@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 {3069, title = {Bioinspired bio-voltage memristors.}, journal = {Nat Commun}, volume = {11}, year = {2020}, month = {2020 Apr 20}, pages = {1861}, abstract = {

Memristive devices are promising candidates to emulate biological computing. However, the typical switching voltages (0.2-2 V) in previously described devices are much higher than the amplitude in biological counterparts. Here we demonstrate a type of diffusive memristor, fabricated from the protein nanowires harvested from the bacterium Geobacter sulfurreducens, that functions at the biological voltages of 40-100 mV. Memristive function at biological voltages is possible because the protein nanowires catalyze metallization. Artificial neurons built from these memristors not only function at biological action potentials (e.g., 100 mV, 1 ms) but also exhibit temporal integration close to that in biological neurons. The potential of using the memristor to directly process biosensing signals is also demonstrated.

}, keywords = {Action Potentials, Biosensing Techniques, Electricity, Electronics, Equipment Design, Geobacter, Humans, Molecular Dynamics Simulation, Nanotechnology, Nanowires, Neural Networks, Computer, Neurons, Synapses, Wearable Electronic Devices}, issn = {2041-1723}, doi = {10.1038/s41467-020-15759-y}, author = {Fu, Tianda and Liu, Xiaomeng and Gao, Hongyan and Ward, Joy E and Liu, Xiaorong and Yin, Bing and Wang, Zhongrui and Zhuo, Ye and Walker, David J F and Joshua Yang, J and Chen, Jianhan and Lovley, Derek R and Yao, Jun} } @article {3071, title = {Power generation from ambient humidity using protein nanowires.}, journal = {Nature}, volume = {578}, year = {2020}, month = {2020 Feb}, pages = {550-554}, abstract = {

Harvesting energy from the environment offers the promise of clean power for self-sustained systems. Known technologies-such as solar cells, thermoelectric devices and mechanical generators-have specific environmental requirements that restrict where they can be deployed and limit their potential for continuous energy production. The ubiquity of atmospheric moisture offers an alternative. However, existing moisture-based energy-harvesting technologies can produce only intermittent, brief (shorter than 50 seconds) bursts of power in the ambient environment, owing to the lack of a sustained conversion mechanism. Here we show that thin-film devices made from nanometre-scale protein wires harvested from the microbe Geobacter sulfurreducens can generate continuous electric power in the ambient environment. The devices produce a sustained voltage of around 0.5~volts across a 7-micrometre-thick film, with a current density of around 17 microamperes per square centimetre. We find the driving force behind this energy generation to be a self-maintained moisture gradient that forms within the film when the film is exposed to the humidity that is naturally present in air. Connecting several devices linearly scales up the voltage and current to power electronics. Our results demonstrate the feasibility of a continuous energy-harvesting strategy that is less restricted by location or environmental conditions than other sustainable approaches.

}, issn = {1476-4687}, doi = {10.1038/s41586-020-2010-9}, author = {Liu, Xiaomeng and Gao, Hongyan and Ward, Joy E and Liu, Xiaorong and Yin, Bing and Fu, Tianda and Chen, Jianhan and Lovley, Derek R and Yao, Jun} } @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 {3076, title = {Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire.}, journal = {Commun Biol}, volume = {2}, year = {2019}, month = {2019}, pages = {219}, abstract = {

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

}, keywords = {Cryoelectron Microscopy, Cytochromes c, Electron Transport, Fimbriae, Bacterial, Geobacter, Nanowires}, issn = {2399-3642}, doi = {10.1038/s42003-019-0448-9}, author = {Filman, David J and Marino, Stephen F and Ward, Joy E and Yang, Lu and Mester, Zolt{\'a}n and Bullitt, Esther and Lovley, Derek R and Strauss, Mike} } @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 {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 {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 {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 {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 {483, title = {Genes for two multicopper proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes.}, journal = {Microbiology}, volume = {154}, year = {2008}, month = {2008 May}, pages = {1422-35}, abstract = {Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 \% similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.}, keywords = {Acetates, Amino Acid Sequence, Bacterial Outer Membrane Proteins, Electrodes, Ferric Compounds, Gene Deletion, Gene Expression Profiling, Geobacter, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, Sequence Alignment, Sequence Homology, Nucleic Acid, Soil Microbiology, Uranium}, issn = {1350-0872}, doi = {10.1099/mic.0.2007/014365-0}, author = {Holmes, Dawn E and Mester, T{\"u}nde and O{\textquoteright}Neil, Regina A and Perpetua, Lorrie A and Larrahondo, M Juliana and Glaven, Richard and Sharma, Manju L and Ward, Joy E and Nevin, Kelly P and Lovley, Derek R} } @article {515, title = {c-Type cytochromes in Pelobacter carbinolicus.}, journal = {Appl Environ Microbiol}, volume = {72}, year = {2006}, month = {2006 Nov}, pages = {6980-5}, abstract = {Previous studies failed to detect c-type cytochromes in Pelobacter species despite the fact that other close relatives in the Geobacteraceae, such as Geobacter and Desulfuromonas species, have abundant c-type cytochromes. Analysis of the recently completed genome sequence of Pelobacter carbinolicus revealed 14 open reading frames that could encode c-type cytochromes. Transcripts for all but one of these open reading frames were detected in acetoin-fermenting and/or Fe(III)-reducing cells. Three putative c-type cytochrome genes were expressed specifically during Fe(III) reduction, suggesting that the encoded proteins may participate in electron transfer to Fe(III). One of these proteins was a periplasmic triheme cytochrome with a high level of similarity to PpcA, which has a role in Fe(III) reduction in Geobacter sulfurreducens. Genes for heme biosynthesis and system II cytochrome c biogenesis were identified in the genome and shown to be expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of protein extracted from acetoin-fermenting P. carbinolicus cells contained three heme-staining bands which were confirmed by mass spectrometry to be among the 14 predicted c-type cytochromes. The number of cytochrome genes, the predicted amount of heme c per protein, and the ratio of heme-stained protein to total protein were much smaller in P. carbinolicus than in G. sulfurreducens. Furthermore, many of the c-type cytochromes that genetic studies have indicated are required for optimal Fe(III) reduction in G. sulfurreducens were not present in the P. carbinolicus genome. These results suggest that further evaluation of the functions of c-type cytochromes in the Geobacteraceae is warranted.}, keywords = {Bacterial Proteins, Cytochromes c, Deltaproteobacteria, Heme, Polymerase Chain Reaction, Proteomics, Reverse Transcriptase Polymerase Chain Reaction, RNA, Messenger}, issn = {0099-2240}, doi = {10.1128/AEM.01128-06}, author = {Haveman, Shelley A and Holmes, Dawn E and Ding, Yan-Huai R and Ward, Joy E and Didonato, Raymond J 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 {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} }