Microbial nanowires and electroactive biofilms

Microbial nanowires and electroactive biofilms ABSTRACT Geobacter bacteria are the only microorganisms known to produce conductive appendages or pili to electronically connect cells to extracellular electron acceptors such as iron oxide minerals and uranium. The conductive pili also promote cell-cell aggregation and the formation of electroactive biofilms. The hallmark of these electroactive biofilms is electronic heterogeneity, mediated by coordinated interactions between the conductive pili and matrix-associated cytochromes. Collectively, the matrix-associated electron carriers discharge respiratory electrons from cells in multilayered biofilms to electron-accepting surfaces such as iron oxide coatings and electrodes poised at a metabolically oxidizable potential. The presence of pilus nanowires in the electroactive biofilms also promotes the immobilization and reduction of soluble metals, even when present at toxic concentrations. This review summarizes current knowledge about the composition of the electroactive biofilm matrix and the mechanisms that allow the wired Geobacter biofilms to generate electrical currents and participate in metal redox transformations. electricigens, type IV pili, c-type cytochromes, Geobacter, iron reduction, uranium reduction INTRODUCTION The discovery that microbial biofilms can use electrodes buried in the seafloor sediments as the electron acceptor for respiration propelled a number of seminal studies to understand the phylogeny and physiology of electrochemically active microorganisms (Bond et al. 2002; Tender et al. 2002). Current-harvesting biofilms are often enriched in bacteria in the family Geobacteraceae, a group of Deltaproteobacteria that gain energy for growth from the reduction of ferric iron (Fe[III]) minerals (reviewed in Shi et al. 2016). The availability of a sequenced genome (Methé et al. 2003) and a genetic system (Coppi et al. 2001) for the model representative Geobacter sulfurreducens helped advance mechanistic studies and led to the discovery in this organism of conductive protein appendages of the Type IV pili class (Reguera et al. 2005). As in other bacteria, the Geobacter pili are assemblies of primarily a single peptide subunit or pilin (Cologgi et al. 2011). Yet the Geobacter pilin’s structure is divergent and optimized for electron transfer (ET) (Feliciano et al. 2012; Reardon and Mueller 2013). Genes encoding this type of divergent pilin are highly conserved among Geobacter species and form an independent line of descent among bacterial pilins (Reguera et al. 2005). Once assembled, the Geobacter pilins form fibers that, in the absence of metals and redox cofactors, conduct electrons at rates that greatly exceed the rates of cellular respiration (Lampa-Pastirk et al. 2016). The conductive pili of Geobacter bacteria are decorated with flexible, surface motifs that promote electrostatic interactions with extracellular electron acceptors and their reduction (Feliciano, Steidl and Reguera 2015). During the reduction of Fe(III) oxides, the pili solubilize some of the metal as Fe(II) but also form magnetite, a mixed Fe(III)-Fe(II) magnetic mineral that remains bound to the pilus filaments (Fig. 1). The soluble Fe(II) provides the electron donor for Fe(II) oxidizers, which cycle the iron. The semiconducting properties of the magnetite product can promote interspecies ET and the syntrophic coupling of the metabolism of Geobacter cells with nitrate reducers (Kato, Hashimoto and Watanabe 2012a) (Fig. 1). Magnetite also stimulates electro-syntrophic interactions between Geobacter spp. and methanogens (Kato, Hashimoto and Watanabe 2012b). Indeed, environmental surveys of soils where electro-syntrophic interactions between Geobacter and methanogens are predicted to occur show a high representation of Geobacter pilin genes and transcripts (Holmes et al. 2017). This suggests that the pili are directly or indirectly (via magnetite) mediating interspecies ET reactions with the syntrophic partners. Figure 1. View largeDownload slide Illustration of reactions mediated by the Geobacter pili (red arrows show direction of electron flow). The two panels show a colorized electron micrograph of a cell of G. sulfurreducens and its monolateral pili during the oxidation of acetate to CO2. The left panel shows the cell during the reduction of the pili-bound Fe(III) oxides (brown mineral particles) to soluble ferrous iron (Fe(II)aq) and magnetite (black mineral particles). The right panel shows interspecies ET from G. sulfurreducens to Thiobacillus denitrificans via the pili-bound magnetite particles, which couple the oxidation of acetate to the reduction of nitrate to ammonia. Figure 1. View largeDownload slide Illustration of reactions mediated by the Geobacter pili (red arrows show direction of electron flow). The two panels show a colorized electron micrograph of a cell of G. sulfurreducens and its monolateral pili during the oxidation of acetate to CO2. The left panel shows the cell during the reduction of the pili-bound Fe(III) oxides (brown mineral particles) to soluble ferrous iron (Fe(II)aq) and magnetite (black mineral particles). The right panel shows interspecies ET from G. sulfurreducens to Thiobacillus denitrificans via the pili-bound magnetite particles, which couple the oxidation of acetate to the reduction of nitrate to ammonia. The pilus nanowires also promote cell–cell aggregation and biofilm formation on various surfaces, including Fe(III) oxide coatings and electrodes poised at a metabolically oxidizing potential (Reguera et al. 2006; Reguera et al. 2007). The biofilm matrix is rich in c-type cytochromes (c-Cyts) and requires the nanowire network to electronically connect the cells and maintain optimal rates of ET in multilayered communities (Steidl, Lampa-Pastirk and Reguera 2016). This allows the biofilms to continue to grow at micrometer distances from an underlying electron-accepting surface and to harvest current from the collective oxidative metabolism of the biofilm cells (Reguera et al. 2006; Reguera et al. 2007). Electroactive biofilms are important members of the environmental consortia that decompose organic matter. The high conservation in Geobacter genomes of acetate transporters and pathways for its oxidation (Butler, Young and Lovley 2010) suggests an active role for the biofilms at the removal of acetate, a common feedback inhibitor of bacterial fermentations whose accumulation would otherwise reduce the rates of organic matter decomposition. Electroactive biofilms of the genetically tractable representative G. sulfurreducens can also metabolize common fermentation products such as lactate and formate, though less efficiently (Speers and Reguera 2012b). Yet adaptive evolution and genetic engineering has been applied to custom tailor the physiology of current-harvesting biofilms and improve their robustness during the removal of fermentation inhibitors (Speers and Reguera 2012a; Speers, Young and Reguera 2014; Awate et al. 2017). Electrodes functionalized with the electroactive biofilms can then be retrofitted into fermenters to remove unwanted byproducts, prevent their accumulation to inhibitory concentrations and maintain an optimal pH (Awate et al. 2017). These systems are essentially a fermenter retrofitted as a microbial electrolysis cell (MEC), an electrochemical system where a small energy input is used to harvest electrons at the anode and react them with protons at the cathode to make H2 (Geelhoed, Hamelers and Stams 2010). By mimicking biofilm syntrophic interactions that drive organic matter decomposition, electro-fermentations address many of the challenges that reduce the economic feasibility of otherwise attractive fermentations (e.g. batch-to-batch variability, low productivity and low product yields) and also reduce the cost associated with the purification of the targeted product (Awate et al. 2017). Electroactive biofilms formed on Fe(III) oxide minerals also contribute indirectly to the cycling of many elements. Fe(III) oxide minerals are widespread in nature and are particularly abundant in sediments and soils (>10 mmol per kg dry matter) (Straub, Kappler and Schink 2005). They provide Geobacter cells with an electron acceptor for respiration and a surface for biofilm formation (Reguera et al. 2007). The soluble Fe(II) released from the reduction of the oxides can in turn initiate the abiotic reduction of other metals such as chromium (Fendorf, weilinga and Hansel 2000), arsenic (Langner and Inskeep 2000), selenium (Myneni, Tokunaga and Brown 1997) and uranium (Jang, Dempsey and Burgos 2007). Although these soluble metals can reach concentrations that would otherwise kill planktonic cells, the conductive biofilm matrix provides cells with a physical and chemical barrier for metal immobilization and reduction (Cologgi et al. 2014). Uranium-reducing biofilms of G. sulfurreducens capitalize, for example, on the metal-binding properties of the conductive pili to immobilize and mineralize the uranyl cation in the areas of the biofilm most exposed to the surrounding milieu (Cologgi et al. 2014). This strategy prevents the permeation of the soluble cation deeper into the biofilm and enhances the collective catalytic activities of the cells during the reduction of the radionuclide (Cologgi et al. 2014). The study of Geobacter biofilms is therefore of interest for the development of improved strategies for the in situ bioremediation of environments impacted by uranium and other metal contaminants. Harnessing the catalytic activities of electroactive biofilms will ultimately require deep understanding of how biofilm cells transfer electrons across the surface-attached multilayered community. This process has been well studied in bacteria whose mechanism of ET relies on the secretion of soluble redox-active molecules such as redox-active antibiotics in Pseudomonas spp. (Hernandez, Kappler and Newman 2004) and soluble electron shuttles in Shewanella spp. (Lies et al. 2005; Marsili et al. 2008; von Canstein et al. 2008). The type of mediator used for biofilm electroactivity determines the ecological niches they occupy (Sturm et al. 2012). Pseudomonas aeruginosa, for example, secretes redox-active antibiotics termed phenazines, whose antimicrobial activity can prevent opportunistic partners from 'sampling the goods' (Pierson and Pierson 2010). Phenazines can also quench O2 and enhance anaerobic survival (Wang, Kern and Newman 2010). Furthermore, they can function as electron shuttles and stimulate current production when added exogenously to electrode-associated biofilms (Rabaey et al. 2005). Extracellular ET in Shewanella oneidensis MR-1, on the other hand, involves interactions between secreted flavin cofactors and specialized outer membrane c-Cyts (reviewed in Shi et al. 2016). This bacterium was originally reported to produce 'nanowires' for extracellular ET (Gorby et al. 2006; El-Naggar et al. 2010). The MR-1 'nanowires' were wrongly assumed to be conductive pili, as in Geobacter, although the conductivity of MR-1 pilus filaments had been ruled out earlier (Reguera et al. 2005). It has now been established that the MR-1 'nanowires' are outer membrane extensions formed by the fusion of outer membrane vesicles (Pirbadian et al. 2014). They contain membrane-bound clusters of c-Cyts whose diffusion could allow for charge transport via collision-exchange mechanisms (Subramanian et al. 2018). However, their ability to support extracellular ET and respiration in vivo is yet to be demonstrated. Secreted flavins are in fact the primary mechanism for the reduction of insoluble electron acceptors by MR-1 (Kotloski and Gralnick 2013). Once secreted, the soluble cofactors become public goods and can be used by other organisms, including those lacking mediators (Prokhorova et al. 2017). Perhaps not surprisingly, biofilm ET by MR-1 is slow compared to the wired biofilms of Geobacter (Renslow et al. 2013a). For additional information about mediator-based electroactive biofilms the reader is referred to other review articles (Borole et al. 2011; Sporer et al. 2017). This minireview focuses on Geobacter biofilms as a paradigm of wired electroactive biofilms. The article does not intend to provide an exhaustive coverage of literature in the field of current-harvesting biofilms grown in electrochemical devices, which is particularly large and covers studies with little to no environmental relevance. For information about electrochemical systems driven by electrode-associated biofilms the reader is referred to two recent review articles (Prevoteau and Rabaey 2017; Lewis et al. 2018). This minireview highlights instead studies that the author sees as having the most significant impact on environmental aspects of electroactive biofilms and microbial nanowires, particularly metal reduction. The article summarizes first what is known about extracellular ET in planktonic cells of Geobacter and uses this model as a reference to discuss how cells build a heterogeneous electroactive biofilm matrix. The last section explores critical gaps of knowledge that in the opinion of the author are more likely to provide novel insights into the environmental roles of electroactive biofilms and biotechnological applications harnessed from them and their components. A PLANKTONIC VIEW OF EXTRACELLULAR ET IN GEOBACTER The hallmark of the physiology of Geobacter bacteria is their ability to gain energy for growth by coupling the oxidation of acetate and other organic or inorganic (e.g., H2) electron donors to the reduction of Fe(III) oxides (Fig. 1). The insoluble nature of the electron acceptor has selected for a complex respiratory network that electronically connects the cell to its environment. Much of what is known about this respiratory machinery comes from genetic studies in planktonic cultures of G. sulfurreducens, which serves as a genetically tractable model representative of the family (Coppi et al. 2001). Genes encoding specific respiratory components can, in principle, be inactivated to assess its effect in extracellular ET. Yet, in G. sulfurreducens, mutations in components of the respiratory chain are often pleiotropic and lead to compensatory effects that can mask the true phenotype (Kim et al. 2005; Kim et al. 2006; Kim and Lovley 2008; Cologgi et al. 2011; Richter, Sandler and Weis 2012; Steidl, Lampa-Pastirk and Reguera 2016). Thus, caution must be exerted when interpreting the phenotype of pleiotropic mutants as well as mutants not evaluated for potential compensatory effects (Vargas et al. 2013; Levar et al. 2014; Zacharoff, Chan and Bond 2016; Levar et al. 2017). Role of extracytoplasmic c-type cytochromes (c-Cyts) Figure 2 shows proteins proposed to mediate ET across the cell envelope of G. sulfurreducens and their approximate midpoint potential (normalized versus the standard hydrogen electrode or SHE for comparisons). The membrane-bound NADH dehydrogenase complex (NADH1) separates the electrons from the protons carried in the cytoplasmic NADH pool, pumps the protons into the periplasm and generates the proton motive force that drives ATP synthesis by the ATPase synthase complex. The electrons flow from NADH1 to the menaquinone pool (MQ), the lipophilic redox-active molecules of the inner membrane. Two putative quinol oxidases (CbcL and ImcH) could accept electrons from the MQ pool as a function of the potential of the extracellular electron acceptor (low for CbcL and high for ImcH) (Levar et al. 2014; Zacharoff, Chan and Bond 2016). CbcL (c- and b-type cytochrome for low potential) is a periplasmic 9-heme c-Cyt anchored to the membrane through a HydC/FdnI domain (Zacharoff, Chan and Bond 2016), which many hydrogenase and formate dehydrogenase enzymes use as MQ oxidoreductases (Jormakka et al. 2002). ImcH is a heptaheme c-Cyt containing a putative membrane region homologous to the NapC/NirT domains that mediate the transfer of electrons from the MQ pool to periplasmic carriers (Levar et al. 2014). The genetic inactivation of CbcL or ImcH prevents the reduction of electron acceptors with potentials less or greater than –0.1 V versus SHE, respectively (Levar et al. 2017). This suggests that these two quinol oxidases function as electrochemical gates, tuning ET across the cell envelope to the redox potential of the extracellular electron acceptor by yet unknown mechanisms. This dual path has been proposed to allow cells to respire a wide range of Fe(III) oxides available in the environment and to adapt to the changes in potential (from high to low) that the mineral oxides experience in the course of their reduction (Levar et al. 2017). Figure 2. View largeDownload slide Electron transport pathway across the cell envelope of G. sulfurreducens during the reduction of ferrihydrite to Fe2+. The position of the electron carriers against a redox scale (left) is based on reported midpoint potentials normalized versus the standard hydrogen electrode (SHE), except for ImcH, whose midpotential has not been experimentally calculated and is, therefore, placed arbitrarily on the scale (indicated with a star [*] symbol). Abbreviations: IM, inner membrane; OM, outer membrane; NADH1, NADH dehydrogenase; MQ, menaquinone; CbcL and ImcH, putative quinol oxidases for the reduction of acceptors with low and high potentials, respectively; PpcAD and OmcBS, c-Cyts of the periplasm and outer membrane, respectively. OmcB is shown as part of a porin-cytochrome complex (Pcc). Figure 2. View largeDownload slide Electron transport pathway across the cell envelope of G. sulfurreducens during the reduction of ferrihydrite to Fe2+. The position of the electron carriers against a redox scale (left) is based on reported midpoint potentials normalized versus the standard hydrogen electrode (SHE), except for ImcH, whose midpotential has not been experimentally calculated and is, therefore, placed arbitrarily on the scale (indicated with a star [*] symbol). Abbreviations: IM, inner membrane; OM, outer membrane; NADH1, NADH dehydrogenase; MQ, menaquinone; CbcL and ImcH, putative quinol oxidases for the reduction of acceptors with low and high potentials, respectively; PpcAD and OmcBS, c-Cyts of the periplasm and outer membrane, respectively. OmcB is shown as part of a porin-cytochrome complex (Pcc). Genetic studies have also identified periplasmic and outer membrane c-Cyts that could electronically connect the inner membrane carriers to the extracellular electron acceptor (Fig. 2). PpcA is a periplasmic triheme cytochrome proposed to function as an intermediary electron carrier to terminal Fe(III) reductases in the outer membrane (Lloyd et al. 2003). It is also one of the few c-Cyts of G. sulfurreducens that is highly conserved in otherGeobacter genomes (Butler, Young and Lovley 2010). PpcA and its homolog PpcD have also been proposed to contribute to the proton electrochemical membrane gradient that drives ATP synthesis (Pessanha et al. 2006; Morgado et al. 2010). The range of potentials that could support energy transduction by these two periplasmic carriers spans almost 100 mV (–0.167 to –0.109 V in PpcA and –0.202 to –0.146 V in PpcD) (Morgado et al. 2010). Two other periplasmic c-Cyts (PpcB and PpcE) do not appear to couple electron and proton transfer and have heme groups with negative, yet slightly different, reduction potentials (Morgado et al. 2010). Collectively, the periplasmic c-Cyts span a large range of potentials that could facilitate ET from membrane carriers to outer membrane c-Cyts such as OmcB (Magnuson et al. 2001; Leang, Coppi and Lovley 2003; Qian et al. 2007). OmcB is part of a trans-outer membrane porin-cytochrome complex (Pcc) required for optimal growth rates with ferrihydrite (Liu et al. 2014b). Pcc complexes comprise an outer membrane c-Cyt (OmcB or OmcC), a periplasmic c-Cyt (OmaB or OmaC) and a porin-like outer membrane protein (OmbB or OmbC) (Liu et al. 2014a; Liu et al. 2015). The two Pcc complexes (OmbB-OmaB-OmcB and OmbC-OmaC-OmcC) appear to have overlapping roles in Fe(III) reduction (Liu et al. 2015). The association of the outer membrane c-Cyt OmcB with other proteins to form a Pcc complex assumes that OmcB is exposed sufficiently on the external side of the outer membrane to bind and transfer electrons to the extracellular electron acceptor (Liu et al. 2014b). However, OmcB is not readily immunodetected on the cell surface, suggesting it is deeply embedded in the outer membrane (Qian et al. 2007). Furthermore, genetic inactivation of OmcB initially impairs the ability of the cells to grow with ferrihydrite but the mutant cells eventually adapt (Leang, Coppi and Lovley 2003; Leang et al. 2005). In contrast, the outer membrane c-Cyt OmcS is easily sheared from the cell surface, consistent with its exposure to the extracellular environment, and it is required for the reduction of Fe(III) oxides (Mehta et al. 2005). This makes OmcS more likely to function as a terminal Fe(III) reductase. Flavin species (riboflavin and flavin mononucleotide) could facilitate this last step of ET (Okamoto et al. 2014b). Flavin cofactors have been detected in planktonic cultures of G. sulfurreducens at levels too low to function as electron shuttles for respiration (Okamoto et al. 2014b). Yet even at low concentrations flavin species could bind outer-membrane c-Cyts and enhance the rate of ET between the cell surface and solid electron acceptors (Okamoto et al. 2014b). This 'flavin-bound cofactor' model assumes that, as in S. oneidensis MR-1, the bound flavins catalyze a one-electron reaction that shifts the redox potential of c-Cyts to accelerate the rates of ET to the electron acceptor (Okamoto et al. 2014a). Experimental evidence to support this model is however yet to be produced. The c-Cyt path presented in Fig. 2 shows electrons flowing through a favorable redox gradient from the NAD+/NADH pair in the cytoplasm (midpoint potential [Em] versus SHE, –0.365 V to –0.320 V) (Thauer, Jungermann and Decker 1977; Saleh et al. 2011), to the MQ/MQH2 couple (Em = –0.260 V vs SHE) (Kishi et al. 2017) and to one of the putative quinol oxidases (CbcL’s Em = −0.15 V vs SHE) (Zacharoff, Chan and Bond 2016). The reduction potential of ferrihydrite to Fe2+ ranges from –0.1 to +0.1 V (Thamdrup 2000). Thus, the periplasmic and outer membrane electron carriers would be expected to have potentials within this narrow range of −0.15 V (CbcL) to +0.1 V (maximum for ferrihydrite). Yet the midpoint potential (vs SHE) of PpcA is −0.167 V (Lloyd et al. 2003), OmcB’s is −0.19 V (Magnuson et al. 2001), and OmcS’ is −0.212 V (Qian et al. 2011). Hence, the path from the periplasmic to the outer membrane c-Cyts is increasingly negative and would seemingly require energy expenditure for electron discharge. This 'uphill' path may reflect discrepancies between in vitro and in vivo potentials. The former were calculated experimentally for the free proteins and did not consider protein–protein interactions and other in vivo variables such as the ratio of oxidized/reduced species, which can significantly change (sometimes by 200 mV) the biological potential that drives electron transport (Thauer, Jungermann and Decker 1977). Indeed, the amount of energy extracted by Geobacter cells during Fe(III) oxide respiration depends on the potential difference between NADH and the inner membrane and/or periplasmic carriers rather than the oxides (Bird, Bonnefoy and Newman 2011). Thus, electron transport beyond the inner membrane and/or periplasm does not appear to require energy expenditure. Electrons could also diffuse from areas of high to low concentration, hopping between redox cofactors of periplasmic and outer membrane carriers, until they reach the extracellular electron acceptor, similarly to the redox-gradient model that has been proposed to drive ET by matrix-associated c-Cyts in electroactive biofilms (Snider et al. 2012). This mechanism could also allow the periplasmic and outer membrane c-Cyts to store electrons in the cell envelope until electronic contact with the electron acceptor has been made and the electrons can be discharged. The notion that extracytoplasmic c-Cyts function as a capacitor is further supported by the fact that Geobacter genomes often encode a great number of these c-Cyts yet sequence conservation among the genes is weak (Butler, Young and Lovley 2010). Furthermore, the c-Cyt content increases in cells of G. sulfurreducens grown under electron acceptor-limitation, allowing for the storage per cell of up to ∼107 electrons produced from the oxidation of electron donors (Esteve-Nunez et al. 2008). Conductive pili make the electrical connection with extracellular electron acceptors In contrast to the poor conservation of c-Cyts genes among Geobacter species (Butler, Young and Lovley 2010), the genes encoding the components of the Type IV pilus apparatus are highly conserved in this and other genera in the order Desulfuromonadales (Reguera et al. 2005; Holmes et al. 2016). This high conservation is consistent with the role of Geobacter pili as terminal reductases for the extracellular reduction of Fe(III) oxides (Reguera et al. 2005) and the uranyl cation (Cologgi et al. 2011). Indeed, deleting the gene encoding the pilin peptide in G. sulfurreducens (Reguera et al. 2005) or in Geobacter metallireducens (Tremblay et al. 2012) prevents growth with Fe(III) oxides as sole electron acceptor. This mutation was later found to be pleiotropic and to lead to defects in c-Cyt expression and secretion (Cologgi et al. 2011; Steidl, Lampa-Pastirk and Reguera 2016). To unmask the true contribution of the pili to extracellular ET, my group constructed a mutant (Tyr3) carrying alanine replacements in the pilin’s three tyrosines that reduced the conductivity of the assembled pili fivefold without affecting the expression of c-Cyts or cell piliation (Steidl, Lampa-Pastirk and Reguera 2016). The increased electrical resistance of the pili led to severe growth defects with Fe(III) oxides, even though some low levels of the metal chelator nitrilotriacetic acid (NTA) were present in the cultures to support the initial growth of the cells in the presence of the oxides (Feliciano, Steidl and Reguera 2015). However, the Tyr3 mutant was chemically rescued in Fe(III) oxides supplemented with concentrations of NTA high enough to bypass the need of the cells to use the pili to establish direct electronic contact with the electron acceptor (Feliciano, Steidl and Reguera 2015). Despite containing a divergent pilin, Geobacter genomes encode highly conserved components of the canonical Type IV pilus biosynthetic apparatus (Speers et al. 2016). As in other bacteria, the Geobacter pilin is synthesized as a precursor with a signal peptide that is recognized and cleaved by a dedicated signal peptidase (PilD) in the inner membrane (Richter, Sandler and Weis 2012). The processed pilins are stored in the inner membrane until ready for assembly by a complex apparatus that spans the multilayered cell envelope (Fig. 3). The pilus fiber grows by polymerizing the pilins at its base and protrudes through the outer membrane via a dedicated PilQ porin secretin. Pilin assembly is energized by a conserved PilB ATPase (Steidl, Lampa-Pastirk and Reguera 2016) and is reversible, that is, the pilus fiber can be retracted in a reaction energized by the PilT4 ATPase (Speers et al. 2016). Deletion of the gene encoding the PilT4 retraction motor in G. sulfurreducens does not affect the expression of c-Cyts but the mutant cells are hyperpiliated and severely impaired in the reduction of Fe(III) oxides (Speers et al. 2016). The hyperpiliation of the PilT4 mutant also promotes cell–cell aggregation and the formation of biofilms that, despite being denser than the wild type, have reduced respiratory rates per cell (Speers et al. 2016). Thus, the dynamic protrusion and retraction of the pili is critical for respiration in planktonic and biofilm cells. Figure 3. View largeDownload slide Dynamics of the pilus of G. sulfurreducens during the reduction of extracellular electron acceptors. The pilus apparatus includes the PilC protein (lavender) that anchors accessory proteins at the base of the pilus, the PilM protein that connects PilC to the lateral PilNO subcomplexes (light blue), and the PilP lipoprotein (lavender) that connects the PilNO subcomplexes to the PilQ secretin ring on the outer membrane (green). The PilB and PilT4 (right) ATPases that energize pilin polymerization and depolymerization, respectively, are highlighted in the pilus protrusion (left) and retraction (right) panels. Also shown are Fe(III) oxide particles (in brown), their reduced product—magnetite—(in black), and the accumulation of reduced c-Cyts (maroon) that could discharge electrons at the base of the pilus. Figure 3. View largeDownload slide Dynamics of the pilus of G. sulfurreducens during the reduction of extracellular electron acceptors. The pilus apparatus includes the PilC protein (lavender) that anchors accessory proteins at the base of the pilus, the PilM protein that connects PilC to the lateral PilNO subcomplexes (light blue), and the PilP lipoprotein (lavender) that connects the PilNO subcomplexes to the PilQ secretin ring on the outer membrane (green). The PilB and PilT4 (right) ATPases that energize pilin polymerization and depolymerization, respectively, are highlighted in the pilus protrusion (left) and retraction (right) panels. Also shown are Fe(III) oxide particles (in brown), their reduced product—magnetite—(in black), and the accumulation of reduced c-Cyts (maroon) that could discharge electrons at the base of the pilus. Planktonic cells have been proposed to use pilus retraction to shed off the bound, reduced mineral particles and recycle the pilin peptides in the membrane for a new round of assembly and metal binding and reduction (Speers et al. 2016) (Fig. 3). The antagonistic cycles of pilus protrusion and retraction could also maximize access to the most bioavailable Fe(III) oxide phases (e.g., ferrihydrite), which are unstable and rapidly transform abiotically into more crystalline and more recalcitrant phases (e.g., goethite and hematite) (Lentini, Wankel and Hansel 2012). Similarly, the retraction of the pili could allow cells to detach the reduced uranium mineral that remains bound to the pilus surface after the reduction of the uranyl cation (Cologgi et al. 2011) (Fig. 3). The disassembly rates estimated for other bacterial pili range from 1000 to 1500 pilins per second (Merz, So and Sheetz 2000; Skerker and Berg 2001). Thus, a full grown pilus, which contains 500–1000 pilins (Sauvonnet et al. 2000), could be retracted in one second. Storing the pilins in the membrane also ensures they are readily available for reassembly to start a new round of metal binding and reduction. Further, antagonistic cycles of protrusion and retraction allow the cell to mineralize uranium at a distance from the cell and to do so fast enough to prevent its permeation in the cell envelope. As a result, the extracellular reduction of the uranyl cation by the pili also provides cells with a protective mechanism from its toxicity (Cologgi et al. 2011). Being anchored in the cell envelope, the pilus pathway is well suited to accept electrons from c-Cyts (Fig. 3). Periplasmic c-Cyts such as PpcA are abundant in the cell envelope and, like the components of the Type IV pilus apparatus, they are encoded by genes conserved in Geobacter genomes (Butler, Young and Lovley 2010). PpcA and PpcD also have the potential to contribute to the proton motive force and energy conservation (Pessanha et al. 2006; Morgado et al. 2010). Furthermore, the periplasmic c-Cyts can function as cellular capacitors, storing electrons and allowing for continued proton pumping across the inner membrane and ATP synthesis (Esteve-Nunez et al. 2008). As the c-Cyts store charges, ATP is available to energize the dynamic protrusion and retraction of the pili and facilitate productive interactions between the pili and the extracellular electron acceptors. Once electronic contact with the electron acceptor is established, charges are predicted to move through the pili at rates that do not limit respiration. Individual pilus fibers purified free of metals and redox cofactors transport charges at rates close to ∼1 billion electrons per second (for a 1 µm-long pilus) at the potential difference that exists between the inner membrane and the extracellular Fe(III) oxides (∼100 mV) (Lampa-Pastirk et al. 2016). This is two orders of magnitude greater than the cellular rates of Fe(III) oxide reduction (∼9 million electrons per cell per second) (Lampa-Pastirk et al. 2016). The pili probed for charge transport in vitro (Lampa-Pastirk et al. 2016) were not chemically fixed and retained the structural and electronic features of cell-associated pili (Veazey, Reguera and Tessmer 2011). However, the pili were adsorbed onto a substrate and did not experience the in vivo cycles of protrusion and retraction that are predicted to promote electronic coupling in the pilus protein matrix and to accelerate charge transport (Feliciano, Steidl and Reguera 2015). Furthermore, cells assemble numerous pili monolaterally, concentrating the conductive filaments on one side of the cell to maximize access to the extracellular electron acceptor (Fig. 1). Thus, the Geobacter pili have the attributes needed to provide an efficient respiratory strategy during the reduction of extracellular electron acceptors. Mechanistic understanding of pilus conductivity The carrier mobility calculated for individual pili (3.2 × 10−2 cm2/Vs), which describes how fast the carriers (electrons or holes) move through the pilus protein fiber, is within the orders reported for organic semiconductors (Lampa-Pastirk et al. 2016), but it is orders of magnitude lower than the mobilities (>1 cm2/Vs) that would be required to describe pilus charge transport according to band theory (Polizzi, Skourtis and Beratan 2012). This argues against the metallic model of pilus conduction, a band conduction mechanism that has been proposed to explain the metallic-like temperature and pH response of conductive pili networks probed dried (Malvankar et al. 2011) or in solution (Ing, Nusca and Hochbaum 2017). As the pili samples used in these two studies were not pure, the contribution of contaminants (including c-Cyts) to the metallic-like responses cannot be ruled out. Furthermore, the protein samples were deposited on electrodes following extensive drying protocols (overnight drying or spin coating, respectively), which could significantly affect the native structure of the pili and its native conductive properties. Concerns (Strycharz-Glaven and Tender 2012; Yates et al. 2016) have also been raised about the description of biofilm conductivity as metallic-like due to the pili (Malvankar et al. 2011). Of special note is the fact that the Malvankar study used as a negative control a pilin-deficient mutant that also has defects in c-Cyts required for biofilm electroactivity (Cologgi et al. 2011; Steidl, Lampa-Pastirk and Reguera 2016) and limitations of the electrochemical gating experiments used to measure the temperature and pH response of biofilms grown across electrode gaps (Yates et al. 2016). The model of metallic-like biofilm conductivity is also incongruent with the thermal activation of conductivity reported for living biofilms of G. sulfurreducens (Yates et al. 2015) and the critical role that both c-Cyts and pili play in the mechanistic stratification of electroactive biofilms (Steidl, Lampa-Pastirk and Reguera 2016). Structural support for the metallic model of pilus conductance is also lacking. This model evokes the clustering of the pilins’ aromatic rings in the rigid sandwich-type configurations that allow for π orbital stacking (Malvankar et al. 2011). Although the pilus carrier mobility is sensitive to the formation of intra- and inter-molecular contacts that bring neighboring aromatic rings close together (3.5–5 Å apart), atomic resolution models of the pilus structure refined via molecular dynamics (MD) reveal aromatic dimers in displaced configurations that cannot support π stacking (Feliciano, Steidl and Reguera 2015; Lampa-Pastirk et al. 2016). Figure 4 shows a snapshot of the structure of a pilus fiber optimized in the MD simulations and the paths of aromatic rings (from tyrosines and phenylalanines) that form axially and transversally (Feliciano, Steidl and Reguera 2015). The figure also shows a close up of a pilus region from the MD model (Feliciano, Steidl and Reguera 2015). The inter-aromatic distances range from 3.5 to 8.5 Å, which are optimal for charge hopping (Feliciano, Steidl and Reguera 2015). Some of the aromatic dimers get close enough (3.5–5 Å apart) to form contacts that could promote the tunneling of charges, but all have displaced geometric configurations that cannot support π stacking. Furthermore, in the MD simulations the aromatic contacts never form at the same time, as would be expected for a metallic wire (Feliciano, Steidl and Reguera 2015). Thus, the inter-aromatic distances and geometries of the contacts support a charge hopping model mediated by aromatic residues, in agreement with the thermal activation of pilus conductivity demonstrated experimentally at biologically relevant voltages (± 0.2–0.4 V) (Lampa-Pastirk et al. 2016). It is interesting to note that similar displaced geometric configurations have been reproduced in non-dynamic, rigid models that claimed to support the metallic model of conductance (Xiao et al. 2016). Caution must therefore be exerted when interpreting this (Xiao et al. 2016) and other (Malvankar et al. 2015) modeling studies that do not consider the geometry of the aromatic contacts to support their conclusions, as even small misalignments of the aromatic rings forming the contacts can dramatically decrease the tunneling rates and prevent metallic conduction (Bredas et al. 2002). Figure 4. View largeDownload slide (A) Surface map of a pilus fiber optimized via MD. (B) Clustering of aromatic residues (tyrosines, yellow; phenylalanines, green) in the pilus fiber showing axial and transversal paths for charge transport. (C) 20-Å close up of a pilus region showing the parallel-displaced and T-shaped geometric configurations of the aromatic contacts (tyrosines, yellow; phenylalanines, green). The tyrosine (Y57) exposed on the flexible carboxyl-terminal segment of each pilin is labeled. Figure 4. View largeDownload slide (A) Surface map of a pilus fiber optimized via MD. (B) Clustering of aromatic residues (tyrosines, yellow; phenylalanines, green) in the pilus fiber showing axial and transversal paths for charge transport. (C) 20-Å close up of a pilus region showing the parallel-displaced and T-shaped geometric configurations of the aromatic contacts (tyrosines, yellow; phenylalanines, green). The tyrosine (Y57) exposed on the flexible carboxyl-terminal segment of each pilin is labeled. GEOBACTER BIOFILMS: A PARADIGM IN ELECTRONIC HETEROGENEITY The biofilm matrix is a heterogeneous medium of polysaccharides, nucleic acids, lipids and/or proteins, collectively known as exopolymeric substances (EPS), that surrounds the biofilm cells and attaches the community to a surface (Flemming and Wingender 2010). The chemical composition of the EPS matrix is carefully controlled by the cells to create a hydrated, catalytic microenvironment optimal for nutrient adsorption and assimilation (Flemming and Wingender 2010). This is particularly important for Geobacter bacteria, which form biofilms on surfaces, such as Fe(III) mineral coatings, that also serve as an electron acceptor for respiration (Reguera et al. 2007). In electroactive biofilms, the EPS matrix becomes an electronic medium of c-Cyts and pili whose coordinated interactions permit the collective discharge of respiratory electrons to support the growth of the biofilm cells (Steidl, Lampa-Pastirk and Reguera 2016). The sections below discuss what is known about the composition and electronic properties of the biofilm electron carriers from studies in the model representative G. sulfurreducens. The biofilm c-type cytochromes (c-Cyts) Biofilm cells use the same electron carriers of planktonic cells to transport electrons across the inner membrane and periplasmic space (Fig. 2). Electron transport across the outer membrane, however, appears to require a distinct set of c-Cyts. For example, the outer membrane c-Cyts OmcS (Mehta et al. 2005) and OmcB (Leang et al. 2003), which are important electron carriers during the reduction of Fe(III) oxides by planktonic cells (Fig. 2), can be deleted without impacting biofilm electroactivity (Holmes et al. 2006; Richter et al. 2009). Furthermore, biofilm cells require the expression of a putative Pcc conduit (extABCD) that is not involved in the reduction of Fe(III) oxides by planktonic cells (Chan et al. 2017). The requirement to use biofilm-specific outer membrane c-Cyts likely reflects an adaptive response of the cells to the local biofilm microenvironment, where interactions with matrix-associated electron carriers become the critical step limiting cell growth and respiration. EPS matrices extracted from electroactive biofilms are enriched in the outer membrane c-Cyt OmcZS (Rollefson et al. 2011; Cologgi et al. 2014; Steidl, Lampa-Pastirk and Reguera 2016). OmcZ is expressed and targeted to the outer membrane as a large protein (OmcZL; 50-kDa) and a more abundant, carboxy-terminal cleaved product (OmcZS; 30-kDa), which retains the cytochrome’s 8 hemes (Inoue et al. 2010). OmcZS is released to the electroactive matrix, where it is anchored to the biofilm Xap polysaccharide (Rollefson et al. 2011). Thin sections of current-harvesting biofilms grown on anode electrodes show the preferential location of OmcZS at or closer (<10µm) to the electrode surface (Inoue et al. 2011). This suggests a critical role for this c-Cyt in completing the last step of ET, from the biofilm to the underlying electron-accepting surface. In support of this, the genetic inactivation of OmcZ leads to a significant reduction in biofilm electroactivity (Nevin et al. 2009; Richter et al. 2009; Steidl, Lampa-Pastirk and Reguera 2016). OmcZS is also abundant in the EPS matrix of uranium-reducing biofilms (Cologgi et al. 2014). The large range of redox potentials covered by its eight heme groups (from −0.42 to −0.06 V) permits the in vitro reduction of Fe(III) citrate, Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analog anthraquinone-2,6-disulfonate (AQDS) in addition to U(VI) (Inoue et al. 2010). The pili of the biofilm matrix also promote the binding and reduction of soluble electron acceptors (Cologgi et al. 2014). Hence, OmcZS and the conductive pili make the biofilm matrix an electronic medium well suited to mediate many metal redox transformations. Mechanistic stratification of electroactive biofilms mediated by conductive pili The c-Cyts of biofilms grown on anode electrodes can be reversibly oxidized and reduced by changing the anode potential, consistent with their role as electron carriers (Liu et al. 2011; Liu and Bond 2012). However, there appears to be a maximum biofilm thickness of ∼10 µm that permits the full reduction of the matrix-associated c-Cyt pool (Liu and Bond 2012). This is also the biofilm stratum that concentrates OmcZS closer to the electron-accepting surface (Inoue et al. 2011). Yet electroactive biofilms can grow tens (or even hundreds) of µm thick (Renslow et al. 2013b). As they grow beyond the 10 µm threshold, diffusion constraints limit the ability of the c-Cyts to transfer electrons in the biofilm matrix and reduced c-Cyts progressively accumulate (Robuschi et al. 2013). This leads to sharp decreases in redox potentials in regions of the biofilm further away from the underlying electrode (Babauta et al. 2012) and the establishment of a redox gradient that drives electron transport to the fully functional electron carriers in the biofilm region closer to the electron-accepting surface (Snider et al. 2012). Figure 5 illustrates this in thin (10 µm) versus thick (20 µm) biofilms. The accumulation of reduced c-Cyts in the upper layers of thick biofilms creates a biological paradox: how can cells continue to grow optimally in these upper regions where the matrix c-Cyts are in a reduced state and unable to accept electrons at optimal rates? Cells in this upper biofilm stratum remain viable and contribute to the collective discharge of electrons despite the increases in biofilm thickness (Reguera et al. 2006). This indicates that the cells in these upper regions have alternative pathways for the discharge of respiratory electrons that bypass the electron acceptor limitation imposed by the accumulation of reduced c-Cyts. Figure 5. View largeDownload slide Model of mechanistic stratification in electroactive biofilms mediated by the pilus nanowires. Shown are thin (10 µm) biofilms formed by the wild type (WT) and pili-deficient PilB− strain and thick (20 µm) WT biofilms. The accumulation of reduced c-Cyts (c-Cyts•) in the upper stratum of thick WT biofilms is illustrated in a darker maroon color. The direction of electron flow towards the underlying electron-accepting substratum is indicated with a vertical arrow (dashed arrow, reduced conductivity) Figure 5. View largeDownload slide Model of mechanistic stratification in electroactive biofilms mediated by the pilus nanowires. Shown are thin (10 µm) biofilms formed by the wild type (WT) and pili-deficient PilB− strain and thick (20 µm) WT biofilms. The accumulation of reduced c-Cyts (c-Cyts•) in the upper stratum of thick WT biofilms is illustrated in a darker maroon color. The direction of electron flow towards the underlying electron-accepting substratum is indicated with a vertical arrow (dashed arrow, reduced conductivity) The most obvious alternative pathway for biofilm ET is that involving the pilus nanowires (Fig. 5). The pili are required to grow multilayered electroactive biofilms on both electron-accepting and non-accepting surfaces (Reguera et al. 2006, 2007; Cologgi et al. 2014). Individual pilus fibers have the reach (length of up to tenths of µm) and charge transport capacity (∼1 billion electrons per second) needed to discharge respiratory electrons at µm distances from the cell (Lampa-Pastirk et al. 2016). The thermal activation of pilus charge transport at biologically relevant potentials (Lampa-Pastirk et al. 2016) is also in agreement with the thermal dependence of charge transport demonstrated for living biofilms (Yates et al. 2015). Thus, both c-Cyts and pili have the electronic properties that confer on living electroactive biofilms their distinctive incoherent redox conductivity. The experimental evidence therefore supports a model of biofilm ET involving both c-Cyts and pili as electron carriers. Although the conductive pili have the properties needed to function as electron carriers in the biofilm matrix, genetic studies have been confounded by the pleiotropic effects that pili-inactivating mutations often have on the expression of c-Cyts required for extracellular ET (Juarez et al. 2009; Cologgi et al. 2011; Cologgi et al. 2014). Steidl et al. demonstrated the absence of OmcZS in the matrix of current-harvesting biofilms formed by a pilA-deficient mutant and a pilA-E5A mutant, which expresses pilins with an alanine replacement in an amino acid (E5) required for recognition and alignment of the pilin peptides during assembly (Steidl, Lampa-Pastirk and Reguera 2016). The mutants overexpressed other c-Cyts and were able to grow to a thickness of ∼10 µm, though growth rates and current production were sharply decreased and were similar to OmcZ-deficient mutant biofilms (Steidl, Lampa-Pastirk and Reguera 2016). By contrast, deletion of the PilB ATPase that powers pilin polymerization (Fig. 3) does not affect the expression of c-Cyts in planktonic or biofilm cells, providing the elusive genetic tool needed to assess the contribution of pili to biofilm electroactivity (Fig. 5). The PilB-deficient mutant can be grown to a thickness of ∼10 µm on an electrode poised at an optimal potential and, despite having wild type-levels of OmcZS in the biofilm matrix, its electroactivity is reduced in half (Steidl, Lampa-Pastirk and Reguera 2016). A Tyr3 mutant, which carries alanine replacements in the three pilin’s tyrosines (Feliciano, Steidl and Reguera 2015), produces pili with reduced conductivity and forms biofilms with the same thickness and electroactivity as the PilB-deficient mutant (Steidl, Lampa-Pastirk and Reguera 2016). Thus, although the Tyr3 biofilms have the structural pili network in their matrix, they are limited in their ability to use it to discharge respiratory electrons. The PilB and Tyr3 genetic studies demonstrate that the conductive pili are required for optimal ET even in biofilms grown to a thickness (∼10 µm) that permits the full reduction of the matrix-associated c-Cyts (Steidl, Lampa-Pastirk and Reguera 2016). This suggests that the pili and c-Cyts work coordinately to maintain optimal rates of ET in thin (up to 10 µm) biofilms (Fig. 5). This model is further supported by the cross-regulation of pili assembly and c-Cyt export observed by us (Steidl, Lampa-Pastirk and Reguera 2016) and others (Richter, Sandler and Weis 2012). Regulation may involve interactions between the two isoforms of the PilA pilin precursor (carrying a short or a long leader peptide) and the secretion system that exports outer membrane c-Cyts (Richter, Sandler and Weis 2012). Indeed, mutations that disrupt pilin-pilin interactions (pilA deletion or E5A replacement in PilA) prevent the expression of several c-Cyts and the secretion of OmcZS to the biofilm matrix (Steidl, Lampa-Pastirk and Reguera 2016). This cross-regulation, mediated by pilin interactions, would be analogous to that reported in P. aeruginosa, which relies on interactions between the pilin and components of the general secretion pathway to modulate protein export across the outer membrane (Lu, Motley and Lory 1997). The cross-regulation between pilin and c-Cyt secretion also warns about the multiple phenotypes that pilin-inactivating mutations can have that are not directly caused by the pilus deficiency but, rather, by the absence of critical c-Cyts. For example, a strain of G. sulfurreducens that produces only the short PilA isoform (pilA4 mutant) has defects in pilin secretion and assembly, no observable defects in c-Cyts and forms biofilms whose electroactivity is about half of the wild-type biofilms (Richter, Sandler and Weis 2012), as reported for the PilB-deficient and Tyr3 mutants (Steidl, Lampa-Pastirk and Reguera 2016). Yet a mutant carrying alanine replacements in 5 aromatic residues of the pilin (Aro5 mutant) and grown as anode biofilms under the same conditions as the pilA4 mutant had a more severe defect (Vargas et al. 2013), producing current plots conspicuously similar to those of an OmcZ-deficient mutant (Nevin et al. 2009) or mutants carrying pilin-inactivation mutations that prevent the expression of OmcZS in the biofilm matrix (Steidl, Lampa-Pastirk and Reguera 2016). As the effect of the Aro5 mutation on the expression of c-Cyts has not been assessed, caution is necessary when interpreting these genetic studies. The identification of the PilB-deficient mutation, which inactivates pilin assembly without interfering with pilin interactions needed for c-Cyt export, also allowed to evaluate the contribution of conductive pili across the redox gradient that establishes in thicker (>10 µm) biofilms (Steidl, Lampa-Pastirk and Reguera 2016). As shown in Fig. 5, while it is possible to increase the concentration of the electron donor (acetate) to grow thicker (∼20 µm) wild-type biofilms, the PilB and the Tyr3 mutant biofilms remain thin and unable to grow beyond the threshold 10-µm thickness that limits the availability of matrix-associated c-Cyts as electron acceptors (Steidl, Lampa-Pastirk and Reguera 2016). These results strongly suggest that the conductive pilus network provides the primary mechanism for charge transport from cells in regions of the biofilm where the c-Cyts cannot operate as efficient electron carriers. The c-Cyts of these upper biofilm layers could however function as a capacitor, storing electrons in the biofilm matrix and establishing the redox gradient that drives the flow of electrons to the underlying electron-accepting surface (Snider et al. 2012). These upper regions of the biofilm are also more exposed to the external medium and therefore have the highest concentrations of nutrients, including electron donors for the cell's oxidative metabolism (Renslow et al. 2013b). The pili can grow several micrometers in length (Reguera et al. 2005) and intertwine to form an intercellular conductive network (Veazey, Reguera and Tessmer 2011) and mediate electronic contacts with c-Cyts in the bottom layers of the biofilm. Thus, the conductive pili are suitable to become the primary mechanism that allows cells in electroactive biofilms to continue to grow without compromising the efficiency of charge transport to the underlying electron acceptor. CONCLUSION AND OUTLOOK Despite their inherent heterogeneity, biofilm cells coordinate their activities to increase their metabolic productivity (Halan, Buehler K and Schmid 2012) and achieve greater catalytic rates during the cycling of nutrients (Hall-Stoodley, Costerton and Stoodley 2004). Similarly, Geobacter cells use conductive pili to form a network of nanowires that mechanistically stratifies the biofilms to maintain optimal rates of electron transport across the multilayered communities and grow cells at a distance from an underlying electron acceptor (Steidl, Lampa-Pastirk and Reguera 2016). The model evokes coordinated interactions between the matrix c-Cyts and the conductive pili by yet unknown mechanisms. The retractile nature of the pili could be critical to these interactions. Current harvesting from a mutant deficient in pilus retraction (PilT4-) is similar to the wild type but the mutant cells are hyperpiliated and form denser biofilms (Speers et al. 2016). As a result, the rates of charge discharge per cell are reduced in the mutant biofilms. This suggests that pilus dynamics within the EPS matrix are critical to biofilm electroactivity. This role could be analogous to the critical role that pilus retraction has in enabling sequential rounds of electronic interactions between planktonic cells and extracellular electron acceptors such as Fe(III) oxides (Fig. 3). Yet in the biofilm matrix antagonistic cycles of pilus protrusion and retraction may be necessary to establish productive interactions with c-Cyts, or perhaps other cells, and maintain optimal rates of cellular respiration. Retraction also enables dynamic control of pilus length in the electroactive matrix so as to maintain optimal cell–cell spacing, a critical consideration for coordinated behavior within biofilms (Speers et al. 2016). Regulation of cell–cell distance can prevent mass transport limitations of nutrients diffusing through the biofilm matrix and, in electroactive biofilms, it could also coordinate the interactions of the pili with other matrix-associated electron carriers. Many questions still remain about how the biofilm cells sense and respond to the local redox environment. The physiology of the biofilm cells, as well as interactions between electron carriers in the biofilm matrix, are expected to be influenced by the pH, redox and electrical gradients that naturally establish across electroactive biofilms as their thickness increases (Franks et al. 2009; Babauta et al. 2012). For example, the pH of anode biofilms decreases in regions closer to the electrode surface and could modulate the rates of proton-coupled ET. The “flavin-bound cofactor" model assumes that flavins bind to c-Cyts and simultaneously move protons and electrons to accelerate the rates of ET, making this reaction pH-dependent (Okamoto et al. 2014a). Proton-coupled ET may also promote pilus charge transport. The close proximity of negatively charged residues to the aromatic rings of the pilin’s three tyrosines has been proposed to favor proton-coupled ET reactions (Feliciano, Steidl and Reguera 2015). In this model, the acidic residue is transiently protonated by the neighboring tyrosine to reduce the oxidation potential of the aromatic amino acid and enable fast rates of charge transport at the low potentials that operate in biological systems (Reece and Nocera 2003; Stubbe et al.2003). This mechanism of proton-coupled ET also makes the rates of pilus charge transport pH-dependent. The incoherent conductivity of living electroactive biofilms fits well with the coupled transport of protons and electrons mediated by c-Cyts (Yates et al. 2015). A similar mechanism could enable the short-distance, multistep hopping reactions that have been proposed to occur between pili and c-Cyts (Bonanni et al. 2013). The degree of piliation of the biofilm cells and concentrations of c-Cyts across the biofilms also merits investigation. Mass transport constraints can establish concentration gradients of essential nutrients such as electron donors (Liu et al. 2011; Babauta et al. 2012). As a result, nutrient availability fluctuates spatially in biofilms, influencing the cell physiology and, potentially, the levels of piliation and secretion of c-Cyts. Transcriptional profiling of the pilin-encoding gene (pilA) is not particularly useful to assess piliation because the levels of pilA transcripts do not change substantially in electroactive biofilms (Franks et al. 2010). Thus, transcriptional markers for piliation need to be identified to investigate how cells modulate pili levels as a function of pH, nutrient and redox cues from their microenvironment. Similarly, the regulatory networks that control the coordinated expression of pili and c-Cyts in response to the redox environment remain largely unknown. P. aeruginosa uses the redox state of secreted phenazines to alter the biofilm structure based on the availability of the electron acceptor (oxygen) (Okegbe, Price-Whelan and Dietrich 2014). Similar signal transduction systems could control the expression of pili and c-Cyts to modulate the rates of extracellular ET in Geobacterbiofilms and achieve redox homeostasis. Ecological studies also need to be undertaken to explore the range of metal transformations that can be mediated by microbial nanowires within electroactive biofilms. The physical and chemical microenvironment provided by the biofilm matrix and the unique physiology of the biofilm cells lead to biofilm-specific traits such as resistance to antimicrobials (Mah and O’Toole 2001). This is similarly reflected in the enhanced ability of electroactive biofilms to immobilize and reduce concentrations of metal contaminants that would otherwise kill planktonic cells (Cologgi et al. 2014). In subsurface environments impacted by metal contamination, biofilm formation provides an efficient mean to persist in the sediment and in areas with highest concentrations of nutrients rather than been swept away by the current of the groundwater. Fe(III) oxides are abundant in the subsurface as dispersed coatings associated with inorganic and organic solid matter that range in size from particles smaller than a bacterial cell, such as clays, to larger particles of sand, gravel and rock. Some of these Fe(III)-coated particles may be suspended in the groundwater as a result of underground current flow and other physical disturbances, thereby providing bacteria with an electron acceptor and a surface for biofilm growth. Geobacter bacteria are also important members of the microbial communities that cycle uranium (Mondani et al. 2011) and cadmium (Muehe et al. 2013). Their ubiquity and ability to mediate a wide range of metal redox transformations makes them attractive targets to develop permeable biobarriers for the sustained immobilization of toxic metals. Critical to bioremediation applications is understanding the environmental cues that regulate the formation of electroactive biofilms and the regulatory networks that modulate critical biofilm parameters such as structure and redox activity. Also important are insights into the composition of mixed-species electroactive biofilms. Evidence is indeed emerging for a role of pilus nanowires and/or c-Cyts in mediating electro-syntrophic interactions (Shrestha and Rotaru 2014). Such interactions could be targeted to maximize the catalytic activities of electroactive biofilms in situ and promote long-term stabilization of the reduced metals. FUNDING The author acknowledges support from grant EAR-1629439 from the U.S. National Science Foundation and the USDA National Institute of Food and Agriculture (Hatch project 1011745). Conflict of interest. None declared. REFERENCES Awate B , Steidl RJ , Hamlischer T et al. Stimulation of electro-fermentation in single-chamber microbial electrolysis cells driven by genetically engineered anode biofilms . J Power Sources . 2017 ; 356 : 510 – 18 . Google Scholar CrossRef Search ADS Babauta JT , Nguyen HD , Harrington TD et al. pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer . Biotechnol Bioeng . 2012 ; 109 : 2651 – 62 . Google Scholar CrossRef Search ADS PubMed Bird LJ , Bonnefoy V , Newman DK . Bioenergetic challenges of microbial iron metabolisms . Trends Microbiol . 2011 ; 19 : 330 – 40 . Google Scholar CrossRef Search ADS PubMed Bonanni PS , Bradley DF , Schrott GD et al. Limitations for current production in Geobacter sulfurreducens biofilms . ChemSusChem . 2013 ; 6 : 711 – 20 . Google Scholar CrossRef Search ADS PubMed Bond DR , Holmes DE , Tender LM et al. Electrode-reducing microorganisms that harvest energy from marine sediments . Science . 2002 ; 295 : 483 – 5 . Google Scholar CrossRef Search ADS PubMed Borole AP , Reguera G , Ringeisen B et al. Electroactive biofilms: current status and future research needs . Energy Environ Sci . 2011 ; 4 : 4813 – 34 . Google Scholar CrossRef Search ADS Bredas JL , Calbert JP , da Silva Filho DA et al. Organic semiconductors: a theoretical characterization of the basic parameters governing charge transport . Proc Natl Acad Sci USA . 2002 ; 99 : 5804 – 9 . Google Scholar CrossRef Search ADS PubMed Butler JE , Young ND , Lovley DR . Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes . BMC Genomics . 2010 ; 11 : 40 . Google Scholar CrossRef Search ADS PubMed Chan CH , Levar CE , Jiménez-Otero F et al. Genome scale mutational analysis of Geobacter sulfurreducens reveals distinct molecular mechanisms for respiration and sensing of poised electrodes versus Fe(III) oxides . J Bacteriol . 2017 ; 199 : e00340 – 17 . Google Scholar PubMed Cologgi DL , Lampa-Pastirk S , Speers AM et al. Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism . Proc Natl Acad Sci USA . 2011 ; 108 : 15248 – 52 . Google Scholar CrossRef Search ADS PubMed Cologgi DL , Speers AM , Bullard BA et al. Enhanced uranium immobilization and reduction by Geobacter sulfurreducens biofilms . Appl Environ Microbiol . 2014 ; 80 : 6638 – 46 . Google Scholar CrossRef Search ADS PubMed Coppi MV , Leang C , Sandler SJ et al. Development of a genetic system for Geobacter sulfurreducens . Appl Environ Microbiol . 2001 ; 67 : 3180 – 87 . Google Scholar CrossRef Search ADS PubMed El-Naggar MY , Wanger G , Leung KM et al. Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1 . Proc Natl Acad Sci USA . 2010 ; 107 : 18127 – 31 . Google Scholar CrossRef Search ADS PubMed Esteve-Nunez A , Sosnik J , Visconti P et al. Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens . Environ Microbiol . 2008 ; 10 : 497 – 505 . Google Scholar CrossRef Search ADS PubMed Feliciano GT , da Silva AJR , Reguera G et al. The molecular and electronic structure of the peptide subunit of Geobacter sulfurreducens conductive pili from first principles . J Phys Chem A . 2012 ; 116 : 8023 – 30 . Google Scholar CrossRef Search ADS PubMed Feliciano GT , Steidl RJ , Reguera G . Structural and functional insights into the conductive pili of Geobacter sulfurreducens revealed in molecular dynamics simulations . Phys Chem Chem Phys . 2015 ; 17 : 22217 – 26 . Google Scholar CrossRef Search ADS PubMed Fendorf SE , Weilinga BW , Hansel CM . Chromium transformations in natural environments: the role of biological and abiological processes in chromium (VI) reduction . Int Geol Rev . 2000 ; 42 : 691 – 701 . Google Scholar CrossRef Search ADS Flemming HC , Wingender J . The biofilm matrix . Nat Rev Microbiol . 2010 ; 8 : 623 – 33 . Google Scholar CrossRef Search ADS PubMed Franks AE , Nevin KP , Glaven RH et al. Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens biofilms . ISME J . 2010 ; 4 : 509 – 19 . Google Scholar CrossRef Search ADS PubMed Franks AE , Nevin KP , Jia H et al. Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm . Energy Environ Sci . 2009 ; 2 : 113 – 9 . Google Scholar CrossRef Search ADS Geelhoed JS , Hamelers HV , Stams AJ . Electricity-mediated biological hydrogen production . Curr Opin Microbiol . 2010 ; 13 : 307 – 15 . Google Scholar CrossRef Search ADS PubMed Gorby YA , Yanina S , McLean JS et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms . Proc Natl Acad Sci USA . 2006 ; 103 : 11358 – 63 . Google Scholar CrossRef Search ADS PubMed Halan B , Buehler K , Schmid A . Biofilms as living catalysts in continuous chemical syntheses . Trends Biotechnol . 2012 ; 30 : 453 – 65 . Google Scholar CrossRef Search ADS PubMed Hall-Stoodley L , Costerton JW , Stoodley P . Bacterial biofilms: from the natural environment to infectious diseases . Nat Rev Micro . 2004 ; 2 : 95 – 108 . Google Scholar CrossRef Search ADS Hernandez ME , Kappler A , Newman DK . Phenazines and other redox-active antibiotics promote microbial mineral reduction . Appl Environ Microbiol . 2004 ; 70 : 921 – 28 . Google Scholar CrossRef Search ADS PubMed Holmes DE , Chaudhuri SK , Nevin KP et al. Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens . Environ Microbiol . 2006 ; 8 : 1805 – 15 . Google Scholar CrossRef Search ADS PubMed Holmes DE , Dang Y , Walker DJ et al. The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer . Microb Genom . 2016 ; 2 : e000072 . Google Scholar PubMed Holmes DE , Shrestha PM , Walker DJF et al. Metatranscriptomic evidence for direct interspecies electron transfer between Geobacter and Methanothrix species in methanogenic rice paddy soils . Appl Environ Microbiol . 2017 ; 83 : e00223 – 17 . Google Scholar CrossRef Search ADS PubMed Ing NL , Nusca TD , Hochbaum AI . Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution . Phys Chem Chem Phys . 2017 ; 19 : 21791 – 9 . Google Scholar CrossRef Search ADS PubMed Inoue K , Leang C , Franks AE et al. Specific localization of the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter sulfurreducens . Environ Microbiol Rep . 2011 ; 3 : 211 – 7 . Google Scholar CrossRef Search ADS PubMed Inoue K , Qian X , Morgado L et al. Purification and characterization of OmcZ, an outer-surface, octaheme c-type cytochrome essential for optimal current production by Geobacter sulfurreducens . Appl Environ Microbiol . 2010 ; 76 : 3999 – 4007 . Google Scholar CrossRef Search ADS PubMed Jang JH , Dempsey BA , Burgos WD . Reduction of U(VI) by Fe(II) in the presence of hydrous ferric oxide and hematite: effects of solid transformation, surface coverage, and humic acid . Water Res . 2007 ; 42 : 2269 – 77 . Google Scholar CrossRef Search ADS PubMed Jormakka M , Tornroth S , Byrne B et al. Molecular basis of proton motive force generation: structure of formate dehydrogenase-N . Science . 2002 ; 295 : 1863 – 8 . Google Scholar CrossRef Search ADS PubMed Juarez K , Kim BC , Nevin K et al. PilR, a transcriptional regulator for pilin and other genes required for Fe(III) reduction in Geobacter sulfurreducens . J Mol Microbiol Biotechnol . 2009 ; 16 : 146 – 58 . Google Scholar CrossRef Search ADS PubMed Kato S , Hashimoto K , Watanabe K . Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals . Environ Microbiol . 2012b ; 14 : 1646 – 54 . Google Scholar CrossRef Search ADS Kato S , Hashimoto K , Watanabe K . Microbial interspecies electron transfer via electric currents through conductive minerals . Proc Natl Acad Sci USA . 2012a ; 109 : 10042 – 6 . Google Scholar CrossRef Search ADS Kim BC , Leang C , Ding YH et al. OmcF, a putative c-type monoheme outer membrane cytochrome required for the expression of other outer membrane cytochromes in Geobacter sulfurreducens . J Bacteriol . 2005 ; 187 : 4505 – 13 . Google Scholar CrossRef Search ADS PubMed Kim BC , Lovley DR . Investigation of direct vs. indirect involvement of the c-type cytochrome MacA in Fe(III) reduction by Geobacter sulfurreducens . FEMS Microbiol Lett . 2008 ; 286 : 39 – 44 . Google Scholar CrossRef Search ADS PubMed Kim BC , Qian X , Leang C et al. Two putative c-type multiheme cytochromes required for the expression of OmcB, an outer membrane protein essential for optimal Fe(III) reduction in Geobacter sulfurreducens . J Bacteriol . 2006 ; 188 : 3138 – 42 . Google Scholar CrossRef Search ADS PubMed Kishi S , Saito K , Kato Y et al. Redox potentials of ubiquinone, menaquinone, phylloquinone, and plastoquinone in aqueous solution . Photosynth Res . 2017 ; 134 : 193 – 200 . Google Scholar CrossRef Search ADS PubMed Kotloski NJ , Gralnick JA . Flavin electron shuttles dominate extracellular electron transfer by Shewanella oneidensis . MBio . 2013 ; 4 : e00553 – 12 . Google Scholar CrossRef Search ADS PubMed Lampa-Pastirk S , Veazey JP , Walsh KA et al. Thermally activated charge transport in microbial protein nanowires . Sci Rep . 2016 ; 6 : 23517 . Google Scholar CrossRef Search ADS PubMed Langner HW , Inskeep WP . Microbial reduction of arsenate in the presence of ferrihydrite . Environ Sci Technol . 2000 ; 34 : 3131 – 6 . Google Scholar CrossRef Search ADS Leang C , Adams LA , Chin KJ et al. Adaptation to disruption of the electron transfer pathway for Fe(III) reduction in Geobacter sulfurreducens . J Bacteriol . 2005 ; 187 : 5918 – 26 . Google Scholar CrossRef Search ADS PubMed Leang C , Coppi MV , Lovley DR , OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens . J. Bacteriol . 2003 ; 185 : 7 2096 – 2103 . Google Scholar CrossRef Search ADS PubMed Leang C , Coppi MV , Lovley DR . OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction inGeobacter sulfurreducens . J Bacteriol . 2003 ; 185 : 2096 – 2103 . Google Scholar CrossRef Search ADS PubMed Lentini CJ , Wankel SD , Hansel CM . Enriched iron(III)-reducing bacterial communities are shaped by carbon substrate and iron oxide mineralogy . Frontiers Microbiol . 2012 ; 3 : 404 . Google Scholar CrossRef Search ADS Levar CE , Chan CH , Mehta-Kolte MG et al. An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors . MBio . 2014 ; 5 : e02034 . Google Scholar CrossRef Search ADS PubMed Levar CE , Hoffman CL , Dunshee AJ et al. Redox potential as a master variable controlling pathways of metal reduction by Geobacter sulfurreducens . ISME J . 2017 ; 11 : 741 – 52 . Google Scholar CrossRef Search ADS PubMed Lewis AJ , Campa MF , Hazen TC et al. Unravelling biocomplexity of electroactive biofilms for producing hydrogen from biomass . Microb Biotechnol . 2018 ; 11 : 84 – 97 . Google Scholar CrossRef Search ADS PubMed Lies DP , Hernandez ME , Kappler A et al. Shewanella oneidensis MR-1 uses overlapping pathways for iron reduction at a distance and by direct contact under conditions relevant for biofilms . Appl Environ Microbiol . 2005 ; 71 : 4414 – 26 . Google Scholar CrossRef Search ADS PubMed Liu Y , Bond DR . Long-distance electron transfer by G. sulfurreducens biofilms results in accumulation of reduced c-type cytochromes . ChemSusChem . 2012 ; 5 : 1047 – 53 . Google Scholar CrossRef Search ADS PubMed Liu Y , Fredrickson JK , Zachara JM et al. Direct involvement of ombB, omaB, and omcB genes in extracellular reduction of Fe(III) by Geobacter sulfurreducens PCA . Front Microbiol . 2015 ; 6 : 1075 . Google Scholar PubMed Liu Y , Kim H , Franklin RR et al. Linking spectral and electrochemical analysis to monitor c-type cytochrome redox status in living Geobacter sulfurreducens biofilms . ChemPhysChem . 2011 ; 12 : 2235 – 41 . Google Scholar CrossRef Search ADS PubMed Liu Y , Wang Z , Liu J et al. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA . Environ Microbiol Rep . 2014a ; 6 : 776 – 85 . Google Scholar CrossRef Search ADS Liu Y , Wang Z , Liu J et al. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA . Environ Microbiol Rep . 2014b ; 6 : 776 – 85 . Google Scholar CrossRef Search ADS Lloyd JR , Leang C , Hodges Myerson AL et al. Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens . Biochem J . 2003 ; 369 : 153 – 61 . Google Scholar CrossRef Search ADS PubMed Lu HM , Motley ST , Lory S . Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion . Mol Microbiol . 1997 ; 25 : 247 – 59 . Google Scholar CrossRef Search ADS PubMed Magnuson TS , Isoyama N , Hodges-Myerson AL et al. Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens . Biochem J . 2001 ; 359 : 147 – 52 . Google Scholar CrossRef Search ADS PubMed Mah TF , O’Toole GA . Mechanisms of biofilm resistance to antimicrobial agents . Trends Microbiol . 2001 ; 9 : 34 – 9 . Google Scholar CrossRef Search ADS PubMed Malvankar NS , Vargas M , Nevin K et al. Structural basis for metallic-like conductivity in microbial nanowires . MBio . 2015 ; 6 : e00084 . Google Scholar CrossRef Search ADS PubMed Malvankar NS , Vargas M , Nevin KP et al. Tunable metallic-like conductivity in microbial nanowire networks . Nature Nanotechnol . 2011 ; 6 : 573 – 9 . Google Scholar CrossRef Search ADS Marsili E , Baron DB , Shikhare ID et al. Shewanella secretes flavins that mediate extracellular electron transfer . Proc Natl Acad Sci USA . 2008 ; 105 : 3968 – 73 . Google Scholar CrossRef Search ADS PubMed Mehta T , Coppi MV , Childers SE et al. Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens . Appl Environ Microbiol . 2005 ; 71 : 8634 – 41 . Google Scholar CrossRef Search ADS PubMed Merz AJ , So M , Sheetz MP . Pilus retraction powers bacterial twitching motility . Nature . 2000 ; 407 : 98 – 102 . Google Scholar CrossRef Search ADS PubMed Methé BA , Nelson KE , Eisen JA et al. Genome of Geobacter sulfurreducens: metal reduction in subsurface environments . Science . 2003 ; 302 : 1967 – 9 . Google Scholar CrossRef Search ADS PubMed Mondani L , Benzerara K , Carriere M et al. Influence of uranium on bacterial communities: a comparison of natural uranium-rich soils with controls . PLoS One . 2011 ; 6 : e25771 . Google Scholar CrossRef Search ADS PubMed Morgado L , Bruix M , Pessanha M et al. Thermodynamic characterization of a triheme cytochrome family from Geobacter sulfurreducens reveals mechanistic and functional diversity . Biophys J . 2010 ; 99 : 293 – 301 . Google Scholar CrossRef Search ADS PubMed Muehe EM , Obst M , Hitchcock A et al. Fate of Cd during microbial Fe(III) mineral reduction by a novel and Cd-tolerant Geobacter species . Environ Sci Technol . 2013 ; 47 : 14099 – 109 . Google Scholar CrossRef Search ADS PubMed Myneni SCB , Tokunaga TK , Brown GE Jr . Abiotic Selenium Redox Transformations in the Presence of Fe(II,III) Oxides . Science . 1997 ; 278 : 1106 – 9 . Google Scholar CrossRef Search ADS Nevin KP , Kim BC , Glaven RH et al. ( 2009 ) Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells . PLoS One . 4 : e5628 . Google Scholar CrossRef Search ADS PubMed Okamoto A , Nakamura R , Nealson KH et al. Bound flavin model suggests similar electron-transfer mechanisms in Shewanella and Geobacter . ChemElectroChem . 2014a ; 1 : 1808 – 12 . Google Scholar CrossRef Search ADS Okamoto A , Saito K , Inoue K et al. Uptake of self-secreted flavins as bound cofactors for extracellular electron transfer in Geobacter species . En Environ Sc . 2014b ; 7 : 1357 – 61 . Google Scholar CrossRef Search ADS Okegbe C , Price-Whelan A , Dietrich LE . Redox-driven regulation of microbial community morphogenesis . Curr Opin Microbiol . 2014 ; 18 : 39 – 45 . Google Scholar CrossRef Search ADS PubMed Pessanha M , Morgado L , Louro RO et al. Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens:  evidence for a role played in e-/H+ energy transduction . Biochemistry . 2006 ; 45 : 13910 – 17 . Google Scholar CrossRef Search ADS PubMed Pierson LS 3rd , Pierson EA . Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes . Appl Microbiol Biotechnol . 2010 ; 86 : 1659 – 70 . Google Scholar CrossRef Search ADS PubMed Pirbadian S , Barchinger SE , Leung KM et al. Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components . Proc Natl Acad Sci USA . 2014 ; 111 : 12883 – 8 . Google Scholar CrossRef Search ADS PubMed Polizzi NF , Skourtis SS , Beratan DN . Physical constraints on charge transport through bacterial nanowires . Faraday Discuss . 2012 ; 155 : 43 – 62 . Google Scholar CrossRef Search ADS PubMed Prevoteau A , Rabaey K . Electroactive biofilms for sensing: reflections and perspectives . ACS Sens . 2017 ; 2 : 1072 – 85 . Google Scholar CrossRef Search ADS PubMed Prokhorova A , Sturm-Richter K , Doetsch A et al. Resilience, dynamics, and interactions within a model multispecies exoelectrogenic-biofilm community . Appl Environ Microbiol . 2017 ; 83 . Qian X , Mester T , Morgado L et al. Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens . Biochim Biophys Acta . 2011 ; 1807 : 404 – 12 . Google Scholar CrossRef Search ADS PubMed Qian X , Reguera G , Mester T et al. Evidence that OmcB and OmpB of Geobacter sulfurreducens are outer membrane surface proteins . FEMS Microbiol Lett . 2007 ; 277 : 21 – 7 . Google Scholar CrossRef Search ADS PubMed Rabaey K , Boon N , Hofte M et al. Microbial phenazine production enhances electron transfer in biofuel cells . Environ Sci Technol . 2005 ; 39 : 3401 – 8 . Google Scholar CrossRef Search ADS PubMed Reardon PN , Mueller KT . Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens . J Biol Chem . 2013 ; 288 : 29260 – 6 . Google Scholar CrossRef Search ADS PubMed Reece SY , Nocera DG . Proton-coupled electron transfer in biology: results from synergistic studies in natural and model systems . Annu Rev Biochem . 2009 ; 78 : 673 – 99 . Google Scholar CrossRef Search ADS PubMed Reguera G , McCarthy KD , Mehta T et al. Extracellular electron transfer via microbial nanowires . Nature . 2005 ; 435 : 1098 – 1101 . Google Scholar CrossRef Search ADS PubMed Reguera G , Nevin KP , Nicoll JS et al. Biofilm and nanowire production lead to increased current in microbial fuel cells . Appl Environ Microbiol . 2006 ; 72 : 7345 – 8 . Google Scholar CrossRef Search ADS PubMed Reguera G , Pollina RB , Nicoll JS et al. Possible nonconductive role of Geobacter sulfurreducens pilus nanowires in biofilm formation . J Bacteriol . 2007 ; 189 : 2125 – 7 . Google Scholar CrossRef Search ADS PubMed Renslow R , Babauta J , Kuprat A et al. Modeling biofilms with dual extracellular electron transfer mechanisms . Phys Chem Chem Phys . 2013a ; 15 : 19262 – 83 . Google Scholar CrossRef Search ADS Renslow RS , Babauta JT , Dohnalkova AC et al. Metabolic spatial variability in electrode-respiring Geobacter sulfurreducens biofilms . Energy Environ Sci . 2013b ; 6 : 1827 – 36 . Google Scholar CrossRef Search ADS Richter H , Nevin KP , Jia HF et al. Cyclic voltammetry of biofilms of wild type and mutant Geobacter sulfurreducens on fuel cell anodes indicates possible roles of OmcB, OmcZ, type IV pili, and protons in extracellular electron transfer . Energy Environ Sci . 2009 ; 2 : 506 – 16 . Google Scholar CrossRef Search ADS Richter LV , Sandler SJ , Weis RM . Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation . J Bacteriol . 2012 ; 194 : 2551 – 63 . Google Scholar CrossRef Search ADS PubMed Robuschi L , Tomba JP , Schrott GD et al. Spectroscopic slicing to reveal internal redox gradients in electricity-producing biofilms . Angew Chem Int Ed Engl . 2013 ; 52 : 925 – 8 . Google Scholar CrossRef Search ADS PubMed Rollefson JB , Stephen CS , Tien M et al. Identification of an extracellular polysaccharide network essential for cytochrome anchoring and biofilm formation inGeobacter sulfurreducens . J Bacteriol . 2011 ; 193 : 1023 – 33 . Google Scholar CrossRef Search ADS PubMed Saleh FS , Rahman MR , Okajima T et al. Determination of formal potential of NADH/NAD+ redox couple and catalytic oxidation of NADH using poly(phenosafranin)-modified carbon electrodes . Bioelectrochemistry . 2011 ; 80 : 121 – 7 . Google Scholar CrossRef Search ADS PubMed Sauvonnet N , Vignon G , Pugsley AP et al. Pilus formation and protein secretion by the same machinery in Escherichia coli . EMBO J . 2000 ; 19 : 2221 – 8 . Google Scholar CrossRef Search ADS PubMed Shi L , Dong H , Reguera G et al. Extracellular electron transfer mechanisms between microorganisms and minerals . Nat Rev Microbiol . 2016 ; 14 : 651 – 62 . Google Scholar CrossRef Search ADS PubMed Shrestha PM , Rotaru AE . Plugging in or going wireless: strategies for interspecies electron transfer . Front Microbiol . 2014 ; 5 : 237 . Google Scholar PubMed Skerker JM , Berg HC . Direct observation of extension and retraction of type IV pili . Proc Natl Acad Sci USA . 2001 ; 98 : 6901 – 4 . Google Scholar CrossRef Search ADS PubMed Snider RM , Strycharz-Glaven SM , Tsoi SD et al. Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven . Proc Natl Acad Sci USA . 2012 ; 109 : 15467 – 72 . Google Scholar CrossRef Search ADS PubMed Speers AM , Reguera G . Consolidated bioprocessing of AFEX-pretreated corn stover to ethanol and hydrogen in a microbial electrolysis cell . Environ Sci Technol . 2012a ; 46 : 7875 – 81 . Google Scholar CrossRef Search ADS Speers AM , Reguera G . Electron donors supporting growth and electroactivity of Geobacter sulfurreducens anode biofilms . Appl Environ Microbiol . 2012b ; 78 : 437 – 44 . Google Scholar CrossRef Search ADS Speers AM , Schindler BD , Hwang J et al. Genetic identification of a PilT motor in Geobacter sulfurreducens reveals a role for pilus retraction in extracellular electron transfer . Front Microbiol . 2016 ; 7 : 1578 . Google Scholar CrossRef Search ADS PubMed Speers AM , Young JM , Reguera G . Fermentation of glycerol into ethanol in a microbial electrolysis cell driven by a customized consortium . Environ Sci Technol . 2014 ; 48 : 6350 – 58 . Google Scholar CrossRef Search ADS PubMed Sporer AJ , Kahl LJ , Price-Whelan A et al. Redox-based regulation of bacterial development and behavior . Annu Rev Biochem . 2017 ; 86 : 777 – 97 . Google Scholar CrossRef Search ADS PubMed Steidl R , Lampa-Pastirk S , Reguera G . Mechanistic stratification in electroactive biofilms of Geobacter sulfurreducens mediated by pilus nanowires . Nature Comm . 2016 ; 7 : 12217 . Google Scholar CrossRef Search ADS Straub KL , Kappler A , Schink B . Enrichment and isolation of ferric-iron- and humic-acid-reducing bacteria . Mehods Enzymol . 2005 ; 397 : 58 – 77 . Google Scholar CrossRef Search ADS Strycharz-Glaven SM , Tender LM . Reply to the ‘Comment on “On electrical conductivity of microbial nanowires and biofilms” by N. S. Malvankar, M. T. Tuominen and D. R. Lovley, Energy Environ. Sci., 2012, 5, DOI: 10.1039/c2ee02613a . Energy Environ Sci . 2012 ; 5 : 6250 – 5 . Google Scholar CrossRef Search ADS Stubbe J , Nocera DG , Yee CS et al. Radical initiation in the class I ribonucleotide reductase: long-range proton-coupled electron transfer . Chem Rev . 2003 ; 103 : 2167 – 201 . Google Scholar CrossRef Search ADS PubMed Sturm G , Dolch K , Richter K et al. Metal reducers and reduction targets. A short survey about the distribution of dissimilatory metal reducers and the multitude of terminal electron acceptors . In: Gescher J , Kappler A (eds). Microbial Metal Respiration: From Geochemistry to Potential Applications . Berlin, Heidelberg : Springer , 2012 , 129 – 59 . Subramanian P , Pirbadian S , El-Naggar MY et al. Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography . Proc Natl Acad Sci USA . 2018 ; 115 : E3246 – 55 . Google Scholar CrossRef Search ADS PubMed Tender LM , Reimers CE , Stecher HA 3rd et al. Harnessing microbially generated power on the seafloor . Nat Biotechnol . 2002 ; 20 : 821 – 5 . Google Scholar CrossRef Search ADS PubMed Thamdrup B . Bacterial manganese and iron reduction in aquatic sediments . In: Schink B (ed). Advances in Microbial Ecology . Boston, MA : Springer , 2000 , 41 – 84 . Google Scholar CrossRef Search ADS Thauer RK , Jungermann K , Decker K . Energy conservation in chemotrophic anaerobic bacteria . Bacteriol Rev . 1977 ; 41 : 100 – 80 . Google Scholar PubMed Tremblay P-L , Aklujkar M , Leang C et al. A genetic system forGeobacter metallireducens: role of the flagellin and pilin in the reduction of Fe(III) oxide . Environ Microbiol Reports . 2012 ; 4 : 82 – 8 . Google Scholar CrossRef Search ADS Vargas M , Malvankar NS , Tremblay PL et al. Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens . MBio . 2013 ; 4 : e00105 – 13 . Google Scholar CrossRef Search ADS PubMed Veazey JP , Reguera G , Tessmer SH . Electronic properties of conductive pili of the metal-reducing bacterium Geobacter sulfurreducens probed by scanning tunneling microscopy . Phys Rev E . 2011 ; 84 : 060901 . Google Scholar CrossRef Search ADS von Canstein H , Ogawa J , Shimizu S et al. Secretion of flavins by Shewanella species and their role in extracellular electron transfer . Appl Environ Microbiol . 2008 ; 74 : 615 – 23 . Google Scholar CrossRef Search ADS PubMed Wang Y , Kern SE , Newman DK . Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer . J Bacteriol . 2010 ; 192 : 365 – 9 . Google Scholar CrossRef Search ADS PubMed Xiao K , Malvankar NS , Shu CJ et al. Low energy atomic models suggesting a pilus structure that could account for electrical conductivity of Geobacter sulfurreducens pili . Sci Rep . 2016 ; 6 : 23385 . Google Scholar CrossRef Search ADS PubMed Yates MD , Golden JP , Roy J et al. Thermally activated long range electron transport in living biofilms . Phys Chem Chem Phys . 2015 ; 17 : 32564 – 70 . Google Scholar CrossRef Search ADS PubMed Yates MD , Strycharz-Glaven SM , Golden JP et al. Measuring conductivity of living Geobacter sulfurreducens biofilms . Nat Nanotechnol . 2016 ; 11 : 910 – 13 . Google Scholar CrossRef Search ADS PubMed Zacharoff L , Chan CH , Bond DR . Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens . Bioelectrochemistry . 2016 ; 107 : 7 – 13 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Ecology Oxford University Press

Microbial nanowires and electroactive biofilms

FEMS Microbiology Ecology , Volume Advance Article (7) – May 21, 2018

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Blackwell
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© FEMS 2018.
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0168-6496
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1574-6941
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10.1093/femsec/fiy086
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Abstract

ABSTRACT Geobacter bacteria are the only microorganisms known to produce conductive appendages or pili to electronically connect cells to extracellular electron acceptors such as iron oxide minerals and uranium. The conductive pili also promote cell-cell aggregation and the formation of electroactive biofilms. The hallmark of these electroactive biofilms is electronic heterogeneity, mediated by coordinated interactions between the conductive pili and matrix-associated cytochromes. Collectively, the matrix-associated electron carriers discharge respiratory electrons from cells in multilayered biofilms to electron-accepting surfaces such as iron oxide coatings and electrodes poised at a metabolically oxidizable potential. The presence of pilus nanowires in the electroactive biofilms also promotes the immobilization and reduction of soluble metals, even when present at toxic concentrations. This review summarizes current knowledge about the composition of the electroactive biofilm matrix and the mechanisms that allow the wired Geobacter biofilms to generate electrical currents and participate in metal redox transformations. electricigens, type IV pili, c-type cytochromes, Geobacter, iron reduction, uranium reduction INTRODUCTION The discovery that microbial biofilms can use electrodes buried in the seafloor sediments as the electron acceptor for respiration propelled a number of seminal studies to understand the phylogeny and physiology of electrochemically active microorganisms (Bond et al. 2002; Tender et al. 2002). Current-harvesting biofilms are often enriched in bacteria in the family Geobacteraceae, a group of Deltaproteobacteria that gain energy for growth from the reduction of ferric iron (Fe[III]) minerals (reviewed in Shi et al. 2016). The availability of a sequenced genome (Methé et al. 2003) and a genetic system (Coppi et al. 2001) for the model representative Geobacter sulfurreducens helped advance mechanistic studies and led to the discovery in this organism of conductive protein appendages of the Type IV pili class (Reguera et al. 2005). As in other bacteria, the Geobacter pili are assemblies of primarily a single peptide subunit or pilin (Cologgi et al. 2011). Yet the Geobacter pilin’s structure is divergent and optimized for electron transfer (ET) (Feliciano et al. 2012; Reardon and Mueller 2013). Genes encoding this type of divergent pilin are highly conserved among Geobacter species and form an independent line of descent among bacterial pilins (Reguera et al. 2005). Once assembled, the Geobacter pilins form fibers that, in the absence of metals and redox cofactors, conduct electrons at rates that greatly exceed the rates of cellular respiration (Lampa-Pastirk et al. 2016). The conductive pili of Geobacter bacteria are decorated with flexible, surface motifs that promote electrostatic interactions with extracellular electron acceptors and their reduction (Feliciano, Steidl and Reguera 2015). During the reduction of Fe(III) oxides, the pili solubilize some of the metal as Fe(II) but also form magnetite, a mixed Fe(III)-Fe(II) magnetic mineral that remains bound to the pilus filaments (Fig. 1). The soluble Fe(II) provides the electron donor for Fe(II) oxidizers, which cycle the iron. The semiconducting properties of the magnetite product can promote interspecies ET and the syntrophic coupling of the metabolism of Geobacter cells with nitrate reducers (Kato, Hashimoto and Watanabe 2012a) (Fig. 1). Magnetite also stimulates electro-syntrophic interactions between Geobacter spp. and methanogens (Kato, Hashimoto and Watanabe 2012b). Indeed, environmental surveys of soils where electro-syntrophic interactions between Geobacter and methanogens are predicted to occur show a high representation of Geobacter pilin genes and transcripts (Holmes et al. 2017). This suggests that the pili are directly or indirectly (via magnetite) mediating interspecies ET reactions with the syntrophic partners. Figure 1. View largeDownload slide Illustration of reactions mediated by the Geobacter pili (red arrows show direction of electron flow). The two panels show a colorized electron micrograph of a cell of G. sulfurreducens and its monolateral pili during the oxidation of acetate to CO2. The left panel shows the cell during the reduction of the pili-bound Fe(III) oxides (brown mineral particles) to soluble ferrous iron (Fe(II)aq) and magnetite (black mineral particles). The right panel shows interspecies ET from G. sulfurreducens to Thiobacillus denitrificans via the pili-bound magnetite particles, which couple the oxidation of acetate to the reduction of nitrate to ammonia. Figure 1. View largeDownload slide Illustration of reactions mediated by the Geobacter pili (red arrows show direction of electron flow). The two panels show a colorized electron micrograph of a cell of G. sulfurreducens and its monolateral pili during the oxidation of acetate to CO2. The left panel shows the cell during the reduction of the pili-bound Fe(III) oxides (brown mineral particles) to soluble ferrous iron (Fe(II)aq) and magnetite (black mineral particles). The right panel shows interspecies ET from G. sulfurreducens to Thiobacillus denitrificans via the pili-bound magnetite particles, which couple the oxidation of acetate to the reduction of nitrate to ammonia. The pilus nanowires also promote cell–cell aggregation and biofilm formation on various surfaces, including Fe(III) oxide coatings and electrodes poised at a metabolically oxidizing potential (Reguera et al. 2006; Reguera et al. 2007). The biofilm matrix is rich in c-type cytochromes (c-Cyts) and requires the nanowire network to electronically connect the cells and maintain optimal rates of ET in multilayered communities (Steidl, Lampa-Pastirk and Reguera 2016). This allows the biofilms to continue to grow at micrometer distances from an underlying electron-accepting surface and to harvest current from the collective oxidative metabolism of the biofilm cells (Reguera et al. 2006; Reguera et al. 2007). Electroactive biofilms are important members of the environmental consortia that decompose organic matter. The high conservation in Geobacter genomes of acetate transporters and pathways for its oxidation (Butler, Young and Lovley 2010) suggests an active role for the biofilms at the removal of acetate, a common feedback inhibitor of bacterial fermentations whose accumulation would otherwise reduce the rates of organic matter decomposition. Electroactive biofilms of the genetically tractable representative G. sulfurreducens can also metabolize common fermentation products such as lactate and formate, though less efficiently (Speers and Reguera 2012b). Yet adaptive evolution and genetic engineering has been applied to custom tailor the physiology of current-harvesting biofilms and improve their robustness during the removal of fermentation inhibitors (Speers and Reguera 2012a; Speers, Young and Reguera 2014; Awate et al. 2017). Electrodes functionalized with the electroactive biofilms can then be retrofitted into fermenters to remove unwanted byproducts, prevent their accumulation to inhibitory concentrations and maintain an optimal pH (Awate et al. 2017). These systems are essentially a fermenter retrofitted as a microbial electrolysis cell (MEC), an electrochemical system where a small energy input is used to harvest electrons at the anode and react them with protons at the cathode to make H2 (Geelhoed, Hamelers and Stams 2010). By mimicking biofilm syntrophic interactions that drive organic matter decomposition, electro-fermentations address many of the challenges that reduce the economic feasibility of otherwise attractive fermentations (e.g. batch-to-batch variability, low productivity and low product yields) and also reduce the cost associated with the purification of the targeted product (Awate et al. 2017). Electroactive biofilms formed on Fe(III) oxide minerals also contribute indirectly to the cycling of many elements. Fe(III) oxide minerals are widespread in nature and are particularly abundant in sediments and soils (>10 mmol per kg dry matter) (Straub, Kappler and Schink 2005). They provide Geobacter cells with an electron acceptor for respiration and a surface for biofilm formation (Reguera et al. 2007). The soluble Fe(II) released from the reduction of the oxides can in turn initiate the abiotic reduction of other metals such as chromium (Fendorf, weilinga and Hansel 2000), arsenic (Langner and Inskeep 2000), selenium (Myneni, Tokunaga and Brown 1997) and uranium (Jang, Dempsey and Burgos 2007). Although these soluble metals can reach concentrations that would otherwise kill planktonic cells, the conductive biofilm matrix provides cells with a physical and chemical barrier for metal immobilization and reduction (Cologgi et al. 2014). Uranium-reducing biofilms of G. sulfurreducens capitalize, for example, on the metal-binding properties of the conductive pili to immobilize and mineralize the uranyl cation in the areas of the biofilm most exposed to the surrounding milieu (Cologgi et al. 2014). This strategy prevents the permeation of the soluble cation deeper into the biofilm and enhances the collective catalytic activities of the cells during the reduction of the radionuclide (Cologgi et al. 2014). The study of Geobacter biofilms is therefore of interest for the development of improved strategies for the in situ bioremediation of environments impacted by uranium and other metal contaminants. Harnessing the catalytic activities of electroactive biofilms will ultimately require deep understanding of how biofilm cells transfer electrons across the surface-attached multilayered community. This process has been well studied in bacteria whose mechanism of ET relies on the secretion of soluble redox-active molecules such as redox-active antibiotics in Pseudomonas spp. (Hernandez, Kappler and Newman 2004) and soluble electron shuttles in Shewanella spp. (Lies et al. 2005; Marsili et al. 2008; von Canstein et al. 2008). The type of mediator used for biofilm electroactivity determines the ecological niches they occupy (Sturm et al. 2012). Pseudomonas aeruginosa, for example, secretes redox-active antibiotics termed phenazines, whose antimicrobial activity can prevent opportunistic partners from 'sampling the goods' (Pierson and Pierson 2010). Phenazines can also quench O2 and enhance anaerobic survival (Wang, Kern and Newman 2010). Furthermore, they can function as electron shuttles and stimulate current production when added exogenously to electrode-associated biofilms (Rabaey et al. 2005). Extracellular ET in Shewanella oneidensis MR-1, on the other hand, involves interactions between secreted flavin cofactors and specialized outer membrane c-Cyts (reviewed in Shi et al. 2016). This bacterium was originally reported to produce 'nanowires' for extracellular ET (Gorby et al. 2006; El-Naggar et al. 2010). The MR-1 'nanowires' were wrongly assumed to be conductive pili, as in Geobacter, although the conductivity of MR-1 pilus filaments had been ruled out earlier (Reguera et al. 2005). It has now been established that the MR-1 'nanowires' are outer membrane extensions formed by the fusion of outer membrane vesicles (Pirbadian et al. 2014). They contain membrane-bound clusters of c-Cyts whose diffusion could allow for charge transport via collision-exchange mechanisms (Subramanian et al. 2018). However, their ability to support extracellular ET and respiration in vivo is yet to be demonstrated. Secreted flavins are in fact the primary mechanism for the reduction of insoluble electron acceptors by MR-1 (Kotloski and Gralnick 2013). Once secreted, the soluble cofactors become public goods and can be used by other organisms, including those lacking mediators (Prokhorova et al. 2017). Perhaps not surprisingly, biofilm ET by MR-1 is slow compared to the wired biofilms of Geobacter (Renslow et al. 2013a). For additional information about mediator-based electroactive biofilms the reader is referred to other review articles (Borole et al. 2011; Sporer et al. 2017). This minireview focuses on Geobacter biofilms as a paradigm of wired electroactive biofilms. The article does not intend to provide an exhaustive coverage of literature in the field of current-harvesting biofilms grown in electrochemical devices, which is particularly large and covers studies with little to no environmental relevance. For information about electrochemical systems driven by electrode-associated biofilms the reader is referred to two recent review articles (Prevoteau and Rabaey 2017; Lewis et al. 2018). This minireview highlights instead studies that the author sees as having the most significant impact on environmental aspects of electroactive biofilms and microbial nanowires, particularly metal reduction. The article summarizes first what is known about extracellular ET in planktonic cells of Geobacter and uses this model as a reference to discuss how cells build a heterogeneous electroactive biofilm matrix. The last section explores critical gaps of knowledge that in the opinion of the author are more likely to provide novel insights into the environmental roles of electroactive biofilms and biotechnological applications harnessed from them and their components. A PLANKTONIC VIEW OF EXTRACELLULAR ET IN GEOBACTER The hallmark of the physiology of Geobacter bacteria is their ability to gain energy for growth by coupling the oxidation of acetate and other organic or inorganic (e.g., H2) electron donors to the reduction of Fe(III) oxides (Fig. 1). The insoluble nature of the electron acceptor has selected for a complex respiratory network that electronically connects the cell to its environment. Much of what is known about this respiratory machinery comes from genetic studies in planktonic cultures of G. sulfurreducens, which serves as a genetically tractable model representative of the family (Coppi et al. 2001). Genes encoding specific respiratory components can, in principle, be inactivated to assess its effect in extracellular ET. Yet, in G. sulfurreducens, mutations in components of the respiratory chain are often pleiotropic and lead to compensatory effects that can mask the true phenotype (Kim et al. 2005; Kim et al. 2006; Kim and Lovley 2008; Cologgi et al. 2011; Richter, Sandler and Weis 2012; Steidl, Lampa-Pastirk and Reguera 2016). Thus, caution must be exerted when interpreting the phenotype of pleiotropic mutants as well as mutants not evaluated for potential compensatory effects (Vargas et al. 2013; Levar et al. 2014; Zacharoff, Chan and Bond 2016; Levar et al. 2017). Role of extracytoplasmic c-type cytochromes (c-Cyts) Figure 2 shows proteins proposed to mediate ET across the cell envelope of G. sulfurreducens and their approximate midpoint potential (normalized versus the standard hydrogen electrode or SHE for comparisons). The membrane-bound NADH dehydrogenase complex (NADH1) separates the electrons from the protons carried in the cytoplasmic NADH pool, pumps the protons into the periplasm and generates the proton motive force that drives ATP synthesis by the ATPase synthase complex. The electrons flow from NADH1 to the menaquinone pool (MQ), the lipophilic redox-active molecules of the inner membrane. Two putative quinol oxidases (CbcL and ImcH) could accept electrons from the MQ pool as a function of the potential of the extracellular electron acceptor (low for CbcL and high for ImcH) (Levar et al. 2014; Zacharoff, Chan and Bond 2016). CbcL (c- and b-type cytochrome for low potential) is a periplasmic 9-heme c-Cyt anchored to the membrane through a HydC/FdnI domain (Zacharoff, Chan and Bond 2016), which many hydrogenase and formate dehydrogenase enzymes use as MQ oxidoreductases (Jormakka et al. 2002). ImcH is a heptaheme c-Cyt containing a putative membrane region homologous to the NapC/NirT domains that mediate the transfer of electrons from the MQ pool to periplasmic carriers (Levar et al. 2014). The genetic inactivation of CbcL or ImcH prevents the reduction of electron acceptors with potentials less or greater than –0.1 V versus SHE, respectively (Levar et al. 2017). This suggests that these two quinol oxidases function as electrochemical gates, tuning ET across the cell envelope to the redox potential of the extracellular electron acceptor by yet unknown mechanisms. This dual path has been proposed to allow cells to respire a wide range of Fe(III) oxides available in the environment and to adapt to the changes in potential (from high to low) that the mineral oxides experience in the course of their reduction (Levar et al. 2017). Figure 2. View largeDownload slide Electron transport pathway across the cell envelope of G. sulfurreducens during the reduction of ferrihydrite to Fe2+. The position of the electron carriers against a redox scale (left) is based on reported midpoint potentials normalized versus the standard hydrogen electrode (SHE), except for ImcH, whose midpotential has not been experimentally calculated and is, therefore, placed arbitrarily on the scale (indicated with a star [*] symbol). Abbreviations: IM, inner membrane; OM, outer membrane; NADH1, NADH dehydrogenase; MQ, menaquinone; CbcL and ImcH, putative quinol oxidases for the reduction of acceptors with low and high potentials, respectively; PpcAD and OmcBS, c-Cyts of the periplasm and outer membrane, respectively. OmcB is shown as part of a porin-cytochrome complex (Pcc). Figure 2. View largeDownload slide Electron transport pathway across the cell envelope of G. sulfurreducens during the reduction of ferrihydrite to Fe2+. The position of the electron carriers against a redox scale (left) is based on reported midpoint potentials normalized versus the standard hydrogen electrode (SHE), except for ImcH, whose midpotential has not been experimentally calculated and is, therefore, placed arbitrarily on the scale (indicated with a star [*] symbol). Abbreviations: IM, inner membrane; OM, outer membrane; NADH1, NADH dehydrogenase; MQ, menaquinone; CbcL and ImcH, putative quinol oxidases for the reduction of acceptors with low and high potentials, respectively; PpcAD and OmcBS, c-Cyts of the periplasm and outer membrane, respectively. OmcB is shown as part of a porin-cytochrome complex (Pcc). Genetic studies have also identified periplasmic and outer membrane c-Cyts that could electronically connect the inner membrane carriers to the extracellular electron acceptor (Fig. 2). PpcA is a periplasmic triheme cytochrome proposed to function as an intermediary electron carrier to terminal Fe(III) reductases in the outer membrane (Lloyd et al. 2003). It is also one of the few c-Cyts of G. sulfurreducens that is highly conserved in otherGeobacter genomes (Butler, Young and Lovley 2010). PpcA and its homolog PpcD have also been proposed to contribute to the proton electrochemical membrane gradient that drives ATP synthesis (Pessanha et al. 2006; Morgado et al. 2010). The range of potentials that could support energy transduction by these two periplasmic carriers spans almost 100 mV (–0.167 to –0.109 V in PpcA and –0.202 to –0.146 V in PpcD) (Morgado et al. 2010). Two other periplasmic c-Cyts (PpcB and PpcE) do not appear to couple electron and proton transfer and have heme groups with negative, yet slightly different, reduction potentials (Morgado et al. 2010). Collectively, the periplasmic c-Cyts span a large range of potentials that could facilitate ET from membrane carriers to outer membrane c-Cyts such as OmcB (Magnuson et al. 2001; Leang, Coppi and Lovley 2003; Qian et al. 2007). OmcB is part of a trans-outer membrane porin-cytochrome complex (Pcc) required for optimal growth rates with ferrihydrite (Liu et al. 2014b). Pcc complexes comprise an outer membrane c-Cyt (OmcB or OmcC), a periplasmic c-Cyt (OmaB or OmaC) and a porin-like outer membrane protein (OmbB or OmbC) (Liu et al. 2014a; Liu et al. 2015). The two Pcc complexes (OmbB-OmaB-OmcB and OmbC-OmaC-OmcC) appear to have overlapping roles in Fe(III) reduction (Liu et al. 2015). The association of the outer membrane c-Cyt OmcB with other proteins to form a Pcc complex assumes that OmcB is exposed sufficiently on the external side of the outer membrane to bind and transfer electrons to the extracellular electron acceptor (Liu et al. 2014b). However, OmcB is not readily immunodetected on the cell surface, suggesting it is deeply embedded in the outer membrane (Qian et al. 2007). Furthermore, genetic inactivation of OmcB initially impairs the ability of the cells to grow with ferrihydrite but the mutant cells eventually adapt (Leang, Coppi and Lovley 2003; Leang et al. 2005). In contrast, the outer membrane c-Cyt OmcS is easily sheared from the cell surface, consistent with its exposure to the extracellular environment, and it is required for the reduction of Fe(III) oxides (Mehta et al. 2005). This makes OmcS more likely to function as a terminal Fe(III) reductase. Flavin species (riboflavin and flavin mononucleotide) could facilitate this last step of ET (Okamoto et al. 2014b). Flavin cofactors have been detected in planktonic cultures of G. sulfurreducens at levels too low to function as electron shuttles for respiration (Okamoto et al. 2014b). Yet even at low concentrations flavin species could bind outer-membrane c-Cyts and enhance the rate of ET between the cell surface and solid electron acceptors (Okamoto et al. 2014b). This 'flavin-bound cofactor' model assumes that, as in S. oneidensis MR-1, the bound flavins catalyze a one-electron reaction that shifts the redox potential of c-Cyts to accelerate the rates of ET to the electron acceptor (Okamoto et al. 2014a). Experimental evidence to support this model is however yet to be produced. The c-Cyt path presented in Fig. 2 shows electrons flowing through a favorable redox gradient from the NAD+/NADH pair in the cytoplasm (midpoint potential [Em] versus SHE, –0.365 V to –0.320 V) (Thauer, Jungermann and Decker 1977; Saleh et al. 2011), to the MQ/MQH2 couple (Em = –0.260 V vs SHE) (Kishi et al. 2017) and to one of the putative quinol oxidases (CbcL’s Em = −0.15 V vs SHE) (Zacharoff, Chan and Bond 2016). The reduction potential of ferrihydrite to Fe2+ ranges from –0.1 to +0.1 V (Thamdrup 2000). Thus, the periplasmic and outer membrane electron carriers would be expected to have potentials within this narrow range of −0.15 V (CbcL) to +0.1 V (maximum for ferrihydrite). Yet the midpoint potential (vs SHE) of PpcA is −0.167 V (Lloyd et al. 2003), OmcB’s is −0.19 V (Magnuson et al. 2001), and OmcS’ is −0.212 V (Qian et al. 2011). Hence, the path from the periplasmic to the outer membrane c-Cyts is increasingly negative and would seemingly require energy expenditure for electron discharge. This 'uphill' path may reflect discrepancies between in vitro and in vivo potentials. The former were calculated experimentally for the free proteins and did not consider protein–protein interactions and other in vivo variables such as the ratio of oxidized/reduced species, which can significantly change (sometimes by 200 mV) the biological potential that drives electron transport (Thauer, Jungermann and Decker 1977). Indeed, the amount of energy extracted by Geobacter cells during Fe(III) oxide respiration depends on the potential difference between NADH and the inner membrane and/or periplasmic carriers rather than the oxides (Bird, Bonnefoy and Newman 2011). Thus, electron transport beyond the inner membrane and/or periplasm does not appear to require energy expenditure. Electrons could also diffuse from areas of high to low concentration, hopping between redox cofactors of periplasmic and outer membrane carriers, until they reach the extracellular electron acceptor, similarly to the redox-gradient model that has been proposed to drive ET by matrix-associated c-Cyts in electroactive biofilms (Snider et al. 2012). This mechanism could also allow the periplasmic and outer membrane c-Cyts to store electrons in the cell envelope until electronic contact with the electron acceptor has been made and the electrons can be discharged. The notion that extracytoplasmic c-Cyts function as a capacitor is further supported by the fact that Geobacter genomes often encode a great number of these c-Cyts yet sequence conservation among the genes is weak (Butler, Young and Lovley 2010). Furthermore, the c-Cyt content increases in cells of G. sulfurreducens grown under electron acceptor-limitation, allowing for the storage per cell of up to ∼107 electrons produced from the oxidation of electron donors (Esteve-Nunez et al. 2008). Conductive pili make the electrical connection with extracellular electron acceptors In contrast to the poor conservation of c-Cyts genes among Geobacter species (Butler, Young and Lovley 2010), the genes encoding the components of the Type IV pilus apparatus are highly conserved in this and other genera in the order Desulfuromonadales (Reguera et al. 2005; Holmes et al. 2016). This high conservation is consistent with the role of Geobacter pili as terminal reductases for the extracellular reduction of Fe(III) oxides (Reguera et al. 2005) and the uranyl cation (Cologgi et al. 2011). Indeed, deleting the gene encoding the pilin peptide in G. sulfurreducens (Reguera et al. 2005) or in Geobacter metallireducens (Tremblay et al. 2012) prevents growth with Fe(III) oxides as sole electron acceptor. This mutation was later found to be pleiotropic and to lead to defects in c-Cyt expression and secretion (Cologgi et al. 2011; Steidl, Lampa-Pastirk and Reguera 2016). To unmask the true contribution of the pili to extracellular ET, my group constructed a mutant (Tyr3) carrying alanine replacements in the pilin’s three tyrosines that reduced the conductivity of the assembled pili fivefold without affecting the expression of c-Cyts or cell piliation (Steidl, Lampa-Pastirk and Reguera 2016). The increased electrical resistance of the pili led to severe growth defects with Fe(III) oxides, even though some low levels of the metal chelator nitrilotriacetic acid (NTA) were present in the cultures to support the initial growth of the cells in the presence of the oxides (Feliciano, Steidl and Reguera 2015). However, the Tyr3 mutant was chemically rescued in Fe(III) oxides supplemented with concentrations of NTA high enough to bypass the need of the cells to use the pili to establish direct electronic contact with the electron acceptor (Feliciano, Steidl and Reguera 2015). Despite containing a divergent pilin, Geobacter genomes encode highly conserved components of the canonical Type IV pilus biosynthetic apparatus (Speers et al. 2016). As in other bacteria, the Geobacter pilin is synthesized as a precursor with a signal peptide that is recognized and cleaved by a dedicated signal peptidase (PilD) in the inner membrane (Richter, Sandler and Weis 2012). The processed pilins are stored in the inner membrane until ready for assembly by a complex apparatus that spans the multilayered cell envelope (Fig. 3). The pilus fiber grows by polymerizing the pilins at its base and protrudes through the outer membrane via a dedicated PilQ porin secretin. Pilin assembly is energized by a conserved PilB ATPase (Steidl, Lampa-Pastirk and Reguera 2016) and is reversible, that is, the pilus fiber can be retracted in a reaction energized by the PilT4 ATPase (Speers et al. 2016). Deletion of the gene encoding the PilT4 retraction motor in G. sulfurreducens does not affect the expression of c-Cyts but the mutant cells are hyperpiliated and severely impaired in the reduction of Fe(III) oxides (Speers et al. 2016). The hyperpiliation of the PilT4 mutant also promotes cell–cell aggregation and the formation of biofilms that, despite being denser than the wild type, have reduced respiratory rates per cell (Speers et al. 2016). Thus, the dynamic protrusion and retraction of the pili is critical for respiration in planktonic and biofilm cells. Figure 3. View largeDownload slide Dynamics of the pilus of G. sulfurreducens during the reduction of extracellular electron acceptors. The pilus apparatus includes the PilC protein (lavender) that anchors accessory proteins at the base of the pilus, the PilM protein that connects PilC to the lateral PilNO subcomplexes (light blue), and the PilP lipoprotein (lavender) that connects the PilNO subcomplexes to the PilQ secretin ring on the outer membrane (green). The PilB and PilT4 (right) ATPases that energize pilin polymerization and depolymerization, respectively, are highlighted in the pilus protrusion (left) and retraction (right) panels. Also shown are Fe(III) oxide particles (in brown), their reduced product—magnetite—(in black), and the accumulation of reduced c-Cyts (maroon) that could discharge electrons at the base of the pilus. Figure 3. View largeDownload slide Dynamics of the pilus of G. sulfurreducens during the reduction of extracellular electron acceptors. The pilus apparatus includes the PilC protein (lavender) that anchors accessory proteins at the base of the pilus, the PilM protein that connects PilC to the lateral PilNO subcomplexes (light blue), and the PilP lipoprotein (lavender) that connects the PilNO subcomplexes to the PilQ secretin ring on the outer membrane (green). The PilB and PilT4 (right) ATPases that energize pilin polymerization and depolymerization, respectively, are highlighted in the pilus protrusion (left) and retraction (right) panels. Also shown are Fe(III) oxide particles (in brown), their reduced product—magnetite—(in black), and the accumulation of reduced c-Cyts (maroon) that could discharge electrons at the base of the pilus. Planktonic cells have been proposed to use pilus retraction to shed off the bound, reduced mineral particles and recycle the pilin peptides in the membrane for a new round of assembly and metal binding and reduction (Speers et al. 2016) (Fig. 3). The antagonistic cycles of pilus protrusion and retraction could also maximize access to the most bioavailable Fe(III) oxide phases (e.g., ferrihydrite), which are unstable and rapidly transform abiotically into more crystalline and more recalcitrant phases (e.g., goethite and hematite) (Lentini, Wankel and Hansel 2012). Similarly, the retraction of the pili could allow cells to detach the reduced uranium mineral that remains bound to the pilus surface after the reduction of the uranyl cation (Cologgi et al. 2011) (Fig. 3). The disassembly rates estimated for other bacterial pili range from 1000 to 1500 pilins per second (Merz, So and Sheetz 2000; Skerker and Berg 2001). Thus, a full grown pilus, which contains 500–1000 pilins (Sauvonnet et al. 2000), could be retracted in one second. Storing the pilins in the membrane also ensures they are readily available for reassembly to start a new round of metal binding and reduction. Further, antagonistic cycles of protrusion and retraction allow the cell to mineralize uranium at a distance from the cell and to do so fast enough to prevent its permeation in the cell envelope. As a result, the extracellular reduction of the uranyl cation by the pili also provides cells with a protective mechanism from its toxicity (Cologgi et al. 2011). Being anchored in the cell envelope, the pilus pathway is well suited to accept electrons from c-Cyts (Fig. 3). Periplasmic c-Cyts such as PpcA are abundant in the cell envelope and, like the components of the Type IV pilus apparatus, they are encoded by genes conserved in Geobacter genomes (Butler, Young and Lovley 2010). PpcA and PpcD also have the potential to contribute to the proton motive force and energy conservation (Pessanha et al. 2006; Morgado et al. 2010). Furthermore, the periplasmic c-Cyts can function as cellular capacitors, storing electrons and allowing for continued proton pumping across the inner membrane and ATP synthesis (Esteve-Nunez et al. 2008). As the c-Cyts store charges, ATP is available to energize the dynamic protrusion and retraction of the pili and facilitate productive interactions between the pili and the extracellular electron acceptors. Once electronic contact with the electron acceptor is established, charges are predicted to move through the pili at rates that do not limit respiration. Individual pilus fibers purified free of metals and redox cofactors transport charges at rates close to ∼1 billion electrons per second (for a 1 µm-long pilus) at the potential difference that exists between the inner membrane and the extracellular Fe(III) oxides (∼100 mV) (Lampa-Pastirk et al. 2016). This is two orders of magnitude greater than the cellular rates of Fe(III) oxide reduction (∼9 million electrons per cell per second) (Lampa-Pastirk et al. 2016). The pili probed for charge transport in vitro (Lampa-Pastirk et al. 2016) were not chemically fixed and retained the structural and electronic features of cell-associated pili (Veazey, Reguera and Tessmer 2011). However, the pili were adsorbed onto a substrate and did not experience the in vivo cycles of protrusion and retraction that are predicted to promote electronic coupling in the pilus protein matrix and to accelerate charge transport (Feliciano, Steidl and Reguera 2015). Furthermore, cells assemble numerous pili monolaterally, concentrating the conductive filaments on one side of the cell to maximize access to the extracellular electron acceptor (Fig. 1). Thus, the Geobacter pili have the attributes needed to provide an efficient respiratory strategy during the reduction of extracellular electron acceptors. Mechanistic understanding of pilus conductivity The carrier mobility calculated for individual pili (3.2 × 10−2 cm2/Vs), which describes how fast the carriers (electrons or holes) move through the pilus protein fiber, is within the orders reported for organic semiconductors (Lampa-Pastirk et al. 2016), but it is orders of magnitude lower than the mobilities (>1 cm2/Vs) that would be required to describe pilus charge transport according to band theory (Polizzi, Skourtis and Beratan 2012). This argues against the metallic model of pilus conduction, a band conduction mechanism that has been proposed to explain the metallic-like temperature and pH response of conductive pili networks probed dried (Malvankar et al. 2011) or in solution (Ing, Nusca and Hochbaum 2017). As the pili samples used in these two studies were not pure, the contribution of contaminants (including c-Cyts) to the metallic-like responses cannot be ruled out. Furthermore, the protein samples were deposited on electrodes following extensive drying protocols (overnight drying or spin coating, respectively), which could significantly affect the native structure of the pili and its native conductive properties. Concerns (Strycharz-Glaven and Tender 2012; Yates et al. 2016) have also been raised about the description of biofilm conductivity as metallic-like due to the pili (Malvankar et al. 2011). Of special note is the fact that the Malvankar study used as a negative control a pilin-deficient mutant that also has defects in c-Cyts required for biofilm electroactivity (Cologgi et al. 2011; Steidl, Lampa-Pastirk and Reguera 2016) and limitations of the electrochemical gating experiments used to measure the temperature and pH response of biofilms grown across electrode gaps (Yates et al. 2016). The model of metallic-like biofilm conductivity is also incongruent with the thermal activation of conductivity reported for living biofilms of G. sulfurreducens (Yates et al. 2015) and the critical role that both c-Cyts and pili play in the mechanistic stratification of electroactive biofilms (Steidl, Lampa-Pastirk and Reguera 2016). Structural support for the metallic model of pilus conductance is also lacking. This model evokes the clustering of the pilins’ aromatic rings in the rigid sandwich-type configurations that allow for π orbital stacking (Malvankar et al. 2011). Although the pilus carrier mobility is sensitive to the formation of intra- and inter-molecular contacts that bring neighboring aromatic rings close together (3.5–5 Å apart), atomic resolution models of the pilus structure refined via molecular dynamics (MD) reveal aromatic dimers in displaced configurations that cannot support π stacking (Feliciano, Steidl and Reguera 2015; Lampa-Pastirk et al. 2016). Figure 4 shows a snapshot of the structure of a pilus fiber optimized in the MD simulations and the paths of aromatic rings (from tyrosines and phenylalanines) that form axially and transversally (Feliciano, Steidl and Reguera 2015). The figure also shows a close up of a pilus region from the MD model (Feliciano, Steidl and Reguera 2015). The inter-aromatic distances range from 3.5 to 8.5 Å, which are optimal for charge hopping (Feliciano, Steidl and Reguera 2015). Some of the aromatic dimers get close enough (3.5–5 Å apart) to form contacts that could promote the tunneling of charges, but all have displaced geometric configurations that cannot support π stacking. Furthermore, in the MD simulations the aromatic contacts never form at the same time, as would be expected for a metallic wire (Feliciano, Steidl and Reguera 2015). Thus, the inter-aromatic distances and geometries of the contacts support a charge hopping model mediated by aromatic residues, in agreement with the thermal activation of pilus conductivity demonstrated experimentally at biologically relevant voltages (± 0.2–0.4 V) (Lampa-Pastirk et al. 2016). It is interesting to note that similar displaced geometric configurations have been reproduced in non-dynamic, rigid models that claimed to support the metallic model of conductance (Xiao et al. 2016). Caution must therefore be exerted when interpreting this (Xiao et al. 2016) and other (Malvankar et al. 2015) modeling studies that do not consider the geometry of the aromatic contacts to support their conclusions, as even small misalignments of the aromatic rings forming the contacts can dramatically decrease the tunneling rates and prevent metallic conduction (Bredas et al. 2002). Figure 4. View largeDownload slide (A) Surface map of a pilus fiber optimized via MD. (B) Clustering of aromatic residues (tyrosines, yellow; phenylalanines, green) in the pilus fiber showing axial and transversal paths for charge transport. (C) 20-Å close up of a pilus region showing the parallel-displaced and T-shaped geometric configurations of the aromatic contacts (tyrosines, yellow; phenylalanines, green). The tyrosine (Y57) exposed on the flexible carboxyl-terminal segment of each pilin is labeled. Figure 4. View largeDownload slide (A) Surface map of a pilus fiber optimized via MD. (B) Clustering of aromatic residues (tyrosines, yellow; phenylalanines, green) in the pilus fiber showing axial and transversal paths for charge transport. (C) 20-Å close up of a pilus region showing the parallel-displaced and T-shaped geometric configurations of the aromatic contacts (tyrosines, yellow; phenylalanines, green). The tyrosine (Y57) exposed on the flexible carboxyl-terminal segment of each pilin is labeled. GEOBACTER BIOFILMS: A PARADIGM IN ELECTRONIC HETEROGENEITY The biofilm matrix is a heterogeneous medium of polysaccharides, nucleic acids, lipids and/or proteins, collectively known as exopolymeric substances (EPS), that surrounds the biofilm cells and attaches the community to a surface (Flemming and Wingender 2010). The chemical composition of the EPS matrix is carefully controlled by the cells to create a hydrated, catalytic microenvironment optimal for nutrient adsorption and assimilation (Flemming and Wingender 2010). This is particularly important for Geobacter bacteria, which form biofilms on surfaces, such as Fe(III) mineral coatings, that also serve as an electron acceptor for respiration (Reguera et al. 2007). In electroactive biofilms, the EPS matrix becomes an electronic medium of c-Cyts and pili whose coordinated interactions permit the collective discharge of respiratory electrons to support the growth of the biofilm cells (Steidl, Lampa-Pastirk and Reguera 2016). The sections below discuss what is known about the composition and electronic properties of the biofilm electron carriers from studies in the model representative G. sulfurreducens. The biofilm c-type cytochromes (c-Cyts) Biofilm cells use the same electron carriers of planktonic cells to transport electrons across the inner membrane and periplasmic space (Fig. 2). Electron transport across the outer membrane, however, appears to require a distinct set of c-Cyts. For example, the outer membrane c-Cyts OmcS (Mehta et al. 2005) and OmcB (Leang et al. 2003), which are important electron carriers during the reduction of Fe(III) oxides by planktonic cells (Fig. 2), can be deleted without impacting biofilm electroactivity (Holmes et al. 2006; Richter et al. 2009). Furthermore, biofilm cells require the expression of a putative Pcc conduit (extABCD) that is not involved in the reduction of Fe(III) oxides by planktonic cells (Chan et al. 2017). The requirement to use biofilm-specific outer membrane c-Cyts likely reflects an adaptive response of the cells to the local biofilm microenvironment, where interactions with matrix-associated electron carriers become the critical step limiting cell growth and respiration. EPS matrices extracted from electroactive biofilms are enriched in the outer membrane c-Cyt OmcZS (Rollefson et al. 2011; Cologgi et al. 2014; Steidl, Lampa-Pastirk and Reguera 2016). OmcZ is expressed and targeted to the outer membrane as a large protein (OmcZL; 50-kDa) and a more abundant, carboxy-terminal cleaved product (OmcZS; 30-kDa), which retains the cytochrome’s 8 hemes (Inoue et al. 2010). OmcZS is released to the electroactive matrix, where it is anchored to the biofilm Xap polysaccharide (Rollefson et al. 2011). Thin sections of current-harvesting biofilms grown on anode electrodes show the preferential location of OmcZS at or closer (<10µm) to the electrode surface (Inoue et al. 2011). This suggests a critical role for this c-Cyt in completing the last step of ET, from the biofilm to the underlying electron-accepting surface. In support of this, the genetic inactivation of OmcZ leads to a significant reduction in biofilm electroactivity (Nevin et al. 2009; Richter et al. 2009; Steidl, Lampa-Pastirk and Reguera 2016). OmcZS is also abundant in the EPS matrix of uranium-reducing biofilms (Cologgi et al. 2014). The large range of redox potentials covered by its eight heme groups (from −0.42 to −0.06 V) permits the in vitro reduction of Fe(III) citrate, Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analog anthraquinone-2,6-disulfonate (AQDS) in addition to U(VI) (Inoue et al. 2010). The pili of the biofilm matrix also promote the binding and reduction of soluble electron acceptors (Cologgi et al. 2014). Hence, OmcZS and the conductive pili make the biofilm matrix an electronic medium well suited to mediate many metal redox transformations. Mechanistic stratification of electroactive biofilms mediated by conductive pili The c-Cyts of biofilms grown on anode electrodes can be reversibly oxidized and reduced by changing the anode potential, consistent with their role as electron carriers (Liu et al. 2011; Liu and Bond 2012). However, there appears to be a maximum biofilm thickness of ∼10 µm that permits the full reduction of the matrix-associated c-Cyt pool (Liu and Bond 2012). This is also the biofilm stratum that concentrates OmcZS closer to the electron-accepting surface (Inoue et al. 2011). Yet electroactive biofilms can grow tens (or even hundreds) of µm thick (Renslow et al. 2013b). As they grow beyond the 10 µm threshold, diffusion constraints limit the ability of the c-Cyts to transfer electrons in the biofilm matrix and reduced c-Cyts progressively accumulate (Robuschi et al. 2013). This leads to sharp decreases in redox potentials in regions of the biofilm further away from the underlying electrode (Babauta et al. 2012) and the establishment of a redox gradient that drives electron transport to the fully functional electron carriers in the biofilm region closer to the electron-accepting surface (Snider et al. 2012). Figure 5 illustrates this in thin (10 µm) versus thick (20 µm) biofilms. The accumulation of reduced c-Cyts in the upper layers of thick biofilms creates a biological paradox: how can cells continue to grow optimally in these upper regions where the matrix c-Cyts are in a reduced state and unable to accept electrons at optimal rates? Cells in this upper biofilm stratum remain viable and contribute to the collective discharge of electrons despite the increases in biofilm thickness (Reguera et al. 2006). This indicates that the cells in these upper regions have alternative pathways for the discharge of respiratory electrons that bypass the electron acceptor limitation imposed by the accumulation of reduced c-Cyts. Figure 5. View largeDownload slide Model of mechanistic stratification in electroactive biofilms mediated by the pilus nanowires. Shown are thin (10 µm) biofilms formed by the wild type (WT) and pili-deficient PilB− strain and thick (20 µm) WT biofilms. The accumulation of reduced c-Cyts (c-Cyts•) in the upper stratum of thick WT biofilms is illustrated in a darker maroon color. The direction of electron flow towards the underlying electron-accepting substratum is indicated with a vertical arrow (dashed arrow, reduced conductivity) Figure 5. View largeDownload slide Model of mechanistic stratification in electroactive biofilms mediated by the pilus nanowires. Shown are thin (10 µm) biofilms formed by the wild type (WT) and pili-deficient PilB− strain and thick (20 µm) WT biofilms. The accumulation of reduced c-Cyts (c-Cyts•) in the upper stratum of thick WT biofilms is illustrated in a darker maroon color. The direction of electron flow towards the underlying electron-accepting substratum is indicated with a vertical arrow (dashed arrow, reduced conductivity) The most obvious alternative pathway for biofilm ET is that involving the pilus nanowires (Fig. 5). The pili are required to grow multilayered electroactive biofilms on both electron-accepting and non-accepting surfaces (Reguera et al. 2006, 2007; Cologgi et al. 2014). Individual pilus fibers have the reach (length of up to tenths of µm) and charge transport capacity (∼1 billion electrons per second) needed to discharge respiratory electrons at µm distances from the cell (Lampa-Pastirk et al. 2016). The thermal activation of pilus charge transport at biologically relevant potentials (Lampa-Pastirk et al. 2016) is also in agreement with the thermal dependence of charge transport demonstrated for living biofilms (Yates et al. 2015). Thus, both c-Cyts and pili have the electronic properties that confer on living electroactive biofilms their distinctive incoherent redox conductivity. The experimental evidence therefore supports a model of biofilm ET involving both c-Cyts and pili as electron carriers. Although the conductive pili have the properties needed to function as electron carriers in the biofilm matrix, genetic studies have been confounded by the pleiotropic effects that pili-inactivating mutations often have on the expression of c-Cyts required for extracellular ET (Juarez et al. 2009; Cologgi et al. 2011; Cologgi et al. 2014). Steidl et al. demonstrated the absence of OmcZS in the matrix of current-harvesting biofilms formed by a pilA-deficient mutant and a pilA-E5A mutant, which expresses pilins with an alanine replacement in an amino acid (E5) required for recognition and alignment of the pilin peptides during assembly (Steidl, Lampa-Pastirk and Reguera 2016). The mutants overexpressed other c-Cyts and were able to grow to a thickness of ∼10 µm, though growth rates and current production were sharply decreased and were similar to OmcZ-deficient mutant biofilms (Steidl, Lampa-Pastirk and Reguera 2016). By contrast, deletion of the PilB ATPase that powers pilin polymerization (Fig. 3) does not affect the expression of c-Cyts in planktonic or biofilm cells, providing the elusive genetic tool needed to assess the contribution of pili to biofilm electroactivity (Fig. 5). The PilB-deficient mutant can be grown to a thickness of ∼10 µm on an electrode poised at an optimal potential and, despite having wild type-levels of OmcZS in the biofilm matrix, its electroactivity is reduced in half (Steidl, Lampa-Pastirk and Reguera 2016). A Tyr3 mutant, which carries alanine replacements in the three pilin’s tyrosines (Feliciano, Steidl and Reguera 2015), produces pili with reduced conductivity and forms biofilms with the same thickness and electroactivity as the PilB-deficient mutant (Steidl, Lampa-Pastirk and Reguera 2016). Thus, although the Tyr3 biofilms have the structural pili network in their matrix, they are limited in their ability to use it to discharge respiratory electrons. The PilB and Tyr3 genetic studies demonstrate that the conductive pili are required for optimal ET even in biofilms grown to a thickness (∼10 µm) that permits the full reduction of the matrix-associated c-Cyts (Steidl, Lampa-Pastirk and Reguera 2016). This suggests that the pili and c-Cyts work coordinately to maintain optimal rates of ET in thin (up to 10 µm) biofilms (Fig. 5). This model is further supported by the cross-regulation of pili assembly and c-Cyt export observed by us (Steidl, Lampa-Pastirk and Reguera 2016) and others (Richter, Sandler and Weis 2012). Regulation may involve interactions between the two isoforms of the PilA pilin precursor (carrying a short or a long leader peptide) and the secretion system that exports outer membrane c-Cyts (Richter, Sandler and Weis 2012). Indeed, mutations that disrupt pilin-pilin interactions (pilA deletion or E5A replacement in PilA) prevent the expression of several c-Cyts and the secretion of OmcZS to the biofilm matrix (Steidl, Lampa-Pastirk and Reguera 2016). This cross-regulation, mediated by pilin interactions, would be analogous to that reported in P. aeruginosa, which relies on interactions between the pilin and components of the general secretion pathway to modulate protein export across the outer membrane (Lu, Motley and Lory 1997). The cross-regulation between pilin and c-Cyt secretion also warns about the multiple phenotypes that pilin-inactivating mutations can have that are not directly caused by the pilus deficiency but, rather, by the absence of critical c-Cyts. For example, a strain of G. sulfurreducens that produces only the short PilA isoform (pilA4 mutant) has defects in pilin secretion and assembly, no observable defects in c-Cyts and forms biofilms whose electroactivity is about half of the wild-type biofilms (Richter, Sandler and Weis 2012), as reported for the PilB-deficient and Tyr3 mutants (Steidl, Lampa-Pastirk and Reguera 2016). Yet a mutant carrying alanine replacements in 5 aromatic residues of the pilin (Aro5 mutant) and grown as anode biofilms under the same conditions as the pilA4 mutant had a more severe defect (Vargas et al. 2013), producing current plots conspicuously similar to those of an OmcZ-deficient mutant (Nevin et al. 2009) or mutants carrying pilin-inactivation mutations that prevent the expression of OmcZS in the biofilm matrix (Steidl, Lampa-Pastirk and Reguera 2016). As the effect of the Aro5 mutation on the expression of c-Cyts has not been assessed, caution is necessary when interpreting these genetic studies. The identification of the PilB-deficient mutation, which inactivates pilin assembly without interfering with pilin interactions needed for c-Cyt export, also allowed to evaluate the contribution of conductive pili across the redox gradient that establishes in thicker (>10 µm) biofilms (Steidl, Lampa-Pastirk and Reguera 2016). As shown in Fig. 5, while it is possible to increase the concentration of the electron donor (acetate) to grow thicker (∼20 µm) wild-type biofilms, the PilB and the Tyr3 mutant biofilms remain thin and unable to grow beyond the threshold 10-µm thickness that limits the availability of matrix-associated c-Cyts as electron acceptors (Steidl, Lampa-Pastirk and Reguera 2016). These results strongly suggest that the conductive pilus network provides the primary mechanism for charge transport from cells in regions of the biofilm where the c-Cyts cannot operate as efficient electron carriers. The c-Cyts of these upper biofilm layers could however function as a capacitor, storing electrons in the biofilm matrix and establishing the redox gradient that drives the flow of electrons to the underlying electron-accepting surface (Snider et al. 2012). These upper regions of the biofilm are also more exposed to the external medium and therefore have the highest concentrations of nutrients, including electron donors for the cell's oxidative metabolism (Renslow et al. 2013b). The pili can grow several micrometers in length (Reguera et al. 2005) and intertwine to form an intercellular conductive network (Veazey, Reguera and Tessmer 2011) and mediate electronic contacts with c-Cyts in the bottom layers of the biofilm. Thus, the conductive pili are suitable to become the primary mechanism that allows cells in electroactive biofilms to continue to grow without compromising the efficiency of charge transport to the underlying electron acceptor. CONCLUSION AND OUTLOOK Despite their inherent heterogeneity, biofilm cells coordinate their activities to increase their metabolic productivity (Halan, Buehler K and Schmid 2012) and achieve greater catalytic rates during the cycling of nutrients (Hall-Stoodley, Costerton and Stoodley 2004). Similarly, Geobacter cells use conductive pili to form a network of nanowires that mechanistically stratifies the biofilms to maintain optimal rates of electron transport across the multilayered communities and grow cells at a distance from an underlying electron acceptor (Steidl, Lampa-Pastirk and Reguera 2016). The model evokes coordinated interactions between the matrix c-Cyts and the conductive pili by yet unknown mechanisms. The retractile nature of the pili could be critical to these interactions. Current harvesting from a mutant deficient in pilus retraction (PilT4-) is similar to the wild type but the mutant cells are hyperpiliated and form denser biofilms (Speers et al. 2016). As a result, the rates of charge discharge per cell are reduced in the mutant biofilms. This suggests that pilus dynamics within the EPS matrix are critical to biofilm electroactivity. This role could be analogous to the critical role that pilus retraction has in enabling sequential rounds of electronic interactions between planktonic cells and extracellular electron acceptors such as Fe(III) oxides (Fig. 3). Yet in the biofilm matrix antagonistic cycles of pilus protrusion and retraction may be necessary to establish productive interactions with c-Cyts, or perhaps other cells, and maintain optimal rates of cellular respiration. Retraction also enables dynamic control of pilus length in the electroactive matrix so as to maintain optimal cell–cell spacing, a critical consideration for coordinated behavior within biofilms (Speers et al. 2016). Regulation of cell–cell distance can prevent mass transport limitations of nutrients diffusing through the biofilm matrix and, in electroactive biofilms, it could also coordinate the interactions of the pili with other matrix-associated electron carriers. Many questions still remain about how the biofilm cells sense and respond to the local redox environment. The physiology of the biofilm cells, as well as interactions between electron carriers in the biofilm matrix, are expected to be influenced by the pH, redox and electrical gradients that naturally establish across electroactive biofilms as their thickness increases (Franks et al. 2009; Babauta et al. 2012). For example, the pH of anode biofilms decreases in regions closer to the electrode surface and could modulate the rates of proton-coupled ET. The “flavin-bound cofactor" model assumes that flavins bind to c-Cyts and simultaneously move protons and electrons to accelerate the rates of ET, making this reaction pH-dependent (Okamoto et al. 2014a). Proton-coupled ET may also promote pilus charge transport. The close proximity of negatively charged residues to the aromatic rings of the pilin’s three tyrosines has been proposed to favor proton-coupled ET reactions (Feliciano, Steidl and Reguera 2015). In this model, the acidic residue is transiently protonated by the neighboring tyrosine to reduce the oxidation potential of the aromatic amino acid and enable fast rates of charge transport at the low potentials that operate in biological systems (Reece and Nocera 2003; Stubbe et al.2003). This mechanism of proton-coupled ET also makes the rates of pilus charge transport pH-dependent. The incoherent conductivity of living electroactive biofilms fits well with the coupled transport of protons and electrons mediated by c-Cyts (Yates et al. 2015). A similar mechanism could enable the short-distance, multistep hopping reactions that have been proposed to occur between pili and c-Cyts (Bonanni et al. 2013). The degree of piliation of the biofilm cells and concentrations of c-Cyts across the biofilms also merits investigation. Mass transport constraints can establish concentration gradients of essential nutrients such as electron donors (Liu et al. 2011; Babauta et al. 2012). As a result, nutrient availability fluctuates spatially in biofilms, influencing the cell physiology and, potentially, the levels of piliation and secretion of c-Cyts. Transcriptional profiling of the pilin-encoding gene (pilA) is not particularly useful to assess piliation because the levels of pilA transcripts do not change substantially in electroactive biofilms (Franks et al. 2010). Thus, transcriptional markers for piliation need to be identified to investigate how cells modulate pili levels as a function of pH, nutrient and redox cues from their microenvironment. Similarly, the regulatory networks that control the coordinated expression of pili and c-Cyts in response to the redox environment remain largely unknown. P. aeruginosa uses the redox state of secreted phenazines to alter the biofilm structure based on the availability of the electron acceptor (oxygen) (Okegbe, Price-Whelan and Dietrich 2014). Similar signal transduction systems could control the expression of pili and c-Cyts to modulate the rates of extracellular ET in Geobacterbiofilms and achieve redox homeostasis. Ecological studies also need to be undertaken to explore the range of metal transformations that can be mediated by microbial nanowires within electroactive biofilms. The physical and chemical microenvironment provided by the biofilm matrix and the unique physiology of the biofilm cells lead to biofilm-specific traits such as resistance to antimicrobials (Mah and O’Toole 2001). This is similarly reflected in the enhanced ability of electroactive biofilms to immobilize and reduce concentrations of metal contaminants that would otherwise kill planktonic cells (Cologgi et al. 2014). In subsurface environments impacted by metal contamination, biofilm formation provides an efficient mean to persist in the sediment and in areas with highest concentrations of nutrients rather than been swept away by the current of the groundwater. Fe(III) oxides are abundant in the subsurface as dispersed coatings associated with inorganic and organic solid matter that range in size from particles smaller than a bacterial cell, such as clays, to larger particles of sand, gravel and rock. Some of these Fe(III)-coated particles may be suspended in the groundwater as a result of underground current flow and other physical disturbances, thereby providing bacteria with an electron acceptor and a surface for biofilm growth. Geobacter bacteria are also important members of the microbial communities that cycle uranium (Mondani et al. 2011) and cadmium (Muehe et al. 2013). Their ubiquity and ability to mediate a wide range of metal redox transformations makes them attractive targets to develop permeable biobarriers for the sustained immobilization of toxic metals. Critical to bioremediation applications is understanding the environmental cues that regulate the formation of electroactive biofilms and the regulatory networks that modulate critical biofilm parameters such as structure and redox activity. Also important are insights into the composition of mixed-species electroactive biofilms. Evidence is indeed emerging for a role of pilus nanowires and/or c-Cyts in mediating electro-syntrophic interactions (Shrestha and Rotaru 2014). Such interactions could be targeted to maximize the catalytic activities of electroactive biofilms in situ and promote long-term stabilization of the reduced metals. FUNDING The author acknowledges support from grant EAR-1629439 from the U.S. National Science Foundation and the USDA National Institute of Food and Agriculture (Hatch project 1011745). Conflict of interest. None declared. REFERENCES Awate B , Steidl RJ , Hamlischer T et al. Stimulation of electro-fermentation in single-chamber microbial electrolysis cells driven by genetically engineered anode biofilms . J Power Sources . 2017 ; 356 : 510 – 18 . Google Scholar CrossRef Search ADS Babauta JT , Nguyen HD , Harrington TD et al. pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer . Biotechnol Bioeng . 2012 ; 109 : 2651 – 62 . Google Scholar CrossRef Search ADS PubMed Bird LJ , Bonnefoy V , Newman DK . Bioenergetic challenges of microbial iron metabolisms . Trends Microbiol . 2011 ; 19 : 330 – 40 . Google Scholar CrossRef Search ADS PubMed Bonanni PS , Bradley DF , Schrott GD et al. Limitations for current production in Geobacter sulfurreducens biofilms . ChemSusChem . 2013 ; 6 : 711 – 20 . Google Scholar CrossRef Search ADS PubMed Bond DR , Holmes DE , Tender LM et al. Electrode-reducing microorganisms that harvest energy from marine sediments . Science . 2002 ; 295 : 483 – 5 . Google Scholar CrossRef Search ADS PubMed Borole AP , Reguera G , Ringeisen B et al. Electroactive biofilms: current status and future research needs . Energy Environ Sci . 2011 ; 4 : 4813 – 34 . Google Scholar CrossRef Search ADS Bredas JL , Calbert JP , da Silva Filho DA et al. Organic semiconductors: a theoretical characterization of the basic parameters governing charge transport . Proc Natl Acad Sci USA . 2002 ; 99 : 5804 – 9 . Google Scholar CrossRef Search ADS PubMed Butler JE , Young ND , Lovley DR . Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes . BMC Genomics . 2010 ; 11 : 40 . Google Scholar CrossRef Search ADS PubMed Chan CH , Levar CE , Jiménez-Otero F et al. Genome scale mutational analysis of Geobacter sulfurreducens reveals distinct molecular mechanisms for respiration and sensing of poised electrodes versus Fe(III) oxides . J Bacteriol . 2017 ; 199 : e00340 – 17 . Google Scholar PubMed Cologgi DL , Lampa-Pastirk S , Speers AM et al. Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism . Proc Natl Acad Sci USA . 2011 ; 108 : 15248 – 52 . Google Scholar CrossRef Search ADS PubMed Cologgi DL , Speers AM , Bullard BA et al. Enhanced uranium immobilization and reduction by Geobacter sulfurreducens biofilms . Appl Environ Microbiol . 2014 ; 80 : 6638 – 46 . Google Scholar CrossRef Search ADS PubMed Coppi MV , Leang C , Sandler SJ et al. Development of a genetic system for Geobacter sulfurreducens . Appl Environ Microbiol . 2001 ; 67 : 3180 – 87 . Google Scholar CrossRef Search ADS PubMed El-Naggar MY , Wanger G , Leung KM et al. Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1 . Proc Natl Acad Sci USA . 2010 ; 107 : 18127 – 31 . Google Scholar CrossRef Search ADS PubMed Esteve-Nunez A , Sosnik J , Visconti P et al. Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens . Environ Microbiol . 2008 ; 10 : 497 – 505 . Google Scholar CrossRef Search ADS PubMed Feliciano GT , da Silva AJR , Reguera G et al. The molecular and electronic structure of the peptide subunit of Geobacter sulfurreducens conductive pili from first principles . J Phys Chem A . 2012 ; 116 : 8023 – 30 . Google Scholar CrossRef Search ADS PubMed Feliciano GT , Steidl RJ , Reguera G . Structural and functional insights into the conductive pili of Geobacter sulfurreducens revealed in molecular dynamics simulations . Phys Chem Chem Phys . 2015 ; 17 : 22217 – 26 . Google Scholar CrossRef Search ADS PubMed Fendorf SE , Weilinga BW , Hansel CM . Chromium transformations in natural environments: the role of biological and abiological processes in chromium (VI) reduction . Int Geol Rev . 2000 ; 42 : 691 – 701 . Google Scholar CrossRef Search ADS Flemming HC , Wingender J . The biofilm matrix . Nat Rev Microbiol . 2010 ; 8 : 623 – 33 . Google Scholar CrossRef Search ADS PubMed Franks AE , Nevin KP , Glaven RH et al. Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens biofilms . ISME J . 2010 ; 4 : 509 – 19 . Google Scholar CrossRef Search ADS PubMed Franks AE , Nevin KP , Jia H et al. Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm . Energy Environ Sci . 2009 ; 2 : 113 – 9 . Google Scholar CrossRef Search ADS Geelhoed JS , Hamelers HV , Stams AJ . Electricity-mediated biological hydrogen production . Curr Opin Microbiol . 2010 ; 13 : 307 – 15 . Google Scholar CrossRef Search ADS PubMed Gorby YA , Yanina S , McLean JS et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms . Proc Natl Acad Sci USA . 2006 ; 103 : 11358 – 63 . Google Scholar CrossRef Search ADS PubMed Halan B , Buehler K , Schmid A . Biofilms as living catalysts in continuous chemical syntheses . Trends Biotechnol . 2012 ; 30 : 453 – 65 . Google Scholar CrossRef Search ADS PubMed Hall-Stoodley L , Costerton JW , Stoodley P . Bacterial biofilms: from the natural environment to infectious diseases . Nat Rev Micro . 2004 ; 2 : 95 – 108 . Google Scholar CrossRef Search ADS Hernandez ME , Kappler A , Newman DK . Phenazines and other redox-active antibiotics promote microbial mineral reduction . Appl Environ Microbiol . 2004 ; 70 : 921 – 28 . Google Scholar CrossRef Search ADS PubMed Holmes DE , Chaudhuri SK , Nevin KP et al. Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens . Environ Microbiol . 2006 ; 8 : 1805 – 15 . Google Scholar CrossRef Search ADS PubMed Holmes DE , Dang Y , Walker DJ et al. The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer . Microb Genom . 2016 ; 2 : e000072 . Google Scholar PubMed Holmes DE , Shrestha PM , Walker DJF et al. Metatranscriptomic evidence for direct interspecies electron transfer between Geobacter and Methanothrix species in methanogenic rice paddy soils . Appl Environ Microbiol . 2017 ; 83 : e00223 – 17 . Google Scholar CrossRef Search ADS PubMed Ing NL , Nusca TD , Hochbaum AI . Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution . Phys Chem Chem Phys . 2017 ; 19 : 21791 – 9 . Google Scholar CrossRef Search ADS PubMed Inoue K , Leang C , Franks AE et al. Specific localization of the c-type cytochrome OmcZ at the anode surface in current-producing biofilms of Geobacter sulfurreducens . Environ Microbiol Rep . 2011 ; 3 : 211 – 7 . Google Scholar CrossRef Search ADS PubMed Inoue K , Qian X , Morgado L et al. Purification and characterization of OmcZ, an outer-surface, octaheme c-type cytochrome essential for optimal current production by Geobacter sulfurreducens . Appl Environ Microbiol . 2010 ; 76 : 3999 – 4007 . Google Scholar CrossRef Search ADS PubMed Jang JH , Dempsey BA , Burgos WD . Reduction of U(VI) by Fe(II) in the presence of hydrous ferric oxide and hematite: effects of solid transformation, surface coverage, and humic acid . Water Res . 2007 ; 42 : 2269 – 77 . Google Scholar CrossRef Search ADS PubMed Jormakka M , Tornroth S , Byrne B et al. Molecular basis of proton motive force generation: structure of formate dehydrogenase-N . Science . 2002 ; 295 : 1863 – 8 . Google Scholar CrossRef Search ADS PubMed Juarez K , Kim BC , Nevin K et al. PilR, a transcriptional regulator for pilin and other genes required for Fe(III) reduction in Geobacter sulfurreducens . J Mol Microbiol Biotechnol . 2009 ; 16 : 146 – 58 . Google Scholar CrossRef Search ADS PubMed Kato S , Hashimoto K , Watanabe K . Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals . Environ Microbiol . 2012b ; 14 : 1646 – 54 . Google Scholar CrossRef Search ADS Kato S , Hashimoto K , Watanabe K . Microbial interspecies electron transfer via electric currents through conductive minerals . Proc Natl Acad Sci USA . 2012a ; 109 : 10042 – 6 . Google Scholar CrossRef Search ADS Kim BC , Leang C , Ding YH et al. OmcF, a putative c-type monoheme outer membrane cytochrome required for the expression of other outer membrane cytochromes in Geobacter sulfurreducens . J Bacteriol . 2005 ; 187 : 4505 – 13 . Google Scholar CrossRef Search ADS PubMed Kim BC , Lovley DR . Investigation of direct vs. indirect involvement of the c-type cytochrome MacA in Fe(III) reduction by Geobacter sulfurreducens . FEMS Microbiol Lett . 2008 ; 286 : 39 – 44 . Google Scholar CrossRef Search ADS PubMed Kim BC , Qian X , Leang C et al. Two putative c-type multiheme cytochromes required for the expression of OmcB, an outer membrane protein essential for optimal Fe(III) reduction in Geobacter sulfurreducens . J Bacteriol . 2006 ; 188 : 3138 – 42 . Google Scholar CrossRef Search ADS PubMed Kishi S , Saito K , Kato Y et al. Redox potentials of ubiquinone, menaquinone, phylloquinone, and plastoquinone in aqueous solution . Photosynth Res . 2017 ; 134 : 193 – 200 . Google Scholar CrossRef Search ADS PubMed Kotloski NJ , Gralnick JA . Flavin electron shuttles dominate extracellular electron transfer by Shewanella oneidensis . MBio . 2013 ; 4 : e00553 – 12 . Google Scholar CrossRef Search ADS PubMed Lampa-Pastirk S , Veazey JP , Walsh KA et al. Thermally activated charge transport in microbial protein nanowires . Sci Rep . 2016 ; 6 : 23517 . Google Scholar CrossRef Search ADS PubMed Langner HW , Inskeep WP . Microbial reduction of arsenate in the presence of ferrihydrite . Environ Sci Technol . 2000 ; 34 : 3131 – 6 . Google Scholar CrossRef Search ADS Leang C , Adams LA , Chin KJ et al. Adaptation to disruption of the electron transfer pathway for Fe(III) reduction in Geobacter sulfurreducens . J Bacteriol . 2005 ; 187 : 5918 – 26 . Google Scholar CrossRef Search ADS PubMed Leang C , Coppi MV , Lovley DR , OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens . J. Bacteriol . 2003 ; 185 : 7 2096 – 2103 . Google Scholar CrossRef Search ADS PubMed Leang C , Coppi MV , Lovley DR . OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction inGeobacter sulfurreducens . J Bacteriol . 2003 ; 185 : 2096 – 2103 . Google Scholar CrossRef Search ADS PubMed Lentini CJ , Wankel SD , Hansel CM . Enriched iron(III)-reducing bacterial communities are shaped by carbon substrate and iron oxide mineralogy . Frontiers Microbiol . 2012 ; 3 : 404 . Google Scholar CrossRef Search ADS Levar CE , Chan CH , Mehta-Kolte MG et al. An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors . MBio . 2014 ; 5 : e02034 . Google Scholar CrossRef Search ADS PubMed Levar CE , Hoffman CL , Dunshee AJ et al. Redox potential as a master variable controlling pathways of metal reduction by Geobacter sulfurreducens . ISME J . 2017 ; 11 : 741 – 52 . Google Scholar CrossRef Search ADS PubMed Lewis AJ , Campa MF , Hazen TC et al. Unravelling biocomplexity of electroactive biofilms for producing hydrogen from biomass . Microb Biotechnol . 2018 ; 11 : 84 – 97 . Google Scholar CrossRef Search ADS PubMed Lies DP , Hernandez ME , Kappler A et al. Shewanella oneidensis MR-1 uses overlapping pathways for iron reduction at a distance and by direct contact under conditions relevant for biofilms . Appl Environ Microbiol . 2005 ; 71 : 4414 – 26 . Google Scholar CrossRef Search ADS PubMed Liu Y , Bond DR . Long-distance electron transfer by G. sulfurreducens biofilms results in accumulation of reduced c-type cytochromes . ChemSusChem . 2012 ; 5 : 1047 – 53 . Google Scholar CrossRef Search ADS PubMed Liu Y , Fredrickson JK , Zachara JM et al. Direct involvement of ombB, omaB, and omcB genes in extracellular reduction of Fe(III) by Geobacter sulfurreducens PCA . Front Microbiol . 2015 ; 6 : 1075 . Google Scholar PubMed Liu Y , Kim H , Franklin RR et al. Linking spectral and electrochemical analysis to monitor c-type cytochrome redox status in living Geobacter sulfurreducens biofilms . ChemPhysChem . 2011 ; 12 : 2235 – 41 . Google Scholar CrossRef Search ADS PubMed Liu Y , Wang Z , Liu J et al. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA . Environ Microbiol Rep . 2014a ; 6 : 776 – 85 . Google Scholar CrossRef Search ADS Liu Y , Wang Z , Liu J et al. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA . Environ Microbiol Rep . 2014b ; 6 : 776 – 85 . Google Scholar CrossRef Search ADS Lloyd JR , Leang C , Hodges Myerson AL et al. Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens . Biochem J . 2003 ; 369 : 153 – 61 . Google Scholar CrossRef Search ADS PubMed Lu HM , Motley ST , Lory S . Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion . Mol Microbiol . 1997 ; 25 : 247 – 59 . Google Scholar CrossRef Search ADS PubMed Magnuson TS , Isoyama N , Hodges-Myerson AL et al. Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens . Biochem J . 2001 ; 359 : 147 – 52 . Google Scholar CrossRef Search ADS PubMed Mah TF , O’Toole GA . Mechanisms of biofilm resistance to antimicrobial agents . Trends Microbiol . 2001 ; 9 : 34 – 9 . Google Scholar CrossRef Search ADS PubMed Malvankar NS , Vargas M , Nevin K et al. Structural basis for metallic-like conductivity in microbial nanowires . MBio . 2015 ; 6 : e00084 . Google Scholar CrossRef Search ADS PubMed Malvankar NS , Vargas M , Nevin KP et al. Tunable metallic-like conductivity in microbial nanowire networks . Nature Nanotechnol . 2011 ; 6 : 573 – 9 . Google Scholar CrossRef Search ADS Marsili E , Baron DB , Shikhare ID et al. Shewanella secretes flavins that mediate extracellular electron transfer . Proc Natl Acad Sci USA . 2008 ; 105 : 3968 – 73 . Google Scholar CrossRef Search ADS PubMed Mehta T , Coppi MV , Childers SE et al. Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens . Appl Environ Microbiol . 2005 ; 71 : 8634 – 41 . Google Scholar CrossRef Search ADS PubMed Merz AJ , So M , Sheetz MP . Pilus retraction powers bacterial twitching motility . Nature . 2000 ; 407 : 98 – 102 . Google Scholar CrossRef Search ADS PubMed Methé BA , Nelson KE , Eisen JA et al. Genome of Geobacter sulfurreducens: metal reduction in subsurface environments . Science . 2003 ; 302 : 1967 – 9 . Google Scholar CrossRef Search ADS PubMed Mondani L , Benzerara K , Carriere M et al. Influence of uranium on bacterial communities: a comparison of natural uranium-rich soils with controls . PLoS One . 2011 ; 6 : e25771 . Google Scholar CrossRef Search ADS PubMed Morgado L , Bruix M , Pessanha M et al. Thermodynamic characterization of a triheme cytochrome family from Geobacter sulfurreducens reveals mechanistic and functional diversity . Biophys J . 2010 ; 99 : 293 – 301 . Google Scholar CrossRef Search ADS PubMed Muehe EM , Obst M , Hitchcock A et al. Fate of Cd during microbial Fe(III) mineral reduction by a novel and Cd-tolerant Geobacter species . Environ Sci Technol . 2013 ; 47 : 14099 – 109 . Google Scholar CrossRef Search ADS PubMed Myneni SCB , Tokunaga TK , Brown GE Jr . Abiotic Selenium Redox Transformations in the Presence of Fe(II,III) Oxides . Science . 1997 ; 278 : 1106 – 9 . Google Scholar CrossRef Search ADS Nevin KP , Kim BC , Glaven RH et al. ( 2009 ) Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells . PLoS One . 4 : e5628 . Google Scholar CrossRef Search ADS PubMed Okamoto A , Nakamura R , Nealson KH et al. Bound flavin model suggests similar electron-transfer mechanisms in Shewanella and Geobacter . ChemElectroChem . 2014a ; 1 : 1808 – 12 . Google Scholar CrossRef Search ADS Okamoto A , Saito K , Inoue K et al. Uptake of self-secreted flavins as bound cofactors for extracellular electron transfer in Geobacter species . En Environ Sc . 2014b ; 7 : 1357 – 61 . Google Scholar CrossRef Search ADS Okegbe C , Price-Whelan A , Dietrich LE . Redox-driven regulation of microbial community morphogenesis . Curr Opin Microbiol . 2014 ; 18 : 39 – 45 . Google Scholar CrossRef Search ADS PubMed Pessanha M , Morgado L , Louro RO et al. Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens:  evidence for a role played in e-/H+ energy transduction . Biochemistry . 2006 ; 45 : 13910 – 17 . Google Scholar CrossRef Search ADS PubMed Pierson LS 3rd , Pierson EA . Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes . Appl Microbiol Biotechnol . 2010 ; 86 : 1659 – 70 . Google Scholar CrossRef Search ADS PubMed Pirbadian S , Barchinger SE , Leung KM et al. Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components . Proc Natl Acad Sci USA . 2014 ; 111 : 12883 – 8 . Google Scholar CrossRef Search ADS PubMed Polizzi NF , Skourtis SS , Beratan DN . Physical constraints on charge transport through bacterial nanowires . Faraday Discuss . 2012 ; 155 : 43 – 62 . Google Scholar CrossRef Search ADS PubMed Prevoteau A , Rabaey K . Electroactive biofilms for sensing: reflections and perspectives . ACS Sens . 2017 ; 2 : 1072 – 85 . Google Scholar CrossRef Search ADS PubMed Prokhorova A , Sturm-Richter K , Doetsch A et al. Resilience, dynamics, and interactions within a model multispecies exoelectrogenic-biofilm community . Appl Environ Microbiol . 2017 ; 83 . Qian X , Mester T , Morgado L et al. Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens . Biochim Biophys Acta . 2011 ; 1807 : 404 – 12 . Google Scholar CrossRef Search ADS PubMed Qian X , Reguera G , Mester T et al. Evidence that OmcB and OmpB of Geobacter sulfurreducens are outer membrane surface proteins . FEMS Microbiol Lett . 2007 ; 277 : 21 – 7 . Google Scholar CrossRef Search ADS PubMed Rabaey K , Boon N , Hofte M et al. Microbial phenazine production enhances electron transfer in biofuel cells . Environ Sci Technol . 2005 ; 39 : 3401 – 8 . Google Scholar CrossRef Search ADS PubMed Reardon PN , Mueller KT . Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens . J Biol Chem . 2013 ; 288 : 29260 – 6 . Google Scholar CrossRef Search ADS PubMed Reece SY , Nocera DG . Proton-coupled electron transfer in biology: results from synergistic studies in natural and model systems . Annu Rev Biochem . 2009 ; 78 : 673 – 99 . Google Scholar CrossRef Search ADS PubMed Reguera G , McCarthy KD , Mehta T et al. Extracellular electron transfer via microbial nanowires . Nature . 2005 ; 435 : 1098 – 1101 . Google Scholar CrossRef Search ADS PubMed Reguera G , Nevin KP , Nicoll JS et al. Biofilm and nanowire production lead to increased current in microbial fuel cells . Appl Environ Microbiol . 2006 ; 72 : 7345 – 8 . Google Scholar CrossRef Search ADS PubMed Reguera G , Pollina RB , Nicoll JS et al. Possible nonconductive role of Geobacter sulfurreducens pilus nanowires in biofilm formation . J Bacteriol . 2007 ; 189 : 2125 – 7 . Google Scholar CrossRef Search ADS PubMed Renslow R , Babauta J , Kuprat A et al. Modeling biofilms with dual extracellular electron transfer mechanisms . Phys Chem Chem Phys . 2013a ; 15 : 19262 – 83 . Google Scholar CrossRef Search ADS Renslow RS , Babauta JT , Dohnalkova AC et al. Metabolic spatial variability in electrode-respiring Geobacter sulfurreducens biofilms . Energy Environ Sci . 2013b ; 6 : 1827 – 36 . Google Scholar CrossRef Search ADS Richter H , Nevin KP , Jia HF et al. Cyclic voltammetry of biofilms of wild type and mutant Geobacter sulfurreducens on fuel cell anodes indicates possible roles of OmcB, OmcZ, type IV pili, and protons in extracellular electron transfer . Energy Environ Sci . 2009 ; 2 : 506 – 16 . Google Scholar CrossRef Search ADS Richter LV , Sandler SJ , Weis RM . Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation . J Bacteriol . 2012 ; 194 : 2551 – 63 . Google Scholar CrossRef Search ADS PubMed Robuschi L , Tomba JP , Schrott GD et al. Spectroscopic slicing to reveal internal redox gradients in electricity-producing biofilms . Angew Chem Int Ed Engl . 2013 ; 52 : 925 – 8 . Google Scholar CrossRef Search ADS PubMed Rollefson JB , Stephen CS , Tien M et al. Identification of an extracellular polysaccharide network essential for cytochrome anchoring and biofilm formation inGeobacter sulfurreducens . J Bacteriol . 2011 ; 193 : 1023 – 33 . Google Scholar CrossRef Search ADS PubMed Saleh FS , Rahman MR , Okajima T et al. Determination of formal potential of NADH/NAD+ redox couple and catalytic oxidation of NADH using poly(phenosafranin)-modified carbon electrodes . Bioelectrochemistry . 2011 ; 80 : 121 – 7 . Google Scholar CrossRef Search ADS PubMed Sauvonnet N , Vignon G , Pugsley AP et al. Pilus formation and protein secretion by the same machinery in Escherichia coli . EMBO J . 2000 ; 19 : 2221 – 8 . Google Scholar CrossRef Search ADS PubMed Shi L , Dong H , Reguera G et al. Extracellular electron transfer mechanisms between microorganisms and minerals . Nat Rev Microbiol . 2016 ; 14 : 651 – 62 . Google Scholar CrossRef Search ADS PubMed Shrestha PM , Rotaru AE . Plugging in or going wireless: strategies for interspecies electron transfer . Front Microbiol . 2014 ; 5 : 237 . Google Scholar PubMed Skerker JM , Berg HC . Direct observation of extension and retraction of type IV pili . Proc Natl Acad Sci USA . 2001 ; 98 : 6901 – 4 . Google Scholar CrossRef Search ADS PubMed Snider RM , Strycharz-Glaven SM , Tsoi SD et al. Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven . Proc Natl Acad Sci USA . 2012 ; 109 : 15467 – 72 . Google Scholar CrossRef Search ADS PubMed Speers AM , Reguera G . Consolidated bioprocessing of AFEX-pretreated corn stover to ethanol and hydrogen in a microbial electrolysis cell . Environ Sci Technol . 2012a ; 46 : 7875 – 81 . Google Scholar CrossRef Search ADS Speers AM , Reguera G . Electron donors supporting growth and electroactivity of Geobacter sulfurreducens anode biofilms . Appl Environ Microbiol . 2012b ; 78 : 437 – 44 . Google Scholar CrossRef Search ADS Speers AM , Schindler BD , Hwang J et al. Genetic identification of a PilT motor in Geobacter sulfurreducens reveals a role for pilus retraction in extracellular electron transfer . Front Microbiol . 2016 ; 7 : 1578 . Google Scholar CrossRef Search ADS PubMed Speers AM , Young JM , Reguera G . Fermentation of glycerol into ethanol in a microbial electrolysis cell driven by a customized consortium . Environ Sci Technol . 2014 ; 48 : 6350 – 58 . Google Scholar CrossRef Search ADS PubMed Sporer AJ , Kahl LJ , Price-Whelan A et al. Redox-based regulation of bacterial development and behavior . Annu Rev Biochem . 2017 ; 86 : 777 – 97 . Google Scholar CrossRef Search ADS PubMed Steidl R , Lampa-Pastirk S , Reguera G . Mechanistic stratification in electroactive biofilms of Geobacter sulfurreducens mediated by pilus nanowires . Nature Comm . 2016 ; 7 : 12217 . Google Scholar CrossRef Search ADS Straub KL , Kappler A , Schink B . Enrichment and isolation of ferric-iron- and humic-acid-reducing bacteria . Mehods Enzymol . 2005 ; 397 : 58 – 77 . Google Scholar CrossRef Search ADS Strycharz-Glaven SM , Tender LM . Reply to the ‘Comment on “On electrical conductivity of microbial nanowires and biofilms” by N. S. Malvankar, M. T. Tuominen and D. R. Lovley, Energy Environ. Sci., 2012, 5, DOI: 10.1039/c2ee02613a . Energy Environ Sci . 2012 ; 5 : 6250 – 5 . Google Scholar CrossRef Search ADS Stubbe J , Nocera DG , Yee CS et al. Radical initiation in the class I ribonucleotide reductase: long-range proton-coupled electron transfer . Chem Rev . 2003 ; 103 : 2167 – 201 . Google Scholar CrossRef Search ADS PubMed Sturm G , Dolch K , Richter K et al. Metal reducers and reduction targets. A short survey about the distribution of dissimilatory metal reducers and the multitude of terminal electron acceptors . In: Gescher J , Kappler A (eds). Microbial Metal Respiration: From Geochemistry to Potential Applications . Berlin, Heidelberg : Springer , 2012 , 129 – 59 . Subramanian P , Pirbadian S , El-Naggar MY et al. Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography . Proc Natl Acad Sci USA . 2018 ; 115 : E3246 – 55 . Google Scholar CrossRef Search ADS PubMed Tender LM , Reimers CE , Stecher HA 3rd et al. Harnessing microbially generated power on the seafloor . Nat Biotechnol . 2002 ; 20 : 821 – 5 . Google Scholar CrossRef Search ADS PubMed Thamdrup B . Bacterial manganese and iron reduction in aquatic sediments . In: Schink B (ed). Advances in Microbial Ecology . Boston, MA : Springer , 2000 , 41 – 84 . Google Scholar CrossRef Search ADS Thauer RK , Jungermann K , Decker K . Energy conservation in chemotrophic anaerobic bacteria . Bacteriol Rev . 1977 ; 41 : 100 – 80 . Google Scholar PubMed Tremblay P-L , Aklujkar M , Leang C et al. A genetic system forGeobacter metallireducens: role of the flagellin and pilin in the reduction of Fe(III) oxide . Environ Microbiol Reports . 2012 ; 4 : 82 – 8 . Google Scholar CrossRef Search ADS Vargas M , Malvankar NS , Tremblay PL et al. Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens . MBio . 2013 ; 4 : e00105 – 13 . Google Scholar CrossRef Search ADS PubMed Veazey JP , Reguera G , Tessmer SH . Electronic properties of conductive pili of the metal-reducing bacterium Geobacter sulfurreducens probed by scanning tunneling microscopy . Phys Rev E . 2011 ; 84 : 060901 . Google Scholar CrossRef Search ADS von Canstein H , Ogawa J , Shimizu S et al. Secretion of flavins by Shewanella species and their role in extracellular electron transfer . Appl Environ Microbiol . 2008 ; 74 : 615 – 23 . Google Scholar CrossRef Search ADS PubMed Wang Y , Kern SE , Newman DK . Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer . J Bacteriol . 2010 ; 192 : 365 – 9 . Google Scholar CrossRef Search ADS PubMed Xiao K , Malvankar NS , Shu CJ et al. Low energy atomic models suggesting a pilus structure that could account for electrical conductivity of Geobacter sulfurreducens pili . Sci Rep . 2016 ; 6 : 23385 . Google Scholar CrossRef Search ADS PubMed Yates MD , Golden JP , Roy J et al. Thermally activated long range electron transport in living biofilms . Phys Chem Chem Phys . 2015 ; 17 : 32564 – 70 . Google Scholar CrossRef Search ADS PubMed Yates MD , Strycharz-Glaven SM , Golden JP et al. Measuring conductivity of living Geobacter sulfurreducens biofilms . Nat Nanotechnol . 2016 ; 11 : 910 – 13 . Google Scholar CrossRef Search ADS PubMed Zacharoff L , Chan CH , Bond DR . Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens . Bioelectrochemistry . 2016 ; 107 : 7 – 13 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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FEMS Microbiology EcologyOxford University Press

Published: May 21, 2018

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