Original Article

Electrically conductive pili from pilin genes of phylogenetically diverse microorganisms

Received:
Revised:
Accepted:
Published online:

Abstract

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 (>100 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.

  • Subscribe to The ISME Journal for full access:

    $547

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. , , , . (2016). Conductivity of individual Geobacter pili. RSC Adv 6: 8354–8357.

  2. , . (2014) The phylum Deferribacteres and the genus Caldithrix. In: al. ERe (ed.). The Prokaryotes—Other Major Lineages of Bacteria and the Archaea. Springer-Verlag: Berlin, Germany, pp 595–611.

  3. , , , , , et al. (2013). Conductive filaments produced by Aeromonas hydrophila. Adv Mater Res 825: 210–213.

  4. , , , . (2001). Development of a genetic system for Geobacter sulfurreducens. Appl Environ Microbiol 67: 3180–3187.

  5. , , , , , et al. (2016). Production of electrically-conductive nanoscale filaments by sulfate- reducing bacteria in the microbial fuel cell. Bioresour Technol 201: 61–67.

  6. , , . (2015). Structural and functional insights into the conductive pili of Geobacter sulfurreducens revealed in molecular dynamics simulations. Phys Chem Chem Phys 17: 22217–22226.

  7. . (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.

  8. , , , . (1990). Flexistipes sinusarabici, a novel genus and species of eubacteria occurring in the Atlantis II deep brines of the Red Sea. Arch Microbiol 154: 120–126.

  9. , , , , , . (2013). A thermophilic gram-negative nitrate-reducing bacterium, Calditerrivibrio nitroreducens, exhibiting electricity generation capability. Environ Sci Technol 47: 12583–12590.

  10. , , , , , . (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345.

  11. . (2004). Metagenomics: application of genomics to uncultured microorganisms. MIcrobiol Mol Biol Rev 68: 669–685.

  12. , , , . (2016). The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microb Genom 2: e000072.

  13. , . (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30: 772–780.

  14. , , , , , et al. (2016). Candidatus Desulfofervidus auxilii, a hydrogenotrophic sulfate-reducing bacterium involved in the thermophilic anaerobic oxidation of methane. Environ Microbiol 18: 3073–3091.

  15. , , . (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870–1874.

  16. , , , , , et al. (2016). Thermally activated charge transport in microbial protein nanowires. Sci Rep 6: 23517.

  17. , , , , . (2013). Engineering Geobacter sulfurreducens to produce a highly cohesive conductive matrix with enhanced capacity for current production. Energy Environ Sci 6: 1901–1908.

  18. , . (2014). Type IV pili of Acidithiobacillus ferrooxidans can transfer electrons from extracellular electron donors. J Basic Microbiol 54: 226–231.

  19. , , , , , . (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.

  20. , , . (2004). Dissimilatory Fe(III) and Mn(IV) reduction. Adv Microb Physiol 49: 219–286.

  21. . (2011). Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of energy-related contamination. Energy Environ Sci 4: 4896–4906.

  22. . (2012). Electromicrobiology. Ann Rev Microbol 66: 391–409.

  23. , . (2015). Seeing is believing: novel imaging techniques help clarify microbial nanowire structure and function. Environ Microbiol 7: 2209–2215.

  24. . (2017a). Happy together: microbial communities that hook up to swap electrons. ISME J 11: 327–336.

  25. . (2017b). e-Biologics: fabrication of sustainable electronics with ‘green’ biological materials. mBio 8: e00695-17.

  26. . (2017c). Syntrophy goes electric: direct interspecies electron transfer (DIET). Ann Rev Microbiol 71: 643–664.

  27. , . (2005). An algorithm for progressive multiple alignment of sequences with insertions. Proc Natl Acad Sci USA 102: 10557–10562.

  28. , , , , , et al. (2011). Tunable metallic-like conductivity in nanostructured biofilms comprised of microbial nanowires. Nat Nanotechnol 6: 573–579.

  29. , , . (2012). Biofilm conductivity is a decisive variable for high-current-density Geobacter sulfurreducens microbial fuel cells. Energy Environ Sci 5: 5790–5797.

  30. , . (2014). Microbial nanowires for bioenergy applications. Curr Opin Biotechnol 27: 88–95.

  31. , , , . (2014). Visualization of charge propagation along individual pili proteins using ambient electrostatic force microscopy. Nat Nanotechnol 9: 1012–1017.

  32. , , , , , et al. (2015). Structural basis for metallic-like conductivity in microbial nanowires. mBio 6: e00084–00015.

  33. , , , , , et al. (2016). Complete genome sequence of Desulfurivibrio alkaliphilus strain AHT2T, a haloalkaliphilic sulfidogen from Egyptian hypersaline alkaline lakes. Stand Genomic Sci 11: 67.

  34. , , , , , et al. (2011). Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates. mBio 2: e00159–00111.

  35. , , , , , et al. (2016). Long-distance electron transfer by cable bacteria in aquifer sediments. ISME J 10: 2010–2019.

  36. , , , , , et al. (2009). Anode biofilm transcriptomics reveals outer surface components essential for high current power production in Geobacter sulfurreducens fuel cells. PLoS One 4: e5628.

  37. , , , , , et al. (2011). Electrosynthesis of organic compounds from carbon dioxide is catalyzed by a diversity of acetogenic microorganisms. Appl Environ Microbiol 77: 2882–2886.

  38. , , , , , . (2005). Extracellular electron transfer via microbial nanowires. Nature 435: 1098–1101.

  39. , , , , , . (2006). Biofilm and nanowire production leads to increased current in Geobacter sulfurreducens fuel cells. Appl Environ Microbiol 72: 7345–7348.

  40. , , , , . (2014a). Direct interspecies electron transfer during syntrophic growth of Geobacter metallireducens and Methanosarcina barkeri on ethanol. Appl Environ Microbiol 80: 4599–4605.

  41. , , , , , et al. (2014b). A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane. Energy Environ Sci 7: 408–415.

  42. , , , . (2015). Link between capacity for current production and syntrophic growth in Geobacter species. Front Microbiol 6: 744.

  43. , , , . (2015). GUIDANCE2: accurate detection of unreliable alignment regions accounting for theuncertainty of multiple parameters. Nucleic Acids Res 43: W7–W14.

  44. , , . (2014). Genomic analysis of bacterial porin-cytochrome gene clusters. Front Microbiol 5: 657.

  45. , , , , , et al. (2016). Extracellular electron transfer mechanisms between microorganisms and minerals. Nat Rev Microbiol 14: 651–662.

  46. , , , , , . (2013). Transcriptomic and genetic analysis of direct interspecies electron transfer. Appl Environ Microbiol 79: 2397–2404.

  47. , , , , , et al. (2014). Correlation between microbial community and granule conductivity in anaerobic bioreactors for brewery wastewater treatment. Bioresource Tech 174: 306–310.

  48. , , , . (2008). Dethiobacter alkaliphilus gen. nov. sp. nov., and Desulfurivibrio alkaliphilus gen. nov. sp. nov.: two novel representatives of reductive sulfur cycle from soda lakes. Extremophiles 12: 431–439.

  49. , , , , , . (2010). Direct exchange of electrons within aggregates of an evolved syntrophic co-culture of anaerobic bacteria. Science 330: 1413–1415.

  50. , , , , , et al. (2016a). Synthetic biological protein nanowires with high conductivity. Small 12: 4481–4485.

  51. , , , , , et al. (2016b). The low conductivity of Geobacter uraniireducens pili suggests a diversity of extracellular electron transfer mechanisms in the genus Geobacter. Front Microbiol 7: 980.

  52. , , , , , et al. (2017). Expressing the Geobacter metallireducens PilA in Geobacter sulfurreducens yields pili with exceptional conductivity. mBio 8: e02203–e02216.

  53. , , , . (2012). A genetic system for Geobacter metallireducens: role of flagella and pili in extracellular electron transfer. Environ Microbiol Rep 4: 82–88.

  54. , , , , , et al. (2013). Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens. mBio 4: e00105–e00113.

  55. , , , , , et al. (2015). Microbial carbon metabolism associated with electrogenic sulphur oxidation in coastal sediments. ISME J 9: 1966–1978.

  56. , , , , , . (2015). Electron transport through electrically conductive nanofilaments in Rhodopseudomonas palustris strain RP2. RSC Adv 5: 100790–100798.

  57. , , , , . (2015). Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria. Nature 526: 587–590.

  58. , . (2001). A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol 18: 691–699.

  59. , , , , , . (2016). Low energy atomic models suggesting a pilus structure that could account for electrical conductivity along the length of Geobacter sulfurreducens pili. Sci Rep 6: 23385.

  60. , , , , , et al. (2009). Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells. Biosens Bioelectron 24: 3498–3503.

Download references

Acknowledgements

This research was supported by Office of Naval Research Grants N000141310549 and N000141612526.

Author information

Author notes

    • Ramesh Y Adhikari

    Current Address: Department of Physics, Jacksonville University, Jacksonville, FL, USA.

Affiliations

  1. Department of Microbiology, University of Massachusetts, Amherst, MA, USA

    • David JF Walker
    • , Dawn E Holmes
    • , Joy E Ward
    • , Trevor L Woodard
    • , Kelly P Nevin
    •  & Derek R Lovley
  2. Department of Physics, University of Massachusetts, Amherst, MA, USA

    • Ramesh Y Adhikari
  3. Department of Physical and Biological Sciences, Western New England University, Springfield, MA, USA

    • Dawn E Holmes
  4. Department of Physics, Jacksonville University, Jacksonville, FL, USA

    • Ramesh Y Adhikari

Authors

  1. Search for David JF Walker in:

  2. Search for Ramesh Y Adhikari in:

  3. Search for Dawn E Holmes in:

  4. Search for Joy E Ward in:

  5. Search for Trevor L Woodard in:

  6. Search for Kelly P Nevin in:

  7. Search for Derek R Lovley in:

Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to Derek R Lovley.

Supplementary information

Supplementary Information accompanies this paper on The ISME Journal website (http://www.nature.com/ismej)