@article {3045, title = {H Is a Major Intermediate in Corrosion of Iron.}, journal = {mBio}, volume = {14}, year = {2023}, month = {2023 Apr 25}, pages = {e0007623}, abstract = {

Desulfovibrio vulgaris has been a primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe have been proposed. We investigated Fe oxidation with a mutant of in which hydrogenase genes were deleted. The hydrogenase mutant grew as well as the parental strain with lactate as the electron donor, but unlike the parental strain, it was not able to grow on H. The parental strain reduced sulfate with Fe as the sole electron donor, but the hydrogenase mutant did not. H accumulated over time in Fe cultures of the hydrogenase mutant and sterile controls but not in parental strain cultures. Sulfide stimulated H production in uninoculated controls apparently by both reacting with Fe to generate H and facilitating electron transfer from Fe to H. Parental strain supernatants did not accelerate H production from Fe, ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe and via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe and either riboflavin or anthraquinone-2,6-disulfonate, and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe as the electron donor. The results demonstrate that primarily accepts electrons from Fe via H as an intermediary electron carrier. These findings clarify the interpretation of previous corrosion studies and suggest that H-mediated electron transfer is an important mechanism for iron corrosion under sulfate-reducing conditions. Microbial corrosion of iron in the presence of sulfate-reducing microorganisms is economically significant. There is substantial debate over how microbes accelerate iron corrosion. Tools for genetic manipulation have only been developed for a few Fe(III)-reducing and methanogenic microorganisms known to corrode iron and in each case those microbes were found to accept electrons from Fe via direct electron transfer. However, iron corrosion is often most intense in the presence of sulfate-reducing microbes. The finding that Desulfovibrio vulgaris relies on H to shuttle electrons between Fe and cells revives the concept, developed in some of the earliest studies on microbial corrosion, that sulfate reducers consumption of H is a major microbial corrosion mechanism. The results further emphasize that direct Fe-to-microbe electron transfer has yet to be rigorously demonstrated in sulfate-reducing microbes.

}, keywords = {Corrosion, Desulfovibrio, Desulfovibrio vulgaris, Hydrogenase, Iron, Lactic Acid, Oxidation-Reduction, Sulfates}, issn = {2150-7511}, doi = {10.1128/mbio.00076-23}, author = {Woodard, Trevor L and Ueki, Toshiyuki and Lovley, Derek R} } @article {3047, title = {Genetic Manipulation of Desulfovibrio ferrophilus and Evaluation of Fe(III) Oxide Reduction Mechanisms.}, journal = {Microbiol Spectr}, volume = {10}, year = {2022}, month = {2022 Dec 21}, pages = {e0392222}, abstract = {

The sulfate-reducing microbe Desulfovibrio ferrophilus is of interest due to its relatively rare ability to also grow with Fe(III) oxide as an electron acceptor and its rapid corrosion of metallic iron. Previous studies have suggested multiple agents for extracellular electron exchange including a soluble electron shuttle, electrically conductive pili, and outer surface multiheme -type cytochromes. However, the previous lack of a strategy for genetic manipulation of limited mechanistic investigations. We developed an electroporation-mediated transformation method that enabled replacement of genes of interest with an antibiotic resistance gene via double-crossover homologous recombination. Genes were identified that are essential for flagellum-based motility and the expression of the two types of pili. Disrupting flagellum-based motility or expression of either of the two pili did not inhibit Fe(III) oxide reduction, nor did deleting genes for multiheme -type cytochromes predicted to be associated with the outer membrane. Although redundancies in cytochrome or pilus function might explain some of these phenotypes, overall, the results are consistent with primarily reducing Fe(III) oxide via an electron shuttle. The finding that is genetically tractable not only will aid in elucidating further details of its mechanisms for Fe(III) oxide reduction but also provides a new experimental approach for developing a better understanding of some of its other unique features, such as the ability to corrode metallic iron at high rates and accept electrons from negatively poised electrodes. is an important pure culture model for Fe(III) oxide reduction and the corrosion of iron-containing metals in anaerobic marine environments. This study demonstrates that is genetically tractable, an important advance for elucidating the mechanisms by which it interacts with extracellular electron acceptors and donors. The results demonstrate that there is not one specific outer surface multiheme -type cytochrome that is essential for Fe(III) oxide reduction. This finding, coupled with the lack of apparent porin-cytochrome conduits encoded in the genome and the finding that deleting genes for pilus and flagellum expression did not inhibit Fe(III) oxide reduction, suggests that has adopted strategies for extracellular electron exchange that are different from those of intensively studied electroactive microbes like and species. Thus, the ability to genetically manipulate is likely to lead to new mechanistic concepts in electromicrobiology.

}, keywords = {Cytochromes, Electron Transport, Ferric Compounds, Iron, Oxidation-Reduction, Oxides}, issn = {2165-0497}, doi = {10.1128/spectrum.03922-22}, author = {Ueki, Toshiyuki and Woodard, Trevor L and Lovley, Derek R} } @article {3048, title = {Microbial biofilms for electricity generation from water evaporation and power to wearables.}, journal = {Nat Commun}, volume = {13}, year = {2022}, month = {2022 Jul 28}, pages = {4369}, abstract = {

Employing renewable materials for fabricating clean energy harvesting devices can further improve sustainability. Microorganisms can be mass produced with renewable feedstocks. Here, we demonstrate that it is possible to engineer microbial biofilms as a cohesive, flexible material for long-term continuous electricity production from evaporating water. Single biofilm sheet (~40 {\textmu}m thick) serving as the functional component in an electronic device continuously produces power density (~1 μW/cm) higher than that achieved with thicker engineered materials. The energy output is comparable to that achieved with similar sized biofilms catalyzing current production in microbial fuel cells, without the need for an organic feedstock or maintaining cell viability. The biofilm can be sandwiched between a pair of mesh electrodes for scalable device integration and current production. The devices maintain the energy production in ionic solutions and can be used as skin-patch devices to harvest electricity from sweat and moisture on skin to continuously power wearable devices. Biofilms made from different microbial species show generic current production from water evaporation. These results suggest that we can harness the ubiquity of biofilms in nature as additional sources of biomaterial for evaporation-based electricity generation in diverse aqueous environments.

}, keywords = {Bioelectric Energy Sources, Biofilms, Electricity, Electrodes, Water, Wearable Electronic Devices}, issn = {2041-1723}, doi = {10.1038/s41467-022-32105-6}, author = {Liu, Xiaomeng and Ueki, Toshiyuki and Gao, Hongyan and Woodard, Trevor L and Nevin, Kelly P and Fu, Tianda and Fu, Shuai and Sun, Lu and Lovley, Derek R and Yao, Jun} } @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 {3062, title = {Stainless steel corrosion via direct iron-to-microbe electron transfer by Geobacter species.}, journal = {ISME J}, volume = {15}, year = {2021}, month = {2021 Oct}, pages = {3084-3093}, abstract = {

Microbial corrosion of iron-based materials is a substantial economic problem. A mechanistic understanding is required to develop mitigation strategies, but previous mechanistic studies have been limited to investigations with relatively pure Fe(0), which is not a common structural material. We report here that the mechanism for microbial corrosion of stainless steel, the metal of choice for many actual applications, can be significantly different from that~for Fe(0). Although H is often an intermediary electron carrier between the metal and microbes during Fe(0) corrosion, we found that H is not abiotically produced from stainless steel, making this corrosion mechanism unlikely. Geobacter sulfurreducens and Geobacter metallireducens, electrotrophs that are known to directly accept electrons from other microbes or electrodes, extracted electrons from stainless steel via direct iron-to-microbe electron transfer. Genetic modification to prevent H consumption did not negatively impact on stainless steel corrosion. Corrosion was inhibited when genes for outer-surface cytochromes that are key electrical contacts were deleted. These results indicate that a common model of microbial Fe(0) corrosion~by hydrogenase-positive microbes, in which H serves as an intermediary electron carrier between the metal surface and the microbe, may not apply to the microbial corrosion of stainless steel. However, direct iron-to-microbe electron transfer is a~feasible route for stainless steel corrosion.

}, keywords = {Corrosion, Electrons, Geobacter, Iron, Stainless Steel}, issn = {1751-7370}, doi = {10.1038/s41396-021-00990-2}, author = {Tang, Hai-Yan and Yang, Chuntian and Ueki, Toshiyuki and Pittman, Conor C and Xu, Dake and Woodard, Trevor L and Holmes, Dawn E and Gu, Tingyue and Wang, Fuhui and Lovley, Derek R} } @article {3070, title = {An Chassis for Production of Electrically Conductive Protein Nanowires.}, journal = {ACS Synth Biol}, volume = {9}, year = {2020}, month = {2020 Mar 20}, pages = {647-654}, abstract = {

pilin-based electrically conductive protein nanowires (e-PNs) are a revolutionary electronic material. They offer novel options for electronic sensing applications and have the remarkable ability to harvest electrical energy from atmospheric humidity. However, technical constraints limit mass cultivation and genetic manipulation of . Therefore, we designed a strain of to express e-PNs by introducing a plasmid that contained an inducible operon with genes for type IV pili biogenesis machinery and a synthetic gene designed to yield a peptide monomer that could be assembled into e-PNs. The e-PNs expressed in and harvested with a simple filtration method had the same diameter (3 nm) and conductance as e-PNs expressed in . These results, coupled with the robustness of for mass cultivation and the extensive toolbox for genetic manipulation, greatly expand the opportunities for large-scale fabrication of novel e-PNs.

}, keywords = {Electric Conductivity, Escherichia coli, Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Graphite, Microorganisms, Genetically-Modified, Microscopy, Atomic Force, Nanowires, Operon, Protein Engineering}, issn = {2161-5063}, doi = {10.1021/acssynbio.9b00506}, author = {Ueki, Toshiyuki and Walker, David J F and Woodard, Trevor L and Nevin, Kelly P and Nonnenmann, Stephen S and Lovley, Derek R} } @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 {3075, title = {Decorating the Outer Surface of Microbially Produced Protein Nanowires with Peptides.}, journal = {ACS Synth Biol}, volume = {8}, year = {2019}, month = {2019 Aug 16}, pages = {1809-1817}, abstract = {

The potential applications of electrically conductive protein nanowires (e-PNs) harvested from might be greatly expanded if the outer surface of the wires could be modified to confer novel sensing capabilities or to enhance binding to other materials. We developed a simple strategy for functionalizing e-PNs with surface-exposed peptides. The gene for the monomer that assembles into e-PNs was modified to add peptide tags at the carboxyl terminus of the monomer. Strains of were constructed that fabricated synthetic e-PNs with a six-histidine "His-tag" or both the His-tag and a nine-peptide "HA-tag" exposed on the outer surface. Addition of the peptide tags did not diminish e-PN conductivity. The abundance of HA-tag in e-PNs was controlled by placing expression of the gene for the synthetic monomer with the HA-tag under transcriptional regulation. These studies suggest broad possibilities for tailoring e-PN properties for diverse applications.

}, keywords = {Carboxy-Lyases, Ethylene Glycols, Molecular Structure, Nanowires, Oxygenases, Peptides, Phenylalanine Ammonia-Lyase, Plasmids, Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Styrenes}, issn = {2161-5063}, doi = {10.1021/acssynbio.9b00131}, author = {Ueki, Toshiyuki and Walker, David J F and Tremblay, Pier-Luc and Nevin, Kelly P and Ward, Joy E and Woodard, Trevor L and Nonnenmann, Stephen S and Lovley, Derek R} } @article {3081, title = {Conductive Composite Materials Fabricated from Microbially Produced Protein Nanowires.}, journal = {Small}, volume = {14}, year = {2018}, month = {2018 Nov}, pages = {e1802624}, abstract = {

Protein-based electronic materials have numerous potential advantages with respect to sustainability and biocompatibility over electronic materials that are synthesized using harsh chemical processes and/or which contain toxic components. The microorganism Geobacter sulfurreducens synthesizes electrically conductive protein nanowires (e-PNs) with high aspect ratios (3 nm {\texttimes} 10-30 {\textmu}m) from renewable organic feedstocks. Here, the integration of G. Sulfurreducens e-PNs into poly(vinyl alcohol) (PVA) as a host polymer matrix is described. The resultant e-PN/PVA composites exhibit conductivities comparable to PVA-based composites containing synthetic nanowires. The relationship between e-PN density and conductivity of the resultant composites is consistent with percolation theory. These e-PNs confer conductivity to the composites even under extreme conditions, with the highest conductivities achieved from materials prepared at pH 1.5 and temperatures greater than 100 {\textdegree}C. These results demonstrate that e-PNs represent viable and sustainable nanowire compositions for the fabrication of electrically conductive composite materials.

}, keywords = {Geobacter, Nanocomposites, Nanowires, Polymers}, issn = {1613-6829}, doi = {10.1002/smll.201802624}, author = {Sun, Yun-Lu and Tang, Hai-Yan and Ribbe, Alexander and Duzhko, Volodimyr and Woodard, Trevor L and Ward, Joy E and Bai, Ying and Nevin, Kelly P and Nonnenmann, Stephen S and Russell, Thomas and Emrick, Todd and Lovley, Derek R} } @article {3083, title = {Construction of a Strain With Exceptional Growth on Cathodes.}, journal = {Front Microbiol}, volume = {9}, year = {2018}, month = {2018}, pages = {1512}, abstract = {

Insoluble extracellular electron donors are important sources of energy for anaerobic respiration in biogeochemical cycling and in diverse practical applications. The previous lack of a genetically tractable model microorganism that could be grown to high densities under anaerobic conditions in pure culture with an insoluble extracellular electron donor has stymied efforts to better understand this form of respiration. We report here on the design of a strain of , designated strain ACL, which grows as thick (ca. 35 μm) confluent biofilms on graphite cathodes poised at -500 mV ( Ag/AgCl) with fumarate as the electron acceptor. Sustained maximum current consumption rates were >0.8 A/m, which is >10-fold higher than the current consumption of the wild-type strain. The improved function on the cathode was achieved by introducing genes for an ATP-dependent citrate lyase, completing the complement of enzymes needed for a reverse TCA cycle for the synthesis of biosynthetic precursors from carbon dioxide. Strain ACL provides an important model organism for elucidating the mechanisms for effective anaerobic growth with an insoluble extracellular electron donor and may offer unique possibilities as a chassis for the introduction of synthetic metabolic pathways for the production of commodities with electrons derived from electrodes.

}, issn = {1664-302X}, doi = {10.3389/fmicb.2018.01512}, author = {Ueki, Toshiyuki and Nevin, Kelly P and Woodard, Trevor L and Aklujkar, Muktak A and Holmes, Dawn E and Lovley, Derek R} } @article {3087, title = {Electrically conductive pili from pilin genes of phylogenetically diverse microorganisms.}, journal = {ISME J}, volume = {12}, year = {2018}, month = {2018 Jan}, pages = {48-58}, abstract = {

The possibility that bacteria other than Geobacter species might contain genes for electrically conductive pili (e-pili) was investigated by heterologously expressing pilin genes of interest in Geobacter sulfurreducens. Strains of G. sulfurreducens producing high current densities, which are only possible with e-pili, were obtained with pilin genes from Flexistipes sinusarabici, Calditerrivibrio nitroreducens and Desulfurivibrio alkaliphilus. The conductance of pili from these strains was comparable to native G. sulfurreducens e-pili. The e-pili derived from C. nitroreducens, and D. alkaliphilus pilin genes are the first examples of relatively long (>100 amino acids) pilin monomers assembling into e-pili. The pilin gene from Candidatus Desulfofervidus auxilii did not yield e-pili, suggesting that the hypothesis that this sulfate reducer wires itself with e-pili to methane-oxidizing archaea to enable anaerobic methane oxidation should be reevaluated. A high density of aromatic amino acids and a lack of substantial aromatic-free gaps along the length of long pilins may be important characteristics leading to e-pili. This study demonstrates a simple method to screen pilin genes from difficult-to-culture microorganisms for their potential to yield e-pili; reveals new sources for biologically based electronic materials; and suggests that a wide phylogenetic diversity of microorganisms may use e-pili for extracellular electron exchange.

}, keywords = {Deltaproteobacteria, Electric Conductivity, Fimbriae Proteins, Fimbriae, Bacterial, Methane, Oxidation-Reduction, Phylogeny}, issn = {1751-7370}, doi = {10.1038/ismej.2017.141}, author = {Walker, David Jf and Adhikari, Ramesh Y and Holmes, Dawn E and Ward, Joy E and Woodard, Trevor L and Nevin, Kelly P 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 {3099, title = {Enhancing anaerobic digestion of complex organic waste with carbon-based conductive materials.}, journal = {Bioresour Technol}, volume = {220}, year = {2016}, month = {2016 Nov}, pages = {516-522}, abstract = {

The aim of this work was to study the methanogenic metabolism of dog food, a food waste surrogate, in laboratory-scale reactors with different carbon-based conductive materials. Carbon cloth, carbon felt, and granular activated carbon all permitted higher organic loading rates and promoted faster recovery of soured reactors than the control reactors. Microbial community analysis revealed that specific and substantial enrichments of Sporanaerobacter and Methanosarcina were present on the carbon cloth surface. These results, and the known ability of Sporanaerobacter species to transfer electrons to elemental sulfur, suggest that Sporanaerobacter species can participate in direct interspecies electron transfer with Methanosarcina species when carbon cloth is available as an electron transfer mediator.

}, keywords = {Anaerobiosis, Animals, Bacteria, Bioreactors, Carbon, Carbon Fiber, Charcoal, Dogs, Electric Conductivity, Fatty Acids, Volatile, Hydrogen-Ion Concentration, Methane, Organic Chemicals, Waste Products}, issn = {1873-2976}, doi = {10.1016/j.biortech.2016.08.114}, author = {Dang, Yan and Holmes, Dawn E and Zhao, Zhiqiang and Woodard, Trevor L and Zhang, Yaobin and Sun, Dezhi and Wang, Li-Ying and Nevin, Kelly P and Lovley, Derek R} } @article {3103, title = {Expanding the Diet for DIET: Electron Donors Supporting Direct Interspecies Electron Transfer (DIET) in Defined Co-Cultures.}, journal = {Front Microbiol}, volume = {7}, year = {2016}, month = {2016}, pages = {236}, abstract = {

Direct interspecies electron transfer (DIET) has been recognized as an alternative to interspecies H2 transfer as a mechanism for syntrophic growth, but previous studies on DIET with defined co-cultures have only documented DIET with ethanol as the electron donor in the absence of conductive materials. Co-cultures of Geobacter metallireducens and Geobacter sulfurreducens metabolized propanol, butanol, propionate, and butyrate with the reduction of fumarate to succinate. G. metallireducens utilized each of these substrates whereas only electrons available from DIET supported G. sulfurreducens respiration. A co-culture of G. metallireducens and a strain of G. sulfurreducens that could not metabolize acetate oxidized acetate with fumarate as the electron acceptor, demonstrating that acetate can also be syntrophically metabolized via DIET. A co-culture of G. metallireducens and Methanosaeta harundinacea previously shown to syntrophically convert ethanol to methane via DIET metabolized propanol or butanol as the sole electron donor, but not propionate or butyrate. The stoichiometric accumulation of propionate or butyrate in the propanol- or butanol-fed cultures demonstrated that M. harundinaceae could conserve energy to support growth solely from electrons derived from DIET. Co-cultures of G. metallireducens and Methanosarcina barkeri could also incompletely metabolize propanol and butanol and did not metabolize propionate or butyrate as sole electron donors. These results expand the range of substrates that are known to be syntrophically metabolized through DIET, but suggest that claims of propionate and butyrate metabolism via DIET in mixed microbial communities warrant further validation.

}, issn = {1664-302X}, doi = {10.3389/fmicb.2016.00236}, author = {Wang, Li-Ying and Nevin, Kelly P and Woodard, Trevor L and Mu, Bo-Zhong and Lovley, Derek R} } @article {3098, title = {Genetic switches and related tools for controlling gene expression and electrical outputs of Geobacter sulfurreducens.}, journal = {J Ind Microbiol Biotechnol}, volume = {43}, year = {2016}, month = {2016 Nov}, pages = {1561-1575}, abstract = {

Physiological studies and biotechnology applications of Geobacter species have been limited by a lack of genetic tools. Therefore, potential additional molecular strategies for controlling metabolism were explored. When the gene for citrate synthase, or acetyl-CoA transferase, was placed under the control of a LacI/IPTG regulator/inducer system, cells grew on acetate only in the presence of IPTG. The TetR/AT system could also be used to control citrate synthase gene expression and acetate metabolism. A strain that required IPTG for growth on D-lactate was constructed by placing the gene for D-lactate dehydrogenase under the control of the LacI/IPTG system. D-Lactate served as an inducer in a strain in which a D-lactate responsive promoter and transcription repressor were used to control citrate synthase expression. Iron- and potassium-responsive systems were successfully incorporated to regulate citrate synthase expression and growth on acetate. Linking the appropriate degradation tags on the citrate synthase protein made it possible to control acetate metabolism with either the endogenous ClpXP or exogenous Lon protease and tag system. The ability to control current output from Geobacter biofilms and the construction of an AND logic gate for acetate metabolism suggested that the tools developed may be applicable for biosensor and biocomputing applications.

}, keywords = {Acetates, Acetyl Coenzyme A, Citrate (si)-Synthase, Electric Conductivity, Gene Expression Regulation, Geobacter, Isopropyl Thiogalactoside, L-Lactate Dehydrogenase, Lac Repressors, Promoter Regions, Genetic, Transferases}, issn = {1476-5535}, doi = {10.1007/s10295-016-1836-5}, author = {Ueki, Toshiyuki and Nevin, Kelly P and Woodard, Trevor L and Lovley, Derek R} } @article {3100, title = {The Low Conductivity of Geobacter uraniireducens Pili Suggests a Diversity of Extracellular Electron Transfer Mechanisms in the Genus Geobacter.}, journal = {Front Microbiol}, volume = {7}, year = {2016}, month = {2016}, pages = {980}, abstract = {

Studies on the mechanisms for extracellular electron transfer in Geobacter species have primarily focused on Geobacter sulfurreducens, but the poor conservation of genes for some electron transfer components within the Geobacter genus suggests that there may be a diversity of extracellular electron transport strategies among Geobacter species. Examination of the gene sequences for PilA, the type IV pilus monomer, in Geobacter species revealed that the PilA sequence of Geobacter uraniireducens was much longer than that of G. sulfurreducens. This is of interest because it has been proposed that the relatively short PilA sequence of G. sulfurreducens is an important feature conferring conductivity to G. sulfurreducens pili. In order to investigate the properties of the G. uraniireducens pili in more detail, a strain of G. sulfurreducens that expressed pili comprised the PilA of G. uraniireducens was constructed. This strain, designated strain GUP, produced abundant pili, but generated low current densities and reduced Fe(III) very poorly. At pH 7, the conductivity of the G. uraniireducens pili was 3 {\texttimes} 10(-4) S/cm, much lower than the previously reported 5 {\texttimes} 10(-2) S/cm conductivity of G. sulfurreducens pili at the same pH. Consideration of the likely voltage difference across pili during Fe(III) oxide reduction suggested that G. sulfurreducens pili can readily accommodate maximum reported rates of respiration, but that G. uraniireducens pili are not sufficiently conductive to be an effective mediator of long-range electron transfer. In contrast to G. sulfurreducens and G. metallireducens, which require direct contact with Fe(III) oxides in order to reduce them, G. uraniireducens reduced Fe(III) oxides occluded within microporous beads, demonstrating that G. uraniireducens produces a soluble electron shuttle to facilitate Fe(III) oxide reduction. The results demonstrate that Geobacter species may differ substantially in their mechanisms for long-range electron transport and that it is important to have information beyond a phylogenetic affiliation in order to make conclusions about the mechanisms by which Geobacter species are transferring electrons to extracellular electron acceptors.

}, issn = {1664-302X}, doi = {10.3389/fmicb.2016.00980}, author = {Tan, Yang and Adhikari, Ramesh Y and Malvankar, Nikhil S and Ward, Joy E and Nevin, Kelly P and Woodard, Trevor L and Smith, Jessica A and Snoeyenbos-West, Oona L and Franks, Ashley E and Tuominen, Mark T 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 {3101, title = {Synthetic Biological Protein Nanowires with High Conductivity.}, journal = {Small}, volume = {12}, year = {2016}, month = {2016 Sep}, pages = {4481-5}, abstract = {

Genetic modification to add tryptophan to PilA, the monomer for the electrically conductive pili of Geobacter sulfurreducens, yields conductive protein filaments 2000-fold more conductive than the wild-type pili while cutting the diameter in half to 1.5 nm.

}, keywords = {Amino Acid Sequence, Electric Conductivity, Fimbriae, Bacterial, Geobacter, Nanowires, Proteins, Tryptophan}, issn = {1613-6829}, doi = {10.1002/smll.201601112}, author = {Tan, Yang and Adhikari, Ramesh Y and Malvankar, Nikhil S and Pi, Shuang and Ward, Joy E and Woodard, Trevor L and Nevin, Kelly P and Xia, Qiangfei and Tuominen, Mark T and Lovley, Derek R} } @article {3105, title = {Link between capacity for current production and syntrophic growth in Geobacter species.}, journal = {Front Microbiol}, volume = {6}, year = {2015}, month = {2015}, pages = {744}, abstract = {

Electrodes are unnatural electron acceptors, and it is yet unknown how some Geobacter species evolved to use electrodes as terminal electron acceptors. Analysis of different Geobacter species revealed that they varied in their capacity for current production. Geobacter metallireducens and G. hydrogenophilus generated high current densities (ca. 0.2 mA/cm(2)), comparable to G. sulfurreducens. G. bremensis, G. chapellei, G. humireducens, and G. uraniireducens, produced much lower currents (ca. 0.05 mA/cm(2)) and G. bemidjiensis was previously found to not produce current. There was no correspondence between the effectiveness of current generation and Fe(III) oxide reduction rates. Some high-current-density strains (G. metallireducens and G. hydrogenophilus) reduced Fe(III)-oxides as fast as some low-current-density strains (G. bremensis, G. humireducens, and G. uraniireducens) whereas other low-current-density strains (G. bemidjiensis and G. chapellei) reduced Fe(III) oxide as slowly as G. sulfurreducens, a high-current-density strain. However, there was a correspondence between the ability to produce higher currents and the ability to grow syntrophically. G. hydrogenophilus was found to grow in co-culture with Methanosarcina barkeri, which is capable of direct interspecies electron transfer (DIET), but not with Methanospirillum hungatei capable only of H2 or formate transfer. Conductive granular activated carbon (GAC) stimulated metabolism of the G. hydrogenophilus - M. barkeri co-culture, consistent with electron exchange via DIET. These findings, coupled with the previous finding that G. metallireducens and G. sulfurreducens are also capable of DIET, suggest that evolution to optimize DIET has fortuitously conferred the capability for high-density current production to some Geobacter species.

}, issn = {1664-302X}, doi = {10.3389/fmicb.2015.00744}, author = {Rotaru, Amelia-Elena and Woodard, Trevor L and Nevin, Kelly P and Lovley, Derek R} } @article {3107, title = {Protozoan grazing reduces the current output of microbial fuel cells.}, journal = {Bioresour Technol}, volume = {193}, year = {2015}, month = {2015 Oct}, pages = {8-14}, abstract = {

Several experiments were conducted to determine whether protozoan grazing can reduce current output from sediment microbial fuel cells. When marine sediments were amended with eukaryotic inhibitors, the power output from the fuel cells increased 2-5-fold. Quantitative PCR showed that Geobacteraceae sequences were 120 times more abundant on anodes from treated fuel cells compared to untreated fuel cells, and that Spirotrichea sequences in untreated fuel cells were 200 times more abundant on anode surfaces than in the surrounding sediments. Defined studies with current-producing biofilms of Geobacter sulfurreducens and pure cultures of protozoa demonstrated that protozoa that were effective in consuming G. sulfurreducens reduced current production up to 91\% when added to G. sulfurreducens fuel cells. These results suggest that anode biofilms are an attractive food source for protozoa and that protozoan grazing can be an important factor limiting the current output of sediment microbial fuel cells.

}, keywords = {Bioelectric Energy Sources, Biofilms, Electricity, Electrodes, Eukaryota, Geobacter, Geologic Sediments}, issn = {1873-2976}, doi = {10.1016/j.biortech.2015.06.056}, author = {Holmes, Dawn E and Nevin, Kelly P and Snoeyenbos-West, Oona L and Woodard, Trevor L and Strickland, Justin N 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 {3115, title = {Converting carbon dioxide to butyrate with an engineered strain of Clostridium ljungdahlii.}, journal = {mBio}, volume = {5}, year = {2014}, month = {2014 Oct 21}, pages = {e01636-14}, abstract = {

Microbial conversion of carbon dioxide to organic commodities via syngas metabolism or microbial electrosynthesis is an attractive option for production of renewable biocommodities. The recent development of an initial genetic toolbox for the acetogen Clostridium ljungdahlii has suggested that C. ljungdahlii may be an effective chassis for such conversions. This possibility was evaluated by engineering a strain to produce butyrate, a valuable commodity that is not a natural product of C. ljungdahlii metabolism. Heterologous genes required for butyrate production from acetyl-coenzyme A (CoA) were identified and introduced initially on plasmids and in subsequent strain designs integrated into the C. ljungdahlii chromosome. Iterative strain designs involved increasing translation of a key enzyme by modifying a ribosome binding site, inactivating the gene encoding the first step in the conversion of acetyl-CoA to acetate, disrupting the gene which encodes the primary bifunctional aldehyde/alcohol dehydrogenase for ethanol production, and interrupting the gene for a CoA transferase that potentially represented an alternative route for the production of acetate. These modifications yielded a strain in which ca. 50 or 70\% of the carbon and electron flow was diverted to the production of butyrate with H2 or CO as the electron donor, respectively. These results demonstrate the possibility of producing high-value commodities from carbon dioxide with C. ljungdahlii as the catalyst. Importance: The development of a microbial chassis for efficient conversion of carbon dioxide directly to desired organic products would greatly advance the environmentally sustainable production of biofuels and other commodities. Clostridium ljungdahlii is an effective catalyst for microbial electrosynthesis, a technology in which electricity generated with renewable technologies, such as solar or wind, powers the conversion of carbon dioxide and water to organic products. Other electron donors for C. ljungdahlii include carbon monoxide, which can be derived from industrial waste gases or the conversion of recalcitrant biomass to syngas, as well as hydrogen, another syngas component. The finding that carbon and electron flow in C. ljungdahlii can be diverted from the production of acetate to butyrate synthesis is an important step toward the goal of renewable commodity production from carbon dioxide with this organism.

}, keywords = {Acetyl Coenzyme A, Butyrates, Carbon Dioxide, Clostridium, Metabolic Engineering, Metabolic Flux Analysis, Metabolic Networks and Pathways, Recombinant Proteins}, issn = {2150-7511}, doi = {10.1128/mBio.01636-14}, author = {Ueki, Toshiyuki and Nevin, Kelly P and Woodard, Trevor L and Lovley, Derek R} } @article {699, title = {Spatial heterogeneity of bacterial communities in sediments from an infiltration basin receiving highway runoff.}, journal = {Microb Ecol}, volume = {64}, year = {2012}, month = {2012 Aug}, pages = {461-73}, abstract = {The bacterial community diversity of highway runoff-contaminated sediment that had undergone 19 years of acetate-based de-icing agents addition followed by three years of acetate-free de-icing agents was investigated. Analysis of 26 sediment samples from two drilled soil cores by means of 16S rDNA PCR generated 3,402 clones, indicating an overall high bacterial diversity, with no prominent members within the communities. Sequence analyses provided evidences that each sediment sample displayed a specific structure bacterial community. Proteobacteria-affiliated clones (58\% and 43\% for the two boreholes) predominated in all samples, followed by Actinobacteria (12\% and 16\%), Firmicutes (7\% and 12\%) and Chloroflexi (7\% and 11\%). The subsurface geochemistry complemented the molecular methods to further distinguish ambient and contaminant plume zones. Principal component analysis revealed that the levels of Fe(II) and dissolved oxygen were strongly correlated with bacterial communities. At elevated Fe(II) levels, sequences associated with anaerobic bacteria were detected in high levels. As iron levels declined and oxygen levels increased below the plume bottom, there was a gradual shift in the community structure toward the increase of aerobic bacteria.}, issn = {1432-184X}, doi = {10.1007/s00248-012-0026-x}, author = {Rotaru, Camelia and Woodard, Trevor L and Choi, Seokyoon and Nevin, Kelly P} } @article {424, title = {Electrosynthesis of organic compounds from carbon dioxide is catalyzed by a diversity of acetogenic microorganisms.}, journal = {Appl Environ Microbiol}, volume = {77}, year = {2011}, month = {2011 May}, pages = {2882-6}, abstract = {Microbial electrosynthesis, a process in which microorganisms use electrons derived from electrodes to reduce carbon dioxide to multicarbon, extracellular organic compounds, is a potential strategy for capturing electrical energy in carbon-carbon bonds of readily stored and easily distributed products, such as transportation fuels. To date, only one organism, the acetogen Sporomusa ovata, has been shown to be capable of electrosynthesis. The purpose of this study was to determine if a wider range of microorganisms is capable of this process. Several other acetogenic bacteria, including two other Sporomusa species, Clostridium ljungdahlii, Clostridium aceticum, and Moorella thermoacetica, consumed current with the production of organic acids. In general acetate was the primary product, but 2-oxobutyrate and formate also were formed, with 2-oxobutyrate being the predominant identified product of electrosynthesis by C. aceticum. S. sphaeroides, C. ljungdahlii, and M. thermoacetica had high (>80\%) efficiencies of electrons consumed and recovered in identified products. The acetogen Acetobacterium woodii was unable to consume current. These results expand the known range of microorganisms capable of electrosynthesis, providing multiple options for the further optimization of this process.}, keywords = {Acetobacterium, Carbon Dioxide, Clostridium, Electrodes, Electrons, Moorella, Organic Chemicals, Oxidation-Reduction, Veillonellaceae}, issn = {1098-5336}, doi = {10.1128/AEM.02642-10}, author = {Nevin, Kelly P and Hensley, Sarah A and Franks, Ashley E and Summers, Zarath M and Ou, Jianhong and Woodard, Trevor L and Snoeyenbos-West, Oona L and Lovley, Derek R} } @article {435, title = {Microbial electrosynthesis: feeding microbes electricity to convert carbon dioxide and water to multicarbon extracellular organic compounds.}, journal = {MBio}, volume = {1}, year = {2010}, month = {2010}, abstract = {The possibility of providing the acetogenic microorganism Sporomusa ovata with electrons delivered directly to the cells with a graphite electrode for the reduction of carbon dioxide to organic compounds was investigated. Biofilms of S. ovata growing on graphite cathode surfaces consumed electrons with the reduction of carbon dioxide to acetate and small amounts of 2-oxobutyrate. Electrons appearing in these products accounted for over 85\% of the electrons consumed. These results demonstrate that microbial production of multicarbon organic compounds from carbon dioxide and water with electricity as the energy source is feasible.}, keywords = {Bioelectric Energy Sources, Carbon Dioxide, Electricity, Organic Chemicals, Veillonellaceae, Water}, issn = {2150-7511}, doi = {10.1128/mBio.00103-10}, author = {Nevin, Kelly P and Woodard, Trevor L and Franks, Ashley E and Summers, Zarath M and Lovley, Derek R} } @article {451, title = {Molecular analysis of phosphate limitation in Geobacteraceae during the bioremediation of a uranium-contaminated aquifer.}, journal = {ISME J}, volume = {4}, year = {2010}, month = {2010 Feb}, pages = {253-66}, abstract = {Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified by microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high-affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU upregulated the most. Quantitative PCR analysis of pstB and phoU transcript levels in G. sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium-bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve because of the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.}, keywords = {Biodegradation, Environmental, Fresh Water, Gene Expression Regulation, Bacterial, Geobacter, Phosphates, Uranium, Water Pollutants}, issn = {1751-7370}, doi = {10.1038/ismej.2009.115}, author = {N{\textquoteright}guessan, Lucie A and Elifantz, Hila and Nevin, Kelly P and Mouser, Paula J and Meth{\'e}, Barbara and Woodard, Trevor L and Manley, Kimberly and Williams, Kenneth H and Wilkins, Michael J and Larsen, Joern T and Long, Philip E 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 {700, title = {Characterizing regulation of metabolism in Geobacter sulfurreducens through genome-wide expression data and sequence analysis.}, journal = {OMICS}, volume = {12}, year = {2008}, month = {2008 Mar}, pages = {33-59}, abstract = {Geobacteraceae are a family of metal reducing bacteria with important applications in bioremediation and electricity generation. G. sulfurreducens is a representative of Geobacteraceae that has been extensively studied with the goal of extending the understanding of this family of organisms for optimizing their practical applications. Here, we have analyzed gene expression data from 10 experiments involving environmental and genetic perturbations and have identified putative transcription factor binding sites (TFBS) involved in regulating key aspects of metabolism. Specifically, we considered data from both a subset of 10 microarray experiments (7 of 10) and all 10 experiments. The expression data from these two sets were independently clustered, and the upstream regions of genes and operons from the clusters in both sets were used to identify TFBS using the AlignACE program. This analysis resulted in the identification of motifs upstream of several genes involved in central metabolism, sulfate assimilation, and energy metabolism, as well as genes potentially encoding acetate permease. Further, similar TFBS were identified from the analysis of both sets, suggesting that these TFBS are significant in the regulation of metabolism in G. sulfurreducens. In addition, we have utilized microarray data to derive condition specific constraints on the capacity of key enzymes in central metabolism. We have incorporated these constraints into the metabolic model of G. sulfurreducens and simulated Fe(II)-limited growth. The resulting prediction was consistent with data, suggesting that regulatory constraints are important for simulating growth phenotypes in nonoptimal environments.}, keywords = {Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Models, Genetic, Oligonucleotide Array Sequence Analysis, Sequence Analysis, DNA, Transcription, Genetic}, issn = {1536-2310}, doi = {10.1089/omi.2007.0043}, author = {Mahadevan, Radhakrishnan and Yan, Bin and Postier, Brad and Nevin, Kelly P and Woodard, Trevor L and O{\textquoteright}Neil, Regina and Coppi, Maddalena V and Meth{\'e}, Barbara A and Krushkal, Julia} } @article {488, title = {Gene transcript analysis of assimilatory iron limitation in Geobacteraceae during groundwater bioremediation.}, journal = {Environ Microbiol}, volume = {10}, year = {2008}, month = {2008 May}, pages = {1218-30}, abstract = {Limitations on the availability of Fe(III) as an electron acceptor are thought to play an important role in restricting the growth and activity of Geobacter species during bioremediation of contaminated subsurface environments, but the possibility that these organisms might also be limited in the subsurface by the availability of iron for assimilatory purposes was not previously considered because copious quantities of Fe(II) are produced as the result of Fe(III) reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of a three-gene cluster consisting of homologues of two iron-dependent regulators, fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II) uptake protein. This cluster appears to be conserved among members of the Geobacteraceae and was detected in several environments. Expression of the fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat cultures. The number of Geobacteraceae feoB transcripts in groundwater samples from a site undergoing in situ uranium bioremediation was relatively high until the concentration of dissolved Fe(II) increased near the end of the field experiment. These results suggest that, because much of the Fe(II) is sequestered in solid phases, Geobacter species, which have a high requirement for iron for iron-sulfur proteins, may be limited by the amount of iron available for assimilatory purposes. These results demonstrate the ability of transcript analysis to reveal previously unsuspected aspects of the in situ physiology of microorganisms in subsurface environments.}, keywords = {Bacterial Proteins, Biodegradation, Environmental, Culture Media, Ferric Compounds, Ferrous Compounds, Fresh Water, Gene Expression Regulation, Bacterial, Geobacter, Iron, Multigene Family, Phylogeny, Polymerase Chain Reaction, Repressor Proteins, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Uranium, Water Pollution, Radioactive}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2007.01537.x}, author = {O{\textquoteright}Neil, Regina A and Holmes, Dawn E and Coppi, Maddalena V and Adams, Lorrie A and Larrahondo, M Juliana and Ward, Joy E and Nevin, Kelly P and Woodard, Trevor L and Vrionis, Helen A and N{\textquoteright}guessan, Lucie A and Lovley, Derek R} } @article {475, title = {Graphite electrode as a sole electron donor for reductive dechlorination of tetrachlorethene by Geobacter lovleyi.}, journal = {Appl Environ Microbiol}, volume = {74}, year = {2008}, month = {2008 Oct}, pages = {5943-7}, abstract = {The possibility that graphite electrodes can serve as the direct electron donor for microbially catalyzed reductive dechlorination was investigated with Geobacter lovleyi. In an initial evaluation of whether G. lovleyi could interact electronically with graphite electrodes, cells were provided with acetate as the electron donor and an electrode as the sole electron acceptor. Current was produced at levels that were ca. 10-fold lower than those previously reported for Geobacter sulfurreducens under similar conditions, and G. lovleyi anode biofilms were correspondingly thinner. When an electrode poised at -300 mV (versus a standard hydrogen electrode) was provided as the electron donor, G. lovleyi effectively reduced fumarate to succinate. The stoichiometry of electrons consumed to succinate produced was 2:1, the ratio expected if the electrode served as the sole electron donor for fumarate reduction. G. lovleyi effectively reduced tetrachloroethene (PCE) to cis-dichloroethene with a poised electrode as the sole electron donor at rates comparable to those obtained when acetate serves as the electron donor. Cells were less abundant on the electrodes when the electrodes served as an electron donor than when they served as an electron acceptor. PCE was not reduced in controls without cells or when the current supply to cells was interrupted. These results demonstrate that G. lovleyi can use a poised electrode as a direct electron donor for reductive dechlorination of PCE. The ability to colocalize dechlorinating microorganisms with electrodes has several potential advantages for bioremediation of subsurface chlorinated contaminants, especially in source zones where electron donor delivery is challenging and often limits dechlorination.}, keywords = {Acetic Acid, Biofilms, Biomass, Electricity, Electrodes, Electrons, Ethylene Dichlorides, Fumarates, Geobacter, Graphite, Microscopy, Electron, Scanning, Succinic Acid, Tetrachloroethylene}, issn = {1098-5336}, doi = {10.1128/AEM.00961-08}, author = {Strycharz, Sarah M and Woodard, Trevor L and Johnson, Jessica P and Nevin, Kelly P and Sanford, Robert A and L{\"o}ffler, Frank E and Lovley, Derek R} } @article {503, title = {Genome-wide expression profiling in Geobacter sulfurreducens: identification of Fur and RpoS transcription regulatory sites in a relGsu mutant.}, journal = {Funct Integr Genomics}, volume = {7}, year = {2007}, month = {2007 Jul}, pages = {229-55}, abstract = {Rel(Gsu) is the single Geobacter sulfurreducens homolog of RelA and SpoT proteins found in many organisms. These proteins are involved in the regulation of levels of guanosine 3{\textquoteright}, 5{\textquoteright} bispyrophosphate, ppGpp, a molecule that signals slow growth and stress response under nutrient limitation in bacteria. We used information obtained from genome-wide expression profiling of the rel(Gsu) deletion mutant to identify putative regulatory sites involved in transcription networks modulated by Rel(Gsu) or ppGpp. Differential gene expression in the rel(Gsu) deletion mutant, as compared to the wild type, was available from two growth conditions, steady state chemostat cultures and stationary phase batch cultures. Hierarchical clustering analysis of these two datasets identified several groups of operons that are likely co-regulated. Using a search for conserved motifs in the upstream regions of these co-regulated operons, we identified sequences similar to Fur- and RpoS-regulated sites. These findings suggest that Fur- and RpoS-dependent gene expression in G. sulfurreducens is affected by Rel(Gsu)-mediated signaling.}, keywords = {Bacterial Proteins, Base Sequence, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genome, Bacterial, Geobacter, Ligases, Mutation, Operon, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid, Repressor Proteins, Sigma Factor, Transcription, Genetic}, issn = {1438-793X}, doi = {10.1007/s10142-007-0048-5}, author = {Krushkal, Julia and Yan, Bin and DiDonato, Laurie N and Puljic, Marko and Nevin, Kelly P and Woodard, Trevor L and Adkins, Ronald M and Meth{\'e}, Barbara A and Lovley, Derek R} } @article {499, title = {Involvement of Geobacter sulfurreducens SfrAB in acetate metabolism rather than intracellular, respiration-linked Fe(III) citrate reduction.}, journal = {Microbiology}, volume = {153}, year = {2007}, month = {2007 Oct}, pages = {3572-85}, abstract = {A soluble ferric reductase, SfrAB, which catalysed the NADPH-dependent reduction of chelated Fe(III), was previously purified from the dissimilatory Fe(III)-reducing micro-organism Geobacter sulfurreducens, suggesting that reduction of chelated forms of Fe(III) might be cytoplasmic. However, metabolically active spheroplast suspensions could not catalyse acetate-dependent Fe(III) citrate reduction, indicating that periplasmic and/or outer-membrane components were required for Fe(III) citrate reduction. Furthermore, phenotypic analysis of an SfrAB knockout mutant suggested that SfrAB was involved in acetate metabolism rather than respiration-linked Fe(III) reduction. The mutant could not grow via the reduction of either Fe(III) citrate or fumarate when acetate was the electron donor but could grow with either acceptor if either hydrogen or formate served as the electron donor. Following prolonged incubation in acetate : fumarate medium in the absence of hydrogen and formate, an {\textquoteright}acetate-adapted{\textquoteright} SfrAB-null strain was isolated that was capable of growth on acetate : fumarate medium but not acetate : Fe(III) citrate medium. Comparison of gene expression in this strain with that of the wild-type revealed upregulation of a potential NADPH-dependent ferredoxin oxidoreductase as well as genes involved in energy generation and amino acid uptake, suggesting that NADPH homeostasis and the tricarboxylic acid (TCA) cycle were perturbed in the {\textquoteright}acetate-adapted{\textquoteright} SfrAB-null strain. Membrane and soluble fractions prepared from the {\textquoteright}acetate-adapted{\textquoteright} strain were depleted of NADPH-dependent Fe(III), viologen and quinone reductase activities. These results indicate that cytoplasmic, respiration-linked reduction of Fe(III) by SfrAB in vivo is unlikely and suggest that deleting SfrAB may interfere with growth via acetate oxidation by interfering with NADP regeneration.}, keywords = {Acetates, Amino Acid Transport Systems, Bacterial Proteins, Cell Membrane, Citric Acid Cycle, Cytoplasm, Energy Metabolism, Ferric Compounds, Formic Acids, Fumarates, Gene Deletion, Gene Expression Profiling, Geobacter, Hydrogen, NADH, NADPH Oxidoreductases, Oligonucleotide Array Sequence Analysis}, issn = {1350-0872}, doi = {10.1099/mic.0.2007/006478-0}, author = {Coppi, Maddalena V and O{\textquoteright}Neil, Regina A and Leang, Ching and Kaufmann, Franz and Meth{\'e}, Barbara A and Nevin, Kelly P and Woodard, Trevor L and Liu, Anna and Lovley, Derek R} } @article {504, title = {Prolixibacter bellariivorans gen. nov., sp. nov., a sugar-fermenting, psychrotolerant anaerobe of the phylum Bacteroidetes, isolated from a marine-sediment fuel cell.}, journal = {Int J Syst Evol Microbiol}, volume = {57}, year = {2007}, month = {2007 Apr}, pages = {701-7}, abstract = {A Gram-negative, non-motile, filamentous, rod-shaped, non-spore-forming bacterium (strain F2(T)) was isolated from the surface of an electricity-harvesting electrode incubated in marine sediments. Strain F2(T) does not contain c-type cytochromes, flexirubin or carotenoids. It is a facultative anaerobe that can ferment sugars by using a mixed acid fermentation pathway and it can grow over a wide range of temperatures (4-42 degrees C). The DNA G+C (44.9 mol\%) content and chemotaxonomic characteristics (major fatty acids, a-15 : 0 and 15 : 0) were consistent with those of species within the phylum Bacteroidetes. Phylogenetic analysis of the 16S rRNA nucleotide and elongation factor G amino acid sequences indicated that strain F2(T) represents a unique phylogenetic cluster within the phylum Bacteroidetes. On the basis of 16S rRNA gene sequence phylogeny, the closest relative available in pure culture, Alkaliflexus imshenetskii, is only 87.5 \% similar to strain F2(T). Results from physiological, biochemical and phylogenetic analyses showed that strain F2(T) should be classified as a novel genus and species within the phylum Bacteroidetes, for which the name Prolixibacter bellariivorans gen. nov., sp. nov. is proposed. The type strain is F2(T) (=ATCC BAA-1284(T)=JCM 13498(T)).}, keywords = {Bacteroidetes, Carbohydrate Metabolism, Cold Temperature, DNA, Bacterial, DNA, Ribosomal, Energy-Generating Resources, Geologic Sediments, Molecular Sequence Data, Phylogeny, RNA, Ribosomal, 16S, Seawater}, issn = {1466-5026}, doi = {10.1099/ijs.0.64296-0}, author = {Holmes, Dawn E and Nevin, Kelly P and Woodard, Trevor L and Peacock, Aaron D and Lovley, Derek R} } @article {506, title = {Reclassification of Trichlorobacter thiogenes as Geobacter thiogenes comb. nov.}, journal = {Int J Syst Evol Microbiol}, volume = {57}, year = {2007}, month = {2007 Mar}, pages = {463-6}, abstract = {Reclassification of the species Trichlorobacter thiogenes as Geobacter thiogenes comb. nov. is proposed on the basis of physiological traits and phylogenetic position. Characteristics additional to those provided in the original description revealed that the type strain (strain K1(T)=ATCC BAA-34(T)=JCM 14045(T)) has the ability to use Fe(III) as an electron acceptor for acetate oxidation and has an electron donor and acceptor profile typical of a Geobacter species, contains abundant c-type cytochromes, and has a temperature optimum of 30 degrees C and a pH optimum near pH 7.0; traits typical of members of the genus Geobacter. Phylogenetic analysis of nifD, recA, gyrB, rpoB, fusA and 16S rRNA genes further indicated that T. thiogenes falls within the Geobacter cluster of the family Geobacteraceae. Based on extensive phylogenetic evidence and the fact that T. thiogenes has the hallmark physiological characteristics of a Geobacter species, Trichlorobacter thiogenes should be reclassified as a member of the genus Geobacter.}, keywords = {DNA, Bacterial, DNA, Ribosomal, Genes, Bacterial, Geobacter, Phylogeny, RNA, Ribosomal, 16S, Temperature}, issn = {1466-5026}, doi = {10.1099/ijs.0.63408-0}, author = {Nevin, Kelly P and Holmes, Dawn E and Woodard, Trevor L and Covalla, Sean F 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} } @article {513, title = {Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens.}, journal = {Environ Microbiol}, volume = {8}, year = {2006}, month = {2006 Oct}, pages = {1805-15}, abstract = {Whole-genome analysis of gene expression in Geobacter sulfurreducens revealed 474 genes with transcript levels that were significantly different during growth with an electrode as the sole electron acceptor versus growth on Fe(III) citrate. The greatest response was a more than 19-fold increase in transcript levels for omcS, which encodes an outer-membrane cytochrome previously shown to be required for Fe(III) oxide reduction. Quantitative reverse transcription polymerase chain reaction and Northern analyses confirmed the higher levels of omcS transcripts, which increased as power production increased. Deletion of omcS inhibited current production that was restored when omcS was expressed in trans. Transcript expression and genetic analysis suggested that OmcE, another outer-membrane cytochrome, is also involved in electron transfer to electrodes. Surprisingly, genes for other proteins known to be important in Fe(III) reduction such as the outer-membrane c-type cytochrome, OmcB, and the electrically conductive pilin "nanowires" did not have higher transcript levels on electrodes, and deletion of the relevant genes did not inhibit power production. Changes in the transcriptome suggested that cells growing on electrodes were subjected to less oxidative stress than cells growing on Fe(III) citrate and that a number of genes annotated as encoding metal efflux proteins or proteins of unknown function may be important for growth on electrodes. These results demonstrate for the first time that it is possible to evaluate gene expression, and hence the metabolic state, of microorganisms growing on electrodes on a genome-wide basis and suggest that OmcS, and to a lesser extent OmcE, are important in electron transfer to electrodes. This has important implications for the design of electrode materials and the genetic engineering of microorganisms to improve the function of microbial fuel cells.}, keywords = {Bacterial Outer Membrane Proteins, Blotting, Northern, Cytochromes c, Electrodes, Electrophysiology, Gene Expression Regulation, Bacterial, Geobacter, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Reverse Transcriptase Polymerase Chain Reaction, RNA, Bacterial, RNA, Messenger}, issn = {1462-2912}, doi = {10.1111/j.1462-2920.2006.01065.x}, author = {Holmes, Dawn E and Chaudhuri, Swades K and Nevin, Kelly P and Mehta, Teena and Meth{\'e}, Barbara A and Liu, Anna and Ward, Joy E and Woodard, Trevor L and Webster, Jennifer and Lovley, Derek R} } @article {532, title = {Geobacter bemidjiensis sp. nov. and Geobacter psychrophilus sp. nov., two novel Fe(III)-reducing subsurface isolates.}, journal = {Int J Syst Evol Microbiol}, volume = {55}, year = {2005}, month = {2005 Jul}, pages = {1667-74}, abstract = {Fe(III)-reducing isolates were recovered from two aquifers in which Fe(III) reduction is known to be important. Strain Bem(T) was enriched from subsurface sediments collected in Bemidji, MN, USA, near a site where Fe(III) reduction is important in aromatic hydrocarbon degradation. Strains P11, P35(T) and P39 were isolated from the groundwater of an aquifer in Plymouth, MA, USA, in which Fe(III) reduction is important because of long-term inputs of acetate as a highway de-icing agent to the subsurface. All four isolates were Gram-negative, slightly curved rods that grew best in freshwater media. Strains P11, P35(T) and P39 exhibited motility via means of monotrichous flagella. Analysis of the 16S rRNA and nifD genes indicated that all four strains are delta-proteobacteria and members of the Geobacter cluster of the Geobacteraceae. Differences in phenotypic and phylogenetic characteristics indicated that the four isolates represent two novel species within the genus Geobacter. All of the isolates coupled the oxidation of acetate to the reduction of Fe(III) [iron(III) citrate, amorphous iron(III) oxide, iron(III) pyrophosphate and iron(III) nitrilotriacetate]. All four strains utilized ethanol, lactate, malate, pyruvate and succinate as electron donors and malate and fumarate as electron acceptors. Strain Bem(T) grew fastest at 30 degrees C, whereas strains P11, P35(T) and P39 grew equally well at 17, 22 and 30 degrees C. In addition, strains P11, P35(T) and P39 were capable of growth at 4 degrees C. The names Geobacter bemidjiensis sp. nov. (type strain Bem(T)=ATCC BAA-1014(T)=DSM 16622(T)=JCM 12645(T)) and Geobacter psychrophilus sp. nov. (strains P11, P35(T) and P39; type strain P35(T)=ATCC BAA-1013(T)=DSM 16674(T)=JCM 12644(T)) are proposed.}, keywords = {Bacterial Proteins, Bacterial Typing Techniques, Cold Temperature, DNA, Bacterial, DNA, Ribosomal, Ferric Compounds, Fresh Water, Genes, rRNA, Geobacter, Geologic Sediments, Massachusetts, Minnesota, Molecular Sequence Data, Oxidation-Reduction, RNA, Ribosomal, 16S, Species Specificity, Water Supply}, issn = {1466-5026}, doi = {10.1099/ijs.0.63417-0}, author = {Nevin, Kelly P and Holmes, Dawn E and Woodard, Trevor L and Hinlein, Erich S and Ostendorf, David W and Lovley, Derek R} } @article {529, title = {Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 Nov}, pages = {6870-7}, abstract = {The Geobacteraceae citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of Geobacteraceae. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of Geobacter metabolism was evaluated in pure culture studies and in four different Geobacteraceae-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of Geobacter sulfurreducens grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene, gltA, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes recA, rpoD, and proC remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of gltA. The expression of gltA was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect gltA transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of Geobacteraceae by tracking the expression of the citrate synthase gene.}, keywords = {Acetates, Citrate (si)-Synthase, Deltaproteobacteria, DNA, Ribosomal, Electrodes, Ferric Compounds, Fresh Water, Geobacter, Geologic Sediments, Petroleum, Phylogeny, RNA, Ribosomal, 16S, Uranium, Water Pollutants, Chemical, Water Pollutants, Radioactive}, issn = {0099-2240}, doi = {10.1128/AEM.71.11.6870-6877.2005}, author = {Holmes, Dawn E and Nevin, Kelly P and O{\textquoteright}Neil, Regina A and Ward, Joy E and Adams, Lorrie A and Woodard, Trevor L and Vrionis, Helen A and Lovley, Derek R} }