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Membrane permeabilization underlies the enhancement of extracellular bioactivity in Shewanella oneidensis by a membrane-spanning conjugated oligoelectrolyte

  • Applied microbial and cell physiology
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Abstract

A stilbene-based membrane spanning conjugated oligoelectrolyte 4,4′-bis(4′-N,N-bis(6″-(N,N,N-trimethyl ammonium) hexyl) amino)-styryl) stilbene tetraiodide (DSSN+) has been reported to be able to interact with bacterial cells and enhance their bioelectricity generation in bioelectrochemical devices, although the mechanism remains elusive. The goal of this study was to elucidate the impacts of DSSN+ on extracellular bioactivity and the underlying mechanism. Specifically, extracellular ferrihydrite reduction by Shewanella oneidensis was used to evaluate the influence of cell-DSSN+ interaction. Our results show that DSSN+ enhanced ferrihydrite reduction by S. oneidensis in a growth-dependent manner. The incorporation of DSSN+ into S. oneidensis cell membrane increased the extracellular concentration of redox shuttles, i.e., flavins, and extracellular enzyme activities without significantly decreasing cell viability. The findings suggested that membrane permeabilization is the dominant mechanism for the enhancement of extracellular bioactivity in S. oneidensis by DSSN+. We further demonstrated that the interaction between DSSN+ and S. oneidensis cells enhanced biofilm formation and stability without compromising the overall biofilm activity. Taken together, our results suggest that membrane spanning conjugated oligoelectrolytes, of which DSSN+ is one of many possible molecular structures, may be applied to enhance extracellular bioactivity in bacteria toward more efficient biofilm-based biocatalysis.

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References

  • Alcalde M, Ferrer M, Plou FJ (2007) Environmental biocatalysis: from remediation with enzymes to novel green processes. Biocatal Biotransform 25(2–4):113–113

    Article  CAS  Google Scholar 

  • Belchik SM, Kennedy DW, Dohnalkova AC, Wang Y, Sevinc PC, Wu H, Lin Y, Lu HP, Fredrickson JK, Shi L (2011) Extracellular reduction of hexavalent chromium by cytochromes MtrC and OmcA of Shewanella oneidensis MR-1. Appl Environ Microbiol 77(12):4035–41

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Beuling E, Van Dusschoten D, Lens P, Van Den Heuvel J, Van As H, Ottengraf S (1998) Characterization of the diffusive properties of biofilms using pulsed field gradient-nuclear magnetic resonance. Biotechnol Bioeng 60(3):283–291

    Article  PubMed  CAS  Google Scholar 

  • Branda SS, Vik Å, Friedman L, Kolter R (2005) Biofilms: the matrix revisited. Trends Microbiol 13(1):20–26

    Article  PubMed  CAS  Google Scholar 

  • Bretschger O, Obraztsova A, Sturm CA, Chang IS, Gorby YA, Reed SB, Culley DE, Reardon CL, Barua S, Romine MF (2007) Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants. Appl Environ Microbiol 73(21):7003–7012

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Cao B, Ahmed B, Kennedy DW, Wang Z, Shi L, Marshall MJ, Fredrickson JK, Isern NG, Majors PD, Beyenal H (2011) Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms to U(VI) immobilization. Environ Sci Technol 45(13):5483–90

    Article  PubMed  CAS  Google Scholar 

  • Chen C-Y, Nace G, Irwin P (2003) A 6×6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli. J Microbiol Methods 55:475–479

    Article  PubMed  CAS  Google Scholar 

  • Chen JJ, Chen W, He H, Li DB, Li WW, Xiong L, Yu HQ (2013) Manipulation of microbial extracellular electron transfer by changing molecular structure of phenazine-type redox mediators. Environ Sci Technol 47(2):1033–1039

    Article  PubMed  CAS  Google Scholar 

  • Clark ME, He Z, Redding AM, Joachimiak MP, Keasling JD, Zhou J, Arkin AP, Mukhopadhyay A, Fields MW (2012) Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: Carbon and energy flow contribute to the distinct biofilm growth state. BMC Genomics 13(1):138

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Coursolle D, Gralnick JA (2012) Reconstruction of extracellular respiratory pathways for iron (III) reduction in Shewanella oneidensis strain MR-1. Front Microbiol 3:56

    Article  PubMed  PubMed Central  Google Scholar 

  • Coursolle D, Baron DB, Bond DR, Gralnick JA (2010) The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. J Bacteriol 192(2):467–474

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Covington ED, Gelbmann CB, Kotloski NJ, Gralnick JA (2010) An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis. Mol Microbiol 78(2):519–532

    Article  PubMed  CAS  Google Scholar 

  • Du J, Thomas AW, Chen XF, Garner LE, Vandenberg CA, Bazan GC (2013) Increased ion conductance across mammalian membranes modified with conjugated oligoelectrolytes. Chem Commun 49(83):9624–9626

    Article  CAS  Google Scholar 

  • Flemming H-C, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8(9):623–633

    PubMed  CAS  Google Scholar 

  • Fredrickson JK, Zachara JM (2008) Electron transfer at the microbe-mineral interface: a grand challenge in biogeochemistry. Geobiology 6(3):245–253

    Article  PubMed  CAS  Google Scholar 

  • Fredrickson JK, Romine MF, Beliaev AS, Auchtung JM, Driscoll ME, Gardner TS, Nealson KH, Osterman AL, Pinchuk G, Reed JL, Rodionov DA, Rodrigues JLM, Saffarini DA, Serres MH, Spormann AM, Zhulin IB, Tiedje JM (2008) Towards environmental systems biology of Shewanella. Nat Rev Microbiol 6(8):592–603

    Article  PubMed  CAS  Google Scholar 

  • Garner LE, Park J, Dyar SM, Chworos A, Sumner JJ, Bazan GC (2010) Modification of the optoelectronic properties of membranes via insertion of amphiphilic phenylenevinylene oligoelectrolytes. J Am Chem Soc 132(29):10042–10052

    Article  PubMed  CAS  Google Scholar 

  • Garner LE, Thomas AW, Sumner JJ, Harvey SP, Bazan GC (2012) Conjugated oligoelectrolytes increase current response and organic contaminant removal in wastewater microbial fuel cells. Energ Environ Sci 5(11):9449–9452

    Article  CAS  Google Scholar 

  • Gross R, Hauer B, Otto K, Schmid A (2007) Microbial biofilms: new catalysts for maximizing productivity of long–term biotransformations. Biotechnol Bioeng 98(6):1123–1134

    Article  PubMed  CAS  Google Scholar 

  • Halan B, Buehler K, Schmid A (2012) Biofilms as living catalysts in continuous chemical syntheses. Trends Biotechnol 30(9):453–465

    Article  PubMed  CAS  Google Scholar 

  • Hartmann M, Jung D (2010) Biocatalysis with enzymes immobilized on mesoporous hosts: the status quo and future trends. J Mater Chem 20(5):844–857

    Article  CAS  Google Scholar 

  • Hinks J, Wang Y, Poh WH, Donose BC, Thomas AW, Wuertz S, Loo SC, Bazan GC, Kjelleberg SL, Mu Y, Seviour TW (2014) Modeling the membrane insertion of molecules designed for transmembrane electron transfer. Langmuir 30(9):2429–2440

    Article  PubMed  CAS  Google Scholar 

  • Hou H, Chen X, Thomas AW, Catania C, Kirchhofer ND, Garner LE, Han A, Bazan GC (2013) Conjugated oligoelectrolytes increase power generation in E. coli microbial fuel cells. Adv Mater 25(11):1593–1597

    Article  PubMed  CAS  Google Scholar 

  • Kiely PD, Regan JM, Logan BE (2011) The electric picnic: synergistic requirements for exoelectrogenic microbial communities. Curr Opin Biotechnol 22(3):378–385

    Article  PubMed  CAS  Google Scholar 

  • Kotloski NJ, Gralnick JA (2013) Flavin electron shuttles dominate extracellular electron transfer by Shewanella oneidensis. mBio 4(1):e00553–12

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Leon R, Fernandes P, Pinheiro H, Cabral J (1998) Whole-cell biocatalysis in organic media. Enzyme Microb Technol 23(7):483–500

    Article  CAS  Google Scholar 

  • Leresche JE, Meyer H-P (2006) Chemocatalysis and biocatalysis (biotransformation): some thoughts of a chemist and of a biotechnologist. Org Proc Res Develop 10(3):572–580

    Article  CAS  Google Scholar 

  • Lewandowski Z, Beyenal H (2007) Fundamentals of biofilm research. CRC Press

  • Lies DP, Hernandez ME, Kappler A, Mielke RE, Gralnick JA, Newman DK (2005) Shewanella oneidensis MR-1 uses overlapping pathways for iron reduction at a distance and by direct contact under conditions relevant for biofilms. Appl Environ Microbiol 71(8):4414–4426

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Liu XW, Sun XF, Chen JJ, Huang YX, Xie JF, Li WW, Sheng GP, Zhang YY, Zhao F, Lu R, Yu HQ (2013) Phenothiazine derivative-accelerated microbial extracellular electron transfer in bioelectrochemical system. Sci Rep 3:1616

    PubMed  PubMed Central  Google Scholar 

  • Marshall MJ, Beliaev AS, Dohnalkova AC, Kennedy DW, Shi L, Wang Z, Boyanov MI, Lai B, Kemner KM, McLean JS, Reed SB, Culley DE, Bailey VL, Simonson CJ, Saffarini DA, Romine MF, Zachara JM, Fredrickson JK (2006) c-Type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis. PLoS Biol 4(9):e268

    Article  PubMed  PubMed Central  Google Scholar 

  • Marshall MJ, Plymale AE, Kennedy DW, Shi L, Wang Z, Reed SB, Dohnalkova AC, Simonson CJ, Liu C, Saffarini DA (2008) Hydrogenase–and outer membrane c–type cytochrome–facilitated reduction of technetium (VII) by Shewanella oneidensis MR–1. Environ Microbiol 10(1):125–136

    PubMed  CAS  Google Scholar 

  • Marsili E, Baron DB, Shikhare ID, Coursolle D, Gralnick JA, Bond DR (2008) Shewanella secretes flavins that mediate extracellular electron transfer. Proc Natl Acad Sci U S A 105(10):3968–3973

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Mohanty A, Kathawala MH, Zhang J, Chen WN, Loo JSC, Kjelleberg S, Yang L, Cao B (2013) Biogenic tellurium nanorods as a novel antivirulence agent inhibiting pyoverdine production in Pseudomonas aeruginosa. Biotechnol Bioeng 111(5):858–865

    Article  PubMed  Google Scholar 

  • Myers CR, Nealson KH (1988) Microbial reduction of manganese oxides: Interactions with iron and sulfur. Geochim Cosmochim Act 52(11):2727–2732

    Article  CAS  Google Scholar 

  • Nikolova P, Ward OP (1993) Whole cell biocatalysis in nonconventional media. J Ind Microbiol 12(2):76–86

    Article  PubMed  CAS  Google Scholar 

  • Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF (2007) Stability of biocatalysts. Curr Opin Chem Biol 11(2):220–225

    Article  PubMed  CAS  Google Scholar 

  • Renslow RS, Majors PD, McLean JS, Fredrickson JK, Ahmed B, Beyenal H (2010) In situ effective diffusion coefficient profiles in live biofilms using pulsed–field gradient nuclear magnetic resonance. Biotechnol Bioeng 106(6):928–937

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Richardson DJ, Butt JN, Fredrickson JK, Zachara JM, Shi L, Edwards MJ, White G, Baiden N, Gates AJ, Marritt SJ (2012) The ‘porin–cytochrome’model for microbe–to–mineral electron transfer. Mol Microbiol 85(2):201–212

    Article  PubMed  CAS  Google Scholar 

  • Rosche B, Li XZ, Hauer B, Schmid A, Buehler K (2009) Microbial biofilms: a concept for industrial catalysis? Trends Biotechnol 27(11):636–643

    Article  PubMed  CAS  Google Scholar 

  • Santacoloma PA, Gr S, Gernaey KV, Woodley JM (2010) Multienzyme-catalyzed processes: next-generation biocatalysis. Org Proc Res Develop 15(1):203–212

    Article  Google Scholar 

  • Shi L, Richardson DJ, Wang Z, Kerisit SN, Rosso KM, Zachara JM, Fredrickson JK (2009) The roles of outer membrane cytochromes of Shewanella and Geobacter in extracellular electron transfer. Environ Microbiol Rep 1(4):220–227

    Article  PubMed  CAS  Google Scholar 

  • Simonides W, Zaremba R, Van Hardeveld C, Van der Laarse W (1988) A nonenzymatic method for the determination of picomole amounts of lactate using HPLC: its application to single muscle fibers. Anal Biochem 169(2):268–273

    Article  PubMed  CAS  Google Scholar 

  • Singh R, Paul D, Jain RK (2006) Biofilms: Implications in bioremediation. Trends Microbiol 14(9):389–397

    Article  PubMed  CAS  Google Scholar 

  • Stewart PS (2003) Diffusion in biofilms. J Bacteriol 185(5):1485–1491

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Thormann KM, Saville RM, Shukla S, Pelletier DA, Spormann AM (2004) Initial phases of biofilm formation in Shewanella oneidensis MR-1. J Bacteriol 186(23):8096–8104

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH, Goss RJM (2011) Engineering biofilms for biocatalysis. ChemBioChem 12(9):1391–1395

    Article  PubMed  CAS  Google Scholar 

  • Wang Z, Liu C, Wang X, Marshall MJ, Zachara JM, Rosso KM, Dupuis M, Fredrickson JK, Heald S, Shi L (2008) Kinetics of reduction of Fe (III) complexes by outer membrane cytochromes MtrC and OmcA of Shewanella oneidensis MR-1. Appl Environ Microbiol 74(21):6746–6755

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Wang VB, Du J, Chen X, Thomas AW, Kirchhofer ND, Garner LE, Maw MT, Poh WH, Hinks J, Wuertz S, Kjellenberg S, Zhang Q, Loo JS, Bazan GC (2013) Improving charge collection in Escherichia coli–carbon electrode devices with conjugated oligoelectrolytes. Phys Chem Chem Phys 15(16):5867–5872

    Article  PubMed  CAS  Google Scholar 

  • Wang VB, Kirchhofer ND, Chen X, Tan MYL, Sivakumar K, Cao B, Zhang Q, Kjelleberg S, Bazan GC, Loo JSC, Marsili E (2014) Comparison of flavins and a conjugated oligoelectrolyte in stimulating extracellular electron transport from Shewanella oneidensis MR-1. Electrochem Commun 41:55–58

    Article  CAS  Google Scholar 

  • Wu C, Cheng YY, Li BB, Li WW, Li DB, Yu HQ (2013) Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1. Bioresource Technol 136:711–714

    Article  CAS  Google Scholar 

  • Yong YC, Yu YY, Yang Y, Liu J, Wang JY, Song H (2013) Enhancement of extracellular electron transfer and bioelectricity output by synthetic porin. Biotechnol Bioeng 110(2):408–416

    Article  PubMed  CAS  Google Scholar 

  • Yu YY, Chen HL, Yong YC, Kim DH, Song H (2011) Conductive artificial biofilm dramatically enhances bioelectricity production in Shewanella-inoculated microbial fuel cells. Chem Commun 47(48):12825–12827

    Article  CAS  Google Scholar 

  • Zhang Y, Ng CK, Cohen Y, Cao B (2014) Cell growth and protein expression of Shewanella oneidensis in biofilms and hydrogel-entrapped cultures. Mol BioSys 10(5):1035–1042

    Article  Google Scholar 

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Acknowledgments

We thank Anee Mohanty, Weijie Liu, and Manisha Mukherjee for their assistance in RNA extraction and qPCR; Chun Kiat Ng and Yuanzhao Ding for flow cell biofilms and CLSM image analyses. We also thank Stefan Wuertz and Liang Yang for their useful discussion and advices. This research was supported by the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Programme, Singapore Centre on Environmental Life Sciences Engineering (SCELSE) (M433C70000.M4220001.C70) and M4330005.C70) and a Start-up Grant (M4080847.030) from College of Engineering, Nanyang Technological University, Singapore. The work at UCSB was supported by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the US Government, and no official endorsement should be inferred.

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Authors and Affiliations

  1. Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, 637551, Singapore

    Krishnakumar Sivakumar, Victor Bochuan Wang, Staffan Kjelleberg, Say Chye Joachim Loo & Bin Cao

  2. Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore

    Krishnakumar Sivakumar

  3. School of Civil and Environmental Engineering, Nanyang Technological University, Singapore, Singapore

    Bin Cao

  4. School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore

    Victor Bochuan Wang & Say Chye Joachim Loo

  5. School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

    Staffan Kjelleberg

  6. School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia

    Staffan Kjelleberg

  7. Department of Materials and Chemistry & Biochemistry, University of California, Santa Barbara, CA, USA

    Xiaofen Chen & Guillermo C. Bazan

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  1. Krishnakumar Sivakumar
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  2. Victor Bochuan Wang
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  3. Xiaofen Chen
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  4. Guillermo C. Bazan
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  6. Say Chye Joachim Loo
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  7. Bin Cao
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Correspondence to Bin Cao.

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Sivakumar, K., Wang, V.B., Chen, X. et al. Membrane permeabilization underlies the enhancement of extracellular bioactivity in Shewanella oneidensis by a membrane-spanning conjugated oligoelectrolyte. Appl Microbiol Biotechnol 98, 9021–9031 (2014). https://doi.org/10.1007/s00253-014-5973-3

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