Electrically Conductive Pili from Pilin Genes of Phylogenetically Diverse Microorganisms

The ISME Journal (2018) 12, 48–58 © 2018 International Society for Microbial Ecology All rights reserved 1751-7362/18 www.nature.com/ismej ORIGINAL ARTICLE Electrically conductive pili from pilin genes of phylogenetically diverse microorganisms David JF Walker1, Ramesh Y Adhikari2,4,5, Dawn E Holmes1,3, Joy E Ward1, Trevor L Woodard1, Kelly P Nevin1 and Derek R Lovley1 1Department of Microbiology, University of Massachusetts, Amherst, MA, USA; 2Department of Physics, University of Massachusetts, Amherst, MA, USA; 3Department of Physical and Biological Sciences, Western New England University, Springfield, MA, USA and 4Department of Physics, Jacksonville University, Jacksonville, FL, USA The possibility that bacteria other than Geobacter species might contain genes for electrically conductive pili (e-pili) was investigated by heterologously expressing pilin genes of interest in Geobacter sulfurreducens. Strains of G. sulfurreducens producing high current densities, which are only possible with e-pili, were obtained with pilin genes from Flexistipes sinusarabici, Calditerrivibrio nitroreducens and Desulfurivibrio alkaliphilus. The conductance of pili from these strains was comparable to native G. sulfurreducens e-pili. The e-pili derived from C. nitroreducens, and D. alkaliphilus pilin genes are the first examples of relatively long (4100 amino acids) pilin monomers assembling into e-pili. The pilin gene from Candidatus Desulfofervidus auxilii did not yield e-pili, suggesting that the hypothesis that this sulfate reducer wires itself with e-pili to methane-oxidizing archaea to enable anaerobic methane oxidation should be reevaluated. A high density of aromatic amino acids and a lack of substantial aromatic-free gaps along the length of long pilins may be important characteristics leading to e-pili. This study demonstrates a simple method to screen pilin genes from difficult-to-culture microorganisms for their potential to yield e-pili; reveals new sources for biologically based electronic materials; and suggests that a wide phylogenetic diversity of microorganisms may use e-pili for extracellular electron exchange. The ISME Journal (2018) 12, 48–58; doi:10.1038/ismej.2017.141; published online 5 September 2017 Introduction that the e-pili of both G. sulfurreducens and G. metallireducens are sufficiently conductive along Electrically conductive pili (e-pili) can have an their length, under physiologically relevant condi- important role in the biogeochemical cycling of tions, to account for maximum potential rates of carbon and metals and have potential applications ‘ ’ extracellular electron transfer (Adhikari et al., 2016; as green electronic materials (Lovley, 2011; Lampa-Pastirk et al., 2016; Tan et al., 2016b, 2017). Malvankar and Lovley, 2014; Lovley and Geobacter strains that lack e-pili, or express pili with Malvankar, 2015; Shi et al., 2016; Lovley, 2017a, diminished conductivities, are incapable of direct b, c). Therefore, a better understanding of the interspecies electron transfer (DIET) and Fe(III) oxide diversity of microorganisms capable of producing reduction (Reguera et al., 2005; Summers et al., e-pili has many potential benefits. To date, e-pili 2010; Tremblay et al., 2012; Shrestha et al., 2013; have been most intensively studied in Geobacter Vargas et al., 2013; Liu et al., 2014; Rotaru et al., sulfurreducens and the closely related Geobacter 2014a, b; Tan et al., 2016b). e-Pili are also required metallireducens (Reguera et al., 2005; Malvankar for Geobacter species to generate high current et al., 2011, 2014, 2015; Vargas et al., 2013; densities when electrodes serve as the sole electron Feliciano et al., 2015; Adhikari et al., 2016; Lampa- acceptor (Reguera et al., 2006; Vargas et al., 2013; Liu Pastirk et al., 2016; Xiao et al., 2016; Tan et al., et al., 2014; Tan et al., 2016b). Increasing e-pili 2016b, 2017). These studies have demonstrated expression increases the conductivity of electrode biofilms and their current output (Yi et al., 2009; Correspondence: DR Lovley, Department of Microbiology, Uni- Malvankar et al., 2011, 2012; Leang et al., 2013). versity of Massachusetts, Morrill IV N Science Center, Amherst, Aromatic amino acids are key components in MA 01003, USA. electron transfer along the length of G. sulfurredu- E-mail: [email protected] cens e-pili (Vargas et al., 2013; Adhikari et al., 2016; 5 Current Address: Department of Physics, Jacksonville University, Lampa-Pastirk et al., 2016; Tan et al., 2016a, 2017). Jacksonville, FL, USA. Received 17 March 2017; revised 17 June 2017; accepted 25 July There is debate whether the aromatic amino acids 2017; published online 5 September 2017 contribute to a traditional electron-hopping electron Diverse electrically conductive pili DF Walker et al 49 transport mechanism or whether overlapping π–π syntrophically and pili-like filaments were abundant orbitals of the aromatics confer a metallic-like in the co-culture (Wegener et al., 2015). It was conductivity similar to that observed in synthetic proposed that the pili of the sulfate reducer were conducting polymers (Malvankar et al., 2011, 2014, conductive (Wegener et al., 2015), but this was not 2015; Vargas et al., 2013; Malvankar and Lovley, verified, possibly due to difficulties in obtaining 2014; Lovley and Malvankar, 2015; Lampa-Pastirk sufficient biomass. et al., 2016). However, the uncertainty over these The properties of proteins encoded in genomes of mechanistic details should not obscure the fact that difficult-to-culture or as-yet-uncultured microorgan- long-range electron transport via e-pili is a remark- isms can often be determined by expressing the able strategy for long-range biological electron genes of interest in more readily cultivated micro- transfer. organisms (Handelsman, 2004). It is possible to It has been speculated that the relatively small size screen pilin genes to determine whether they have (61 amino acids) of the G. sulfurreducens pilin is a the potential to yield pilin proteins that can assemble key feature contributing to its assembly into e-pili into e-pili by heterologously expressing the pilin (Reguera et al., 2005; Malvankar et al., 2015; Holmes gene of interest in G. sulfurreducens in place of the et al., 2016). Pili comprised of larger pilins, such as native G. sulfurreducens pilin gene (Vargas et al., those found in Shewanella oneidensis (Reguera 2013; Liu et al., 2014; Tan et al., 2016a, b, 2017). et al., 2005), Pseudomonas aeruginosa (Reguera Only if the pilin gene yields e-pili is G. sulfurredu- et al., 2005; Liu et al., 2014) and G. uraniireducens cens then capable of producing high current den- (Tan et al., 2016b) have poor conductivity. On the sities on graphite electrodes. For example, pili of basis of these results, it was proposed that smaller P. aeruginosa are poorly conductive (Reguera et al., pilins might permit the tight packing of aromatic 2005) and a strain of G. sulfurreducens expressing amino acids required for conductivity (Malvankar the pilin gene of P. aeruginosa produced low current et al., 2015; Xiao et al., 2016). However, previously densities (Liu et al., 2014). Expression of the pilin investigated poorly conductive pili comprised of gene from G. metallireducens in G. sulfurreducens longer pilins have either (1) a lower overall density yielded e-pili that were even more conductive than of aromatic amino acids than those found in e-pili the e-pili originating from the native G. sulfurredu- (that is, P. aeruginosa and S. oneidensis) or (2) a large cens pilin gene (Tan et al., 2017). In contrast, the gap within the pilin sequence devoid of aromatic pilin gene of G. uraniireducens, which is phylogen- amino acids, such as observed in the pilin of etically distinct from the G. sulfurreducens and G. uraniireducens (Figure 1). These considerations G. metallireducens pilin genes (Holmes et al., suggest that the conductivity of a broader diversity of 2016), yielded poorly conductive pili in G. sulfurre- pili should be investigated. ducens (Tan et al., 2016b). As expected, the G. Evaluating pili conductivity is technically challen- sulfurreducens strain expressing the G. uraniiredu- ging, especially with difficult-to-culture microorgan- cens pilin genes produced low current densities (Tan isms. For example, it was suggested that e-pili were et al., 2016b) as did a G. sulfurreducens strain involved in DIET in an enriched consortium that expressing a synthetic pilin gene designed to yield contained a methane-oxidizing archaeon and its poorly conductive pili (Vargas et al., 2013). The poor sulfate-reducing partner (Wegener et al., 2015). The conductivity of the G. uraniireducens pili was sulfate-reducing partner highly expressed a pilin consistent with the finding that G. uraniireducens gene when the two microorganisms were growing uses an electron shuttle for Fe(III) reduction and is Figure 1 Comparison of aromatic amino-acid placement (red rectangles) within mature pilin protein sequences of G. sulfurreducens, G. metallireducens, F. sinusarabici, C. nitroreducens, D. alkaliphilus, S. oneidensis, P. aeruginosa, G. uraniireducens and Candidatus Desulfofervidus auxilii. Also shown are the total number of amino acids, the number of aromatic amino acids and the percentage of total amino acids that are aromatic. The ISME Journal Diverse electrically conductive pili DF Walker et al 50 incapable of producing high current densities on sequences. All positions with o95% site coverage graphite electrodes (Rotaru et al., 2015; Tan et al., were eliminated.

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