Skip to main content
PMC home page
NASA Author Manuscripts logoLink to NASA Author Manuscripts
. Author manuscript; available in PMC: 2021 Jan 3.
Published in final edited form as: Environ Microbiol Rep. 2019 Nov 21;12(1):49–57. doi: 10.1111/1758-2229.12809

Phylogenetic and structural diversity of aromatically dense pili from environmental metagenomes

Marcus S Bray 1,2,†, Jieying Wu 1, Cory C Padilla 1,‡, Frank J Stewart 1, David A Fowle 3, Cynthia Henny 4, Rachel L Simister 5,6, Katharine J Thompson 5,6, Sean A Crowe 2,5,6,7, Jennifer B Glass 1,2,*
PMCID: PMC7779115  NIHMSID: NIHMS1657953  PMID: 31701641

Summary

Electroactive type IV pili, or e-pili, are used by some microbial species for extracellular electron transfer. Recent studies suggest that e-pili may be more phylogenetically and structurally diverse than previously assumed. Here, we used updated aromatic density thresholds (≥9.8% aromatic amino acids, ≤22-aa aromatic gaps and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57) to search for putative e-pilin genes in metagenomes from diverse ecosystems with active microbial metal cycling. Environmental putative e-pilins were diverse in length and phylogeny, and included truncated e-pilins in Geobacter spp., as well as longer putative e-pilins in Fe(II)-oxidizing Betaproteobacteria and Zetaproteobacteria.

Introduction

Electroactive microbes transport electrons through cell membranes into the extracellular environment (Sydow et al., 2014; Koch and Harnisch, 2016; Logan et al., 2019). These microbes play important roles in biogeochemical cycles in soils and sediments, bioremediation of toxic metals and energy generation in microbial fuel cells (Lovley, 1991; Lovley and Coates, 1997; Logan, 2009; Lovley, 2011; Mahadevan et al., 2011). Electroactive Deltaproteobacteria in the genus Geobacter (order Desulfuromonadales) perform long-range extracellular electron transfer (EET) through electroactive pili (e-pili), composed of e-pilin structural subunits (Lovley, 2017; Lovley and Walker, 2019). Geobacter use e-pili for Fe(III) respiration, direct interspecies electron transfer (DIET), and growth on anodes (Reguera et al., 2005; Reguera et al., 2006; Rotaru et al., 2014).

Geobacter e-pili belong to the larger family of type IV-a pilins (T4aPs), which are broadly distributed in Bacteria and Archaea (Imam et al., 2011; Giltner et al., 2012; Berry and Pelicic, 2015). T4aPs have evolved to perform diverse cellular functions, including twitching motility, attachment and genetic transformation. Most characterized Geobacter e-pilins are truncated versions of canonical T4aPs (Holmes et al., 2016). Type II (or ‘pseudopilin’) proteins are structurally similar to, but phylogenetically distinct from T4aPs, and assemble into type II secretion (T2S) systems instead of pili (Ayers et al., 2010).

Aromatic amino acid density seems to be essential for efficient electron transport in e-pili (Vargas et al., 2013; Liu et al., 2014; Liu et al., 2019). The close packing of aromatic residues within the pilus likely facilitates EET (Reardon and Mueller, 2013; Feliciano et al., 2015; Lovley, 2017). In particular, Phe1, Tyr24, and Tyr27 are key residues (Xiao et al., 2016), and Tyr32, Phe51, and Tyr57 also play important roles (Liu et al., 2019). The most conductive e-pilus measured to date is that of Geobacter metallireducens, which contains pilins that are 59 aa in mature length (after signal peptide sequence removal at the prepilin cleavage site) and comprised of 15.3% aromatics and no aromatic-free gaps >22 aa (Table S1). The G. metallireducens e-pilus is 5000 times more conductive than the Geobacter sulfurreducens e-pilus, which has pilins that are 61 aa in mature length and comprised of 9.8% aromatics and no aromatic-free gaps >22 aa (Tan et al., 2017). The G. sulfurreducens e-pilus is 100 times more conductive than the Geobacter uraniireducens pilus, which contains much longer pilins (193 aa), 9.1% aromatics, and a 53 aa aromatic-free gap (Tan et al., 2016). Non-electroactive T4aPs are thought to be incapable of electroactivity due to insufficient aromatic residue packing (Feliciano et al., 2015; Malvankar et al., 2015; Kolappan et al., 2016). To our knowledge, the most aromaticrich predicted e-pilus belongs to Desulfobacula phenolica (16.9%; Holmes et al., 2016).

Multiheme cytochromes (MHCs) are also involved in EET. Outer membrane MHCs move electrons from the periplasm into the extracellular environment (Aklujkar et al., 2013). The hexaheme OmcS can localize with Geobacter e-pili (Leang et al., 2010; Vargas et al., 2013; Liu et al., 2014). Conductive filaments comprised solely of OmcS were recovered from outer-membrane preparations of G. sulfurreducens grown in microbial fuel cells (Filman et al., 2019; Wang et al., 2019), but substantial evidence suggests that e-pilins in wild-type Geobacter cultures are comprised of PilA (Lovley and Walker, 2019).

Recently, the phylogenetic and structural diversity of e-pili has expanded beyond Geobacter spp. with the discovery of strongly conductive pili in clades outside of Geobacter genera, including Syntrophus aciditrophicus (Deltaproteobacteria/Syntrophobacterales), Desulfurivibrio alkaliphilus (Deltaproteobacteria/Desulfobacterales), Calditerrivibrio nitroreducens (Deferribacteres), and the archaeon Methanospirillum hungatei (Euryarchaeota/Methanomicrobiales) (Walker et al., 2018, 2019a, 2019b) (Table S1). Pilin genes in these four microbes are much longer (110–182 aa) than in Geobacter spp. but have similar aromaticity (11%–13%) and similar maximum aromatic-free gaps (22–35 aa). Pili from Desulfofervidus auxilii, Shewanella oneidensis, and Pseudomonas aeruginosa with minimal conductance have lower aromaticity (5.6%–6.8%) and larger aromatic-free gaps (42–52 aa; Reguera et al., 2005; Liu et al., 2014; Walker et al., 2018). Therefore, it seems that aromatic density, defined here as percentage of aromatic amino acids and spacing of aromatic residues in the pilin sequence, is the key factor for identifying putative e-pilins based on sequence similarity (Walker et al., 2019a). In this study, we searched metagenomes from metal-rich environments and enrichment cultures for putative e-pilins based on aromatic density and spacing.

Results

Aromatic density and spacing distinguishes e-pilins from non-conductive T4aPs

We obtained published sequences for seven biochemically confirmed e-pilins, four non-conductive pilins (Table S1) and 35 functionally verified attachment/motility/competence T4aPs (Table S2). Biochemically confirmed e-pilins had mature lengths of 59–182 aa, 9.8%–16.9% aromatics, and maximum aromatic-free gaps of 22–35 aa (Fig. 1; Table S1). Pilins implicated in functions other than long-range EET had 93–208 aa mature lengths, 3.5%–11.0% aromatics, and 22–75 aa aromatic-free gaps (Fig. 1; Table S2). Sequence alignments showed that all bacterial e-pilins contained Phe1, Tyr24, Tyr27, and Tyr/Phe51. Most also contained an aromatic amino acid (Tyr or Phe) at residues 32, 50, and 57. Therefore, we used ≥9.8% aromatics, ≤22-aa aromatic-free gap, and the presence of aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57 as a conservative threshold for predicting putative e-pilins from metagenomes, consistent with thresholds established by Walker et al. (2019a). Using these thresholds, two T4aPs in Table S2 were predicted to be conductive: G. sulfurreducens OxpG, which forms a T2S system required for reduction of insoluble Fe(III) (Mehta et al., 2006), and Dichelobacter nodosus PilE, which is required for extracellular protease secretion and competence (Han et al., 2007).

Fig. 1.

Fig. 1.

Open in a new tab

Basis for distinguishing e-pilins from other pilins. Top: Alignment showing the location of aromatic residues in each pilin tested for conductivity in previous studies (Table S1). Dark horizontal lines indicate 42–53 aa aromatic-free gaps in non-conductive pilins. Conserved N-terminal aromatic residues in bacterial e-pilins are indicated by vertical boxes. All bacterial e-pilins contained F-1, Y-24, Y-27, Y/F-51, Y/F-50 and/or Y/F-51, and H/Y/F-32 and/or Y/F-57. The only N-terminal residues shared by archaeal and bacterial e-pilins were F-1 and Y-57. Bottom: Relationship between gap size and percentage of aromatic amino acids in the mature pilin peptide for four types of pilins, which was used to establish conservative criteria for identifying putative e-pilins in environmental metagenomes. Therefore, we used ≥9.8% aromatics and ≤22-aa aromatic-free gap (boxed area labelled ‘conductive e-pilins’), and the presence of aromatic amino acids at residues as a conservative threshold for predicting putative e-pilins from metagenomes, consistent with thresholds established by Walker et al. (2019a). Additional information about each pilin is in Tables S1 and S2.

Putative e-pilins are present in ferruginous environments

We used the G. sulfurreducens e-pilin to query metagenomic contigs or metagenome-assembled genomes (MAGs) from environments with conditions amenable to metal respiration. We included metagenomes from ferruginous sediments from two lakes, Lake Matano and Lake Towuti, in the Malili Lakes system on Sulawesi, Indonesia, and the ferruginous water column from Kabuno Bay, Lake Kivu, Democratic Republic of Congo. These permanently stratified tropical lakes host one of the largest ferruginous environments on modern Earth with abundant iron-cycling microbes likely capable of EET (Crowe et al., 2007; Vuillemin et al., 2016). Other environments included deep groundwaters from Sweden (Asop Hard Rock), Japan (Horonobe Underground Laboratory), USA (Rifle, Colorado) and the North Atlantic (North Pond marine aquifer). We also included putative e-pilins from year-long laboratory incubations inoculated with Lake Matano sediment amended with Fe(III) or Mn(III) (see Experimental Procedures).

We screened the retrieved amino acid sequences for T4Ps using Pilfind (Imam et al., 2011), and the aromatic density thresholds established above (≥9.8% aromatic amino acids, ≤22-aa aromatic gaps and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57). After partial sequences were removed, we recovered putative e-pilins ranging from 58 to 162 aa mature length with 9.8%–15.5% aromatic density (Table S3; Supplemental Data File).

Widening the phylogenetic diversity of putative e-pilins

To determine the phylogenetic diversity of environmental e-pilins, we constructed a maximum likelihood tree from an alignment of the T4aP amino acid sequences described above, as well as additional predicted Deltaproteobacteria e-pilins from cultured species (Holmes et al., 2016; Walker et al., 2018) and BLAST searches (Fig. 2). Methanospirillum hungatei e-pilin was used as the outgroup. The T4aP phylogeny was broadly consistent with previous findings (Holmes et al., 2016; Walker et al., 2018). All truncated e-pilins and all confirmed bacterial e-pilins clustered with Deltaproteobacteria. Non-conductive Gammaproteobacteria pilins and T2S pseudopilins fell on separate branches. Truncated Desulfuromonadales e-pilins (∼60 aa) formed their own branch within the Deltaproteobacteria cluster. Other branches on the Deltaproteobacteria cluster contained recently discovered e-pilins from Desulfobacterales, Deferribacteres, and Syntrophobacterales. Roughly half of environmental putative e-pilins clustered with Deltaproteobacteria, including two putative e-pilins from native Lake Matano sediment and six putative e-pilins from >1 year anoxic incubations of Lake Matano sediments with Fe(III) oxides (Table S3; Supplemental Data File). Putative e-pilins from marine Zetaproteobacteria (Mariprofundus micogutta and two MAGs from the North Pond marine subsurface aquifer) and Nitrospinae (Crystal Geyser, Utah, USA) also clustered with Deltaproteobacteria e-pilins.

Fig. 2.

Fig. 2.

Open in a new tab

Maximum likelihood phylogenetic tree of pilin sequences. Methanomicrobiales archaellum was used as the outgroup. Asterisks indicate putative e-pilins from enrichment cultures. Dashed lines indicate sequences that do not meet the criteria for e-pili. Double lines indicate that the branch length was shortened to fit inside the figure boundaries. The gene order for Betaproteobacteria putative e-pilins (pilE) is shown. See Supplemental Data File for details on each sequence and full names of species. Table S2 provides characteristics of type IV pilins and type II secretion (T2S) pseudopilins involved in functions other than EET. Environmental abbreviations: A, Asop; CG, Crystal Geyser; K, Kabuno; M, Matano; Mc, McNutt; R, Rifle; T, Towuti.

Approximately half of environmental putative e-pilins fell outside the Deltaproteobacteria cluster on the T4aP phylogeny (Fig. 2). Eight unique putative e-pilin sequences (found 29 times in Kabuno Bay metagenomes), and one e-pilin from McNutt Creek (Georgia, USA), formed a distinct phylogenetic cluster with pilE genes from cultured Betaproteobacteria (Gallionella, Leptothrix, Methylotenera, Sulfuricella, Thauera, and Dechloromonas), Gallionellales MAGs from groundwater, and a Rhodocyclales MAG from Lake Matano enrichment cultures (311FMe.001; NCBI genome accession VAUH01000000). Betaproteobacteria PilE sequences in this clade contained 10.1%–13.5% aromatics, ≤22-aa aromatic-free gaps and key aromatic residues at positions 1, 24, 27, 50, 51, and 57. In all cases, putative Betaproteobacteria pilE genes were followed by fimT-pilVWXY1, which encode minor pilin assembly proteins (Nguyen et al., 2015).

Several putative environmental e-pilins clustered with non-conductive pilins from Deltaproteobacteria, Gammaproteobacteria, and Firmicutes. These putative e-pilins were from MAGs belonging to the candidate phylum Dependentiae (formerly TM6) from Rifle groundwater, Alteromonas NORP73 from North Pond marine subsurface aquifer, Gammaproteobacteria HGW15 from Horonobe Underground Laboratory, and Proteobacteria CG-11 from Crystal Geyser. The G. sulfurreducens OxpG, two sequences from Lake Matano enrichment cultures, and Omnitrophica sequences from Crystal Geyser were located on the same branch as the outgroup. Omnitrophica have been implicated in anaerobic respiration with metals (Hernsdorf et al., 2017) or sulfite (Anantharaman et al., 2018). To assess potential capacity for metal reduction, we searched MAGs that contained putative e-pilins for outer membrane/extracellular MHCs. Notably, the Omnitrophica MAG contained 10 putative MHCs located adjacent to each other in the genome, three of which were predicted to be extracellular or outer-membrane MHCs, each with 11 or 13 hemes (Fig. S1).

Discussion

We recovered genes that meet in silico requirements for conductivity based on aromatic density and spacing, both inside and outside of the well-established Deltaproteobacteria cluster. Our phylogenetic analyses suggest that the Deltaproteobacteria e-pilin genes have undergone more extensive horizontal gene transfer (HGT) than previously known. Our results suggest that truncated e-pilins are limited to the Deltaproteobacteria cluster, whereas predicted e-pilins outside of Deltaproteobacteria were full-length. In addition to their previously recognized HGT to several Deferribacteres species (Holmes et al., 2016; Walker et al., 2018), we found putative e-pilins that clustered with Deltaproteobacteria in MAGs from Nitrospinae and Zetaproteobacteria. Nitrospinae are chemoautotrophic nitrite oxidizers that have not, to our knowledge, previously been implicated in EET. Zetaproteobacteria, the dominant marine Fe(II) oxidizers, were known to possess pilA genes, but the gene products were previously classified as non-conductive because they are >100 aa in length (He et al., 2017). Given the recent discovery of conductive e-pili with >100 aa (Walker et al., 2018), the possible occurrence of e-pilins in Zetaproteobacteria such as Mariprofundus micogutta needs to be re-evaluated.

Outside of the Deltaproteobacteria cluster, several putative e-pilin genes clustered with non-conductive Gammaproteobacteria pilins. Alteromonas are known to reduce Fe(III) and form electroactive biofilms (Vandecandelaere et al., 2008), but have not previously, to our knowledge, been found to possess e-pilins. The findings suggest that non-conductive full-length pilins may be capable of evolving conductive properties, although this awaits experimental validation.

Putative e-pilins were also found associated with clades not previously known to possess e-pili. Kabuno Bay metagenomes contained abundant e-pilin sequences most similar to those found in metabolically diverse Betaproteobacteria genera, including Gallionella, Leptothrix, Methylotenera, Sulfuricella, Thauera, and Dechloromonas. These putative e-pilin genes were classified as pilE and were followed by genes involved in minor pilus assembly. Putative Betaproteobacteria e-pili genes were also found in other groundwater MAGs, including Crystal Geyser, where Gallionellaceae are among the most abundant bacteria (Probst et al., 2018).

While the aromatically dense pilins in this study met the bioinformatic thresholds for e-pili, it is possible that they are used for another function, such as DIET (Holmes et al., 2017; Walker et al., 2019a) or cellular detection of solid surfaces via electrical communication (Lovley, 2017). Evaluation of the conductivity of the putative e-pilins awaits testing by genetic complementation of ΔpilA in G. sulfurreducens, as in Walker et al. (2018).

Conclusions

This study identified putative e-pilins in the environment using aromatic density and gaps as the predictive tool, building off of previous studies that established the conductivity of longer PilA proteins (Walker et al., 2018). The sequences we recovered suggest that e-pilins are both phylogenetically and structurally diverse. We conclude that e-pili may be composed of pilin monomers of a variety of lengths and aromatic densities, and that diverse bacteria, including Fe(II)-oxidizing Betaproteobacteria and Zetaproteobacteria, may use e-pili for EET or possibly other unknown functions.

Experimental procedures

Sampling and enrichment of Lake Matano sediment

Two sediment cores were obtained from 590 m water depth in Lake Matano, Sulawesi Island, Indonesia in May 2010 (2°28′ S, 121°20′ E, in situ sediment temperature ∼27°C) and stored under anoxic conditions. The sediments were mixed with anoxic freshwater media in a 1:5 ratio in an anoxic chamber and dispensed in stoppered serum bottles, as in Bray et al. (2017). Cultures were amended first with goethite and later with ferrihydrite. They were incubated for 490 days at 30°C, with multiple transfers, each time diluting the original sediment with freshwater media. Sediment had been diluted over 1000-fold by the time DNA was extracted for sequencing. Details on metagenomes from 395-day anoxic enrichments of Lake Matano sediment incubated with Mn(III) pyrophosphate are reported in a separate publication (Szeinbaum et al., 2019).

DNA extraction and metagenome sequencing, assembly, binning and annotation

Community DNA from Lake Matano sediment enrichments was extracted from 2 g samples and purified using a PowerSoil Isolation Kit and UltraClean® 15 Purification Kit (formerly MO BIO Laboratories, now Qiagen, Carlsbad, CA, USA) following the manufacturer’s protocol. Indexed libraries were created from purified community DNA using the NexteraXT DNA Sample Prep kit (Illumina, San Diego, CA, USA) following manufacturer’s instructions. Libraries were pooled and sequenced on two runs of an Illumina MiSeq using a 500 cycle (paired end 250 × 250 bp) kit. Illumina reads were quality trimmed using Trim Galore! (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with a quality score and minimum length cut-off of Q25 and 100 bp respectively, and merged with FLASH with the shortest overlap of 25 bp. Barcoded sequences were de-multiplexed, trimmed (length cut-off 100 bp) and filtered to remove low quality reads (average Phred score < 25) using Trim Galore!. Forward and reverse reads were assembled using SPAdes (Nurk et al., 2013) with the ‘meta’ option. The number of contigs, contig length, GC content, N50 and L50 assembly statistics were calculated with metaQUAST (Mikheenko et al., 2015). Raw sequence reads and all genomic bins were deposited in NCBI under the accession number PRJNA505658.

E-pilin identification from microbial metagenomes

Environmental metagenomes and MAGs were downloaded from IMG-JGI and NCBI (see Table S1 for taxon object IDs). For all metagenomes, Prodigal (Hyatt et al., 2010) was used to predict genes from contig files and write them to amino acid FASTA files. Amino acid sequences from MAGs were downloaded directly from NCBI. Predicted protein files were then used as databases for protein BLAST, using the G. sulfurreducens PilA protein as query. Hits with a bit score greater than 55 were pulled from the databases. These recovered sequences were then further verified as T4P using Pilfind (http://signalfind.org/pilfind.html), a web tool that identifies type IV pilin signal sequences (Imam et al., 2011). Pilin amino acid sequences were then run through a python script that calculated the mature pilin length, percent aromatic amino acids and aromatic free gaps (https://github.com/GlassLabGT/Python-scripts). Partial genes were retained if truncated on the N-terminus before the signal peptide and removed if truncated on the C-terminus. Remaining sequences were manually screened for the presence of aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57.

Pilin multiple sequence alignment and phylogenetic analysis

Identified pilin amino acid sequences were aligned using MUSCLE and a maximum likelihood tree was constructed using MEGA. The alignment is provided as Supporting Information. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model (Jones et al., 1992). Archaeal pili from M. hungatei and two other Methanomicrobiales were used for the outgroup. The tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. There were 52 positions total in the final data set. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).

MHC analysis

Groundwater MAGs in which we identified aromatically dense pilins were further probed for the presence of MHC proteins. Amino acids files were run through the ‘cytochrome_stats.py’ described in Badalamenti et al. (2016) available at https://github.com/bondlab/scripts, which identifies proteins with three or more cytochrome-binding motifs (Cxx(x)CH).

Supplementary Material

Supplemental Material

Table S1. Characteristics of predicted or confirmed e-pilin proteins, and non-conductive pilins, in order of increasing aromatic density, from cultured isolates, based on Holmes et al., 2016 and Walker et al., 2018. These proteins listed as ‘U’ are predicted to infer long-range electron transport capability in the listed species, based on the criteria listed in the paper. Biochemically confirmed e-pilins are bolded.

Table S2. Characteristics of type IV pilins and pseudopilins involved in functions other than long-range electron transport, in order of increasing aromatic density. A: attachment; C: competence; F: fibril assembly; M: motility; S: secretion of proteins.

Table S3. Putative e-pilin genes identified from environmental metagenomes and long-term enrichment of Lake Matano sediment in the presence of metal oxides. Putative e-pilins are defined as containing (≥9.8% aromatic amino acids, ≤22-aa aromatic gaps, and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57. See Supplemental Data File for sequence and details about each putative e-pilin.

Fig. S1. Putative operon with multiple extracellular multiheme cytochromes found in Candidatus Omnitrophica bacterium CG11_big_fil_rev_8_21_14_0_20_42_13. Numbers represent number of cytochromes in each gene product. Predicted protein locations based on PSORTb analysis. E: extracellular; OM: outer membrane; P: periplasmic; C: cytoplasmic; CM: cytoplasmic membrane. NCBI accession numbers for each protein (PIQ89515-PIQ89529) are labelled beneath each gene.

Appendix S1. Supporting Information.

ACKNOWLEDGEMENTS

This research was funded by NASA Exobiology grant NNX14AJ87G with support from the NASA Astrobiology Institute (NNA15BB03A). S.A.C., R.L.S., and K.J.T. were supported through NSERC Discovery grant 0487, CFI projects 229652 and 36071, and the Canada Research Chairs program. We thank Bianca Costa, Miles Mobley, Benjamin Reed, and Johnny Striepen for assistance with laboratory incubations, and Sean Elliott and Betül Kaçar for helpful discussions. This research was carried out with permission from the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia (Ritekdikti), the Natural Resources Conservation Center (BKSDA), and The government of Luwu Timur of Sulawesi, Indonesia. We also acknowledge support from the director of the Research Center for Limnology (RCL)-Indonesian Institute of Sciences (LIPI), Tri Widiyanto (RCL-LIPI), Aan Diyanto (RCL-LIPI) and staff of RCL-LIPI.

Footnotes

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

References

  1. Aklujkar M, Coppi M, Leang C, Kim B, Chavan M, Perpetua L, et al. (2013) Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens. Microbiology 159: 515–535. [DOI] [PubMed] [Google Scholar]
  2. Anantharaman K, Hausmann B, Jungbluth SP, Kantor RS, Lavy A, Warren LA, et al. (2018) Expanded diversity of microbial groups that shape the dissimilatory sulfur cycle. ISME J 12: 1715–1728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ayers M, Howell PL, and Burrows LL (2010) Architecture of the type II secretion and type IV pilus machineries. Future Microbiol 5: 1203–1218. [DOI] [PubMed] [Google Scholar]
  4. Badalamenti JP, Summers ZM, Chan CH, Gralnick JA, and Bond DR (2016) Isolation and genomic characterization of ‘Desulfuromonas soudanensis WTL’, a metal-and electrode-respiring bacterium from anoxic deep subsurface brine. Front Microbiol 7: 913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berry J-L, and Pelicic V. (2015) Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol Rev 39: 134–154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bray MS, Wu J, Reed BC, Kretz CB, Belli KM, Simister RL, et al. (2017) Shifting microbial communities sustain multi-year iron reduction and methanogenesis in ferruginous sediment incubations. Geobiology 15: 678–689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crowe SA, O’Neill AH, Kulczycki E, Weisener CG, Roberts JA, and Fowle DA (2007) Reductive dissolution of trace metals from sediments. Geomicrobiol J 24: 157–165. [Google Scholar]
  8. Feliciano G, Steidl R, and Reguera G. (2015) Structural and functional insights into the conductive pili of Geobacter sulfurreducens revealed in molecular dynamics simulations. Phys Chem Chem Phys 17: 22217–22226. [DOI] [PubMed] [Google Scholar]
  9. Filman DJ, Marino SF, Ward JE, Yang L, Mester Z, Bullitt E, et al. (2019) Cryo-EM reveals the structural basis of long-range electron transport in a cytochromebased bacterial nanowire. Commun Biol 2: 219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Giltner CL, Nguyen Y, and Burrows LL (2012) Type IV pilin proteins: versatile molecular modules. Microbiol Mol Biol Rev 76: 740–772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Han X, Kennan RM, Parker D, Davies JK, and Rood JI (2007) Type IV fimbrial biogenesis is required for protease secretion and natural transformation in Dichelobacter nodosus. J Bacteriol 189: 5022–5033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. He S, Barco RA, Emerson D, and Roden EE (2017) Comparative genomic analysis of neutrophilic iron (II) oxidizer genomes for candidate genes in extracellular electron transfer. Front Microbiol 8: 1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hernsdorf AW, Amano Y, Miyakawa K, Ise K, Suzuki Y, Anantharaman K, et al. (2017) Potential for microbial H2 and metal transformations associated with novel bacteria and archaea in deep terrestrial subsurface sediments. ISME J 11: 1915–1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holmes DE, Dang Y, Walker DJ, and Lovley DR (2016) The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microb Genomics 2: e000072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holmes DE, Shrestha PM, Walker DJ, Dang Y, Nevin KP, Woodard TL, and Lovley DR (2017) Meta-transcriptomic evidence for direct interspecies electron transfer between Geobacter and Methanothrix species in methanogenic rice paddy soils. Appl Environ Microbiol 83: e00223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, and Hauser LJ (2010) Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11: 119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Imam S, Chen Z, Roos DS, and Pohlschröder M. (2011) Identification of surprisingly diverse type IV pili, across a broad range of Gram-positive bacteria. PloS One 6: e28919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones DT, Taylor WR, and Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Bioinformatics 8: 275–282. [DOI] [PubMed] [Google Scholar]
  19. Koch C, and Harnisch F. (2016) Is there a specific ecological niche for electroactive microorganisms? ChemElectroChem 3: 1282–1295. [Google Scholar]
  20. Kolappan S, Coureuil M, Yu X, Nassif X, Egelman EH, and Craig L. (2016) Structure of the Neisseria meningitidis type IV pilus. Nat Commun 7: 13015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kumar S, Stecher G, and Tamura K. (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leang C, Qian X, Mester T, and Lovley DR (2010) Alignment of the c-type cytochrome OmcS along pili of Geobacter sulfurreducens. Appl Environ Microbiol 76: 4080–4084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu X, Tremblay P-L, Malvankar NS, Nevin KP, Lovley DR, and Vargas M. (2014) A Geobacter sulfurreducens strain expressing Pseudomonas aeruginosa type IV pili localizes OmcS on pili but is deficient in Fe (III) oxide reduction and current production. Appl Environ Microbiol 80: 1219–1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu X, Wang S, Xu A, Zhang L, Liu H, and Ma LZ (2019) Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa. Appl Microbiol Biotechnol 103: 1535–1544. [DOI] [PubMed] [Google Scholar]
  25. Logan BE (2009) Exoelectrogenic bacteria that power microbial fuel cells. Nat Rev Microbiol 7: 375–381. [DOI] [PubMed] [Google Scholar]
  26. Logan BE, Rossi R, and Saikaly PE (2019) Electroactive microorganisms in bioelectrochemical systems. Nat Rev Microbiol 17: 307–319. [DOI] [PubMed] [Google Scholar]
  27. Lovley DR (1991) Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol Rev 55: 259–287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lovley DR (2011) Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of energy-related contamination. Energ Environ Sci 4: 4896–4906. [Google Scholar]
  29. Lovley DR (2017) Electrically conductive pili: biological function and potential applications in electronics. Curr Opin Electrochem 4: 190–198. [Google Scholar]
  30. Lovley DR, and Coates JD (1997) Bioremediation of metal contamination. Curr Opin Biotechnol 8: 285–289. [DOI] [PubMed] [Google Scholar]
  31. Lovley DR, and Walker DJF (2019) Geobacter protein nanowires. Front Microbiol 10: 2078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mahadevan R, Palsson BØ, and Lovley DR (2011) In situ to in silico and back: elucidating the physiology and ecology of Geobacter spp. using genome-scale modelling. Nat Rev Microbiol 9: 39–50. [DOI] [PubMed] [Google Scholar]
  33. Malvankar NS, Vargas M, Nevin K, Tremblay P-L, Evans-Lutterodt K, Nykypanchuk D, et al. (2015) Structural basis for metallic-like conductivity in microbial nanowires. MBio 6: e00084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mehta T, Childers SE, Glaven R, Lovley DR, and Mester T. (2006) A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens. Microbiology 152: 2257–2264. [DOI] [PubMed] [Google Scholar]
  35. Mikheenko A, Saveliev V, and Gurevich A. (2015) MetaQUAST: evaluation of metagenome assemblies. Bioinformatics 32: 1088–1090. [DOI] [PubMed] [Google Scholar]
  36. Nguyen Y, Sugiman-Marangos S, Harvey H, Bell SD, Charlton CL, Junop MS, and Burrows LL (2015) Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin. J Biol Chem 290: 601–611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nurk S, Bankevich A, Antipov D, Gurevich AA, Korobeynikov A, Lapidus A, et al. (2013) Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J Comput Biol 20: 714–737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Probst AJ, Ladd B, Jarett JK, Geller-McGrath DE, Sieber CM, Emerson JB, et al. (2018) Differential depth distribution of microbial function and putative symbionts through sediment-hosted aquifers in the deep terrestrial subsurface. Nat Microbiol 3: 328–336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reardon PN, and Mueller KT (2013) Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. J Biol Chem 288: 29260–29266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, and Lovley DR (2005) Extracellular electron transfer via microbial nanowires. Nature 435: 1098–1101. [DOI] [PubMed] [Google Scholar]
  41. Reguera G, Nevin KP, Nicoll JS, Covalla SF, Woodard TL, and Lovley DR (2006) Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells. Appl Environ Microbiol 72: 7345–7348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rotaru A-E, Shrestha PM, Liu F, Markovaite B, Chen S, Nevin KP, and Lovley DR (2014) Direct interspecies electron transfer between Geobacter metallireducens and Methanosarcina barkeri. Appl Environ Microbiol 80: 4599–4605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sydow A, Krieg T, Mayer F, Schrader J, and Holtmann D. (2014) Electroactive bacteria—molecular mechanisms and genetic tools. Appl Microbiol Biotechnol 98: 8481–8495. [DOI] [PubMed] [Google Scholar]
  44. Szeinbaum N, Nunn BL, Cavazos AR, Crowe SA, Stewart FJ, DiChristina TJ, et al. (2019) Novel insights into the taxonomic diversity and molecular mechanisms of bacterial Mn(III) reduction BioRXiv. 10.1101/695007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tan Y, Adhikari RY, Malvankar NS, Ward JE, Nevin KP, Woodard TL, et al. (2016) The low conductivity of Geobacter uraniireducens pili suggests a diversity of extracellular electron transfer mechanisms in the genus Geobacter. Front Microbiol 7: 980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tan Y, Adhikari RY, Malvankar NS, Ward JE, Woodard TL, Nevin KP, and Lovley DR (2017) Expressing the Geobacter metallireducens PilA in Geobacter sulfurreducens yields pili with exceptional conductivity. MBio 8: e02203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vandecandelaere I, Nercessian O, Segaert E, Achouak W, Mollica A, Faimali M, et al. (2008) Alteromonas genovensis sp. nov., isolated from a marine electroactive biofilm and emended description of Alteromonas macleodii Baumann et al. 1972 (approved lists 1980). Int J Syst Evol Microbiol 58: 2589–2596. [DOI] [PubMed] [Google Scholar]
  48. Vargas M, Malvankar NS, Tremblay P-L, Leang C, Smith JA, Patel P, et al. (2013) Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens. MBio 4: e00105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vuillemin A, Friese A, Alawi M, Henny C, Nomosatryo S, Wagner D, et al. (2016) Geomicrobiological features of ferruginous sediments from Lake Towuti, Indonesia. Front Microbiol 7: 1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Walker DJ, Nevin KP, Holmes DE, Rotaru A-E, Ward JE, Woodard TL, et al. (2019a) Syntrophus conductive pili demonstrate that common hydrogen-donating syntrophs can have a direct electron transfer option. BioRXiv 10.1101/479683 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Walker DJF, Adhikari RY, Holmes DE, Ward JE, Woodard TL, Nevin KP, and Lovley DR (2018) Electrically conductive pili from pilin genes of phylogenetically diverse microorganisms. ISME J 12: 48–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Walker DJF, Martz E, Holmes DE, Zhou Z, Nonnenmann SS, and Lovley DR (2019b) The archaellum of Methanospirillum hungatei is electrically conductive. mBio 10: e00579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wang F, Gu Y, O’Brien JP, Sophia MY, Yalcin SE, Srikanth V, et al. (2019) Structure of microbial nanowires reveals stacked hemes that transport electrons over micrometers. Cell 177: 361–369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Xiao K, Malvankar NS, Shu C, Martz E, Lovley DR, and Sun X. (2016) Low energy atomic models suggesting a pilus structure that could account for electrical conductivity of Geobacter sulfurreducens pili. Sci Rep 6: 23385. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material

Table S1. Characteristics of predicted or confirmed e-pilin proteins, and non-conductive pilins, in order of increasing aromatic density, from cultured isolates, based on Holmes et al., 2016 and Walker et al., 2018. These proteins listed as ‘U’ are predicted to infer long-range electron transport capability in the listed species, based on the criteria listed in the paper. Biochemically confirmed e-pilins are bolded.

Table S2. Characteristics of type IV pilins and pseudopilins involved in functions other than long-range electron transport, in order of increasing aromatic density. A: attachment; C: competence; F: fibril assembly; M: motility; S: secretion of proteins.

Table S3. Putative e-pilin genes identified from environmental metagenomes and long-term enrichment of Lake Matano sediment in the presence of metal oxides. Putative e-pilins are defined as containing (≥9.8% aromatic amino acids, ≤22-aa aromatic gaps, and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57. See Supplemental Data File for sequence and details about each putative e-pilin.

Fig. S1. Putative operon with multiple extracellular multiheme cytochromes found in Candidatus Omnitrophica bacterium CG11_big_fil_rev_8_21_14_0_20_42_13. Numbers represent number of cytochromes in each gene product. Predicted protein locations based on PSORTb analysis. E: extracellular; OM: outer membrane; P: periplasmic; C: cytoplasmic; CM: cytoplasmic membrane. NCBI accession numbers for each protein (PIQ89515-PIQ89529) are labelled beneath each gene.

Appendix S1. Supporting Information.

NIHMS1657953-supplement-Supplemental_Material.pdf (408.1KB, pdf)

RESOURCES