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The role of pili in Fe(iii) oxide reduction was studied with Geobacter sulfurreducens because a genetic system9 and the complete genome sequence10 are available. As expected from previous studies6, G. sulfurreducens produced pili during growth on Fe(iii) oxide (Fig. 1a) but not on soluble Fe(iii) (Fig. 1b), and the pili were localized to one side of the cell. The formation of pili could also be induced during growth on the alternative electron acceptor fumarate if the cells were grown at the suboptimal temperature of 25 °C (Fig. 2a), indicating that pilin production in G. sulfurreducens might be growth-regulated as it is in other bacteria11.

Figure 1: Geobacter sulfurreducens pili.
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a, b, Transmisssion electron micrographs of cells grown with poorly crystalline Fe(iii) oxides (a) or soluble Fe(iii) citrate (b). Arrows indicate pili. Scale bars, 0.5 µm. c, Phylogenetic distance tree derived from amino-terminal amino acid sequence alignments (see Supplementary Information and Supplementary Fig. S1) showing the relationship of annotated G. sulfurreducens pilin-domain proteins, encoded by GSU1496 and GSU1776, and pilins (blue branches) and pseudopilins (green branches) of other bacteria. d, Genomic organization of pilus biosynthesis genes surrounding GSU1496 in G. sulfurreducens and the pilA gene in M. xanthus17.

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Figure 2: Transmission electron microscopy analyses.
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Shown are cells of a wild-type strain (a), a pilA-deficient mutant strain (b) and a complemented mutant strain (c) of G. sulfurreducens. Cells were grown in medium with acetate and fumarate at 25 °C to induce the formation of pili, then negatively stained. Insets in a and c show details of pili produced by the wild-type and complemented mutant strains, respectively. Scale bars, 0.2 µm.

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The genome sequence of G. sulfurreducens contained two open reading frames (ORFs), GSU1496 and GSU1776, predicted to code for pilin domain proteins with the conserved amino-terminal amino acid characteristics of type IV pilins12. Phylogenetic analyses placed the protein encoded by ORF GSU1776 among bacterial pseudopilins of type II secretion systems, and subsequent studies have confirmed the role of this gene, termed oxpG, in protein secretion to the outer membrane13. The protein encoded by ORF GSU1496 formed an independent line of descent along with pilin subunits of other members of the Geobacteraceae such as Geobacter metallireducens and Pelobacter propionicus (Fig. 1c). The predicted length of these Geobacter pilin proteins was considerably shorter than other bacterial pilins (see Supplementary Fig. S1) and was restricted to the highly conserved N-terminal domain of bacterial type IV pilins, which functions in inner membrane insertion, signal processing and pilin polymerization, and forms the central helical core of the pilus filament12,14. The degree of conservation of geopilins at this region was lower than other bacterial pilins and, as a result, geopilins were phylogenetically distant from other bacterial pilins, including a type IV pilin of another metal reducer, Shewanella oneidensis (Fig. 1c). Homologues of genes required for the formation and assembly of pili in other Gram-negative bacteria15,16 are upstream of the Geobacter pilA gene (Fig. 1d), in a genetic arrangement similar to that of the pili genes in Myxococcus xanthus17, a δ-proteobacterium distantly related to Geobacter. These results indicate that the GSU1496 gene encodes a pilin subunit, which was designated PilA.

When pilA was deleted, G. sulfurreducens failed to produce pili (Fig. 2b) and could no longer reduce insoluble electron acceptors such as poorly crystalline Fe(iii) oxides (Fig. 3) and Mn(iv) oxides (data not shown). In contrast, the mutant could reduce soluble electron acceptors, such as fumarate and Fe(iii) citrate as well as the wild type. The mutant also grew in medium containing Fe(iii) oxide if the chelator nitrilotriacetate was added to solubilize some of the Fe(iii) or in the presence of anthraquinone-2,6-disulfonate (AQDS) (Supplementary Fig. S2). AQDS serves as a soluble electron shuttle and transfers electrons between the cell surface and the surface of the Fe(iii) oxide18, alleviating the need for direct contact for Fe(iii) oxide reduction5. Complementation of the pilA mutation with a functional copy of the pilA gene in trans restored the capacity for assembling pili (Fig. 2c) and for Fe(iii) oxide reduction (Fig. 3a). These results showed that G. sulfurreducens required the assembly of functional pili to reduce insoluble Fe(iii) oxides.

Figure 3: Effect of a mutation in pilin production on reduction of Fe( iii ) oxide and attachment.
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a, Cells (open symbols) of the wild-type (circles), ΔpilA mutant (triangles) and complemented ΔpilA mutant (squares) strains and Fe(ii) produced from Fe(iii) reduction (filled symbols). Plus signs, Fe(ii) in uninoculated medium; crosses, cells in uninoculated medium. b, Biomass of cells attached to Fe(iii) oxide-coated coverslips over time; inset, confocal scanning laser microscopy of biomass attached in the first 24 h to the Fe(iii) oxide, which is at the bottom of each image. Scale bar, 20 µm. Solid bars, wild type (WT); open bars, ΔpilA mutant (PilA-). Error bars show s.d. c, d, Transmission electron micrographs of fumarate-grown WT (c) and PilA- (d) cells amended with Fe(iii) oxides (indicated by arrows). Scale bars, 0.5 µm. Inset in (c) shows WT pili intertwined with Fe(iii) oxides.

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One known function of type IV pili in other microorganisms is establishing contact with surfaces7,8. Fe(iii) oxides are typically smaller than G. sulfurreducens cells (Fig. 1a), but it was possible to quantify the potential for attachment of G. sulfurreducens to Fe(iii) by inoculating fumarate-grown cells into medium in which Fe(iii) oxide, attached to glass coverslips, was provided as the sole electron acceptor. Within the first 24 h, the cells of the pilA-deficient strain that were added initially attached to Fe(iii) oxides as well as the wild type (Fig. 3b), but whereas the wild type grew on the Fe(iii) oxide, as indicated by an increase in biomass on the Fe(iii) oxide over the next 24 h, the pilA mutant could not grow, as shown by a decrease in biomass (Fig. 3b). The pilA-deficient mutant did grow on the surface if fumarate was provided as an alternative electron acceptor (data not shown). These results showed that pili are not required for Fe(iii) oxides to attach to cells and confirmed the necessity for pili for growth with Fe(iii) oxides as the sole electron acceptor. Further evaluation of the nature of the association of the Fe(iii) oxides with the cells revealed that, when Fe(iii) oxides were added to fumarate-grown cells, the outer surface of the pilA-deficient mutant still had the ability to bind Fe(iii) oxides (Fig. 3d) but in the wild type there was substantial association of Fe(iii) oxides with the pili (Fig. 3c).

It has previously been proposed that Geobacter's pili might mediate surface motility, which might aid G. sulfurreducens in locating Fe(iii) or Mn(vi) oxides1,2,6, but no twitching motility of the wild-type cells was observed on glass surfaces coated with Fe(iii) oxide. Furthermore, deleting a putative pilT gene (Supplementary Information), which is required for twitching motility in other organisms19, had no effect on Fe(iii) oxide reduction.

These results indicated that the pili might have a more direct role in electron transfer to Fe(iii) oxides. To evaluate this we measured the electrical conductivity through the pili. Pili and other proteins released from the outer surface of G. sulfurreducens grown with fumarate (Supplementary Fig. S3) were immobilized on a graphite surface and analysed with an atomic force microscope (AFM) equipped with a conductive tip and electronics that permitted mapping of the local conductance from the tip to the substrate (Fig. 4). Topographic analysis revealed pili as well as other, unidentified, more globular, proteins which were also sheared off the outer cell surface (Fig. 4a). When a voltage was applied to the tip there was a strong current response along the pilus filament, which was positive when a positive voltage was applied and negative with a negative voltage (Fig. 4b, c). In contrast, the non-pilin proteins had no detectable conductivity and in instances in which the non-pilin proteins covered the pili filaments they insulated the pili from the conductive tip. This general response, initially observed in relatively large-scale scans (Fig. 4a–c), was even clearer in cross-sections in which high current was associated with the slight increase in topography associated with the pilus filament, but the higher topography, associated with non-pilin material, had no detectable current (Fig. 4d, middle panel). A scan across a portion of the pilin filament overlain by other material also yielded no detectable current (Fig. 4d, bottom panel). Current line scans generated after applying different voltages while scanning the same region of a pilus demonstrated a linear, ohmic, correspondence between current and voltage applied (Fig. 4e, f). When similar studies were performed with pili from the metal reducer Shewanella oneidensis or the non-metal reducer Pseudomonas aeruginosa (Supplementary Fig. S4 and S5), no conductance was detected.

Figure 4: Conducting-probe atomic force microscopy.
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a, Topography of a pilus (indicated by arrows) and non-pilin globular proteins. b, c, Current image (b) of the same field when a slow, triangular sweep bias voltage (c) was applied to the tip. d, Image showing both the topography and current of a pilus (top); the top and bottom white lines show the locations of the scans in the middle and bottom panels, respectively. Thick line (blue open circles where visible), height; red line, current. e, Results from disabling the slow axis to repeatedly scan horizontally across the same portion of a pilus filament. The apparent increased width of the pilus is an artefact of this form of scanning. f, Correspondence between current and applied voltage. Scale bars, 100 nm.

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These results show that the pili of G. sulfurreducens are highly conductive. This indicates that G. sulfurreducens requires pili in order to reduce Fe(iii) oxides because pili are the electrical connection between the cell and the surface of the Fe(iii) oxides. This contrasts with the nearly universal concept that outer-membrane cytochromes are the proteins that transfer electrons to Fe(iii) oxide in Fe(iii) reducers1,3. However, the outer-membrane cytochrome model for Fe(iii) reduction has serious limitations. For example, whereas Geobacter and Desulfuromonas species in the Geobacteraceae family of Fe(iii)-reducing microorganisms contain abundant c-type cytochromes, no c-type cytochromes could be detected in Pelobacter species4, which are phylogenetically intertwined with Geobacter and Desulfuromonas species20,21. Yet Pelobacter species—which, like Geobacter species, contain pili localized on one side of the cell—also are capable of reducing Fe(iii) oxides4.

The conductive pili provide the opportunity to extend electron transfer capabilities well beyond the outer surface of the cells, which might be especially important in soils in which Fe(iii) oxides exist as heterogeneously dispersed coatings on clays and other particulate matter. The pilus apparatus is anchored in the periplasm and outer membrane of Gram-negative cells, thus offering the possibility that pili accept electrons from periplasmic and/or outer membrane electron transfer proteins. These intermediary electron transfer proteins need not be the same in all organisms, which is consistent with the differences in cytochrome content and/or composition in different Fe(iii) reducers1. The likely function of the pili is to complete the circuit between these various intermediary electron carriers and the Fe(iii) oxide.

In addition to serving as a conduit for electron transfer to Fe(iii) oxides, pili could conceivably be involved in other electron transfer reactions. For example, pili of individual Geobacter cells are often intertwined, raising the possibility of cell-to-cell electron transfer through pili. These biologically produced nanowires might be useful in nanoelectronic applications22,23, with the possibility of genetically modifying pilin structure and/or composition to generate nanowires with different functionalities.

Methods

Bacterial strains and culture conditions

All G. sulfurreducens strains were isogenic with the wild-type strain PCA (ATCC 51573). A PilA- mutant strain was generated by replacement of the +61 to +159 coding region of the pilA gene (GSU1496) with a chloramphenicol cassette, as described previously9. The pilA mutation was complemented in trans by introducing plasmid pRG5-pilA, a pRG5 derivative24 carrying a wild-type copy of the coding region of pilA.

Cells were routinely cultured at 30 or 25 °C under strictly anaerobic conditions in freshwater medium supplemented with acetate as electron donor, with fumarate, Fe(iii)-citrate or poorly crystalline Fe(iii) oxides (100 mM) as the electron acceptor25, and in the presence of chloramphenicol (15 µg ml-1) or spectinomycin (150–300 µg ml-1) for cultures of the PilA- and pRG5-pilA strains, respectively. Rates of Fe(iii) oxide reduction were determined by measuring the production of Fe(ii), and growth was determined as cell counts of acridine-orange-stained cells. For attachment assays, cells were grown in the presence of coverslips coated with Fe(iii) oxide; the attached biomass was determined with crystal violet (see Supplementary Information).

Conducting-probe atomic force microscopy analyses

Pili and other outer-surface proteins that were sheared from the cell surface (see Supplementary Information) were left to adsorb for 20 min on the surface of freshly cleaved, highly oriented pyrolytic graphite, fixed with 1% glutaraldehyde for 5 min, washed twice with double deionized water and blotted dry. Samples were examined with a Veeco Dimension 3100 AFM equipped with a Nanoscope IV controller and a SAM III signal access module to enable electrical interfacing with the tip. A gold-coated AFM tip (nominal spring constant 0.06 N m-1; Veeco Inc.) was used for the imaging. The AFM was operated in contact mode with simultaneous tip–substrate conductivity mapping. While imaging, a slow ‘triangle-sweep’ bias voltage was applied to the tip in reference to the graphite surface by using a low-noise battery-powered ramping circuit. Current was measured with a DL Instruments 1211 current preamplifier.