Cytochrome OmcS Is Not Essential for Extracellular Electron Transport via Conductive Pili in Geobacter sulfurreducens Strain KN400.

The multi-heme c-type cytochrome OmcS is one of the central components used for extracellular electron transport in the Geobacter sulfurreducens strain DL-1, but its role in other microbes, including other strains of G. sulfurreducens, is currently a matter of debate. Therefore, we investigated the function of OmcS in the G. sulfurreducens strain KN400, which is even more effective in extracellular electron transfer than the DL-1 strain. We found that deleting omcS from strain KN400 did not negatively impact the rate of Fe(III) oxide reduction and that the cells expressed conductive filaments. Replacing the wild-type pilin gene with the aro-5 pilin gene eliminated the OmcS-deficient strain's ability to transport electrons to insoluble electron acceptors and diminished filament conductivity. These results are consistent with the concept that electrically conductive pili are the primary conduit for long-range electron transfer in G. sulfurreducens and closely related species. These findings, coupled with the lack of OmcS homologs in other microbes capable of extracellular electron transfer, suggest that OmcS is not a common critical component for extracellular electron transfer. IMPORTANCE OmcS has been widely studied and noted to be one of the key components for extracellular electron exchange by the Geobacter sulfurreducens strain DL-1. However, the true importance of OmcS warrants further investigation because it is well known that few bacteria, even within the Geobacteraceae family, contain OmcS homologs, and many bacteria that are capable of extracellular electron transfer lack an abundance of any type of outer surface c-type cytochrome. In addition, there is debate about the importance of OmcS filaments in the mechanism of extracellular electron transport to insoluble electron acceptors by G. sulfurreducens. It has been suggested that filaments comprised of OmcS rather than e-pili are the predominant conductive filaments expressed by G. sulfurreducens. However, the results presented here, along with multiple other sources of evidence, indicate that OmcS filaments cannot be the primary, conductive, protein nanowires expressed by G. sulfurreducens.

Solvent-Induced Assembly of Microbial Protein Nanowires into Superstructured Bundles.

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 Geobacter sulfurreducens and Escherichia coli. 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.

Extracellular Electron Exchange Capabilities of Desulfovibrio ferrophilus and Desulfopila corrodens.

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, Desulfovibrio ferrophilus and Desulfopila corrodens are not able to directly extract electrons from solid-phase electron-donating surfaces. D. ferrophilus grew with Fe(III) as the electron acceptor, but Dp. corrodens did not. D. ferrophilus 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 D. ferrophilus 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 c-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 D. ferrophilus has adopted a novel combination of strategies to enable extracellular electron transport, which may be of biogeochemical and technological significance.

Conductive Composite Materials Fabricated from Microbially Produced Protein Nanowires.

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 × 10-30 µm) 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 °C. These results demonstrate that e-PNs represent viable and sustainable nanowire compositions for the fabrication of electrically conductive composite materials.

Metatranscriptomic Evidence for Direct Interspecies Electron Transfer between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils.

The possibility that Methanothrix (formerly Methanosaeta) and Geobacter 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 Geobacter sulfurreducens 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 Geobacter species, but sequences most closely related to putative e-pilin genes from genera such as Desulfobacterium, Deferribacter, Geoalkalibacter, and Desulfobacula, were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with Geobacter species, and the finding that Geobacter 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 Geobacter genes. In addition, homologs of genes encoding OmcS, a c-type cytochrome associated with the e-pili of G. sulfurreducens and required for DIET, were also highly expressed in the soils. Methanothrix 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 CO2 reduction in Methanothrix Thus, these results are consistent with a model in which Geobacter species were providing electrons to Methanothrix species for methane production through electrical connections of e-pili.IMPORTANCEMethanothrix 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 Methanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution that Methanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when Methanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp., and possibly other methanogens, via DIET.

Synthetic Biological Protein Nanowires with High Conductivity.

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.

Genetic switches and related tools for controlling gene expression and electrical outputs of Geobacter sulfurreducens.

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.

Electrobiocorrosion by microbes without outer-surface cytochromes.

Anaerobic microbial corrosion of iron-containing metals causes extensive economic damage. Some microbes are capable of direct metal-to-microbe electron transfer (electrobiocorrosion), but the prevalence of electrobiocorrosion among diverse methanogens and acetogens is poorly understood because of a lack of tools for their genetic manipulation. Previous studies have suggested that respiration with 316L  stainless steel as the electron donor is indicative of electrobiocorrosion, because, unlike pure Fe0, 316L  stainless steel does not abiotically generate H2 as an intermediary electron carrier. Here, we report that all of the methanogens (Methanosarcina vacuolata, Methanothrix soehngenii, and Methanobacterium strain IM1) and acetogens (Sporomusa ovata and Clostridium ljungdahlii) evaluated respired with pure Fe0 as the electron donor, but only M. vacuolata, Mx. soehngenii, and S. ovata were capable of stainless steel electrobiocorrosion. The electrobiocorrosive methanogens required acetate as an additional energy source in order to produce methane from stainless steel. Cocultures of S. ovata and Mx. soehngenii demonstrated how acetogens can provide acetate to methanogens during corrosion. Not only was Methanobacterium strain IM1 not capable of electrobiocorrosion, but it also did not accept electrons from Geobacter metallireducens, an effective electron-donating partner for direct interspecies electron transfer to all methanogens that can directly accept electrons from Fe0. The finding that M. vacuolata, Mx. soehngenii, and S. ovata are capable of electrobiocorrosion, despite a lack of the outer-surface c-type cytochromes previously found to be important in other electrobiocorrosive microbes, demonstrates that there are multiple microbial strategies for making electrical contact with Fe0.

H2 Is a Major Intermediate in Desulfovibrio vulgaris Corrosion of Iron.

Desulfovibrio vulgaris has been a primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe0 have been proposed. We investigated Fe0 oxidation with a mutant of D. vulgaris 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 H2. The parental strain reduced sulfate with Fe0 as the sole electron donor, but the hydrogenase mutant did not. H2 accumulated over time in Fe0 cultures of the hydrogenase mutant and sterile controls but not in parental strain cultures. Sulfide stimulated H2 production in uninoculated controls apparently by both reacting with Fe0 to generate H2 and facilitating electron transfer from Fe0 to H+. Parental strain supernatants did not accelerate H2 production from Fe0, ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe0 and D. vulgaris via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe0 and either riboflavin or anthraquinone-2,6-disulfonate, and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe0 as the electron donor. The results demonstrate that D. vulgaris primarily accepts electrons from Fe0 via H2 as an intermediary electron carrier. These findings clarify the interpretation of previous D. vulgaris corrosion studies and suggest that H2-mediated electron transfer is an important mechanism for iron corrosion under sulfate-reducing conditions. IMPORTANCE 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 Fe0 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 H2 to shuttle electrons between Fe0 and cells revives the concept, developed in some of the earliest studies on microbial corrosion, that sulfate reducers consumption of H2 is a major microbial corrosion mechanism. The results further emphasize that direct Fe0-to-microbe electron transfer has yet to be rigorously demonstrated in sulfate-reducing microbes.

Detrimental impact of the Geobacter metallireducens type VI secretion system on direct interspecies electron transfer.

Direct interspecies electron transfer (DIET) is important in anaerobic communities of environmental and practical significance. Other than the need for close physical contact for electrical connections, the interactions of DIET partners are poorly understood. Type VI secretion systems (T6SSs) typically kill competitive microbes. Surprisingly, Geobacter metallireducens highly expressed T6SS genes when DIET-based co-cultures were initiated with Geobacter sulfurreducens. T6SS gene expression was lower when the electron shuttle anthraquinone-2,6-disulfonate was added to alleviate the need for interspecies contact. Disruption of hcp, the G. metallireducens gene for the main T6SS needle-tube protein subunit, and the most highly upregulated gene in DIET-grown cells eliminated the long lag periods required for the initiation of DIET. The mutation did not aid DIET in the presence of granular-activated carbon (GAC), consistent with the fact that DIET partners do not make physical contact when electrically connected through conductive materials. The hcp-deficient mutant also established DIET quicker with Methanosarcina barkeri. However, the mutant also reduced Fe(III) oxide faster than the wild-type strain, a phenotype not expected from the loss of the T6SS. Quantitative PCR revealed greater gene transcript abundance for key components of extracellular electron transfer in the hcp-deficient mutant versus the wild-type strain, potentially accounting for the faster Fe(III) oxide reduction and impact on DIET. The results highlight that interspecies interactions beyond electrical connections may influence DIET effectiveness. The unexpected increase in the expression of genes for extracellular electron transport components when hcp was deleted emphasizes the complexities in evaluating the electromicrobiology of highly adaptable Geobacter species. IMPORTANCE Direct interspecies electron transfer is an alternative to the much more intensively studied process of interspecies H2 transfer as a mechanism for microbes to share electrons during the cooperative metabolism of energy sources. DIET is an important process in anaerobic soils and sediments generating methane, a significant greenhouse gas. Facilitating DIET can accelerate and stabilize the conversion of organic wastes to methane biofuel in anaerobic digesters. Therefore, a better understanding of the factors controlling how fast DIET partnerships are established is expected to lead to new strategies for promoting this bioenergy process. The finding that when co-cultured with G. sulfurreducens, G. metallireducens initially expressed a type VI secretion system, a behavior not conducive to interspecies cooperation, illustrates the complexity of establishing syntrophic relationships.

Spatial heterogeneity of bacterial communities in sediments from an infiltration basin receiving highway runoff.

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.

Genetic Manipulation of Desulfovibrio ferrophilus and Evaluation of Fe(III) Oxide Reduction Mechanisms.

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 D. ferrophilus extracellular electron exchange including a soluble electron shuttle, electrically conductive pili, and outer surface multiheme c-type cytochromes. However, the previous lack of a strategy for genetic manipulation of D. ferrophilus limited mechanistic investigations. We developed an electroporation-mediated transformation method that enabled replacement of D. ferrophilus 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 D. ferrophilus 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 c-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 D. ferrophilus primarily reducing Fe(III) oxide via an electron shuttle. The finding that D. ferrophilus 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. IMPORTANCE Desulfovibrio ferrophilus 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 D. ferrophilus 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 D. ferrophilus c-type cytochrome that is essential for Fe(III) oxide reduction. This finding, coupled with the lack of apparent porin-cytochrome conduits encoded in the D. ferrophilus genome and the finding that deleting genes for pilus and flagellum expression did not inhibit Fe(III) oxide reduction, suggests that D. ferrophilus has adopted strategies for extracellular electron exchange that are different from those of intensively studied electroactive microbes like Shewanella and Geobacter species. Thus, the ability to genetically manipulate D. ferrophilus is likely to lead to new mechanistic concepts in electromicrobiology.

Microbial biofilms for electricity generation from water evaporation and power to wearables.

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 µm thick) serving as the functional component in an electronic device continuously produces power density (~1 µW/cm2) 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.

Electrosynthesis of organic compounds from carbon dioxide is catalyzed by a diversity of acetogenic microorganisms.

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.

Generation of High Current Densities in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor.

Geobacter sulfurreducens is commonly employed as a model for the study of extracellular electron transport mechanisms in the Geobacter species. Deletion of pilB, 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 pilB deletion was not demonstrated directly but was inferred from the observation that pilB deletion mutants produced lower current densities than wild-type cells. Here, we report that deleting pilB 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 pilB was deleted. These results suggest that a reinterpretation of the results of previous studies on G. sulfurreducens pilB deletion strains may be necessary. IMPORTANCE 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.

Stainless steel corrosion via direct iron-to-microbe electron transfer by Geobacter species.

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 H2 is often an intermediary electron carrier between the metal and microbes during Fe(0) corrosion, we found that H2 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 H2 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 H2 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.

An Escherichia coli Chassis for Production of Electrically Conductive Protein Nanowires.

Geobacter sulfurreducens' 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 G. sulfurreducens. Therefore, we designed a strain of Escherichia coli to express e-PNs by introducing a plasmid that contained an inducible operon with E. coli 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 E. coli and harvested with a simple filtration method had the same diameter (3 nm) and conductance as e-PNs expressed in G. sulfurreducens. These results, coupled with the robustness of E. coli for mass cultivation and the extensive E. coli toolbox for genetic manipulation, greatly expand the opportunities for large-scale fabrication of novel e-PNs.

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

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

Decorating the Outer Surface of Microbially Produced Protein Nanowires with Peptides.

The potential applications of electrically conductive protein nanowires (e-PNs) harvested from Geobacter sulfurreducens 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 G. sulfurreducens gene for the monomer that assembles into e-PNs was modified to add peptide tags at the carboxyl terminus of the monomer. Strains of G. sulfurreducens 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.

Gene transcript analysis of assimilatory iron limitation in Geobacteraceae during groundwater bioremediation.

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.