Abstract
Bioelectrical nanowires as ecomaterials have great potential on environmental applications. A wide range of bacteria can express type IV pili (T4P), which are long protein fibers assembled from PilA. The T4P of Geobacter sulfurreducens are well known as “microbial nanowires,” yet T4P of Pseudomonas aeruginosa (PaT4P) was believed to be poorly conductive. P. aeruginosa is an aerobic and electrochemically active bacterium. Its T4P have been known to be responsible for surface attachment, twitching motility and biofilm formation. Here, we show that PaT4P can be highly conductive while assembled by a truncated P. aeruginosa PilA (PaPilA) containing only N-terminus 61 amino acids. Furthermore, increasing the number of aromatic amino acids in the PaPilA1–61 significantly enhances the conductivity of pili and the bioelectricity output of P. aeruginosa in microbial fuel cell system, suggesting a potential application of PaT4P as a conductive nanomaterial. The N-terminal region of PilA from diverse eubacteria is highly conserved, implying a general way to synthesize highly conductive microbial nanowires and to increase the bioelectricity output of microbial fuel cell.
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References
Adhikari RY, Malvankar NS, Tuominen MT, Lovley DR (2016) Conductivity of individual Geobacter pili. RSC Adv 6(10):8354–8357. https://doi.org/10.1039/c5ra28092c
Bachman J (2013) Site-directed mutagenesis. Methods Enzymol 529:241–248. https://doi.org/10.1016/b978-0-12-418687-3.00019-7
Baynham PJ, Ramsey DM, Gvozdyev BV, Cordonnier EM, Wozniak DJ (2006) The Pseudomonas aeruginosa ribbon-helix-helix DNA-binding protein AlgZ (AmrZ) controls twitching motility and biogenesis of type IV pili. J Bacteriol 188(1):132–140. https://doi.org/10.1128/JB.188.1.132-140.2006
Cologgi DL, Lampa-Pastirk S, Speers AM, Kelly SD, Reguera G (2011) Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism. Proc Natl Acad Sci U S A 108(37):15248–15252. https://doi.org/10.1073/pnas.1108616108
Craig L, Li J (2008) Type IV pili: paradoxes in form and function. Curr Opin Struct Biol 18(2):267–277. https://doi.org/10.1016/j.sbi.2007.12.009
Craig L, Pique ME, Tainer JA (2004) Type IV pilus structure and bacterial pathogenicity. Nat Rev Microbiol 2(5):363–378. https://doi.org/10.1038/nrmicro885
Craig L, Taylor RK, Pique ME, Adair BD, Arvai AS, Singh M, Lloyd SJ, Shin DS, Getzoff ED, Yeager M, Forest KT, Tainer JA (2003) Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin. Mol Cell 11(5):1139–1150
Dietrich LE, Price-Whelan A, Petersen A, Whiteley M, Newman DK (2006) The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol 61(5):1308–1321. https://doi.org/10.1111/j.1365-2958.2006.05306.x
Ding H, Li Y, Lu A, Jin S, Quan C, Wang C, Wang X, Zeng C, Yan Y (2010) Photocatalytically improved azo dye reduction in a microbial fuel cell with rutile-cathode. Bioresour Technol 101(10):3500–3505. https://doi.org/10.1016/j.biortech.2009.11.107
Giltner CL, Habash M, Burrows LL (2010) Pseudomonas aeruginosa minor pilins are incorporated into type IV pili. J Mol Biol 398(3):444–461. https://doi.org/10.1016/j.jmb.2010.03.028
Giltner CL, Nguyen Y, Burrows LL (2012) Type IV pilin proteins: versatile molecular modules. Microbiol Mol Biol Rev 76(4):740–772. https://doi.org/10.1128/MMBR.00035-12
Guo X, Gorodetsky AA, Hone J, Barton JK, Nuckolls C (2008) Conductivity of a single DNA duplex bridging a carbon nanotube gap. Nat Nanotechnol 3(3):163–167. https://doi.org/10.1038/nnano.2008.4
Holloway BW (1955) Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol 13(3):572–581. https://doi.org/10.1099/00221287-13-3-572
Holmes DE, Dang Y, Walker DJ, Lovley DR (2016) The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microb Genom 2(8):e000072. https://doi.org/10.1099/mgen.0.000072
Jensen SE, Fecycz IT, Campbell JN (1980) Nutritional factors controlling exocellular protease production by Pseudomonas aeruginosa. J Bacteriol 144(2):844–847
Kus JV, Tullis E, Cvitkovitch DG, Burrows LL (2004) Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology 150(Pt 5):1315–1326. https://doi.org/10.1099/mic.0.26822-0
Lampa-Pastirk S, Veazey JP, Walsh KA, Feliciano GT, Steidl RJ, Tessmer SH, Reguera G (2016) Thermally activated charge transport in microbial protein nanowires. Sci Rep 6:23517. https://doi.org/10.1038/srep23517
Li Y, Li H (2014) Type IV pili of Acidithiobacillus ferrooxidans can transfer electrons from extracellular electron donors. J Basic Microbiol 54(3):226–231. https://doi.org/10.1002/jobm.201200300
Liu X, Tremblay PL, Malvankar NS, Nevin KP, Lovley DR, 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(3):1219–1224. https://doi.org/10.1128/AEM.02938-13
Lovley DR (2017) e-Biologics: fabrication of sustainable electronics with “green” biological materials. MBio 8(3):e00695. Doi:https://doi.org/10.1128/mBio.00695-17
Ma L, Jackson KD, Landry RM, Parsek MR, Wozniak DJ (2006) Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the Psl polysaccharide in adhesion and maintaining biofilm structure postattachment. J Bacteriol 188(23):8213–8221. https://doi.org/10.1128/JB.01202-06
Malvankar NS, Lovley DR (2012) Microbial nanowires: a new paradigm for biological electron transfer and bioelectronics. ChemSusChem 5(6):1039–1046. https://doi.org/10.1002/cssc.201100733
Malvankar NS, Vargas M, Nevin K, Tremblay PL, Evans-Lutterodt K, Nykypanchuk D, Martz E, Tuominen MT, Lovley DR (2015) Structural basis for metallic-like conductivity in microbial nanowires. MBio 6(2):e00084. https://doi.org/10.1128/mBio.00084-15
Malvankar NS, Vargas M, Nevin KP, Franks AE, Leang C, Kim BC, Inoue K, Mester T, Covalla SF, Johnson JP, Rotello VM, Tuominen MT, Lovley DR (2011) Tunable metallic-like conductivity in microbial nanowire networks. Nat Nanotechnol 6(9):573–579. https://doi.org/10.1038/nnano.2011.119
Malvankar NS, Yalcin SE, Tuominen MT, Lovley DR (2014) Visualization of charge propagation along individual pili proteins using ambient electrostatic force microscopy. Nat Nanotechnol 9(12):1012–1017. https://doi.org/10.1038/nnano.2014.236
Nguyen Y, Jackson SG, Aidoo F, Junop M, Burrows LL (2010) Structural characterization of novel Pseudomonas aeruginosa type IV pilins. J Mol Biol 395(3):491–503. https://doi.org/10.1016/j.jmb.2009.10.070
O’Toole GA (2011) Microtiter dish biofilm formation assay. J Vis Exp 47:2437. https://doi.org/10.3791/2437
Pelicic V (2008) Type IV pili: e pluribus unum? Mol Microbiol 68(4):827–837. https://doi.org/10.1111/j.1365-2958.2008.06197.x
Qiu D, Damron FH, Mima T, Schweizer HP, Yu HD (2008) PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria. Appl Environ Microbiol 74(23):7422–7426. https://doi.org/10.1128/AEM.01369-08
Rabaey K, Boon N, Hofte M, Verstraete W (2005) Microbial phenazine production enhances electron transfer in biofuel cells. Environ Sci Technol 39(9):3401–3408
Rashid MH, Kornberg A (2000) Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97(9):4885–4890. https://doi.org/10.1073/pnas.060030097
Reardon PN, Mueller KT (2013) Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. J Biol Chem 288(41):29260–29266. https://doi.org/10.1074/jbc.M113.498527
Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, Lovley DR (2005) Extracellular electron transfer via microbial nanowires. Nature 435(7045):1098–1101. https://doi.org/10.1038/nature03661
Seker UO, Chen AY, Citorik RJ, Lu TK (2017) Synthetic biogenesis of bacterial amyloid nanomaterials with tunable inorganic-organic interfaces and electrical conductivity. ACS Synth Biol 6(2):266–275. https://doi.org/10.1021/acssynbio.6b00166
Shen HB, Yong XY, Chen YL, Liao ZH, Si RW, Zhou J, Wang SY, Yong YC, OuYang PK, Zheng T (2014) Enhanced bioelectricity generation by improving pyocyanin production and membrane permeability through sophorolipid addition in Pseudomonas aeruginosa-inoculated microbial fuel cells. Bioresour Technol 167:490–494. https://doi.org/10.1016/j.biortech.2014.05.093
Shih C, Museth AK, Abrahamsson M, Blanco-Rodriguez AM, Di Bilio AJ, Sudhamsu J, Crane BR, Ronayne KL, Towrie M, Vlcek A Jr, Richards JH, Winkler JR, Gray HB (2008) Tryptophan-accelerated electron flow through proteins. Science 320(5884):1760–1762. https://doi.org/10.1126/science.1158241
Steidl RJ, Lampa-Pastirk S, Reguera G (2016) Mechanistic stratification in electroactive biofilms of Geobacter sulfurreducens mediated by pilus nanowires. Nat Commun 7:12217. https://doi.org/10.1038/ncomms12217
Sund CJ, McMasters S, Crittenden SR, Harrell LE, Sumner JJ (2007) Effect of electron mediators on current generation and fermentation in a microbial fuel cell. Appl Microbiol Biotechnol 76(3):561–568. https://doi.org/10.1007/s00253-007-1038-1
Sure S, Torriero AA, Gaur A, Li LH, Chen Y, Tripathi C, Adholeya A, Ackland ML, Kochar M (2015) Inquisition of Microcystis aeruginosa and Synechocystis nanowires: characterization and modelling. Antonie Van Leeuwenhoek 108(5):1213–1225. https://doi.org/10.1007/s10482-015-0576-2
Touhami A, Jericho MH, Boyd JM, Beveridge TJ (2006) Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy. J Bacteriol 188(2):370–377. https://doi.org/10.1128/JB.188.2.370-377.2006
Vargas M, Malvankar NS, Tremblay PL, Leang C, Smith JA, Patel P, Snoeyenbos-West O, Nevin KP, Lovley DR (2013) Aromatic amino acids required for pili conductivity and long-range extracellular electron transport in Geobacter sulfurreducens. MBio 4(2):e00105–e00113. https://doi.org/10.1128/mBio.00105-13
Vinckx T, Wei Q, Matthijs S, Cornelis P (2010) The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin. Microbiology 156(Pt 3):678–686. https://doi.org/10.1099/mic.0.031971-0
Walker DJ, Adhikari RY, Holmes DE, Ward JE, Woodard TL, Nevin KP, Lovley DR (2018) Electrically conductive pili from pilin genes of phylogenetically diverse microorganisms. ISME J 12(1):48–58. https://doi.org/10.1038/ismej.2017.141
Wang S, Parsek MR, Wozniak DJ, Ma LZ (2013a) A spider web strategy of type IV pili-mediated migration to build a fibre-like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms. Environ Microbiol 15(8):2238–2253. https://doi.org/10.1111/1462-2920.12095
Wang VB, Chua SL, Cao B, Seviour T, Nesatyy VJ, Marsili E, Kjelleberg S, Givskov M, Tolker-Nielsen T, Song H, Loo JS, Yang L (2013b) Engineering PQS biosynthesis pathway for enhancement of bioelectricity production in Pseudomonas aeruginosa microbial fuel cells. PLoS One 8(5):e63129. https://doi.org/10.1371/journal.pone.0063129
Watson SM, Pike AR, Pate J, Houlton A, Horrocks BR (2014) DNA-templated nanowires: morphology and electrical conductivity. Nanoscale 6(8):4027–4037. https://doi.org/10.1039/c3nr06767j
Wei Q, Ma LZ (2013) Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int J Mol Sci 14(10):20983–21005. https://doi.org/10.3390/ijms141020983
Whitchurch CB, Erova TE, Emery JA, Sargent JL, Harris JM, Semmler AB, Young MD, Mattick JS, Wozniak DJ (2002) Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility. J Bacteriol 184(16):4544–4554
Yong XY, Feng J, Chen YL, Shi DY, Xu YS, Zhou J, Wang SY, Xu L, Yong YC, Sun YM, Shi CL, PK OY, Zheng T (2014a) Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell. Biosens Bioelectron 56:19–25. https://doi.org/10.1016/j.bios.2013.12.058
Yong XY, Shi DY, Chen YL, Feng J, Xu L, Zhou J, Wang SY, Yong YC, Sun YM, OuYang PK, Zheng T (2014b) Enhancement of bioelectricity generation by manipulation of the electron shuttles synthesis pathway in microbial fuel cells. Bioresour Technol 152:220–224. 10.1016/j.biortech.2013.10.086
Zhao K, Tseng BS, Beckerman B, Jin F, Gibiansky ML, Harrison JJ, Luijten E, Parsek MR, Wong GCL (2013) Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms. Nature 497(7449):388–391. https://doi.org/10.1038/nature12155
Acknowledgments
We sincerely thank Dr. Mohamed Y. El-Naggar and Dr. Sahand Pirbadian at the University of Southern California for the helpful comments on the manuscript and the CP-AFM experiments respectively, Dr. Daniel J. Wozniak at the Ohio State University for the anti-PaPilA antibody, Dr. Anhuai Lu and Dr. Hongrui Ding at the Peking University for the MFC configuration, Dr. Cong Liu at the Jiangsu Normal University for the G. sulfurreducens PCA strain, and Dr. Qing Wei at the Institute of Microbiology, Chinese Academy of Sciences for the manuscript preparation.
Funding
This study was funded by the National Basic Research Program of China (973) (2014CB846002 for LM) and by the National Natural Science Foundation of China (31570126 to LM and 31770152 to SW).
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Liu, X., Wang, S., Xu, A. et al. Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa. Appl Microbiol Biotechnol 103, 1535–1544 (2019). https://doi.org/10.1007/s00253-018-9484-5
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DOI: https://doi.org/10.1007/s00253-018-9484-5