pilin genes from genera such as , , , and , were also detected. Approximately 17\% of all metagenomic and metatranscriptomic bacterial sequences clustered with species, and the finding that spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed genes. In addition, homologs of genes encoding OmcS, a -type cytochrome associated with the e-pili of and required for DIET, were also highly expressed in the soils. species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO reduction in Thus, these results are consistent with a model in which species were providing electrons to species for methane production through electrical connections of e-pili. species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for in methanogenic rice paddy soils is important because it demonstrates that the contribution that makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with spp., and possibly other methanogens, via DIET.

}, keywords = {Carbon Dioxide, Electron Transport, Fimbriae Proteins, Gene Expression Profiling, Geobacter, Metagenome, Methane, Methanosarcinaceae, Oryza, Soil Microbiology}, issn = {1098-5336}, doi = {10.1128/AEM.00223-17}, author = {Holmes, Dawn E and Shrestha, Pravin M and Walker, David J F and Dang, Yan and Nevin, Kelly P and Woodard, Trevor L and Lovley, Derek R} } @article {3104, title = {Potential enhancement of direct interspecies electron transfer for syntrophic metabolism of propionate and butyrate with biochar in up-flow anaerobic sludge blanket reactors.}, journal = {Bioresour Technol}, volume = {209}, year = {2016}, month = {2016 Jun}, pages = {148-56}, abstract = {

Promoting direct interspecies electron transfer (DIET) to enhance syntrophic metabolism may be a strategy for accelerating the conversion of organic wastes to methane, but microorganisms capable of metabolizing propionate and butyrate via DIET under methanogenic conditions have yet to be identified. In an attempt to establish methanogenic communities metabolizing propionate or butyrate with DIET, enrichments were initiated with up-flow anaerobic sludge blanket (UASB), similar to those that were previously reported to support communities that metabolized ethanol with DIET that relied on direct biological electrical connections. In the absence of any amendments, microbial communities enriched were dominated by microorganisms closely related to pure cultures that are known to metabolize propionate or butyrate to acetate with production of H2. When biochar was added to the reactors there was a substantial enrichment on the biochar surface of 16S rRNA gene sequences closely related to Geobacter and Methanosaeta species known to participate in DIET.

}, keywords = {Acetates, Bioreactors, Butyric Acid, Charcoal, Electron Transport, Geobacter, Methane, Microbial Consortia, Propionates, RNA, Ribosomal, 16S, Sewage, Waste Disposal, Fluid}, issn = {1873-2976}, doi = {10.1016/j.biortech.2016.03.005}, author = {Zhao, Zhiqiang and Zhang, Yaobin and Holmes, Dawn E and Dang, Yan and Woodard, Trevor L and Nevin, Kelly P and Lovley, Derek R} } @article {3128, title = {Magnetite compensates for the lack of a pilin-associated c-type cytochrome in extracellular electron exchange.}, journal = {Environ Microbiol}, volume = {17}, year = {2015}, month = {2015 Mar}, pages = {648-55}, abstract = {

Nanoscale magnetite can facilitate microbial extracellular electron transfer that plays an important role in biogeochemical cycles, bioremediation and several bioenergy strategies, but the mechanisms for the stimulation of extracellular electron transfer are poorly understood. Further investigation revealed that magnetite attached to the electrically conductive pili of Geobacter species in a manner reminiscent of the association of the multi-heme c-type cytochrome OmcS with the pili of Geobacter sulfurreducens. Magnetite conferred extracellular electron capabilities on an OmcS-deficient strain unable to participate in interspecies electron transfer or Fe(III) oxide reduction. In the presence of magnetite wild-type cells repressed expression of the OmcS gene, suggesting that cells might need to produce less OmcS when magnetite was available. The finding that magnetite can compensate for the lack of the electron transfer functions of a multi-heme c-type cytochrome has implications not only for the function of modern microbes, but also for the early evolution of microbial electron transport mechanisms.

}, keywords = {Cytochrome c Group, Electron Transport, Electrons, Ferrosoferric Oxide, Fimbriae Proteins, Fimbriae, Bacterial, Gene Expression Regulation, Bacterial, Geobacter, Heme, Oxides}, issn = {1462-2920}, doi = {10.1111/1462-2920.12485}, author = {Liu, Fanghua and Rotaru, Amelia-Elena and Shrestha, Pravin M and Malvankar, Nikhil S and Nevin, Kelly P and Lovley, Derek R} } @article {3117, title = {Carbon cloth stimulates direct interspecies electron transfer in syntrophic co-cultures.}, journal = {Bioresour Technol}, volume = {173}, year = {2014}, month = {2014 Dec}, pages = {82-86}, abstract = {

This study investigated the possibility that the electrical conductivity of carbon cloth accelerates direct interspecies electron transfer (DIET) in co-cultures. Carbon cloth accelerated metabolism of DIET co-cultures (Geobacter metallireducens-Geobacter sulfurreducens and G.metallireducens-Methanosarcina barkeri) but did not promote metabolism of co-cultures performing interspecies H2 transfer (Desulfovibrio vulgaris-G.sulfurreducens). On the other hand, DIET co-cultures were not stimulated by poorly conductive cotton cloth. Mutant strains lacking electrically conductive pili, or pili-associated cytochromes participated in DIET only in the presence of carbon cloth. In co-cultures promoted by carbon cloth, cells were primarily associated with the cloth although the syntrophic partners were too far apart for cell-to-cell biological electrical connections to be feasible. Carbon cloth seemingly mediated interspecies electron transfer between the distant syntrophic partners. These results suggest that the ability of carbon cloth to accelerate DIET should be considered in anaerobic digester designs that incorporate carbon cloth.

}, keywords = {Carbon, Cell Communication, Coculture Techniques, Electric Conductivity, Electron Transport, Geobacter, Materials Testing, Membranes, Artificial, Microbial Consortia, Oxidation-Reduction, Symbiosis}, issn = {1873-2976}, doi = {10.1016/j.biortech.2014.09.009}, author = {Chen, Shanshan and Rotaru, Amelia-Elena and Liu, Fanghua and Philips, Jo and Woodard, Trevor L and Nevin, Kelly P and Lovley, Derek R} } @article {3127, title = {Constraint-based modeling of carbon fixation and the energetics of electron transfer in Geobacter metallireducens.}, journal = {PLoS Comput Biol}, volume = {10}, year = {2014}, month = {2014 Apr}, pages = {e1003575}, abstract = {

Geobacter species are of great interest for environmental and biotechnology applications as they can carry out direct electron transfer to insoluble metals or other microorganisms and have the ability to assimilate inorganic carbon. Here, we report on the capability and key enabling metabolic machinery of Geobacter metallireducens GS-15 to carry out CO2 fixation and direct electron transfer to iron. An updated metabolic reconstruction was generated, growth screens on targeted conditions of interest were performed, and constraint-based analysis was utilized to characterize and evaluate critical pathways and reactions in G. metallireducens. The novel capability of G. metallireducens to grow autotrophically with formate and Fe(III) was predicted and subsequently validated in vivo. Additionally, the energetic cost of transferring electrons to an external electron acceptor was determined through analysis of growth experiments carried out using three different electron acceptors (Fe(III), nitrate, and fumarate) by systematically isolating and examining different parts of the electron transport chain. The updated reconstruction will serve as a knowledgebase for understanding and engineering Geobacter and similar species.

}, keywords = {Carbon, Electron Transport, Energy Metabolism, Genome, Bacterial, Geobacter, Models, Biological}, issn = {1553-7358}, doi = {10.1371/journal.pcbi.1003575}, author = {Feist, Adam M and Nagarajan, Harish and Rotaru, Amelia-Elena and Tremblay, Pier-Luc and Zhang, Tian and Nevin, Kelly P and Lovley, Derek R and Zengler, Karsten} } @article {3125, title = {Direct interspecies electron transfer between Geobacter metallireducens and Methanosarcina barkeri.}, journal = {Appl Environ Microbiol}, volume = {80}, year = {2014}, month = {2014 Aug}, pages = {4599-605}, abstract = {

Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H2-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H2 as an electron donor but metabolized little of the acetate that P.carbinolicus produced. This suggested that H2, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H2 transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H2 or electrons derived from DIET for CO2 reduction. Furthermore, M. barkeri is genetically tractable,making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

}, keywords = {Biological Transport, Electron Transport, Ethanol, Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Hydrogen, Methane, Methanosarcina barkeri}, issn = {1098-5336}, doi = {10.1128/AEM.00895-14}, author = {Rotaru, Amelia-Elena and Shrestha, Pravin Malla and Liu, Fanghua and Markovaite, Beatrice and Chen, Shanshan and Nevin, Kelly P and Lovley, Derek R} } @article {3126, title = {Going wireless: Fe(III) oxide reduction without pili by Geobacter sulfurreducens strain JS-1.}, journal = {Appl Environ Microbiol}, volume = {80}, year = {2014}, month = {2014 Jul}, pages = {4331-40}, abstract = {

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.

}, keywords = {Adaptation, Physiological, Biofilms, DNA, Bacterial, Electron Transport, Ferric Compounds, Fimbriae Proteins, Fimbriae, Bacterial, Gene Deletion, Geobacter, Oligonucleotide Array Sequence Analysis, Proteomics, Sequence Analysis, DNA}, issn = {1098-5336}, doi = {10.1128/AEM.01122-14}, author = {Smith, Jessica A and Tremblay, Pier-Luc and Shrestha, Pravin Malla and Snoeyenbos-West, Oona L and Franks, Ashley E and Nevin, Kelly P and Lovley, Derek R} } @article {3124, title = {Promoting interspecies electron transfer with biochar.}, journal = {Sci Rep}, volume = {4}, year = {2014}, month = {2014 May 21}, pages = {5019}, abstract = {

Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.

}, keywords = {Charcoal, Coculture Techniques, Electron Transport, Electrons, Ethanol, Geobacter, Methanosarcina barkeri, Soil}, issn = {2045-2322}, doi = {10.1038/srep05019}, author = {Chen, Shanshan and Rotaru, Amelia-Elena and Shrestha, Pravin Malla and Malvankar, Nikhil S and Liu, Fanghua and Fan, Wei and Nevin, Kelly P and Lovley, Derek R} } @article {3142, title = {Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens.}, journal = {mBio}, volume = {4}, year = {2013}, month = {2013 Mar 12}, pages = {e00105-13}, abstract = {

UNLABELLED: It has been proposed that Geobacter sulfurreducens requires conductive pili for long-range electron transport to Fe(III) oxides and for high-density current production in microbial fuel cells. In order to investigate this further, we constructed a strain of G. sulfurreducens, designated Aro-5, which produced pili with diminished conductivity. This was accomplished by modifying the amino acid sequence of PilA, the structural pilin protein. An alanine was substituted for each of the five aromatic amino acids in the carboxyl terminus of PilA, the region in which G. sulfurreducens PilA differs most significantly from the PilAs of microorganisms incapable of long-range extracellular electron transport. Strain Aro-5 produced pili that were properly decorated with the multiheme c-type cytochrome OmcS, which is essential for Fe(III) oxide reduction. However, pili preparations of the Aro-5 strain had greatly diminished conductivity and Aro-5 cultures were severely limited in their capacity to reduce Fe(III) compared to the control strain. Current production of the Aro-5 strain, with a graphite anode serving as the electron acceptor, was less than 10\% of that of the control strain. The conductivity of the Aro-5 biofilms was 10-fold lower than the control strain{\textquoteright}s. These results demonstrate that the pili of G. sulfurreducens must be conductive in order for the cells to be effective in extracellular long-range electron transport.

IMPORTANCE: Extracellular electron transfer by Geobacter species plays an important role in the biogeochemistry of soils and sediments and has a number of bioenergy applications. For example, microbial reduction of Fe(III) oxide is one of the most geochemically significant processes in anaerobic soils, aquatic sediments, and aquifers, and Geobacter organisms are often abundant in such environments. Geobacter sulfurreducens produces the highest current densities of any known pure culture, and close relatives are often the most abundant organisms colonizing anodes in microbial fuel cells that harvest electricity from wastewater or aquatic sediments. The finding that a strain of G. sulfurreducens that produces pili with low conductivity is limited in these extracellular electron transport functions provides further insight into these environmentally significant processes.

}, keywords = {Amino Acids, Aromatic, Bioelectric Energy Sources, Biofilms, Electricity, Electrodes, Electron Transport, Ferric Compounds, Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Graphite}, issn = {2150-7511}, doi = {10.1128/mBio.00105-13}, author = {Vargas, Madeline and Malvankar, Nikhil S and Tremblay, Pier-Luc and Leang, Ching and Smith, Jessica A and Patel, Pranav and Snoeyenbos-West, Oona and Nevin, Kelly P and Lovley, Derek R} } @article {441, title = {A c-type cytochrome and a transcriptional regulator responsible for enhanced extracellular electron transfer in Geobacter sulfurreducens revealed by adaptive evolution.}, journal = {Environ Microbiol}, volume = {13}, year = {2011}, month = {2011 Jan}, pages = {13-23}, abstract = {The stimulation of subsurface microbial metabolism often associated with engineered bioremediation of groundwater contaminants presents subsurface microorganisms, which are adapted for slow growth and metabolism in the subsurface, with new selective pressures. In order to better understand how Geobacter species might adapt to selective pressure for faster metal reduction in the subsurface, Geobacter sulfurreducens was put under selective pressure for rapid Fe(III) oxide reduction. The genomes of two resultant strains with rates of Fe(III) oxide reduction that were 10-fold higher than those of the parent strain were resequenced. Both strains contain either a single base-pair change or a 1 nucleotide insertion in a GEMM riboswitch upstream of GSU1761, a gene coding for the periplasmic c-type cytochrome designated PgcA. GSU1771, a gene coding for a SARP regulator, was also mutated in both strains. Introduction of either of the GEMM riboswitch mutations upstream of pgcA in the wild-type increased the abundance of pgcA transcripts, consistent with increased expression of pgcA in the adapted strains. One of the mutations doubled the rate of Fe(III) oxide reduction. Interruption of GSU1771 doubled the Fe(III) oxide reduction rate. This was associated with an increased in expression of pilA, the gene encoding the structural protein for the pili thought to function as microbial nanowires. The combination of the GSU1771 interruption with either of the pgcA mutations resulted in a strain that reduced Fe(III) as fast as the comparable adapted strain. These results suggest that the accumulation of a small number of beneficial mutations under selective pressure, similar to that potentially present during bioremediation, can greatly enhance the capacity for Fe(III) oxide reduction in G. sulfurreducens. Furthermore, the results emphasize the importance of the c-type cytochrome PgcA and pili in Fe(III) oxide reduction and demonstrate how adaptive evolution studies can aid in the elucidation of complex mechanisms, such as extracellular electron transfer.}, keywords = {Adaptation, Physiological, Biodegradation, Environmental, Cytochrome c Group, DNA, Bacterial, Electron Transport, Evolution, Molecular, Ferric Compounds, Gene Expression Profiling, Genes, Bacterial, Genome, Bacterial, Geobacter, Mutagenesis, Insertional, Mutation, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Riboswitch, Sequence Analysis, DNA}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2010.02302.x}, author = {Tremblay, Pier-Luc and Summers, Zarath M and Glaven, Richard H and Nevin, Kelly P and Zengler, Karsten and Barrett, Christian L and Qiu, Yu and Palsson, Bernhard O and Lovley, Derek R} } @article {437, title = {Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens.}, journal = {Bioelectrochemistry}, volume = {80}, year = {2011}, month = {2011 Feb}, pages = {142-50}, abstract = {Geobacter sulfurreducens is one of the few microorganisms available in pure culture known to directly accept electrons from a negatively poised electrode. Microarray analysis was used to compare gene transcript abundance in biofilms of G. sulfurreducens using a graphite electrode as the sole electron donor for fumarate reduction compared with transcript abundance in biofilms growing on the same material, but not consuming current. Surprisingly, genes for putative cell-electrode connections, such as outer-surface cytochromes and pili, which are highly expressed in current-producing biofilms, were not highly expressed in current-consuming biofilms. Microarray analysis of G. sulfurreducens gene transcript abundance in current-consuming biofilms versus current-producing biofilms gave similar results. In both comparative studies current-consuming biofilms had greater transcript abundance for a gene (GSU3274) encoding a putative monoheme, c-type cytochrome. Deletion of genes for outer-surface proteins previously shown to be essential for optimal electron transfer to electrodes had no impact on electron transfer from electrodes. Deletion of GSU3274 completely inhibited electron transfer from electrodes, but had no impact on electron transfer to electrodes. These differences in gene expression patterns and the impact of gene deletions suggest that the mechanisms for electron transfer from electrodes to G. sulfurreducens differ significantly from the mechanisms for electron transfer to electrodes.}, keywords = {Bacterial Proteins, Biofilms, Cytochromes, Electrodes, Electron Transport, Electrons, Gene Expression, Geobacter, Graphite, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Sequence Deletion}, issn = {1878-562X}, doi = {10.1016/j.bioelechem.2010.07.005}, author = {Strycharz, Sarah M and Glaven, Richard H and Coppi, Maddalena V and Gannon, Sarah M and Perpetua, Lorrie A and Liu, Anna and Nevin, Kelly P and Lovley, Derek R} } @article {462, title = {Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells.}, journal = {PLoS One}, volume = {4}, year = {2009}, month = {2009}, pages = {e5628}, abstract = {The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 microm) biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Bioelectric Energy Sources, Biofilms, Cytochromes, Electrodes, Electron Transport, Fumarates, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genetic Complementation Test, Geobacter, Microscopy, Confocal, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, RNA, Messenger, Up-Regulation}, issn = {1932-6203}, doi = {10.1371/journal.pone.0005628}, author = {Nevin, Kelly P and Kim, Byoung-Chan and Glaven, Richard H and Johnson, Jessica P and Woodard, Trevor L and Meth{\'e}, Barbara A and Didonato, Raymond J and Covalla, Sean F and Franks, Ashley E and Liu, Anna and Lovley, Derek R} } @article {501, title = {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.}, journal = {Appl Environ Microbiol}, volume = {73}, year = {2007}, month = {2007 Aug}, pages = {5347-53}, abstract = {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.}, keywords = {Bacteriological Techniques, Deltaproteobacteria, Electricity, Electron Transport, Ferrous Compounds, In Situ Hybridization, Fluorescence, Microscopy, Confocal, Oxidation-Reduction, RNA, Ribosomal, 16S}, issn = {0099-2240}, doi = {10.1128/AEM.00804-07}, author = {Richter, Hanno and Lanthier, Martin and Nevin, Kelly P and Lovley, Derek R} } @article {514, title = {Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells.}, journal = {Appl Environ Microbiol}, volume = {72}, year = {2006}, month = {2006 Nov}, pages = {7345-8}, abstract = {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.}, keywords = {Acetates, Bioelectric Energy Sources, Biofilms, Electricity, Electrodes, Electron Transport, Fimbriae, Bacterial, Geobacter, Microscopy, Confocal, Mutation, Nanowires}, issn = {0099-2240}, doi = {10.1128/AEM.01444-06}, author = {Reguera, Gemma and Nevin, Kelly P and Nicoll, Julie S and Covalla, Sean F and Woodard, Trevor L and Lovley, Derek R} }