Seeing is Believing: Novel Imaging Techniques Help Clarify Microbial Nanowire
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Structure and Function 1
Accepted Article
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Running Title: Resolving Microbial Nanowire Structure and Function
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Derek R. Lovley1* and Nikhil S. Malvankar1,2
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1
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts.
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Department of Physics, University of Massachusetts, Amherst, Massachusetts.
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*Corresponding author
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Derek R. Lovley
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639 North Pleasant Street,
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203, Morrill Science Center IV North,
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Amherst, MA 01003, USA.
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Phone: 413-545-9651
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Fax: 413-545-1578
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Email: [email protected]://www.geobacter.org/
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12708
1 This article is protected by copyright. All rights reserved.
Accepted Article
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Summary
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Novel imaging approaches have recently helped to clarify the properties of “microbial
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nanowires”. Geobacter sulfurreducens pili are actual wires. They possess metallic-like
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conductivity, which can be attributed to overlapping pi-pi orbitals of key aromatic amino
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acids. Electrostatic force microscopy recently confirmed charge propagation along the
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pili, in a manner similar to carbon nanotubes. The pili are essential for long-range
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electron transport to insoluble electron acceptors and interspecies electron transfer.
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Previous claims that S. oneidensis also produce conductive pili have recently been
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recanted, based on novel live-imaging studies. The putative pili are, in fact, long
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extensions of the cytochrome-rich, outer-membrane and periplasm that, when dried,
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collapse to form filaments with dimensions similar to pili. It has yet to be demonstrated
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whether the cytochrome-to-cytochrome electron hopping documented in the dried
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membrane extensions takes place in intact hydrated membrane extensions or whether the
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membrane extensions enhance electron transport to insoluble electron acceptors such as
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Fe(III) oxides or electrodes. These findings demonstrate that G. sulfurreducens
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conductive pili and the outer-membrane extensions of S. oneidensis are fundamentally
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different in composition, mechanism of electron transport, and physiological role. New
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methods for evaluating filament conductivity will facilitate screening the microbial world
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for nanowires and elucidating their function.
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2 This article is protected by copyright. All rights reserved.
Accepted Article
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Introduction Microbial nanowires are one of the most fascinating microbial structures
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discovered within the last decade. They play an important role in carbon and mineral
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cycling in anaerobic soils and sediments; are a unique strategy for interspecies energy
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exchange; and have practical applications in bioenergy, bioremediation, and
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bioelectronics (Lovley, 2011b; Malvankar and Lovley, 2014). Until recently there has
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been substantial controversy and confusion over the physical properties, composition, and
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function of the microbial nanowires of Geobacter sulfurreducens and Shewanella
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oneidensis, the two microbes that have primarily served as the basis for microbial
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nanowire mechanistic studies (Malvankar and Lovley, 2012). New clarity has now
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emerged, with the aid of novel imaging approaches (Malvankar et al., 2014b; Pirbadian et
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al., 2014).
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Putative S. oneidensis Pili Nanowires are Actually Membrane Extensions A stunning result from new imaging approaches has been the revelation that the
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conductive filaments from S. oneidensis, which were previously thought to be pili are, in
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fact, extensions of the outer membrane and periplasm (Pirbadian et al., 2014). Elegant,
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live fluorescence microscopy revealed the growth of extensions of the outer membrane
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that distort the typical rod shape of the cells with protrusions that in some instances are
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longer than the cell itself. When the outer-membrane extensions are dried for
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conductivity measurements, their diameter shrinks over 10-fold, yielding a material that
3 This article is protected by copyright. All rights reserved.
looks like a filament of ca. 10 nm diameter. This artifact resembles pili, hence the prior
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mistaken identity (Pirbadian et al., 2014).
Accepted Article
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These findings resolve a long, contentious debate over whether S. oneidensis can
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produce conductive pili. Initial studies that examined the pili of S. oneidensis with
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conducting-tip atomic force microscopy concluded that the pili were not conductive
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(Reguera et al., 2005). Subsequent studies contended that S. oneidensis had conductive
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pili (Gorby et al., 2006). Now that it is clear that those later studies (Gorby et al., 2006)
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were evaluating the conductivity of the dried outer-membrane extensions (Pirbadian et
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al., 2014) it appears that the initial finding that S. oneidensis does not produce conductive
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pili (Reguera et al., 2005) was correct.
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Geobacter Nanowires are Type IV Pili In contrast, it was clear from the beginning that G. sulfurreducens nanowires were
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type IV pili. The hypothesis that the G. sulfurreducens pili might function as wires
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resulted from the observation that pili were specifically expressed during growth on
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Fe(III) oxides and other insoluble electron acceptors (Childers et al., 2002); and the fact
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that deleting the gene for PilA, the pilus monomer, inhibited the reduction of Fe(III)
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oxides, but not soluble, chelated Fe(III) (Mehta et al., 2002). These findings, coupled
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with the subsequent observations that: 1) nano-sized Fe(III) oxides specifically associated
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with the pili; and 2) the pili were electrically conductive across their diameter; led to the
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suggestion that the G. sulfurreducens pili functioned as wires to promote long-range
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electron conduction to Fe(III) oxides (Reguera et al., 2005). Further study demonstrated
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that networks of the conductive pili permitted G. sulfurreducens to produce thick,
4 This article is protected by copyright. All rights reserved.
electrically conductive biofilms on the anodes of microbial fuel cells (Reguera et al.,
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2006; Nevin et al., 2009; Malvankar et al., 2011). This probably explains why Geobacter
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species produce the highest current densities of any known microorganism, and typically
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outcompete other microbes when anodes are provided as an electron acceptor in complex
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microbial environments (Lovley et al., 2011; Lovley, 2012). The conductive pili are also
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key conduits for environmentally significant interspecies electrical connections, as
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described below.
Accepted Article
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Cytochrome Electron Hopping in Membrane Filaments but Not Pili When the S. oneidensis membrane extensions are dried, the resultant filaments
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conduct electrons via cytochrome-to-cytochrome electron hopping/tunneling (Gorby et
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al., 2006; El-Naggar et al., 2008; El-Naggar et al., 2010; Leung et al., 2013). It has been
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proposed that a similar electron transport is possible along intact membrane extensions
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under physiologically relevant, hydrated conditions (Pirbadian et al., 2014). This as yet
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untested hypothesis should be evaluated soon, because it is key to elucidating the
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function of the membrane extensions.
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Cytochrome-to-cytochrome electron hopping/tunneling in the dried
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membrane/periplasmic preparations may not be surprising because drying collapses the
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material (Pirbadian et al., 2014), which aggregates proteins (Dohnalkova et al., 2011),
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and could bring the cytochromes close enough for rapid electron exchange. However,
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such close packing of cytochromes (distance of no more than 1-2 nm to permit
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hopping/tunneling (Polizzi et al., 2012)) in a hydrated lipid membrane would be
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unprecedented in a gram-negative microorganism, yielding an outer ‘membrane’ that
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contained more protein than lipid.
Accepted Article
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Fortunately, techniques for directly measuring the conductivity of hydrated
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biological materials are available (Malvankar et al., 2011) and could be adapted for
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measuring the hypothesized conductivity along the membrane extensions under
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physiologically relevant conditions. Furthermore, the hypothesized tight packing of
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cytochromes (Pirbadian et al., 2014) could readily be verified with atomic force
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microscopy techniques, similar to those previous employed to document cytochrome
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spacing on the pili of G. sulfurreducens (Malvankar et al., 2012a).
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The multi-heme c-type cytochrome OmcS is specifically localized on the G.
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sulfurreducens pili (Leang et al., 2010), but atomic force microscopy demonstrated that
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OmcS is spaced too far apart for electron hopping/tunneling along the length of the pili
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(Malvankar et al., 2012a). Denaturing the cytochromes associated with the pili has no
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impact on pili conductivity and the response of pili conductivity to temperature and pH is
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inconsistent with an electron hopping/tunneling mechanism (Malvankar et al., 2011).
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Studies in which heterologous PilA subunits were expressed in G. sulfurreducens further
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demonstrated that stringing OmcS on pili is not sufficient to promote electron transfer
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along the length of the pili (Vargas et al., 2013; Liu et al., 2014b).
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Carbon Nanotube-Like Charge Propagation in G. sulfurreducens Pili Cytochrome-free electron transport along G. sulfurreducens pili was suggested in
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the first studies on microbial nanowires (Reguera et al., 2005) and subsequent studies
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demonstrated that this could be attributed to metallic-like electron conduction along the
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length of the pili (Malvankar et al., 2011; Vargas et al., 2013). However, unequivocal
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visualization of this phenomenon has only recently become available with electrostatic
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force microscopy (EFM), in the first application of this technique to a biological protein
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(Malvankar et al., 2014b).
Accepted Article
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EFM has emerged as a powerful tool to visualize electron distribution and
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conductivity at a molecular level (Heim et al., 2004; Dautel et al., 2008; Melin et al.,
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2010). Charges are injected at a specific point and the flow of injected charges is
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visualized by measuring the local electric force gradients (Melin et al., 2010). EFM
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studies have previously demonstrated (Paillet et al., 2005; Zdrojek et al., 2006; Barboza
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et al., 2008; Melin et al., 2010) that charge injected into carbon nanotubes spreads
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homogeneously along their entire length (Fig. 1A). This is possible because sp2
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hybridization of carbon atoms results in overlapping pi orbitals (Heeger, 2001). The
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delocalization of the electrons in the overlapping pi orbitals confers a metallic-like
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conductivity (Heeger, 2001).
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Remarkably, charge propagates in G. sulfurreducens pili (Fig. 1B) in the same
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manner as in carbon nanotubes (Malvankar et al., 2014b). How is this possible in a
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protein filament? It appears that an ancestor to G. sulfurreducens evolved the typical
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type IV filament structure (Craig et al., 2004; Giltner et al., 2012) to position aromatic
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amino acids close enough for pi-pi overlapping of the aromatics (Malvankar et al., 2011;
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Vargas et al., 2013). This was accomplished by shedding a large portion of the PilA
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monomer. For example, the N-terminal sequences of Pseudomonas aeruginosa and G.
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sulfurreducens PilA are highly conserved, but the carboxyl terminus of the G.
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sulfurreducens is substantially truncated (Liu et al., 2014b). P. aeruginosa pili are not
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conductive (Reguera et al., 2005), even when expressed in G. sulfurreducens (Liu et al.,
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2014b). If five key aromatic amino acids in the carboxyl terminus of G. sulfurreducens
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are replaced with an alanine, pili are produced, with the pili-associated cytochrome OmcS
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properly localized, but the pili conductivity is greatly diminished (Vargas et al., 2013).
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Aro-5, the strain that produces these poorly conductive pili, lacks phenotypes associated
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with long-range electron conduction along the pili, such as the ability to reduce Fe(III)
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oxides or to form conductive biofilms capable of generating high current densities in
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microbial fuel cells (Vargas et al., 2013).
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Direct evidence for the metallic-like conductivity of G. sulfurreducens pili
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includes temperature and pH responses characteristic of metallic-like conductivity and
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inconsistent with electron hopping/tunneling (Malvankar et al., 2011). X-ray diffraction
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has provided structural evidence for pi-pi stacking (Malvankar et al., 2011; Malvankar et
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al., 2014a). The Aro-5 pili lack the diffraction pattern characteristic of pi-pi stacking
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(Malvankar et al., 2014a) and do not propagate charge injected with EFM (Malvankar et
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al., 2014b).
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Thus, the pili of G. sulfurreducens and the dried outer-membrane extensions of S.
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oneidensis have distinct mechanisms for electron transport along the length of the
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filaments. This finding and the fact that they are comprised of completely different
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materials suggests that the same terminology should not be used to describe these
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disparate materials.
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Terminology to Distinguish Distinct Filaments: Nanowires and Nanopods
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The outer-membrane and periplasmic extensions of S. oneidensis were referred to
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as microbial nanowires (Pirbadian et al., 2014). This designation seems premature. The
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membrane extensions have not yet been shown to function as a wire under
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physiologically relevant conditions and there is of yet no evidence that the outer-
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membrane extensions promote long-range electron transport to insoluble electron
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acceptors such as Fe (III) oxides or electrodes.
Accepted Article
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This contrasts with the conductive pili of G. sulfurreducens which function as a
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true wire with metallic-like conductivity and have clearly been demonstrated to enhance
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long-range electron transfer to Fe(III) oxides and through current-producing biofilms, as
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well as to facilitate interspecies electron exchange.
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We propose that, in accordance with the first publication of this term (Reguera et
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al., 2005), ‘microbial nanowires’ be reserved for filaments that have been demonstrated
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to: 1) be conductive along their length under physiologically relevant conditions; and 2)
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play a role in extracellular electron exchange. Using the same term for the S. oneidensis
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filaments in the absence of meeting either of these criteria will only perpetuate the
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confusion in the microbial nanowire field that resulted from the mistaken concept that S.
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oneidensis produced conductive pili like G. sulfurreducens.
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Our suggestion for a descriptor for the S. oneidensis filaments is ‘microbial
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nanopods’ because the S. oneidensis extensions are reminiscent of the pseudopod
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extensions of eukaryotic cells. However, those studying S. oneidensis membrane
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extensions may prefer to propose alternative the new terminology.
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Pili-Mediated Direct Interspecies Electron Transfer
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Accepted Article
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It is rare that microbial phenomena can be visualized with the naked eye, but no
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special imaging equipment was required to see one of the most recently recognized
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manifestations of conductive pili function. Direct interspecies electron transfer (DIET),
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mediated by conductive pili, was first documented in co-cultures of G. metallireducens
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and G. sulfurreducens that adapted to share electrons to promote their mutual metabolism
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and growth (Summers et al., 2010). Only G. metallireducens could utilize the electron
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donor (ethanol) in the medium and only G. sulfurreducens could utilize the fumarate
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provided as an electron acceptor. The co-culture produced visually apparent (1-2 mm
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diameter) aggregates that had metallic-like conductivity (Summers et al., 2010) similar to
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the metallic-like conductivity of G. sulfurreducens current-producing biofilms
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(Malvankar et al., 2011). Multiple lines of evidence have confirmed the role of the pili of
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both Geobacter species in the interspecies electron exchange (Summers et al., 2010;
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Shrestha and Rotaru, 2014).
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Metallic-like conductivity is also observed in Geobacter-rich microbial
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aggregates that form in upflow anaerobic sludge blanket (UASB) digesters treating
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brewery waste (Morita et al., 2011). Metatranscriptomic analysis of a digester, as well as
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transcriptomic and physiological studies with defined co-cultures, revealed that
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Methanosaeta species can accept electrons via DIET for the reduction of carbon dioxide
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to methane (Rotaru et al., 2014b). This is surprising because it was not previously known
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that Methanosaeta, the most prodigious methanogens on Earth (Smith and Ingram-Smith,
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2007), could use carbon dioxide as an electron acceptor, or that methanogens could
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establish direct electrical connections with other microorganisms. Methanosarcina
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barkeri, representative of the Methanosarcina species that are also abundant in many
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submerged soils and sediments, can also accept electrons via DIET (Rotaru et al., 2014a).
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One electrical component of Geobacter-methanogen DIET is the Geobacter conductive
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pili (Rotaru et al., 2014a; Rotaru et al., 2014b). The methanogen electrical connections
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that complete the DIET circuit are as yet unknown.
Accepted Article
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A number of conductive materials can substitute for pili to promote DIET,
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reducing the time required for species to establish electrical connections, and permitting
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pili-deficient strains to participate in DIET. These include biochar, a common soil
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additive (Chen et al., 2014b) and conductive carbon materials that may be used to
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promote anaerobic waste digestion such as carbon cloth (Chen et al., 2014a) and granular
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activated carbon (Liu et al., 2012). Although it was considered that magnetite might also
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serve as a pili substitute for DIET (Kato et al., 2012), subsequent studies have suggested
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that it functions as an outer-surface c-type cytochrome analog (Liu et al., 2014a).
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Conclusions
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The resolution that S. oneidensis conductive filaments are fundamentally different
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than the conductive pili of G. sulfurreducens is expected to greatly accelerate progress in
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the study of microbial nanowires. No longer will it be necessary to try to reconcile
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dramatically different results from studies of these disparate materials. Important
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questions remain, however.
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For S. oneidensis, it remains to be determined whether the outer-membrane
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extensions actually facilitate extracellular electron transport to insoluble electron
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acceptors. Microorganisms produce cellular extensions for a diversity of functions
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(Nickell et al., 2003; Näther et al., 2006; Comolli et al., 2008; Jahn et al., 2008; Wanner
11 This article is protected by copyright. All rights reserved.
et al., 2008; Dubey and Ben-Yehuda, 2011; Lovley, 2011a; Berleman and Auer, 2013;
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McCaig et al., 2013; Comolli and Banfield, 2014; Remis et al., 2014) and S. oneidensis
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grows under a wide diversity of conditions (Hau and Gralnick, 2007; Fredrickson et al.,
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2008). Therefore, it should not be assumed that just because S. oneidensis is capable of
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Fe(III) oxide reduction that the outer-membrane extensions are involved in Fe(III) oxide
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reduction. Direct experimental verification is required, especially given the substantial
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evidence which suggests that S. oneidensis primarily reduces Fe(III) oxide via a soluble
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electron shuttle intermediary, which would obviate the need for wires (Brutinel and
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Gralnick, 2012).
Accepted Article
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In contrast, Geobacter species greatly benefit from microbial nanowires because
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they must directly contact Fe(III) oxides in order to reduce them and the flexible
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nanowires greatly extend the reach of the cell beyond the outer cell surface (Lovley et al.,
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2011). The metallic-like conductivity of G. sulfurreducens pili provides a new paradigm
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for biological electron transport, significantly different from the electron
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hopping/tunneling that characterizes all other forms of biological electron transport that
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have been studied to date. Although several models that have attempted to model the G.
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sulfurreducens pilus structure have questioned whether sufficient pi-pi stacking of
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aromatic amino acids for metallic-like conductivity is possible (Feliciano et al., 2012;
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Boesen and Nielsen, 2013; Bonanni et al., 2013; Reardon and Mueller, 2013), actual
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experimental results trump modeling, which can be based on faulty assumptions, and the
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recent EFM imaging of charge propagation (Malvankar et al., 2014b) further supports the
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abundant experimental evidence (Malvankar et al., 2011; Vargas et al., 2013) supporting
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metallic-like conductivity. At this point, structural studies, potentially taking advantage
12 This article is protected by copyright. All rights reserved.
of recent advances in cryo electron microscopy (Glaeser, 2013; Smith and Rubinstein,
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2014), are more likely to advance understanding of the features that confer metallic-like
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conductivity than will theoretical modeling.
Accepted Article
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However, even with the limited information already available, microbial
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nanowires may now be considered a ‘microbial part’ for synthetic biology applications.
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Genetic manipulations and adaptive evolution have demonstrated that increasing pili
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production can enhance desired applications, such as increasing biofilm conductivity and
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the power output of microbial fuel cells (Yi et al., 2009; Malvankar et al., 2012b; Leang
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et al., 2013). Manipulating the pili structure to increase pi-pi stacking of aromatic amino
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acids, or introducing more conductive non-native amino acids, may have comparable
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practical benefits.
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Improved methods for evaluating the conductivity of microbial filaments under
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physiologically relevant conditions (Malvankar et al., 2011; Malvankar et al., 2014b)
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now make it possible to consider screening the vast diversity of the microbial world for
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microbial nanowires. It appears that conductive pili have only recently evolved within
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the Geobacteraceae. However, given the strong competitive advantage that conductive
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pili appear to confer in Fe(III)-reducing environments (Lovley et al., 2011), convergent
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evolution may have reached a similar conductive solution in diverse microbes. This
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could include Archaea, many of which can use Fe (III) as an electron acceptor (Vargas et
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al., 1998; Kashefi and Lovley, 2003; Yamada et al., 2014), in addition to participating in
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DIET.
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Advancing the study of microbial nanowires will require the continued
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collaboration of microbiologists, biophysicists, structural biologists, biochemists, and
13 This article is protected by copyright. All rights reserved.
ecologists. The potential benefits of such studies include not only a better understanding
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of the natural world, but also the development of exciting new parts for the emerging
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fields of electromicrobiology, bioelectronics, and biocomputing.
Accepted Article
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Acknowledgements
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The authors research on microbial nanowires are supported by Office of Naval Research
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grants N000141210229, N00014-13-1-0550, and N000141310549. The authors declare
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no conflicts of interest.
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transport in a single pentacene monolayer island. Nano Letters 4: 2145-2150.
365
Jahn, U., Gallenberger, M., Junglas, B., Eisenreich, W., Stetter, K.O., Rachel, R., and
366
Huber, H. (2008) Nanoarchaeum equitans and Ignicoccus hospitalis: new insights into a
367
unique, intimate association of two archaea. Journal of Bacteriology 190: 1743-1750.
368
Kashefi, K., and Lovley, D.R. (2003) Extending the Upper Temperature Limit for Life.
369
Science 301: 934.
Accepted Article
352
17 This article is protected by copyright. All rights reserved.
Kato, S., Hashimoto, K., and Watanabe, K. (2012) Microbial interspecies electron
371
transfer via electric currents through conductive minerals. Proceedings of the National
372
Academy of Sciences 109: 10042-10046.
373
Leang, C., Qian, X., Mester, T., and Lovley, D.R. (2010) Alignment of the c-type
374
cytochrome OmcS along pili of Geobacter sulfurreducens. Applied and Environmental
375
Microbiology 76: 4080-4084.
376
Leang, C., Malvankar, N.S., Franks, A.E., Nevin, K.P., and Lovley, D.R. (2013)
377
Engineering Geobacter sulfurreducens to produce a highly cohesive conductive matrix
378
with enhanced capacity for current production. Energy & Environmental Science 6: 1901-
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1908.
380
Leung, K.M., Wanger, G., El-Naggar, M.Y., Gorby, Y., Southam, G., Lau, W.M., and
381
Yang, J. (2013) Shewanella oneidensis MR-1 bacterial nanowires exhibit p-type, tunable
382
electronic behavior. Nano Letters 13: 2407-2411.
383
Liu, F., Rotaru, A.-E., Shrestha, P.M., Malvankar, N.S., Nevin, K.P., and Lovley, D.R.
384
(2012) Promoting direct interspecies electron transfer with activated carbon. Energy &
385
Environmental Science 5: 8982-8989.
386
Liu, F., Rotaru, A.-E., Shrestha, P.M., Malvankar, N.S., Nevin, K.P., and Lovley, D.R.
387
(2014a) Magnetite compensates for the lack of a pilin-associated c-type cytochrome in
388
extracellular electron exchange. Environmental Microbiology doi: 10.1111/1462-
389
2920.12485.
Accepted Article
370
18 This article is protected by copyright. All rights reserved.
Liu, X., Tremblay, P.-L., Malvankar, N.S., Nevin, K.P., Lovley, D.R., and Vargas, M.
391
(2014b) A Geobacter sulfurreducens strain expressing Pseudomonas aeruginosa type IV
392
pili localizes OmcS on pili but is deficient in Fe(III) oxide reduction and current
393
production. Applied and Environmental Microbiology 80: 1219-1224.
394
Lovley, D. (2011a) Reach out and touch someone: potential impact of DIET (direct
395
interspecies energy transfer) on anaerobic biogeochemistry, bioremediation, and
396
bioenergy. Reviews in Environmental Science and Bio/Technology 10: 101-105.
397
Lovley, D.R. (2011b) Live wires: direct extracellular electron exchange for bioenergy
398
and the bioremediation of energy-related contamination. Energy and Environmental
399
Science 4: 4896-4906.
400
Lovley, D.R. (2012) Electromicrobiology. Annual Review of Microbiology 66: 391-409.
401
Lovley, D.R., Ueki, T., Zhang, T., Malvankar, N.S., Shrestha, P.M., Flanagan, K. et al.
402
(2011) Geobacter: The microbe electric’s physiology, ecology, and practical applications.
403
Advances in Microbial Physiology 59: 1-100.
404
Malvankar, N., Vargas, M., Nevin, K.P., Tremblay, P.L., Evans-Lutterodt, K.,
405
Nykypanchuk, D. et al. (2014a) Structural basis underlying the metallic-like conductivity
406
of microbial nanowires. mBio in review.
407
Malvankar, N.S., and Lovley, D.R. (2012) Microbial nanowires: A new paradigm for
408
biological electron transfer and bioelectronics. ChemSusChem 5: 1039-1046.
Accepted Article
390
19 This article is protected by copyright. All rights reserved.
Malvankar, N.S., and Lovley, D.R. (2014) Microbial nanowires for bioenergy
410
applications. Current Opinion in Biotechnology 27: 88-95.
411
Malvankar, N.S., Tuominen, M.T., and Lovley, D.R. (2012a) Lack of cytochrome
412
involvement in long-range electron transport through conductive biofilms and nanowires
413
of Geobacter sulfurreducens. Energy & Environmental Science 5: 8651-8659.
414
Malvankar, N.S., Tuominen, M.T., and Lovley, D.R. (2012b) Biofilm conductivity is a
415
decisive variable for high-current-density microbial fuel cells. Energy & Environmental
416
Science 5: 5790-5797.
417
Malvankar, N.S., Yalcin, S.E., Tuominen, M.T., and Lovley, D.R. (2014b) Visulation of
418
charge propagation along individual pili proteins using ambient electrostatatic force
419
microscopy. Nature Nanotechnology doi: 10.1038/NNANO.2014.236.
420
Malvankar, N.S., Vargas, M., Nevin, K.P., Franks, A.E., Leang, C., Kim, B.C. et al.
421
(2011) Tunable metallic-like conductivity in microbial nanowire networks. Nature
422
Nanotechnology 6: 573-579.
423
McCaig, W.D., Koller, A., and Thanassi, D.G. (2013) Production of outer membrane
424
vesicles and outer membrane tubes by Francisella novicida. Journal of Bacteriology 195:
425
1120-1132.
426
Mehta, T., Childers, S.E., and Lovley, D. (2002) Geobacter sulfurreducens requires pili
427
in order to reduce Fe(III) oxide. In Abstracts of the American Society of Microbiology
428
(ASM) Annual Meeting, Salt Lake City, Utah.
Accepted Article
409
20 This article is protected by copyright. All rights reserved.
Melin, T., Zdrojek, M., and Brunel, D. (2010) Electrostatic Force Microscopy and Kelvin
430
Force Microscopy as a Probe of the Electrostatic and Electronic Properties of Carbon
431
Nanotubes In Scanning Probe Microscopy in Nanoscience and Nanotechnology.
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Bhushan, B. (ed): Springer Berlin Heidelberg, pp. 89-128.
433
Morita, M., Malvankar, N.S., Franks, A.E., Summers, Z.M., Giloteaux, L., Rotaru, A.E.
434
et al. (2011) Potential for direct interspecies electron transfer in methanogenic wastewater
435
digester aggregates. mBio 2: e00159-00111.
436
Näther, D.J., Rachel, R., Wanner, G., and Wirth, R. (2006) Flagella of Pyrococcus
437
furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces,
438
and cell-cell contacts. Journal of Bacteriology 188: 6915-6923.
439
Nevin, K.P., Kim, B.C., Glaven, R.H., Johnson, J.P., Woodard, T.L., Methé, B.A. et al.
440
(2009) Anode biofilm transcriptomics reveals outer surface components essential for high
441
density current production in Geobacter sulfurreducens fuel cells. PLoS ONE 4: e5628.
442
Nickell, S., Hegerl, R., Baumeister, W., and Rachel, R. (2003) Pyrodictium cannulae
443
enter the periplasmic space but do not enter the cytoplasm, as revealed by cryo-electron
444
tomography. Journal of Structural Biology 141: 34-42.
445
Paillet, M., Poncharal, P., and Zahab, A. (2005) Electrostatics of individual single-walled
446
carbon nanotubes investigated by electrostatic force microscopy. Physical Review Letters
447
94: 186801.
448
Pirbadian, S., Barchinger, S.E., Leung, K.M., Byun, H.S., Jangir, Y., Bouhenni, R.A. et
449
al. (2014) Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic
Accepted Article
429
21 This article is protected by copyright. All rights reserved.
extensions of the extracellular electron transport components. Proceedings of the
451
National Academy of Sciences 111: 12883-12888.
452
Polizzi, N.F., Skourtis, S.S., and Beratan, D.N. (2012) Physical constraints on charge
453
transport through bacterial nanowires. Faraday Discussions 155: 43-61.
454
Reardon, P.N., and Mueller, K.T. (2013) Structure of the type IVa major pilin from the
455
electrically conductive bacterial nanowires of Geobacter sulfurreducens. Journal of
456
Biological Chemistry 288: 29260-29266.
457
Reguera, G., McCarthy, K.D., Mehta, T., Nicoll, J.S., Tuominen, M.T., and Lovley, D.R.
458
(2005) Extracellular electron transfer via microbial nanowires. Nature 435: 1098-1101.
459
Reguera, G., Nevin, K.P., Nicoll, J.S., Covalla, S.F., Woodard, T.L., and Lovley, D.R.
460
(2006) Biofilm and nanowire production leads to increased current in Geobacter
461
sulfurreducens fuel cells. Applied and Environmental Microbiology 72: 7345-7348.
462
Remis, J.P., Wei, D., Gorur, A., Zemla, M., Haraga, J., Allen, S. et al. (2014) Bacterial
463
social networks: structure and composition of Myxococcus xanthus outer membrane
464
vesicle chains. Environmental Microbiology 16: 598-610.
465
Rotaru, A.E., Shrestha, P.M., Liu, F., Markovaite, B., Chen, S., Nevin, K.P., and Lovley,
466
D.R. (2014a) Direct interspecies electron transfer between Geobacter metallireducens
467
and Methanosarcina barkeri. Applied and Environmental Microbiology 80:4599-4605.
468
Rotaru, A.E., Shrestha, P.M., Liu, F., Shrestha, M., Shrestha, D., Embree, M. et al.
469
(2014b) A new model for electron flow during anaerobic digestion: direct interspecies
Accepted Article
450
22 This article is protected by copyright. All rights reserved.
electron transfer to Methanosaeta for the reduction of carbon dioxide to methane. Energy
471
and Environmental Science 7: 408-415.
472
Shrestha, P.M., and Rotaru, A.-E. (2014) Plugging in or Going Wireless: Strategies for
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Interspecies Electron Transfer. Frontiers in Microbiology 5: 1-8.
474
Smith, K.S., and Ingram-Smith, C. (2007) Methanosaeta, the forgotten methanogen?
475
Trends in Microbiology 15: 150-155.
476
Smith, M.T.J., and Rubinstein, J.L. (2014) Beyond blob-ology. Science 345: 617-619.
477
Summers, Z.M., Fogarty, H.E., Leang, C., Franks, A.E., Malvankar, N.S., and Lovley,
478
D.R. (2010) Direct exchange of electrons within aggregates of an evolved syntrophic
479
coculture of anaerobic bacteria. Science 330: 1413-1415.
480
Vargas, M., Kashefi, K., Blunt-Harris, E.L., and Lovley, D.R. (1998) Microbiological
481
evidence for Fe(III) reduction on early Earth. Nature 395: 65-67.
482
Vargas, M., Malvankar, N.S., Tremblay, P.L., Leang, C., Smith, J., Patel, P. et al. (2013)
483
Aromatic amino acids required for pili conductivity and long-range extracellular electron
484
transport in Geobacter sulfurreducens. mBio 4: e00105-00113.
485
Wanner, G., Vogl, K., and Overmann, J. (2008) Ultrastructural Characterization of the
486
Prokaryotic Symbiosis in “Chlorochromatium aggregatum”. Journal of Bacteriology
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190: 3721-3730.
Accepted Article
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23 This article is protected by copyright. All rights reserved.
Yamada, C., Kato, S., Kimura, S., Ishii, M., and Igarashi, Y. (2014) Reduction of Fe(III)
489
oxides by phylogenetically and physiologically diverse thermophilic methanogens. FEMS
490
Microbiology Ecology 89: 637-645.
491
Yi, H., Nevin, K.P., Kim, B.C., Franks, A.E., Klimes, A., Tender, L.M., and Lovley, D.R.
492
(2009) Selection of a variant of Geobacter sulfurreducens with enhanced capacity for
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current production in microbial fuel cells. Biosensors and Bioelectronics 24: 3498-3503.
494
Zdrojek, M., Mélin, T., Diesinger, H., Stiévenard, D., Gebicki, W., and Adamowicz, L.
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(2006) Comment on “electrostatics of individual single-walled carbon nanotubes
496
investigated by electrostatic force microscopy”. Physical Review Letters 96: 039703.
Accepted Article
488
497
24 This article is protected by copyright. All rights reserved.
Accepted Article
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499 500
Figure 1. Imaging charge flow with electrostatic force microscopy. (A) Charge
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delocalization in carbon nanotubes [Reprinted with permission from (Zdrojek et al.,
502
2006) Copyright (2006) by the American Physical Society]. The charges were injected at
503
the location designated with the white line. The yellow contrast reveals the propagation
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of the injected charges. (B) Charge propagation in pili of G. sulfurreducens [Figure
505
adapted from (Malvankar et al., 2014b) with permission]. The charges were injected at
506
the location designated with the red arrow. The white contrast reveals the propagation of
507
the injected charges along the pilus, which also propagated into another pilus that was in
25 This article is protected by copyright. All rights reserved.
contact with the charge-injected pilus. Charge did not propagate across a break
509
introduced in the pilus highlighted in red.
Accepted Article
508
26 This article is protected by copyright. All rights reserved.
2
Structure and Function 1
Accepted Article
1
3 4
Running Title: Resolving Microbial Nanowire Structure and Function
5 6
Derek R. Lovley1* and Nikhil S. Malvankar1,2
7
1
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts.
8
2
Department of Physics, University of Massachusetts, Amherst, Massachusetts.
9
10 11 12
*Corresponding author
13
Derek R. Lovley
14
639 North Pleasant Street,
15
203, Morrill Science Center IV North,
16
Amherst, MA 01003, USA.
17
Phone: 413-545-9651
18
Fax: 413-545-1578
19
Email: [email protected]://www.geobacter.org/
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12708
1 This article is protected by copyright. All rights reserved.
Accepted Article
20 21
Summary
22 23
Novel imaging approaches have recently helped to clarify the properties of “microbial
24
nanowires”. Geobacter sulfurreducens pili are actual wires. They possess metallic-like
25
conductivity, which can be attributed to overlapping pi-pi orbitals of key aromatic amino
26
acids. Electrostatic force microscopy recently confirmed charge propagation along the
27
pili, in a manner similar to carbon nanotubes. The pili are essential for long-range
28
electron transport to insoluble electron acceptors and interspecies electron transfer.
29
Previous claims that S. oneidensis also produce conductive pili have recently been
30
recanted, based on novel live-imaging studies. The putative pili are, in fact, long
31
extensions of the cytochrome-rich, outer-membrane and periplasm that, when dried,
32
collapse to form filaments with dimensions similar to pili. It has yet to be demonstrated
33
whether the cytochrome-to-cytochrome electron hopping documented in the dried
34
membrane extensions takes place in intact hydrated membrane extensions or whether the
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membrane extensions enhance electron transport to insoluble electron acceptors such as
36
Fe(III) oxides or electrodes. These findings demonstrate that G. sulfurreducens
37
conductive pili and the outer-membrane extensions of S. oneidensis are fundamentally
38
different in composition, mechanism of electron transport, and physiological role. New
39
methods for evaluating filament conductivity will facilitate screening the microbial world
40
for nanowires and elucidating their function.
41 42
2 This article is protected by copyright. All rights reserved.
Accepted Article
43 44 45
Introduction Microbial nanowires are one of the most fascinating microbial structures
46
discovered within the last decade. They play an important role in carbon and mineral
47
cycling in anaerobic soils and sediments; are a unique strategy for interspecies energy
48
exchange; and have practical applications in bioenergy, bioremediation, and
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bioelectronics (Lovley, 2011b; Malvankar and Lovley, 2014). Until recently there has
50
been substantial controversy and confusion over the physical properties, composition, and
51
function of the microbial nanowires of Geobacter sulfurreducens and Shewanella
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oneidensis, the two microbes that have primarily served as the basis for microbial
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nanowire mechanistic studies (Malvankar and Lovley, 2012). New clarity has now
54
emerged, with the aid of novel imaging approaches (Malvankar et al., 2014b; Pirbadian et
55
al., 2014).
56 57 58
Putative S. oneidensis Pili Nanowires are Actually Membrane Extensions A stunning result from new imaging approaches has been the revelation that the
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conductive filaments from S. oneidensis, which were previously thought to be pili are, in
60
fact, extensions of the outer membrane and periplasm (Pirbadian et al., 2014). Elegant,
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live fluorescence microscopy revealed the growth of extensions of the outer membrane
62
that distort the typical rod shape of the cells with protrusions that in some instances are
63
longer than the cell itself. When the outer-membrane extensions are dried for
64
conductivity measurements, their diameter shrinks over 10-fold, yielding a material that
3 This article is protected by copyright. All rights reserved.
looks like a filament of ca. 10 nm diameter. This artifact resembles pili, hence the prior
66
mistaken identity (Pirbadian et al., 2014).
Accepted Article
65
67
These findings resolve a long, contentious debate over whether S. oneidensis can
68
produce conductive pili. Initial studies that examined the pili of S. oneidensis with
69
conducting-tip atomic force microscopy concluded that the pili were not conductive
70
(Reguera et al., 2005). Subsequent studies contended that S. oneidensis had conductive
71
pili (Gorby et al., 2006). Now that it is clear that those later studies (Gorby et al., 2006)
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were evaluating the conductivity of the dried outer-membrane extensions (Pirbadian et
73
al., 2014) it appears that the initial finding that S. oneidensis does not produce conductive
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pili (Reguera et al., 2005) was correct.
75 76 77
Geobacter Nanowires are Type IV Pili In contrast, it was clear from the beginning that G. sulfurreducens nanowires were
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type IV pili. The hypothesis that the G. sulfurreducens pili might function as wires
79
resulted from the observation that pili were specifically expressed during growth on
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Fe(III) oxides and other insoluble electron acceptors (Childers et al., 2002); and the fact
81
that deleting the gene for PilA, the pilus monomer, inhibited the reduction of Fe(III)
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oxides, but not soluble, chelated Fe(III) (Mehta et al., 2002). These findings, coupled
83
with the subsequent observations that: 1) nano-sized Fe(III) oxides specifically associated
84
with the pili; and 2) the pili were electrically conductive across their diameter; led to the
85
suggestion that the G. sulfurreducens pili functioned as wires to promote long-range
86
electron conduction to Fe(III) oxides (Reguera et al., 2005). Further study demonstrated
87
that networks of the conductive pili permitted G. sulfurreducens to produce thick,
4 This article is protected by copyright. All rights reserved.
electrically conductive biofilms on the anodes of microbial fuel cells (Reguera et al.,
89
2006; Nevin et al., 2009; Malvankar et al., 2011). This probably explains why Geobacter
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species produce the highest current densities of any known microorganism, and typically
91
outcompete other microbes when anodes are provided as an electron acceptor in complex
92
microbial environments (Lovley et al., 2011; Lovley, 2012). The conductive pili are also
93
key conduits for environmentally significant interspecies electrical connections, as
94
described below.
Accepted Article
88
95 96 97
Cytochrome Electron Hopping in Membrane Filaments but Not Pili When the S. oneidensis membrane extensions are dried, the resultant filaments
98
conduct electrons via cytochrome-to-cytochrome electron hopping/tunneling (Gorby et
99
al., 2006; El-Naggar et al., 2008; El-Naggar et al., 2010; Leung et al., 2013). It has been
100
proposed that a similar electron transport is possible along intact membrane extensions
101
under physiologically relevant, hydrated conditions (Pirbadian et al., 2014). This as yet
102
untested hypothesis should be evaluated soon, because it is key to elucidating the
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function of the membrane extensions.
104
Cytochrome-to-cytochrome electron hopping/tunneling in the dried
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membrane/periplasmic preparations may not be surprising because drying collapses the
106
material (Pirbadian et al., 2014), which aggregates proteins (Dohnalkova et al., 2011),
107
and could bring the cytochromes close enough for rapid electron exchange. However,
108
such close packing of cytochromes (distance of no more than 1-2 nm to permit
109
hopping/tunneling (Polizzi et al., 2012)) in a hydrated lipid membrane would be
5 This article is protected by copyright. All rights reserved.
unprecedented in a gram-negative microorganism, yielding an outer ‘membrane’ that
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contained more protein than lipid.
Accepted Article
110
112
Fortunately, techniques for directly measuring the conductivity of hydrated
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biological materials are available (Malvankar et al., 2011) and could be adapted for
114
measuring the hypothesized conductivity along the membrane extensions under
115
physiologically relevant conditions. Furthermore, the hypothesized tight packing of
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cytochromes (Pirbadian et al., 2014) could readily be verified with atomic force
117
microscopy techniques, similar to those previous employed to document cytochrome
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spacing on the pili of G. sulfurreducens (Malvankar et al., 2012a).
119
The multi-heme c-type cytochrome OmcS is specifically localized on the G.
120
sulfurreducens pili (Leang et al., 2010), but atomic force microscopy demonstrated that
121
OmcS is spaced too far apart for electron hopping/tunneling along the length of the pili
122
(Malvankar et al., 2012a). Denaturing the cytochromes associated with the pili has no
123
impact on pili conductivity and the response of pili conductivity to temperature and pH is
124
inconsistent with an electron hopping/tunneling mechanism (Malvankar et al., 2011).
125
Studies in which heterologous PilA subunits were expressed in G. sulfurreducens further
126
demonstrated that stringing OmcS on pili is not sufficient to promote electron transfer
127
along the length of the pili (Vargas et al., 2013; Liu et al., 2014b).
128 129 130
Carbon Nanotube-Like Charge Propagation in G. sulfurreducens Pili Cytochrome-free electron transport along G. sulfurreducens pili was suggested in
131
the first studies on microbial nanowires (Reguera et al., 2005) and subsequent studies
132
demonstrated that this could be attributed to metallic-like electron conduction along the
6 This article is protected by copyright. All rights reserved.
length of the pili (Malvankar et al., 2011; Vargas et al., 2013). However, unequivocal
134
visualization of this phenomenon has only recently become available with electrostatic
135
force microscopy (EFM), in the first application of this technique to a biological protein
136
(Malvankar et al., 2014b).
Accepted Article
133
137
EFM has emerged as a powerful tool to visualize electron distribution and
138
conductivity at a molecular level (Heim et al., 2004; Dautel et al., 2008; Melin et al.,
139
2010). Charges are injected at a specific point and the flow of injected charges is
140
visualized by measuring the local electric force gradients (Melin et al., 2010). EFM
141
studies have previously demonstrated (Paillet et al., 2005; Zdrojek et al., 2006; Barboza
142
et al., 2008; Melin et al., 2010) that charge injected into carbon nanotubes spreads
143
homogeneously along their entire length (Fig. 1A). This is possible because sp2
144
hybridization of carbon atoms results in overlapping pi orbitals (Heeger, 2001). The
145
delocalization of the electrons in the overlapping pi orbitals confers a metallic-like
146
conductivity (Heeger, 2001).
147
Remarkably, charge propagates in G. sulfurreducens pili (Fig. 1B) in the same
148
manner as in carbon nanotubes (Malvankar et al., 2014b). How is this possible in a
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protein filament? It appears that an ancestor to G. sulfurreducens evolved the typical
150
type IV filament structure (Craig et al., 2004; Giltner et al., 2012) to position aromatic
151
amino acids close enough for pi-pi overlapping of the aromatics (Malvankar et al., 2011;
152
Vargas et al., 2013). This was accomplished by shedding a large portion of the PilA
153
monomer. For example, the N-terminal sequences of Pseudomonas aeruginosa and G.
154
sulfurreducens PilA are highly conserved, but the carboxyl terminus of the G.
155
sulfurreducens is substantially truncated (Liu et al., 2014b). P. aeruginosa pili are not
7 This article is protected by copyright. All rights reserved.
conductive (Reguera et al., 2005), even when expressed in G. sulfurreducens (Liu et al.,
157
2014b). If five key aromatic amino acids in the carboxyl terminus of G. sulfurreducens
158
are replaced with an alanine, pili are produced, with the pili-associated cytochrome OmcS
159
properly localized, but the pili conductivity is greatly diminished (Vargas et al., 2013).
160
Aro-5, the strain that produces these poorly conductive pili, lacks phenotypes associated
161
with long-range electron conduction along the pili, such as the ability to reduce Fe(III)
162
oxides or to form conductive biofilms capable of generating high current densities in
163
microbial fuel cells (Vargas et al., 2013).
Accepted Article
156
164
Direct evidence for the metallic-like conductivity of G. sulfurreducens pili
165
includes temperature and pH responses characteristic of metallic-like conductivity and
166
inconsistent with electron hopping/tunneling (Malvankar et al., 2011). X-ray diffraction
167
has provided structural evidence for pi-pi stacking (Malvankar et al., 2011; Malvankar et
168
al., 2014a). The Aro-5 pili lack the diffraction pattern characteristic of pi-pi stacking
169
(Malvankar et al., 2014a) and do not propagate charge injected with EFM (Malvankar et
170
al., 2014b).
171
Thus, the pili of G. sulfurreducens and the dried outer-membrane extensions of S.
172
oneidensis have distinct mechanisms for electron transport along the length of the
173
filaments. This finding and the fact that they are comprised of completely different
174
materials suggests that the same terminology should not be used to describe these
175
disparate materials.
176 177
Terminology to Distinguish Distinct Filaments: Nanowires and Nanopods
8 This article is protected by copyright. All rights reserved.
The outer-membrane and periplasmic extensions of S. oneidensis were referred to
179
as microbial nanowires (Pirbadian et al., 2014). This designation seems premature. The
180
membrane extensions have not yet been shown to function as a wire under
181
physiologically relevant conditions and there is of yet no evidence that the outer-
182
membrane extensions promote long-range electron transport to insoluble electron
183
acceptors such as Fe (III) oxides or electrodes.
Accepted Article
178
184
This contrasts with the conductive pili of G. sulfurreducens which function as a
185
true wire with metallic-like conductivity and have clearly been demonstrated to enhance
186
long-range electron transfer to Fe(III) oxides and through current-producing biofilms, as
187
well as to facilitate interspecies electron exchange.
188
We propose that, in accordance with the first publication of this term (Reguera et
189
al., 2005), ‘microbial nanowires’ be reserved for filaments that have been demonstrated
190
to: 1) be conductive along their length under physiologically relevant conditions; and 2)
191
play a role in extracellular electron exchange. Using the same term for the S. oneidensis
192
filaments in the absence of meeting either of these criteria will only perpetuate the
193
confusion in the microbial nanowire field that resulted from the mistaken concept that S.
194
oneidensis produced conductive pili like G. sulfurreducens.
195
Our suggestion for a descriptor for the S. oneidensis filaments is ‘microbial
196
nanopods’ because the S. oneidensis extensions are reminiscent of the pseudopod
197
extensions of eukaryotic cells. However, those studying S. oneidensis membrane
198
extensions may prefer to propose alternative the new terminology.
199 200
Pili-Mediated Direct Interspecies Electron Transfer
9 This article is protected by copyright. All rights reserved.
Accepted Article
201
It is rare that microbial phenomena can be visualized with the naked eye, but no
202
special imaging equipment was required to see one of the most recently recognized
203
manifestations of conductive pili function. Direct interspecies electron transfer (DIET),
204
mediated by conductive pili, was first documented in co-cultures of G. metallireducens
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and G. sulfurreducens that adapted to share electrons to promote their mutual metabolism
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and growth (Summers et al., 2010). Only G. metallireducens could utilize the electron
207
donor (ethanol) in the medium and only G. sulfurreducens could utilize the fumarate
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provided as an electron acceptor. The co-culture produced visually apparent (1-2 mm
209
diameter) aggregates that had metallic-like conductivity (Summers et al., 2010) similar to
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the metallic-like conductivity of G. sulfurreducens current-producing biofilms
211
(Malvankar et al., 2011). Multiple lines of evidence have confirmed the role of the pili of
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both Geobacter species in the interspecies electron exchange (Summers et al., 2010;
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Shrestha and Rotaru, 2014).
214
Metallic-like conductivity is also observed in Geobacter-rich microbial
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aggregates that form in upflow anaerobic sludge blanket (UASB) digesters treating
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brewery waste (Morita et al., 2011). Metatranscriptomic analysis of a digester, as well as
217
transcriptomic and physiological studies with defined co-cultures, revealed that
218
Methanosaeta species can accept electrons via DIET for the reduction of carbon dioxide
219
to methane (Rotaru et al., 2014b). This is surprising because it was not previously known
220
that Methanosaeta, the most prodigious methanogens on Earth (Smith and Ingram-Smith,
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2007), could use carbon dioxide as an electron acceptor, or that methanogens could
222
establish direct electrical connections with other microorganisms. Methanosarcina
223
barkeri, representative of the Methanosarcina species that are also abundant in many
10 This article is protected by copyright. All rights reserved.
submerged soils and sediments, can also accept electrons via DIET (Rotaru et al., 2014a).
225
One electrical component of Geobacter-methanogen DIET is the Geobacter conductive
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pili (Rotaru et al., 2014a; Rotaru et al., 2014b). The methanogen electrical connections
227
that complete the DIET circuit are as yet unknown.
Accepted Article
224
228
A number of conductive materials can substitute for pili to promote DIET,
229
reducing the time required for species to establish electrical connections, and permitting
230
pili-deficient strains to participate in DIET. These include biochar, a common soil
231
additive (Chen et al., 2014b) and conductive carbon materials that may be used to
232
promote anaerobic waste digestion such as carbon cloth (Chen et al., 2014a) and granular
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activated carbon (Liu et al., 2012). Although it was considered that magnetite might also
234
serve as a pili substitute for DIET (Kato et al., 2012), subsequent studies have suggested
235
that it functions as an outer-surface c-type cytochrome analog (Liu et al., 2014a).
236 237
Conclusions
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The resolution that S. oneidensis conductive filaments are fundamentally different
239
than the conductive pili of G. sulfurreducens is expected to greatly accelerate progress in
240
the study of microbial nanowires. No longer will it be necessary to try to reconcile
241
dramatically different results from studies of these disparate materials. Important
242
questions remain, however.
243
For S. oneidensis, it remains to be determined whether the outer-membrane
244
extensions actually facilitate extracellular electron transport to insoluble electron
245
acceptors. Microorganisms produce cellular extensions for a diversity of functions
246
(Nickell et al., 2003; Näther et al., 2006; Comolli et al., 2008; Jahn et al., 2008; Wanner
11 This article is protected by copyright. All rights reserved.
et al., 2008; Dubey and Ben-Yehuda, 2011; Lovley, 2011a; Berleman and Auer, 2013;
248
McCaig et al., 2013; Comolli and Banfield, 2014; Remis et al., 2014) and S. oneidensis
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grows under a wide diversity of conditions (Hau and Gralnick, 2007; Fredrickson et al.,
250
2008). Therefore, it should not be assumed that just because S. oneidensis is capable of
251
Fe(III) oxide reduction that the outer-membrane extensions are involved in Fe(III) oxide
252
reduction. Direct experimental verification is required, especially given the substantial
253
evidence which suggests that S. oneidensis primarily reduces Fe(III) oxide via a soluble
254
electron shuttle intermediary, which would obviate the need for wires (Brutinel and
255
Gralnick, 2012).
Accepted Article
247
256
In contrast, Geobacter species greatly benefit from microbial nanowires because
257
they must directly contact Fe(III) oxides in order to reduce them and the flexible
258
nanowires greatly extend the reach of the cell beyond the outer cell surface (Lovley et al.,
259
2011). The metallic-like conductivity of G. sulfurreducens pili provides a new paradigm
260
for biological electron transport, significantly different from the electron
261
hopping/tunneling that characterizes all other forms of biological electron transport that
262
have been studied to date. Although several models that have attempted to model the G.
263
sulfurreducens pilus structure have questioned whether sufficient pi-pi stacking of
264
aromatic amino acids for metallic-like conductivity is possible (Feliciano et al., 2012;
265
Boesen and Nielsen, 2013; Bonanni et al., 2013; Reardon and Mueller, 2013), actual
266
experimental results trump modeling, which can be based on faulty assumptions, and the
267
recent EFM imaging of charge propagation (Malvankar et al., 2014b) further supports the
268
abundant experimental evidence (Malvankar et al., 2011; Vargas et al., 2013) supporting
269
metallic-like conductivity. At this point, structural studies, potentially taking advantage
12 This article is protected by copyright. All rights reserved.
of recent advances in cryo electron microscopy (Glaeser, 2013; Smith and Rubinstein,
271
2014), are more likely to advance understanding of the features that confer metallic-like
272
conductivity than will theoretical modeling.
Accepted Article
270
273
However, even with the limited information already available, microbial
274
nanowires may now be considered a ‘microbial part’ for synthetic biology applications.
275
Genetic manipulations and adaptive evolution have demonstrated that increasing pili
276
production can enhance desired applications, such as increasing biofilm conductivity and
277
the power output of microbial fuel cells (Yi et al., 2009; Malvankar et al., 2012b; Leang
278
et al., 2013). Manipulating the pili structure to increase pi-pi stacking of aromatic amino
279
acids, or introducing more conductive non-native amino acids, may have comparable
280
practical benefits.
281
Improved methods for evaluating the conductivity of microbial filaments under
282
physiologically relevant conditions (Malvankar et al., 2011; Malvankar et al., 2014b)
283
now make it possible to consider screening the vast diversity of the microbial world for
284
microbial nanowires. It appears that conductive pili have only recently evolved within
285
the Geobacteraceae. However, given the strong competitive advantage that conductive
286
pili appear to confer in Fe(III)-reducing environments (Lovley et al., 2011), convergent
287
evolution may have reached a similar conductive solution in diverse microbes. This
288
could include Archaea, many of which can use Fe (III) as an electron acceptor (Vargas et
289
al., 1998; Kashefi and Lovley, 2003; Yamada et al., 2014), in addition to participating in
290
DIET.
291
Advancing the study of microbial nanowires will require the continued
292
collaboration of microbiologists, biophysicists, structural biologists, biochemists, and
13 This article is protected by copyright. All rights reserved.
ecologists. The potential benefits of such studies include not only a better understanding
294
of the natural world, but also the development of exciting new parts for the emerging
295
fields of electromicrobiology, bioelectronics, and biocomputing.
Accepted Article
293
296 297
Acknowledgements
298
The authors research on microbial nanowires are supported by Office of Naval Research
299
grants N000141210229, N00014-13-1-0550, and N000141310549. The authors declare
300
no conflicts of interest.
301 302
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488
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Accepted Article
498
499 500
Figure 1. Imaging charge flow with electrostatic force microscopy. (A) Charge
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delocalization in carbon nanotubes [Reprinted with permission from (Zdrojek et al.,
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2006) Copyright (2006) by the American Physical Society]. The charges were injected at
503
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of the injected charges. (B) Charge propagation in pili of G. sulfurreducens [Figure
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adapted from (Malvankar et al., 2014b) with permission]. The charges were injected at
506
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507
the injected charges along the pilus, which also propagated into another pilus that was in
25 This article is protected by copyright. All rights reserved.
contact with the charge-injected pilus. Charge did not propagate across a break
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introduced in the pilus highlighted in red.
Accepted Article
508
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