@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 {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 {3056, title = {Mechanisms for Electron Uptake by Methanosarcina acetivorans during Direct Interspecies Electron Transfer.}, journal = {mBio}, volume = {12}, year = {2021}, month = {2021 Oct 26}, pages = {e0234421}, abstract = {

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here, we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in coculture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in grown in coculture versus pure-culture growth on acetate revealed that transcripts for the outer-surface multiheme type cytochrome MmcA were higher during DIET-based growth. Deletion of inhibited DIET. The high aromatic amino acid content of archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based coculture as well as the archaellum-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based cocultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen. The conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connections on the outer surfaces of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of organisms that play an important role in methane production in soils and sediments participate in DIET.

}, keywords = {Archaeal Proteins, Electron Transport, Electrons, Methane, Methanosarcina, Transcriptome}, issn = {2150-7511}, doi = {10.1128/mBio.02344-21}, author = {Holmes, Dawn E and Zhou, Jinjie and Ueki, Toshiyuki and Woodard, Trevor 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 {3077, title = {Iron Corrosion via Direct Metal-Microbe Electron Transfer.}, journal = {mBio}, volume = {10}, year = {2019}, month = {2019 May 14}, abstract = {

The concept that anaerobic microorganisms can directly accept electrons from Fe(0) has been controversial because direct metal-microbe electron transfer has previously only been indirectly inferred. Fe(0) oxidation was studied with strain ACL, an autotrophic strain that was previously shown to grow with electrons derived from a graphite cathode as the sole electron donor. Strain ACL grew with Fe(0) as the sole electron donor and fumarate as the electron acceptor. However, it appeared that at least a portion of the electron transfer was via H produced nonenzymatically from the oxidation of Fe(0) to Fe(II). H, which accumulated in abiotic controls, was consumed during the growth of strain ACL, the cells were predominately planktonic, and genes for the uptake hydrogenase were highly expressed. Strain ACL was constructed to prevent growth on H or formate by deleting the genes for the uptake of hydrogenase and formate dehydrogenases from strain ACL. Strain ACL also grew with Fe(0) as the sole electron donor, but H accumulated in the culture, and cells heavily colonized Fe(0) surfaces with no visible planktonic growth. Transcriptomics suggested that the outer surface -type cytochromes OmcS and OmcZ were important during growth of strain ACL on Fe(0). Strain ACL did not grow on Fe(0) if the gene for either of these cytochromes was deleted. The specific attachment of strain ACL to Fe(0), coupled with requirements for known extracellular electrical contacts, suggest that direct metal-microbe electron transfer is the most likely option for Fe(0) serving as an electron donor. The anaerobic corrosion of iron structures is expensive to repair and can be a safety and environmental concern. It has been known for over 100 years that the presence of anaerobic respiratory microorganisms can accelerate iron corrosion. Multiple studies have suggested that there are sulfate reducers, methanogens, and acetogens that can directly accept electrons from Fe(0) to support sulfate or carbon dioxide reduction. However, all of the strains studied can also use H as an electron donor for growth, which is known to be abiotically produced from Fe(0). Furthermore, no proteins definitely shown to function as extracellular electrical contacts with Fe(0) were identified. The studies described here demonstrate that direct electron transfer from Fe(0) can support anaerobic respiration. They also map out a simple genetic approach to the study of iron corrosion mechanisms in other microorganisms. A better understanding of how microorganisms promote iron corrosion is expected to lead to the development of strategies that can help reduce adverse impacts from this process.

}, keywords = {Anaerobiosis, Corrosion, Cytochromes, Electron Transport, Formate Dehydrogenases, Geobacter, Iron, Oxidation-Reduction, Oxidoreductases, Transcriptome}, issn = {2150-7511}, doi = {10.1128/mBio.00303-19}, author = {Tang, Hai-Yan and Holmes, Dawn E and Ueki, Toshiyuki and Palacios, Paola A and Lovley, Derek R} } @article {3074, title = {A Membrane-Bound Cytochrome Enables To Conserve Energy from Extracellular Electron Transfer.}, journal = {mBio}, volume = {10}, year = {2019}, month = {2019 Aug 20}, abstract = {

Extracellular electron exchange in species and closely related plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, -type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of makes it an excellent model microbe for evaluating -type cytochrome-dependent extracellular electron transfer in The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of -type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some was previously proposed, but these studies with provide the first genetic evidence for cytochrome-based extracellular electron transfer in The results suggest parallels with Gram-negative bacteria, such as and species, in which multiheme outer-surface -type cytochromes are an essential component for electrical communication with the extracellular environment. offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in with implications not only for methanogens but possibly also for that anaerobically oxidize methane.

}, keywords = {Acetates, Anthraquinones, Cytochromes, Electron Transport, Electrons, Ferric Compounds, Gene Expression Regulation, Archaeal, Gram-Negative Bacteria, Membranes, Mesna, Methane, Methanol, Methanosarcina, Oxidation-Reduction, Oxidoreductases, Transcriptome}, issn = {2150-7511}, doi = {10.1128/mBio.00789-19}, author = {Holmes, Dawn E and Ueki, Toshiyuki and Tang, Hai-Yan and Zhou, Jinjie and Smith, Jessica A and Chaput, Gina 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 {3090, title = {Toward establishing minimum requirements for extracellular electron transfer in Geobacter sulfurreducens.}, journal = {FEMS Microbiol Lett}, volume = {364}, year = {2017}, month = {2017 May 01}, abstract = {

The highly redundant pathways for extracellular electron transfer in Geobacter sulfurreducens must be simplified for this microorganism to serve as an effective chassis for applications such as the development of sensors and biocomputing. Five homologs of the periplasmic c-type cytochromes, PpcA-E, offer the possibility of multiple routes of electron transfer across the periplasm. The presence of a large number of outer membrane c-type cytochromes allows G. sulfurreducens to adapt to disruption of an electron transfer pathway in the outer membrane. A strain in which genes for all five periplasmic cytochromes, PpcA-E, were deleted did not reduce Fe(III). Introducing ppcA under the control of an IPTG-inducible system in the quintuple deletion strain yielded a strain that reduced Fe(III) only in the presence of IPTG. A strain lacking known major outer membrane cytochromes, OmcB, OmcE, OmcS and OmcT, and putative functional homologs of OmcB, did not reduce Fe(III). Introduction of omcB in this septuple deletion strain restored the ability to reduce Fe(III). These results demonstrate that it is possible to trim redundancy from the extracellular electron transfer pathways in G. sulfurreducens in order to construct strains with defined extracellular electron transfer routes.

}, keywords = {Bacterial Outer Membrane Proteins, Bacterial Proteins, Cytochromes c, Electron Transport, Ferric Compounds, Gene Expression Regulation, Bacterial, Geobacter, Oxidation-Reduction, Periplasm}, issn = {1574-6968}, doi = {10.1093/femsle/fnx093}, author = {Ueki, Toshiyuki and DiDonato, Laurie N and Lovley, Derek R} } @article {3106, title = {Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.}, journal = {ISME J}, volume = {10}, year = {2016}, month = {2016 Feb}, pages = {310-20}, abstract = {

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Cytochrome c Group, Electron Transport, Ferric Compounds, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, Proteomics, Sequence Alignment, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2015.113}, author = {Yun, Jiae and Malvankar, Nikhil S and Ueki, Toshiyuki and Lovley, Derek R} } @article {404, title = {Interspecies electron transfer via hydrogen and formate rather than direct electrical connections in cocultures of Pelobacter carbinolicus and Geobacter sulfurreducens.}, journal = {Appl Environ Microbiol}, volume = {78}, year = {2012}, month = {2012 Nov}, pages = {7645-51}, abstract = {

Direct interspecies electron transfer (DIET) is an alternative to interspecies H(2)/formate transfer as a mechanism for microbial species to cooperatively exchange electrons during syntrophic metabolism. To understand what specific properties contribute to DIET, studies were conducted with Pelobacter carbinolicus, a close relative of Geobacter metallireducens, which is capable of DIET. P. carbinolicus grew in coculture with Geobacter sulfurreducens with ethanol as the electron donor and fumarate as the electron acceptor, conditions under which G. sulfurreducens formed direct electrical connections with G. metallireducens. In contrast to the cell aggregation associated with DIET, P. carbinolicus and G. sulfurreducens did not aggregate. Attempts to initiate cocultures with a genetically modified strain of G. sulfurreducens incapable of both H(2) and formate utilization were unsuccessful, whereas cocultures readily grew with mutant strains capable of formate but not H(2) uptake or vice versa. The hydrogenase mutant of G. sulfurreducens compensated, in cocultures, with significantly increased formate dehydrogenase gene expression. In contrast, the transcript abundance of a hydrogenase gene was comparable in cocultures with that for the formate dehydrogenase mutant of G. sulfurreducens or the wild type, suggesting that H(2) was the primary electron carrier in the wild-type cocultures. Cocultures were also initiated with strains of G. sulfurreducens that could not produce pili or OmcS, two essential components for DIET. The finding that P. carbinolicus exchanged electrons with G. sulfurreducens via interspecies transfer of H(2)/formate rather than DIET demonstrates that not all microorganisms that can grow syntrophically are capable of DIET and that closely related microorganisms may use significantly different strategies for interspecies electron exchange.

}, keywords = {Coculture Techniques, Deltaproteobacteria, Electricity, Electron Transport, Electrons, Formates, Geobacter, Hydrogen, Microbial Interactions}, issn = {1098-5336}, doi = {10.1128/AEM.01946-12}, author = {Rotaru, Amelia-Elena and Shrestha, Pravin M and Liu, Fanghua and Ueki, Toshiyuki and Nevin, Kelly and Summers, Zarath M and Lovley, Derek R} }