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Research article

Environmental Microbiology and Engineering

Quorum Quenching Bacteria Isolated from the Sludge of a Wastewater Treatment Plant and Their Application for Controlling Biofilm Formation

A-Leum Kim 1, Son-Young Park 1, Chi-Ho Lee 1, Chung-Hak Lee 2 and Jung-Kee Lee 1*

1Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea, 2School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea

Received: July 7, 2014; Accepted: August 10, 2014

J. Microbiol. Biotechnol. 2014; 24(11): 1574-1582

Published November 28, 2014 https://doi.org/10.4014/jmb.1407.07009

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Bacteria recognize changes in their population density by sensing the concentration of signal molecules, N-acyl-homoserine lactones (AHLs). AHL-mediated quorum sensing (QS) plays a key role in biofilm formation, so the interference of QS, referred to as quorum quenching (QQ), has received a great deal of attention. A QQ strategy can be applied to membrane bioreactors (MBRs) for advanced wastewater treatment to control biofouling. To isolate QQ bacteria that can inhibit biofilm formation, we isolated diverse AHL-degrading bacteria from a laboratory-scale MBR and sludge from real wastewater treatment plants. A total of 225 AHLdegrading bacteria were isolated from the sludge sample by enrichment culture. To identify the enzyme responsible for AHL degradation in QQ bacteria, AHL-degrading activities were analyzed using cell-free lysate, culture supernatant, and whole cells. Afipia sp. and Acinetobacter sp. strains produced the intracellular QQ enzyme, whereas Pseudomonas sp. and Micrococcus sp. produced the extracellular QQ enzyme that was most likely to produce AHLacylase. AHL-degrading activity was observed in whole-cell assay with the Microbacterium sp. and Rhodococcus sp. strains. There has been no report for AHL-degrading capability in the case of Streptococcus sp. and Afipia sp. strains. Finally, inhibition of biofilm formation by isolated QQ bacteria or enzymes was observed on glass slides and 96-well microtiter plates using crystal violet staining. QQ strains or enzymes not only inhibited initial biofilm development but also reduced established biofilms.

Keywords

quorum sensing, quorum quenching, AHL, biofilm, wastewater treatment

References

  1. Bokhove M, Nadal Jimenez P, Quax WJ, Dijkstra BW. 2010. The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket. Proc. Natl. Acad. Sci. USA 107: 686-691.
    Pubmed CrossRef
  2. Cheong SC, Lee CH, Moon YH, Oh HS, Kim SR, Lee SH, et al. 2013. Isolation and identification of indigenous quorum quenching bacteria, Pseudomonas sp. 1A1, for biofouling control in MBR. Ind. Eng. Chem. Res. 31: 10554-10560.
    CrossRef
  3. Chowdhary PK, Keshavan N, Nguyen HQ, Peterson JA, Gonzalez JE, Haines DC. 2007. Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines. Biochemistry 46: 14429-14437.
    Pubmed CrossRef
  4. Cook DM, Li PL, Ruchaud F, Padden S, Farrand SK. 1997. Ti plasmid conjugation is independent of vir: reconstitution of the tra functions from pTiC58 as a binary system. J. Bacteriol. 179: 1291-1297.
    Pubmed
  5. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP. 1998. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280: 295-298.
    Pubmed CrossRef
  6. Dickschat JS. 2010. Quorum sensing and bacterial biofilms. Nat. Prod. Rep. 27: 343-369.
    Pubmed CrossRef
  7. Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813-817.
    Pubmed CrossRef
  8. Eberhard A, Burlingame AL, Eberhard C, Kenyon GL, Nealson KH, Oppenheimer NJ. 1981. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry 20: 2444-2449.
    Pubmed CrossRef
  9. Flemming HC, Neu TR, Wozniak DJ. 2007. T he EPS matrix:the “house of biofilm cells”. J. Bacteriol. 189: 7945-7947.
    Pubmed CrossRef
  10. Fuqua WC, Winans SC, Greenberg EP. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell densityresponsive transcriptional regulators. J. Bacteriol. 176: 269-275.
    Pubmed
  11. Fux CA, Stoodley P, Hall-Stoodley L, Costerton JW. 2003. Bacterial biofilms: a diagnostic and therapeutic challenge. Expert Rev. Anti Infect. Ther. 1: 667-683.
    Pubmed CrossRef
  12. Huang JJ, Petersen A, Whiteley M, Leadbetter JR. 2006. Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1. Appl. Environ. Microbiol. 72: 1190-1197.
    Pubmed CrossRef
  13. Kang BR, Lee JH, Ko SJ, Lee YH, Cha JS, Cho BH, Kim YC. 2004. Degradation of acyl-homoserine lactone molecules by Acinetobacter sp. strain C1010. Can. J. Microbiol. 50: 935-941.
    Pubmed CrossRef
  14. Latifi A, Winson MK, Foglino M, Bycroft BW, Stewart GS, Lazdunski A, Williams P. 1995. Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol. Microbiol. 17: 333-343.
    Pubmed CrossRef
  15. Leadbetter JR, Greenberg EP. 2000. Metabolism of acylhomoserine lactone quorum-sensing signals by Variovorax paradoxus. J. Bacteriol. 182: 6921-6926.
    Pubmed CrossRef
  16. Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK. 2002. Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl. Environ. Microbiol. 68: 3919-3924.
    Pubmed CrossRef
  17. Lin YH, Xu JL, Hu J, Wang LH, Ong SL, Leadbetter JR, Zhang LH. 2003. Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol. Microbiol. 47: 849-860.
    Pubmed CrossRef
  18. Ochiai S, Morohoshi T, Kurabeishi A, Shinozaki M, Fujita H, Sawada I, Ikeda T. 2013. Production and degradation of N-acylhomoserine lactone quorum sensing signal molecules in bacteria isolated from activated sludge. Biosci. Biotechnol. Biochem. 77: 2436-2440.
    Pubmed CrossRef
  19. Oh HS, Yeon KM, Yang CS, Kim SR, Lee CH, Park SY, et al. 2012. Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ. Sci. Technol. 46: 4877-4884.
    Pubmed CrossRef
  20. O’Toole GA, Kolter R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28: 449-461.
    Pubmed CrossRef
  21. Park SY, Hwang BJ, Shin MH, Kim JA, Kim HK, Lee JK. 2006. N-Acylhomoserine lactonase-producing Rhodococcus spp. with different AHL-degrading activities. FEMS Microbiol. Lett. 261: 102-108.
    Pubmed CrossRef
  22. Park SY, Lee SJ, Oh TK, Oh JW, Koo BT, Yum DY, Lee JK. 2003. AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149: 1541-1550.
    Pubmed CrossRef
  23. Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, Iglewski BH, Greenberg EP. 1994. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc. Natl. Acad. Sci. USA 91: 197-201.
    Pubmed CrossRef
  24. Rasmussen TB, Givskov M. 2006. Quorum sensing inhibitors:a bargain of effects. Microbiology 152: 895-904.
    Pubmed CrossRef
  25. Salmond GP, Bycroft BW, Stewart GS, Williams P. 1995. The bacterial ‘enigma’: cracking the code of cell-cell communication. Mol. Microbiol. 16: 615-624.
    Pubmed CrossRef
  26. Shaw PD, Ping G, Daly SL, Cha C, Cronan JE Jr, Rinehart KL, Farrand SK. 1997. Detecting and characterizing N-acylhomoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94: 6036-6041.
    Pubmed CrossRef
  27. Sintim HO, Smith JA, Wang J, Nakayama S, Yan L. 2010. Paradigm shift in discovering next-generation anti-infective agents: targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small molecules. Future Med. Chem. 2: 1005-1035.
    Pubmed CrossRef
  28. Stewart PS, Costerton JW. 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358: 135-138.
    CrossRef
  29. Throup JP, Camara M, Briggs GS, Winson MK, Chhabra SR, Bycroft BW, et al. 1995. Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules. Mol. Microbiol. 17: 345-356.
    Pubmed CrossRef
  30. Uroz S, Chhabra SR, Camara M, Williams P, Oger P, Dessaux Y. 2005. N-Acylhomoserine lactone quorum-sensing molecules are modified and degraded by Rhodococcus erythropolis W2 by both amidolytic and novel oxidoreductase activities. Microbiology 151: 3313-3322.
    Pubmed CrossRef
  31. Uroz S, Oger PM, Chapelle E, Adeline MT, Faure D, Dessaux Y. 2008. A Rhodococcus qsdA-encoded enzyme defines a novel class of large-spectrum quorum-quenching lactonases. Appl. Environ. Microbiol. 74: 1357-1366.
    Pubmed CrossRef
  32. Wang LH, Dong YH, Zhang LH. 2008. Quorum quenching :impact and mechanisms, pp. 379-392. I n Winans SC, Bassler BL (eds.). Chemical Communication Among Bacteria. ASM Press, USA.
  33. Wang WZ, Morohoshi T, Someya N, Ikeda T. 2012. Diversity and distribution of N-acylhomoserine lactone (AHL)-degrading activity and AHL-lactonase (AiiM) in genus Microbacterium. Microbes Environ. 27: 330-333.
    Pubmed CrossRef
  34. Whitehead NA, Barnard AM, Slater H, Simpson NJ, Salmond GP. 2001. Quorum-sensing in gram-negative bacteria. FEMS Microbiol Rev. 25: 365-404.
    Pubmed CrossRef
  35. Whitehead NA, Welch M, Salmond GP. 2001. Silencing the majority. Nat. Biotechnol. 19: 735-736.
    Pubmed CrossRef
  36. Xiong Y, Liu Y. 2010. Biological control of microbial attachment: a promising alternative for mitigating membrane biofouling. Appl. Microbiol. Biotechnol. 86: 825-837.
    Pubmed CrossRef
  37. Yeon KM, Cheong WS, Oh HS, Lee WN, Hwang BK, Lee CH, et al. 2009. Quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment. Environ. Sci. Technol. 43: 380-385.
    Pubmed CrossRef
  38. Zhang L, Murphy PJ, Kerr A, Tate ME. 1993. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362: 446-448.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2014; 24(11): 1574-1582

Published online November 28, 2014 https://doi.org/10.4014/jmb.1407.07009

Copyright © The Korean Society for Microbiology and Biotechnology.

Quorum Quenching Bacteria Isolated from the Sludge of a Wastewater Treatment Plant and Their Application for Controlling Biofilm Formation

A-Leum Kim 1, Son-Young Park 1, Chi-Ho Lee 1, Chung-Hak Lee 2 and Jung-Kee Lee 1*

1Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea, 2School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea

Received: July 7, 2014; Accepted: August 10, 2014

Abstract

Bacteria recognize changes in their population density by sensing the concentration of signal
molecules, N-acyl-homoserine lactones (AHLs). AHL-mediated quorum sensing (QS) plays a
key role in biofilm formation, so the interference of QS, referred to as quorum quenching
(QQ), has received a great deal of attention. A QQ strategy can be applied to membrane
bioreactors (MBRs) for advanced wastewater treatment to control biofouling. To isolate QQ
bacteria that can inhibit biofilm formation, we isolated diverse AHL-degrading bacteria from a
laboratory-scale MBR and sludge from real wastewater treatment plants. A total of 225 AHLdegrading
bacteria were isolated from the sludge sample by enrichment culture. To identify
the enzyme responsible for AHL degradation in QQ bacteria, AHL-degrading activities were
analyzed using cell-free lysate, culture supernatant, and whole cells. Afipia sp. and
Acinetobacter sp. strains produced the intracellular QQ enzyme, whereas Pseudomonas sp. and
Micrococcus sp. produced the extracellular QQ enzyme that was most likely to produce AHLacylase.
AHL-degrading activity was observed in whole-cell assay with the Microbacterium sp.
and Rhodococcus sp. strains. There has been no report for AHL-degrading capability in the case
of Streptococcus sp. and Afipia sp. strains. Finally, inhibition of biofilm formation by isolated
QQ bacteria or enzymes was observed on glass slides and 96-well microtiter plates using
crystal violet staining. QQ strains or enzymes not only inhibited initial biofilm development
but also reduced established biofilms.

Keywords: quorum sensing, quorum quenching, AHL, biofilm, wastewater treatment

References

  1. Bokhove M, Nadal Jimenez P, Quax WJ, Dijkstra BW. 2010. The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket. Proc. Natl. Acad. Sci. USA 107: 686-691.
    Pubmed CrossRef
  2. Cheong SC, Lee CH, Moon YH, Oh HS, Kim SR, Lee SH, et al. 2013. Isolation and identification of indigenous quorum quenching bacteria, Pseudomonas sp. 1A1, for biofouling control in MBR. Ind. Eng. Chem. Res. 31: 10554-10560.
    CrossRef
  3. Chowdhary PK, Keshavan N, Nguyen HQ, Peterson JA, Gonzalez JE, Haines DC. 2007. Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines. Biochemistry 46: 14429-14437.
    Pubmed CrossRef
  4. Cook DM, Li PL, Ruchaud F, Padden S, Farrand SK. 1997. Ti plasmid conjugation is independent of vir: reconstitution of the tra functions from pTiC58 as a binary system. J. Bacteriol. 179: 1291-1297.
    Pubmed
  5. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP. 1998. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280: 295-298.
    Pubmed CrossRef
  6. Dickschat JS. 2010. Quorum sensing and bacterial biofilms. Nat. Prod. Rep. 27: 343-369.
    Pubmed CrossRef
  7. Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813-817.
    Pubmed CrossRef
  8. Eberhard A, Burlingame AL, Eberhard C, Kenyon GL, Nealson KH, Oppenheimer NJ. 1981. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry 20: 2444-2449.
    Pubmed CrossRef
  9. Flemming HC, Neu TR, Wozniak DJ. 2007. T he EPS matrix:the “house of biofilm cells”. J. Bacteriol. 189: 7945-7947.
    Pubmed CrossRef
  10. Fuqua WC, Winans SC, Greenberg EP. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell densityresponsive transcriptional regulators. J. Bacteriol. 176: 269-275.
    Pubmed
  11. Fux CA, Stoodley P, Hall-Stoodley L, Costerton JW. 2003. Bacterial biofilms: a diagnostic and therapeutic challenge. Expert Rev. Anti Infect. Ther. 1: 667-683.
    Pubmed CrossRef
  12. Huang JJ, Petersen A, Whiteley M, Leadbetter JR. 2006. Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1. Appl. Environ. Microbiol. 72: 1190-1197.
    Pubmed CrossRef
  13. Kang BR, Lee JH, Ko SJ, Lee YH, Cha JS, Cho BH, Kim YC. 2004. Degradation of acyl-homoserine lactone molecules by Acinetobacter sp. strain C1010. Can. J. Microbiol. 50: 935-941.
    Pubmed CrossRef
  14. Latifi A, Winson MK, Foglino M, Bycroft BW, Stewart GS, Lazdunski A, Williams P. 1995. Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol. Microbiol. 17: 333-343.
    Pubmed CrossRef
  15. Leadbetter JR, Greenberg EP. 2000. Metabolism of acylhomoserine lactone quorum-sensing signals by Variovorax paradoxus. J. Bacteriol. 182: 6921-6926.
    Pubmed CrossRef
  16. Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK. 2002. Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl. Environ. Microbiol. 68: 3919-3924.
    Pubmed CrossRef
  17. Lin YH, Xu JL, Hu J, Wang LH, Ong SL, Leadbetter JR, Zhang LH. 2003. Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol. Microbiol. 47: 849-860.
    Pubmed CrossRef
  18. Ochiai S, Morohoshi T, Kurabeishi A, Shinozaki M, Fujita H, Sawada I, Ikeda T. 2013. Production and degradation of N-acylhomoserine lactone quorum sensing signal molecules in bacteria isolated from activated sludge. Biosci. Biotechnol. Biochem. 77: 2436-2440.
    Pubmed CrossRef
  19. Oh HS, Yeon KM, Yang CS, Kim SR, Lee CH, Park SY, et al. 2012. Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ. Sci. Technol. 46: 4877-4884.
    Pubmed CrossRef
  20. O’Toole GA, Kolter R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28: 449-461.
    Pubmed CrossRef
  21. Park SY, Hwang BJ, Shin MH, Kim JA, Kim HK, Lee JK. 2006. N-Acylhomoserine lactonase-producing Rhodococcus spp. with different AHL-degrading activities. FEMS Microbiol. Lett. 261: 102-108.
    Pubmed CrossRef
  22. Park SY, Lee SJ, Oh TK, Oh JW, Koo BT, Yum DY, Lee JK. 2003. AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149: 1541-1550.
    Pubmed CrossRef
  23. Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, Iglewski BH, Greenberg EP. 1994. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc. Natl. Acad. Sci. USA 91: 197-201.
    Pubmed CrossRef
  24. Rasmussen TB, Givskov M. 2006. Quorum sensing inhibitors:a bargain of effects. Microbiology 152: 895-904.
    Pubmed CrossRef
  25. Salmond GP, Bycroft BW, Stewart GS, Williams P. 1995. The bacterial ‘enigma’: cracking the code of cell-cell communication. Mol. Microbiol. 16: 615-624.
    Pubmed CrossRef
  26. Shaw PD, Ping G, Daly SL, Cha C, Cronan JE Jr, Rinehart KL, Farrand SK. 1997. Detecting and characterizing N-acylhomoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94: 6036-6041.
    Pubmed CrossRef
  27. Sintim HO, Smith JA, Wang J, Nakayama S, Yan L. 2010. Paradigm shift in discovering next-generation anti-infective agents: targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small molecules. Future Med. Chem. 2: 1005-1035.
    Pubmed CrossRef
  28. Stewart PS, Costerton JW. 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358: 135-138.
    CrossRef
  29. Throup JP, Camara M, Briggs GS, Winson MK, Chhabra SR, Bycroft BW, et al. 1995. Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules. Mol. Microbiol. 17: 345-356.
    Pubmed CrossRef
  30. Uroz S, Chhabra SR, Camara M, Williams P, Oger P, Dessaux Y. 2005. N-Acylhomoserine lactone quorum-sensing molecules are modified and degraded by Rhodococcus erythropolis W2 by both amidolytic and novel oxidoreductase activities. Microbiology 151: 3313-3322.
    Pubmed CrossRef
  31. Uroz S, Oger PM, Chapelle E, Adeline MT, Faure D, Dessaux Y. 2008. A Rhodococcus qsdA-encoded enzyme defines a novel class of large-spectrum quorum-quenching lactonases. Appl. Environ. Microbiol. 74: 1357-1366.
    Pubmed CrossRef
  32. Wang LH, Dong YH, Zhang LH. 2008. Quorum quenching :impact and mechanisms, pp. 379-392. I n Winans SC, Bassler BL (eds.). Chemical Communication Among Bacteria. ASM Press, USA.
  33. Wang WZ, Morohoshi T, Someya N, Ikeda T. 2012. Diversity and distribution of N-acylhomoserine lactone (AHL)-degrading activity and AHL-lactonase (AiiM) in genus Microbacterium. Microbes Environ. 27: 330-333.
    Pubmed CrossRef
  34. Whitehead NA, Barnard AM, Slater H, Simpson NJ, Salmond GP. 2001. Quorum-sensing in gram-negative bacteria. FEMS Microbiol Rev. 25: 365-404.
    Pubmed CrossRef
  35. Whitehead NA, Welch M, Salmond GP. 2001. Silencing the majority. Nat. Biotechnol. 19: 735-736.
    Pubmed CrossRef
  36. Xiong Y, Liu Y. 2010. Biological control of microbial attachment: a promising alternative for mitigating membrane biofouling. Appl. Microbiol. Biotechnol. 86: 825-837.
    Pubmed CrossRef
  37. Yeon KM, Cheong WS, Oh HS, Lee WN, Hwang BK, Lee CH, et al. 2009. Quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment. Environ. Sci. Technol. 43: 380-385.
    Pubmed CrossRef
  38. Zhang L, Murphy PJ, Kerr A, Tate ME. 1993. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362: 446-448.
    Pubmed CrossRef