Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Characterization of Quorum-Quenching Bacteria Isolated from Biofouled Membrane Used in Reverse Osmosis Process

Biofouling이 일어난 역삼투막에서 분리한 쿼럼 저해 세균의 특성

  • Moon, Sooyoung (Department of Environmental Engineering and Energy, Myongji University) ;
  • Huang, Xinxin (Department of Environmental Engineering and Energy, Myongji University) ;
  • Choi, Sung-Chan (Department of Environmental Science and Biotechnology, Hallym University) ;
  • Oh, Young-Sook (Department of Environmental Engineering and Energy, Myongji University)
  • 문수영 (명지대학교 환경에너지공학과) ;
  • ;
  • 최성찬 (한림대학교 환경생명공학과) ;
  • 오영숙 (명지대학교 환경에너지공학과)
  • Received : 2014.03.28
  • Accepted : 2014.05.14
  • Published : 2014.06.30
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Abstract

Acyl homoserine lactone (AHL) lactonase has been proved to be the AHL-degrading enzyme with the highest substrate specificity for AHL molecules and has shown a considerable potential as low-cost and efficient quorum quenching (QQ) technique. However, few studies focused on its inhibitory effect on biofilm formation which is also a quorum sensing (QS)-regulated phenomenon. In this study, QQ activity of six isolates from biofouled reverse osmosis membranes was studied using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 as biosensors under various conditions. All of the isolates belonged to the genus Bacillus and showed QQ activity regardless of the acyl chain length or substitution of AHL molecule. The isolates were capable of significantly inhibiting biofilm formation (46.7-58.3%) by Pseudomonas aeruginosa PAO1 and produced heat-sensitive extracellular QQ substances. The LC-MS analysis of the QQ activity of a selected isolate, RO1S-5, revealed the degradation of N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 AHL) and the production of corresponding acyl homoserine (3-oxo-C12-HS), which indicated the activity of AHL lactonase. The broad AHL substrate range and high substrate specificity suggested that the isolate would be useful for the control of biofilm-related pathogenesis and biofouling in industrial processes.

Acyl homoserine lactone (AHL) 분해효소인 lactonase는 높은 기질 특이성을 지니기 때문에 경제적이고 효율적인 쿼럼 저해 기술로 이용될 가능성을 지니고 있다. 본 연구에서는 Chromobacterium violaceum CV026과 Agrobacterium tumefaciens NTL4를 바이오센서로 이용하여 biofouling이 일어난 역삼투막 시료로부터 쿼럼 센싱과 관련된 생물막 형성을 억제하는 6종의 균주를 분리 연구하였다. 분리된 균주는 모두 Bacillus 속으로 동정되었으며, AHL 분자의 acyl 사슬 길이나 치환 종류에 상관 없이 쿼럼 저해활성을 보여주었다. 균주들은 Pseudomonas aeruginosa PAO1에 의한 생물막 형성을 46.7-58.3% 정도 감소시켰으며 이 때 저해물질은 열처리에 민감한 특성을 보여주었다. 분리 균주 중 RO1S-5를 이용하여 N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 AHL)과 반응시킨 결과, 상응하는 acyl homoserine (3-oxo-C12-HS)이 생성되는 것을 LC-MS로 확인하여 쿼럼 저해가 lactonase 활성에 의한 것임을 규명하였다. AHL 물질에 대한 높은 특이성 등을 감안할 때 분리 균주 RO1S-5는 생물막 형성과 관련된 질병이나 산업공정 중 발생하는 biofouling을 해결하는데 유용하게 쓰일 수 있을 것으로 기대된다.

Keywords

References

  1. Adonizio, A., Kong, K.F., and Mathee, K. 2008. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. Antimicrob. Agents Chemother. 52, 198-203. https://doi.org/10.1128/AAC.00612-07
  2. Ammor, M.S., Michaelidis, C., and Nychas, G.J. 2008. Insights into the role of quorum sensing in food spoilage. J. Food Prot. 71, 1510-1525. https://doi.org/10.4315/0362-028X-71.7.1510
  3. Cao, Y., He, S., Zhou, Z., Zhang, M., Mao, W., Zhang, H., and Yao, B. 2012. Orally administered thermostable N-acyl homoserine lactonase from Bacillus sp. strain AI96 attenuates Aeromonas hydrophila infection in zebrafish. Appl. Environ. Microbiol. 78, 1899-1908. https://doi.org/10.1128/AEM.06139-11
  4. Carlier, A., Uroz, S., Smadja, B., Fray, R., Latour, X., Dessaux, Y., and Faure, D. 2003. The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-acyl homoserine lactonase activity. Appl. Environ. Microbiol. 69, 4989-4993. https://doi.org/10.1128/AEM.69.8.4989-4993.2003
  5. Case, R.J., Labbate, M., and Kjelleberg, S. 2008. AHL-driven quorum-sensing circuits: their frequency and function among the Proteobacteria. ISME J. 2, 345-349. https://doi.org/10.1038/ismej.2008.13
  6. Cha, C., Gao, P., Chen, Y.C., Shaw, P.D., and Farrand, S.K. 1998. Production of acyl- homoserine lactone quorum-sensing signals by Gram-negative plant-associated bacteria. Mol. Plant Microbe Interact. 11, 1119-1129. https://doi.org/10.1094/MPMI.1998.11.11.1119
  7. Chen, F., Gao, Y., Chen, X., Yu, Z., and Li, X. 2013. Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection. Int. J. Mol. Sci. 14, 17477-17500. https://doi.org/10.3390/ijms140917477
  8. Chen, R., Zhou, Z., Cao, Y., Bai, Y., and Yao, B. 2010. High yield expression of an AHL-lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture. Microb. Cell Fact. 9, 39-48. https://doi.org/10.1186/1475-2859-9-39
  9. Cheong, W.S., Lee, C.H., Moon, Y.H., Oh, H.S., Kim, S.R., Lee, S.H., Lee, C.H., and Lee, J.K. 2013. Isolation and identification of indigenous quorum quenching bacteria, Pseudomonas sp. 1A1, for biofouling control in MBR. Ind. Eng. Chem. Res. 52, 10554-10560. https://doi.org/10.1021/ie303146f
  10. Chowdhary, P.K., Keshavan, N., Nguyen, H.Q., Peterson, J.A., Gonzalez, J.E., and Haines, D.C. 2007. Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines. Biochem. 46, 14429-14437. https://doi.org/10.1021/bi701945j
  11. Christiaen, S.E.A., Brackman, G., Nelis, H.J., and Coenye, T. 2011. Isolation and identification of quorum quenching bacteria from environmental samples. J. Microbiol. Methods 87, 213-219. https://doi.org/10.1016/j.mimet.2011.08.002
  12. Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski, B.H., Costerton, J.W., and Greenberg, E.P. 1998. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295-298. https://doi.org/10.1126/science.280.5361.295
  13. Defoirdt, T., Boon, N., Bossier, P., and Verstraete, W. 2004. Disruption of bacterial quorum sensing: an unexplored strategy to fight infections in aquaculture. Aquaculture 240, 69-88. https://doi.org/10.1016/j.aquaculture.2004.06.031
  14. Dong, Y.H., Gusti, A.R., Zhang, Q., Xu, J.L., and Zhang, L.H. 2002. Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68, 1754-1759. https://doi.org/10.1128/AEM.68.4.1754-1759.2002
  15. Dong, Y.H., Wang, L.H., Xu, J.L., Zhang, H.B., Zhang, X.F., and Zhang, L.H. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411, 813-817. https://doi.org/10.1038/35081101
  16. Dong, Y.H., Wang, L.H., and Zhang, L.H. 2007. Quorum-quenching microbial infections: mechanisms and implications. Phil. Trans. R. Soc. B 362, 1201-1211. https://doi.org/10.1098/rstb.2007.2045
  17. Dong, Y.H., Xu, J.L., Li, X.Z., and Zhang, L.H. 2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. USA 97, 3526-3531. https://doi.org/10.1073/pnas.97.7.3526
  18. Dong, Y.H. and Zhang, L.H. 2005. Quorum sensing and quorum-quenching enzymes. J. Microbiol. 43, 101-109.
  19. Donlan, R.M. 2001. Biofilms and device-associated infections. Emerging Infect. Dis. 7, 277-281. https://doi.org/10.3201/eid0702.010226
  20. Estrela, A.B. and Abraham, W.R. 2010. Combining biofilm-controlling compounds and antibiotics as a promising new way to control biofilm infections. Pharmaceuticals 3, 1374-1393. https://doi.org/10.3390/ph3051374
  21. Farrand, S.K., Qin, Y., and Oger, P. 2002. Quorum-sensing system of Agrobacterium plasmids: analysis and utility. Methods Enzymol. 358, 452-484. https://doi.org/10.1016/S0076-6879(02)58108-8
  22. Hentzer, M., Riedel, K., Rasmussen, T.B., Heydorn, A., Andersen, J.B., Parsek, M.R., Rice, S.A., Eberl, L., Molin, S., Hoiby, N., and et al. 2002. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148, 87-102. https://doi.org/10.1099/00221287-148-1-87
  23. Hoffman, L.R., D'Argenio, D.A., MacCoss, M.J., Zhang, Z., Jones, R.A., and Miller, S.I. 2005. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436, 1171-1175. https://doi.org/10.1038/nature03912
  24. Kawaguchi, T., Chen, Y.P., Norman, R.S, and Decho, A.W. 2008. Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay. Appl. Environ. Microbiol. 74, 3667-3671. https://doi.org/10.1128/AEM.02869-07
  25. Kim, J.H., Choi, D.C., Yeon, K.M., Kim, S.R., and Lee, C.H. 2011. Enzyme-immobilized nanofiltration membrane to mitigate biofouling based on quorum quenching. Environ. Sci. Technol. 45, 1601-1607. https://doi.org/10.1021/es103483j
  26. Kim, C., Kim, J., Park, H.Y., Park, H.J., Lee, J.H., Kim, C.K., and Yoon, J. 2008. Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa. Appl. Microbiol. Biotechnol. 80, 37-47. https://doi.org/10.1007/s00253-008-1474-6
  27. Kim, S., Lee, S., Hong, S., Oh, Y., Seoul, M., Kweon, J., and Kim, T. 2009. Biofouling of reverse osmosis membranes: microbial quorum sensing and fouling propensity. Desalination 249, 303-315.
  28. Kiran, S., Sharma, P., Harjai, K., and Capalash, N. 2011. Enzymatic quorum quenching increases antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa. Iran. J. Microbiol. 3, 1-12.
  29. Lee, S.J., Park, S.Y., Lee, J.J., Yum, D.Y., Koo, B.T., and Lee, J.K. 2002. Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl. Environ. Microbiol. 68, 3919-3924. https://doi.org/10.1128/AEM.68.8.3919-3924.2002
  30. Lin, Y.H., Xu, J.L., Hu, J., Wang, L.H., Ong, S.L., Leadbetter, J.R., and Zhang, L.H. 2003. Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol. Microbiol. 47, 849-860. https://doi.org/10.1046/j.1365-2958.2003.03351.x
  31. Liu, X., Jia, J., Popat, R., Ortori, C.A., Li, J., Diggle, S.P., Gao, K., and Camara, M. 2011. Characterisation of two quorum sensing systems in the endophytic Serratia plymuthica strain G3: differential control of motility and biofilm formation according to life-style. BMC Microbiol. 11, 26-37. https://doi.org/10.1186/1471-2180-11-26
  32. Luo, Z.Q., Su, S., and Farrand, S.K. 2003. In situ activation of the quorum-sensing transcription factor TraR by cognate and noncognate acyl-homoserine lactone ligands: kinetics and consequences. J. Bacteriol. 185, 5665-5672. https://doi.org/10.1128/JB.185.19.5665-5672.2003
  33. Mani, A., Hameed, S.S., Ramalingam, S., and Narayanan, M. 2012. Assessment of quorum quenching activity of Bacillus species against Pseudomonas aeruginosa MTCC 2297. Global J. Pharmacol. 6, 118-125.
  34. McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft, B.W., and et al. 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143, 3703-3711. https://doi.org/10.1099/00221287-143-12-3703
  35. Mei, G.Y., Yan, X.X., Turak, A., Luo, Z.Q., and Zhang, L.Q. 2010. AidH, an alpha/beta-hydrolase fold family member from an Ochrobactrum sp. strain, is a novel N-acylhomoserine lactonase. Appl. Environ. Microbiol. 76, 4933-4942. https://doi.org/10.1128/AEM.00477-10
  36. Molina, L., Constantinescu, F., Michel, L., Reimmann, C., Duffy, B., and Defago, G. 2003. Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. FEMS Microbiol. Ecol. 45, 71-81. https://doi.org/10.1016/S0168-6496(03)00125-9
  37. Morohoshi, T., Someya, N., and Ikeda, T. 2009. Novel N-acylhomoserine lactone-degrading bacteria isolated from the leaf surface of Solanum tuberosum and their quorum-quenching properties. Biosci. Biotechnol. Biochem. 73, 2124-2127. https://doi.org/10.1271/bbb.90283
  38. Ng, W.L. and Bassler, B.L. 2009. Bacterial quorum-sensing network architectures. Annu. Rev. Genet. 43, 197-222. https://doi.org/10.1146/annurev-genet-102108-134304
  39. Nithya, C., Aravindraja, C., and Pandian, S.K. 2010. Bacillus pumilus of Palk Bay origin inhibits quorum-sensing-mediated virulence factors in Gram-negative bacteria. Res. Microbiol. 161, 293-304. https://doi.org/10.1016/j.resmic.2010.03.002
  40. Novick, R.P. and Geisinger, E. 2008. Quorum sensing in staphylococci. Annu. Rev. Genet. 42, 541-564. https://doi.org/10.1146/annurev.genet.42.110807.091640
  41. O'Toole, G.A. and Kolter, R. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30, 295-304. https://doi.org/10.1046/j.1365-2958.1998.01062.x
  42. Park, S.Y., Hwang, B.J., Shin, M.H., Kim, J.A., Kim, H.K., and Lee, J.K. 2006. N-acyl homoserine lactonase-producing Rhodococcus spp. with different AHL-degrading activities. FEMS Microbiol. Lett. 261, 102-108. https://doi.org/10.1111/j.1574-6968.2006.00336.x
  43. Park, S.Y., Kang, H.O., Jang, H.S., Lee, J.K., Koo, B.T., and Yum, D.Y. 2005. Identification of extracellular N-acylhomoserine lactone acylase from a Streptomyces sp. and its application to quorum quenching. Appl. Environ. Microbiol. 71, 2632-2641. https://doi.org/10.1128/AEM.71.5.2632-2641.2005
  44. Park, S.Y., Lee, S.J., Oh, T.K., Oh, J.W., Koo, B.T., Yum, D.Y., and Lee, J.K. 2003. AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149, 1541-1550. https://doi.org/10.1099/mic.0.26269-0
  45. Parsek, M.R., Val, D.L., Hanzelka, B.L., Cronan, Jr. J.E., and Greenberg, E.P. 1999. Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA 96, 4360-4365. https://doi.org/10.1073/pnas.96.8.4360
  46. Paul, D., Kim, Y.S., Ponnusamy, K., and Kweon, J.H. 2009. Application of quorum quenching to inhibit biofilm formation. Environ. Eng. Sci. 26, 1319-1324. https://doi.org/10.1089/ees.2008.0392
  47. Pesci, E.C., Pearson, J.P., Seed, P.C., and Iglewski, B.H. 1997. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179, 3127-3132. https://doi.org/10.1128/jb.179.10.3127-3132.1997
  48. Ravn, L., Christensen, A.B., Molin, S., Givskov, M., and Gram, L. 2001. Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics. J. Microbiol. Methods 44, 239-251. https://doi.org/10.1016/S0167-7012(01)00217-2
  49. Reimmann, C., Ginet, N., Michel, L., Keel, C., Michaux, P., Krishnapillai, V., Zala, M., Heurlier, K., Triandafillu, K., Harms, H., and et al. 2002. Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1. Microbiology 148, 923-932. https://doi.org/10.1099/00221287-148-4-923
  50. Romero, M., Martin-Cuadrado, A.B., Roca-Rivada, A., Cabello, A.M., and Otero, A. 2011. Quorum quenching in cultivable bacteria from dense marine coastal microbial communities. FEMS Microbiol. Ecol. 75, 205-217. https://doi.org/10.1111/j.1574-6941.2010.01011.x
  51. Shetye, G.S., Singh, N., Gao, X., Bandyopadhyay, D., Yan, A., and Luk, Y.Y. 2013. Structures and biofilm inhibition activities of brominated furanones for Escherichia coli and Pseudomonas aeruginosa. Med. Chem. Commun. 4, 1079-1084. https://doi.org/10.1039/c3md00059a
  52. Sio, C.F., Otten, L.G., Cool, R.H., Diggle, S.P., Braun, P.G., Bos, R., Daykin, M., Camara, M., Williams, P., and Quax, W.J. 2006. Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1. Infect. Immun. 74, 1673-1682. https://doi.org/10.1128/IAI.74.3.1673-1682.2006
  53. Smith, R.S. and Iglewski, B.H. 2003a. P. aeruginosa quorum-sensing systems and virulence. Curr. Opin. Microbiol. 6, 56-60. https://doi.org/10.1016/S1369-5274(03)00008-0
  54. Smith, R.S. and Iglewski, B.H. 2003b. Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target. J. Clin. Invest. 112, 1460-1465. https://doi.org/10.1172/JCI200320364
  55. Steindler, L. and Venturi, V. 2007. Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors. FEMS Microbiol. Lett. 266, 1-9. https://doi.org/10.1111/j.1574-6968.2006.00501.x
  56. Stewart, P.S. and Costerton, J.W. 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358, 135-138. https://doi.org/10.1016/S0140-6736(01)05321-1
  57. Tang, K., Zhang, Y., Yu, M., Shi, X., Coenye, T., Bossier, P., and Zhang, X.H. 2013. Evaluation of a new high-throughput method for identifying quorum quenching bacteria. Sci. Rep. 3, 2935. https://doi.org/10.1038/srep02935
  58. Ulrich, R.L. 2004. Quorum quenching: enzymatic disruption of N-acylhomoserine lactone-mediated bacterial communication in Burkholderia thailandensis. Appl. Environ. Microbiol. 70, 6173-6180. https://doi.org/10.1128/AEM.70.10.6173-6180.2004
  59. Van Houdt, R. and Michiels, C.W. 2010. Biofilm formation and the food industry, a focus on the bacterial outer surface. J. Appl. Microbiol. 109, 1117-1131. https://doi.org/10.1111/j.1365-2672.2010.04756.x
  60. Wang, L.H., Weng, L.X., Dong, Y.H., and Zhang, L.H. 2004. Specificity and enzyme kinetics of the quorum-quenching N-acyl homoserine lactone lactonase (AHL- lactonase). J. Biol. Chem. 279, 13645-13651. https://doi.org/10.1074/jbc.M311194200
  61. Wevers, E., Moons, P., Van Houdt, R., Lurquin, I., Aertsen, A., and Michiels, C.W. 2009. Quorum sensing and butanediol fermentation affect colonization and spoilage of carrot slices by Serratia plymuthica. Int. J. Food Microbiol. 134, 63-69. https://doi.org/10.1016/j.ijfoodmicro.2008.12.017
  62. Whitehead, N.A., Welch, M., and Salmond, G.P.C. 2001. Silencing the majority. Nature Biotechnol. 19, 735-736. https://doi.org/10.1038/90780
  63. Williams, P. and Camara, M. 2009. Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr. Opin. Microbiol. 12, 182-191. https://doi.org/10.1016/j.mib.2009.01.005
  64. Yates, E.A., Philipp, B., Buckley, C., Atkinson, S., Chhabra, S.R., Sockett, R.E., Goldner, M., Dessaux, Y., Camara, M., Smith, H., and et al. 2002. N-Acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa. Infect. Immun. 70, 5635-5646. https://doi.org/10.1128/IAI.70.10.5635-5646.2002
  65. Yeon, K.M., Lee, C.H., and Kim, J. 2009. Magnetic enzyme carrier for effective biofouling control in the membrane bioreactor based on enzymatic quorum quenching. Environ. Sci. Technol. 43, 7403-7409. https://doi.org/10.1021/es901323k
  66. Zamani, M., Behboudi, K., and Ahmadzadeh, M. 2013. Quorum quenching by Bacillus cereus U92: a double-edged sword in biological control of plant diseases. Biocontrol Sci. Technol. 23, 555-573. https://doi.org/10.1080/09583157.2013.787046
  67. Zhang, L., Ruan, L., Hu, C., Wu, H., Chen, S., Yu, Z., and Sun, M. 2007. Fusion of the genes for AHL-lactonase and S-layer protein in Bacillus thuringiensis increases its ability to inhibit soft rot caused by Erwinia carotovora. Appl. Microbiol. Biotechnol. 74, 667-675. https://doi.org/10.1007/s00253-006-0696-8
  68. Zhu, J., Cha, Y., Zhong, Z., Li, S., and Winans, S.C. 2003. Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii. Appl. Environ. Microbiol. 69, 6949-6953. https://doi.org/10.1128/AEM.69.11.6949-6953.2003