Environmental predictors of electroactive bacterioplankton in small boreal lakes.

Extracellular electron transfer (EET) by electroactive bacteria in anoxic soils and sediments is an intensively researched subject, but EET's function in planktonic ecology has been less considered. Following the discovery of an unexpectedly high prevalence of EET genes in a bog lake's bacterioplankton, we hypothesized that the redox capacities of dissolved organic matter (DOM) enrich for electroactive bacteria by mediating redox chemistry. We developed the bioinformatics pipeline FEET (Find EET) to identify and summarize predicted EET protein-encoding genes from metagenomics data. We then applied FEET to 36 bog and thermokarst lakes and correlated gene occurrence with environmental data to test our predictions. Our results provide indirect evidence that DOM may participate in bacterioplankton EET. We found a similarly high prevalence of genes encoding putative EET proteins in most of these lakes, where oxidative EET strongly correlated with DOM. Numerous novel clusters of multiheme cytochromes that may enable EET were identified. Taxa previously not considered EET-capable were found to carry EET genes. We propose that EET and DOM interactions are of ecologically important to bacterioplankton in small boreal lakes, and that EET, particularly by methylotrophs and anoxygenic phototrophs, should be further studied and incorporated into methane emission models of melting permafrost.

Lack of Specificity in Geobacter Periplasmic Electron Transfer.

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple ΔppcABCDE ΔpgcA mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. IMPORTANCE Many inner and outer membrane cytochromes used by Geobacter for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that Geobacter periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of Geobacter electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.

Structure of Geobacter OmcZ filaments suggests extracellular cytochrome polymers evolved independently multiple times.

While early genetic and low-resolution structural observations suggested that extracellular conductive filaments on metal-reducing organisms such as Geobacter were composed of type IV pili, it has now been established that bacterial c-type cytochromes can polymerize to form extracellular filaments capable of long-range electron transport. Atomic structures exist for two such cytochrome filaments, formed from the hexaheme cytochrome OmcS and the tetraheme cytochrome OmcE. Due to the highly conserved heme packing within the central OmcS and OmcE cores, and shared pattern of heme coordination between subunits, it has been suggested that these polymers have a common origin. We have now used cryo-electron microscopy (cryo-EM) to determine the structure of a third extracellular filament, formed from the Geobacter sulfurreducens octaheme cytochrome, OmcZ. In contrast to the linear heme chains in OmcS and OmcE from the same organism, the packing of hemes, heme:heme angles, and between-subunit heme coordination is quite different in OmcZ. A branched heme arrangement within OmcZ leads to a highly surface exposed heme in every subunit, which may account for the formation of conductive biofilm networks, and explain the higher measured conductivity of OmcZ filaments. This new structural evidence suggests that conductive cytochrome polymers arose independently on more than one occasion from different ancestral multiheme proteins.

Cryo-EM structure of an extracellular Geobacter OmcE cytochrome filament reveals tetrahaem packing.

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens were first observed two decades ago, with genetic and biochemical data suggesting that conductive fibres were type IV pili. Recently, an extracellular conductive filament of G. sulfurreducens was found to contain polymerized c-type cytochrome OmcS subunits, not pilin subunits. Here we report that G. sulfurreducens also produces a second, thinner appendage comprised of cytochrome OmcE subunits and solve its structure using cryo-electron microscopy at ~4.3 Å resolution. Although OmcE and OmcS subunits have no overall sequence or structural similarities, upon polymerization both form filaments that share a conserved haem packing arrangement in which haems are coordinated by histidines in adjacent subunits. Unlike OmcS filaments, OmcE filaments are highly glycosylated. In extracellular fractions from G. sulfurreducens, we detected type IV pili comprising PilA-N and -C chains, along with abundant B-DNA. OmcE is the second cytochrome filament to be characterized using structural and biophysical methods. We propose that there is a broad class of conductive bacterial appendages with conserved haem packing (rather than sequence homology) that enable long-distance electron transport to chemicals or other microbial cells.

The Signaling Pathway That cGAMP Riboswitches Found: Analysis and Application of Riboswitches to Study cGAMP Signaling in Geobacter sulfurreducens.

The Hypr cGAMP signaling pathway was discovered via the function of the riboswitch. In this study, we show the development of a method for affinity capture followed by sequencing to identify non-coding RNA regions that bind nucleotide signals such as cGAMP. The RNAseq of affinity-captured cGAMP riboswitches from the Geobacter sulfurreducens transcriptome highlights general challenges that remain for this technique. Furthermore, by applying riboswitch reporters in vivo, we identify new growth conditions and transposon mutations that affect cGAMP levels in G. sulfurreducens. This work reveals an extensive regulatory network and supports a second functional cGAMP synthase gene in G. sulfurreducens. The activity of the second synthase was validated using riboswitch-based fluorescent biosensors, and is the first known example of an active enzyme with a variant GGDDF motif.

Geobacter sulfurreducens inner membrane cytochrome CbcBA controls electron transfer and growth yield near the energetic limit of respiration.

Geobacter sulfurreducens utilizes extracellular electron acceptors such as Mn(IV), Fe(III), syntrophic partners, and electrodes that vary from +0.4 to -0.3 V versus standard hydrogen electrode (SHE), representing a potential energy span that should require a highly branched electron transfer chain. Here we describe CbcBA, a bc-type cytochrome essential near the thermodynamic limit of respiration when acetate is the electron donor. Mutants-lacking cbcBA ceased Fe(III) reduction at -0.21 V versus SHE, could not transfer electrons to electrodes between -0.21 and -0.28 V, and could not reduce the final 10%-35% of Fe(III) minerals. As redox potential decreased during Fe(III) reduction, cbcBA was induced with the aid of the regulator BccR to become one of the most highly expressed genes in G. sulfurreducens. Growth yield (CFU/mM Fe(II)) was 112% of WT in ∆cbcBA, and deletion of cbcL (an unrelated bc-cytochrome essential near -0.15 V) in ΔcbcBA increased yield to 220%. Together with ImcH, which is required at high redox potentials, CbcBA represents a third cytoplasmic membrane oxidoreductase in G. sulfurreducens. This expanding list shows how metal-reducing bacteria may constantly sense redox potential to adjust growth efficiency in changing environments.

Evidence of a Streamlined Extracellular Electron Transfer Pathway from Biofilm Structure, Metabolic Stratification, and Long-Range Electron Transfer Parameters.

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-µm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

Survival of the first rather than the fittest in a Shewanella electrode biofilm.

For natural selection to operate there must exist heritable variation among individuals that affects their survival and reproduction. Among free-living microbes, where differences in growth rates largely define selection intensities, competitive exclusion is common. However, among surface attached communities, these dynamics become less predictable. If extreme circumstances were to dictate that a surface population is immortal and all offspring must emigrate, the offspring would be unable to contribute to the composition of the population. Meanwhile, the immortals, regardless of reproductive capacity, would remain unchanged in relative abundance. The normal cycle of birth, death, and competitive exclusion would be broken. We tested whether conditions required to set up this idealized scenario can be approximated in a microbial biofilm. Using two differentially-reproducing strains of Shewanella oneidensis grown on an anode as the sole terminal electron acceptor - a system in which metabolism is obligately tied to surface attachment - we found that selection against a slow-growing competitor is drastically reduced. This work furthers understanding of natural selection dynamics in sessile microbial communities, and provides a framework for designing stable microbial communities for industrial and experimental applications.

Novel Microbial Groups Drive Productivity in an Archean Iron Formation.

Deep subsurface environments are decoupled from Earth's surface processes yet diverse, active, and abundant microbial communities thrive in these isolated environments. Microbes inhabiting the deep biosphere face unique challenges such as electron donor/acceptor limitations, pore space/fracture network limitations, and isolation from other microbes within the formation. Of the few systems that have been characterized, it is apparent that nutrient limitations likely facilitate diverse microbe-microbe interactions (i.e., syntrophic, symbiotic, or parasitic) and that these interactions drive biogeochemical cycling of major elements. Here we describe microbial communities living in low temperature, chemically reduced brines at the Soudan Underground Mine State Park, United States. The Soudan Iron mine intersects a massive hematite formation at the southern extent of the Canadian Shield. Fractured rock aquifer brines continuously flow from exploratory boreholes drilled circa 1960 and are enriched in deuterium compared to the global meteoric values, indicating brines have had little contact with surface derived waters, and continually degas low molecular weight hydrocarbons C1-C4. Microbial enrichments suggest that once brines exit the boreholes, oxidation of the hydrocarbons occur. Amplicon sequencing show these borehole communities are low in diversity and dominated by Firmicute and Proteobacteria phyla. From the metagenome assemblies, we recovered approximately thirty genomes with estimated completion over 50%. Analysis of genome taxonomy generally followed the amplicon data, and highlights that several of the genomes represent novel families and genera. Metabolic reconstruction shows two carbon-fixation pathways were dominant, the Wood-Ljungdahl (acetogenesis) and Calvin-Benson-Bassham (via RuBisCo), indicating that inorganic carbon likely enters into the microbial foodweb with differing carbon fractionation potentials. Interestingly, methanogenesis is likely driven by Methanolobus and suggests cycling of methylated compounds and not H2/CO2 or acetate. Furthermore, the abundance of sulfate in brines suggests cryptic sulfur cycling may occur, as we detect possible sulfate reducing and thiosulfate oxidizing microorganisms. Finally, a majority of the microorganisms identified contain genes that would allow them to participate in several element cycles, highlighting that in these deep isolated systems metabolic flexibility may be an important life history trait.

Electrolocation? The evidence for redox-mediated taxis in Shewanella oneidensis.

Shewanella oneidensis is a dissimilatory metal reducing bacterium and model for extracellular electron transfer (EET), a respiratory mechanism in which electrons are transferred out of the cell. In the last 10 years, migration to insoluble electron acceptors for EET has been shown to be nonrandom and tactic, seemingly in the absence of molecular or energy gradients that typically allow for taxis. As the ability to sense, locate, and respire electrodes has applications in bioelectrochemical technology, a better understanding of taxis in S. oneidensis is needed. While the EET conduits of S. oneidensis have been studied extensively, its taxis pathways and their interplay with EET are not yet understood, making investigation into taxis phenomena nontrivial. Since S. oneidensis is a member of an EET-encoding clade, the genetic circuitry of taxis to insoluble acceptors may be conserved. We performed a bioinformatic analysis of Shewanella genomes to identify S. oneidensis chemotaxis orthologs conserved in the genus. In addition to the previously reported core chemotaxis gene cluster, we identify several other conserved proteins in the taxis signaling pathway. We present the current evidence for the two proposed models of EET taxis, "electrokinesis" and flavin-mediated taxis, and highlight key areas in need of further investigation.

A Hybrid Extracellular Electron Transfer Pathway Enhances the Survival of Vibrio natriegens.

Vibrio natriegens is the fastest-growing microorganism discovered to date, making it a useful model for biotechnology and basic research. While it is recognized for its rapid aerobic metabolism, less is known about anaerobic adaptations in V. natriegens or how the organism survives when oxygen is limited. Here, we describe and characterize extracellular electron transfer (EET) in V. natriegens, a metabolism that requires movement of electrons across protective cellular barriers to reach the extracellular space. V. natriegens performs extracellular electron transfer under fermentative conditions with gluconate, glucosamine, and pyruvate. We characterized a pathway in V. natriegens that requires CymA, PdsA, and MtrCAB for Fe(III) citrate and Fe(III) oxide reduction, which represents a hybrid of strategies previously discovered in Shewanella and Aeromonas Expression of these V. natriegens genes functionally complemented Shewanella oneidensis mutants. Phylogenetic analysis of the inner membrane quinol dehydrogenases CymA and NapC in gammaproteobacteria suggests that CymA from Shewanella diverged from Vibrionaceae CymA and NapC. Analysis of sequenced Vibrionaceae revealed that the genetic potential to perform EET is conserved in some members of the Harveyi and Vulnificus clades but is more variable in other clades. We provide evidence that EET enhances anaerobic survival of V. natriegens, which may be the primary physiological function for EET in VibrionaceaeIMPORTANCE Bacteria from the genus Vibrio occupy a variety of marine and brackish niches with fluctuating nutrient and energy sources. When oxygen is limited, fermentation or alternative respiration pathways must be used to conserve energy. In sedimentary environments, insoluble oxide minerals (primarily iron and manganese) are able to serve as electron acceptors for anaerobic respiration by microorganisms capable of extracellular electron transfer, a metabolism that enables the use of these insoluble substrates. Here, we identify the mechanism for extracellular electron transfer in Vibrio natriegens, which uses a combination of strategies previously identified in Shewanella and Aeromonas We show that extracellular electron transfer enhanced survival of V. natriegens under fermentative conditions, which may be a generalized strategy among Vibrio spp. predicted to have this metabolism.

NanoSIMS imaging reveals metabolic stratification within current-producing biofilms.

Metal-reducing bacteria direct electrons to their outer surfaces, where insoluble metal oxides or electrodes act as terminal electron acceptors, generating electrical current from anaerobic respiration. Geobacter sulfurreducens is a commonly enriched electricity-producing organism, forming thick conductive biofilms that magnify total activity by supporting respiration of cells not in direct contact with electrodes. Hypotheses explaining why these biofilms fail to produce higher current densities suggest inhibition by formation of pH, nutrient, or redox potential gradients; but these explanations are often contradictory, and a lack of direct measurements of cellular growth within biofilms prevents discrimination between these models. To address this fundamental question, we measured the anabolic activity of G. sulfurreducens biofilms using stable isotope probing coupled to nanoscale secondary ion mass spectrometry (nanoSIMS). Our results demonstrate that the most active cells are at the anode surface, and that this activity decreases with distance, reaching a minimum 10 µm from the electrode. Cells nearest the electrode continue to grow at their maximum rate in weeks-old biofilms 80-µm-thick, indicating nutrient or buffer diffusion into the biofilm is not rate-limiting. This pattern, where highest activity occurs at the electrode and declines with each cell layer, is present in thin biofilms (<5 µm) and fully grown biofilms (>20 µm), and at different anode redox potentials. These results suggest a growth penalty is associated with respiring insoluble electron acceptors at micron distances, which has important implications for improving microbial electrochemical devices as well as our understanding of syntrophic associations harnessing the phenomenon of microbial conductivity.

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration.

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3',3'-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

Divergent Nrf Family Proteins and MtrCAB Homologs Facilitate Extracellular Electron Transfer in Aeromonas hydrophila.

Extracellular electron transfer (EET) is a strategy for respiration in which electrons generated from metabolism are moved outside the cell to a terminal electron acceptor, such as iron or manganese oxide. EET has primarily been studied in two model systems, Shewanella oneidensis and Geobacter sulfurreducens Metal reduction has also been reported in numerous microorganisms, including Aeromonas spp., which are ubiquitous Gammaproteobacteria found in aquatic ecosystems, with some species capable of pathogenesis in humans and fish. Genomic comparisons of Aeromonas spp. revealed a potential outer membrane conduit homologous to S. oneidensis MtrCAB. While the ability to respire metals and mineral oxides is not widespread in the genus Aeromonas, 90% of the sequenced Aeromonas hydrophila isolates contain MtrCAB homologs. A. hydrophila ATCC 7966 mutants lacking mtrA are unable to reduce metals. Expression of A. hydrophila mtrCAB in an S. oneidensis mutant lacking homologous components restored metal reduction. Although the outer membrane conduits for metal reduction were similar, homologs of the S. oneidensis inner membrane and periplasmic EET components CymA, FccA, and CctA were not found in A. hydrophila We characterized a cluster of genes predicted to encode components related to a formate-dependent nitrite reductase (NrfBCD), here named NetBCD (for Nrf-like electron transfer), and a predicted diheme periplasmic cytochrome, PdsA (periplasmic diheme shuttle). We present genetic evidence that proteins encoded by this cluster facilitate electron transfer from the cytoplasmic membrane across the periplasm to the MtrCAB conduit and function independently from an authentic NrfABCD system. A. hydrophila mutants lacking pdsA and netBCD were unable to reduce metals, while heterologous expression of these genes could restore metal reduction in an S. oneidensis mutant background. EET may therefore allow A. hydrophila and other species of Aeromonas to persist and thrive in iron- or manganese-rich oxygen-limited environments.IMPORTANCE Metal-reducing microorganisms are used for electricity production, bioremediation of toxic compounds, wastewater treatment, and production of valuable compounds. Despite numerous microorganisms being reported to reduce metals, the molecular mechanism has primarily been studied in two model systems, Shewanella oneidensis and Geobacter sulfurreducens We have characterized the mechanism of extracellular electron transfer in Aeromonas hydrophila, which uses the well-studied Shewanella system, MtrCAB, to move electrons across the outer membrane; however, most Aeromonas spp. appear to use a novel mechanism to transfer electrons from the inner membrane through the periplasm and to the outer membrane. The conserved use of MtrCAB in Shewanella spp. and Aeromonas spp. for metal reduction and conserved genomic architecture of metal reduction genes in Aeromonas spp. may serve as genomic markers for identifying metal-reducing microorganisms from genomic or transcriptomic sequencing. Understanding the variety of pathways used to reduce metals can allow for optimization and more efficient design of microorganisms used for practical applications.

Identification of Different Putative Outer Membrane Electron Conduits Necessary for Fe(III) Citrate, Fe(III) Oxide, Mn(IV) Oxide, or Electrode Reduction by Geobacter sulfurreducens.

At least five gene clusters in the Geobacter sulfurreducens genome encode putative "electron conduits" implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single-gene-cluster deletions and all possible multiple-deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III) and Mn(IV) oxides, and graphite electrodes poised at +0.24 V and -0.1 V versus the standard hydrogen electrode (SHE). Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an ΔomcBC background, such as extEFG, were needed to produce defects greater than 50% compared to findings with the wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III) oxide, Mn(IV) oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than the wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth conditions showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of genes encoding known redox proteins or pilus components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens, depending on the available extracellular electron acceptor.IMPORTANCE Gram-negative metal-reducing bacteria utilize electron conduits, chains of redox proteins spanning the outer membrane, to transfer electrons to the extracellular surface. Only one pathway for electron transfer across the outer membrane of Geobacter sulfurreducens has been linked to Fe(III) reduction. However, G. sulfurreducens is able to respire a wide array of extracellular substrates. Here we present the first combinatorial genetic analysis of five different electron conduits via creation of new markerless deletion strains and complementation vectors. Multiple conduit gene clusters appear to have overlapping roles, including two that have never been linked to metal reduction. Another recently described cluster (ExtABCD) was the only electron conduit essential during electrode reduction, a substrate of special importance to biotechnological applications of this organism.

Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides.

Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed Geobacter sulfurreducens transposon-insertion sequencing (Tn-Seq) libraries for growth, with soluble fumarate or an electrode as the electron acceptor. Libraries with >33,000 unique insertions and an average of 9 insertions/kb allowed an assessment of each gene's fitness in a single experiment. Mutations in 1,214 different genomic features impaired growth with fumarate, and the significance of 270 genes unresolved by annotation due to the presence of one or more functional homologs was determined. Tn-Seq analysis of -0.1 V versus standard hydrogen electrode (SHE) electrode-grown cells identified mutations in a subset of genes encoding cytochromes, processing systems for proline-rich proteins, sensory networks, extracellular structures, polysaccharides, and metabolic enzymes that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of select genes identified via Tn-Seq revealed a new putative porin-cytochrome conduit complex (extABCD) crucial for growth with electrodes, which was not required for Fe(III) oxide reduction. In addition, four mutants lacking components of a putative methyl-accepting chemotaxis-cyclic dinucleotide sensing network (esnABCD) were defective in electrode colonization but grew normally with Fe(III) oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to Fe(III) oxides.IMPORTANCE Since metal oxide electron acceptors are insoluble, one hypothesis is that cells sense and reduce metals using the same molecular mechanisms used to form biofilms on electrodes and produce electricity. However, by simultaneously comparing thousands of Geobacter sulfurreducens transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration. This supports an emerging model where G. sulfurreducens recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. These findings provide a possible explanation for studies that correlate electricity generation with syntrophic interspecies electron transfer by Geobacter and reveal many previously unrecognized targets for engineering this useful capability in other organisms.

Redox potential as a master variable controlling pathways of metal reduction by Geobacter sulfurreducens.

Geobacter sulfurreducens uses at least two different pathways to transport electrons out of the inner membrane quinone pool before reducing acceptors beyond the outer membrane. When growing on electrodes poised at oxidizing potentials, the CbcL-dependent pathway operates at or below redox potentials of -0.10 V vs the standard hydrogen electrode, whereas the ImcH-dependent pathway operates only above this value. Here, we provide evidence that G. sulfurreducens also requires different electron transfer proteins for reduction of a wide range of Fe(III)- and Mn(IV)-(oxyhydr)oxides, and must transition from a high- to low-potential pathway during reduction of commonly studied soluble and insoluble metal electron acceptors. Freshly precipitated Fe(III)-(oxyhydr)oxides could not be reduced by mutants lacking the high-potential pathway. Aging these minerals by autoclaving did not change their powder X-ray diffraction pattern, but restored reduction by mutants lacking the high-potential pathway. Mutants lacking the low-potential, CbcL-dependent pathway had higher growth yields with both soluble and insoluble Fe(III). Together, these data suggest that the ImcH-dependent pathway exists to harvest additional energy when conditions permit, and CbcL switches on to allow respiration closer to thermodynamic equilibrium conditions. With evidence of multiple pathways within a single organism, the study of extracellular respiration should consider not only the crystal structure or solubility of a mineral electron acceptor, but rather the redox potential, as this variable determines the energetic reward affecting reduction rates, extents, and final microbial growth yields in the environment.

Geobacter sulfurreducens Extracellular Multiheme Cytochrome PgcA Facilitates Respiration to Fe(III) Oxides But Not Electrodes.

Extracellular cytochromes are hypothesized to facilitate the final steps of electron transfer between the outer membrane of the metal-reducing bacterium Geobacter sulfurreducens and solid-phase electron acceptors such as metal oxides and electrode surfaces during the course of respiration. The triheme c-type cytochrome PgcA exists in the extracellular space of G. sulfurreducens, and is one of many multiheme c-type cytochromes known to be loosely bound to the bacterial outer surface. Deletion of pgcA using a markerless method resulted in mutants unable to transfer electrons to Fe(III) and Mn(IV) oxides; yet the same mutants maintained the ability to respire to electrode surfaces and soluble Fe(III) citrate. When expressed and purified from Shewanella oneidensis, PgcA demonstrated a primarily alpha helical structure, three bound hemes, and was processed into a shorter 41 kDa form lacking the lipodomain. Purified PgcA bound Fe(III) oxides, but not magnetite, and when PgcA was added to cell suspensions of G. sulfurreducens, PgcA accelerated Fe(III) reduction similar to addition of FMN. Addition of soluble PgcA to ΔpgcA mutants also restored Fe(III) reduction. This report highlights a distinction between proteins involved in extracellular electron transfer to metal oxides and poised electrodes, and suggests a specific role for PgcA in facilitating electron transfer at mineral surfaces.

Isolation and Genomic Characterization of 'Desulfuromonas soudanensis WTL', a Metal- and Electrode-Respiring Bacterium from Anoxic Deep Subsurface Brine.

Reaching a depth of 713 m below the surface, the Soudan Underground Iron Mine (Soudan, MN, USA) transects a massive Archaean (2.7 Ga) banded iron formation, providing a remarkably accessible window into the terrestrial deep biosphere. Despite organic carbon limitation, metal-reducing microbial communities are present in potentially ancient anoxic brines continuously emanating from exploratory boreholes on Level 27. Using graphite electrodes deposited in situ as bait, we electrochemically enriched and isolated a novel halophilic iron-reducing Deltaproteobacterium, 'Desulfuromonas soudanensis' strain WTL, from an acetate-fed three-electrode bioreactor poised at +0.24 V (vs. standard hydrogen electrode). Cyclic voltammetry revealed that 'D. soudanensis' releases electrons at redox potentials approximately 100 mV more positive than the model freshwater surface isolate Geobacter sulfurreducens, suggesting that its extracellular respiration is tuned for higher potential electron acceptors. 'D. soudanensis' contains a 3,958,620-bp circular genome, assembled to completion using single-molecule real-time (SMRT) sequencing reads, which encodes a complete TCA cycle, 38 putative multiheme c-type cytochromes, one of which contains 69 heme-binding motifs, and a LuxI/LuxR quorum sensing cassette that produces an unidentified N-acyl homoserine lactone. Another cytochrome is predicted to lie within a putative prophage, suggesting that horizontal gene transfer plays a role in respiratory flexibility among metal reducers. Isolation of 'D. soudanensis' underscores the utility of electrode-based approaches for enriching rare metal reducers from a wide range of habitats.

Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens.

The respiration of metals by the bacterium Geobacter sulfurreducens requires electrons generated by metabolism to pass from the interior of the cell to electron acceptors beyond the cell membranes. The G. sulfurreducens inner membrane multiheme c-type cytochrome ImcH is required for respiration to extracellular electron acceptors with redox potentials greater than -0.1 V vs. SHE, but ImcH is not essential for electron transfer to lower potential acceptors. In contrast, deletion of cbcL, encoding an inner membrane protein consisting of b-type and multiheme c-type cytochrome domains, severely affected reduction of low potential electron acceptors such as Fe(III)-oxides and electrodes poised at -0.1 V vs. SHE. Catalytic cyclic voltammetry of a ΔcbcL strain growing on poised electrodes revealed a 50 mV positive shift in driving force required for electron transfer out of the cell. In non-catalytic conditions, low-potential peaks present in wild type biofilms were absent in ∆cbcL mutants. Expression of cbcL in trans increased growth at low redox potential and restored features to cyclic voltammetry. This evidence supports a model where CbcL is a component of a second electron transfer pathway out of the G. sulfurreducens inner membrane that dominates when redox potential is at or below -0.1 V vs. SHE.