Development of a Sensitive Bioassay Method for Quorum Sensing Inhibitor Screening Using a Recombinant Agrobacterium tumefaciens

  • Kim Yeon Hee (Department of Chemical and Biochemical Engineering and Institute for Environmental Technology and Industry, Pusan National University) ;
  • Kim Young Hee (Department of Chemical and Biochemical Engineering and Institute for Environmental Technology and Industry, Pusan National University) ;
  • Kim Jung Sun (Department of Biotechnology, Dongseo University) ;
  • Park Sunghoon (Department of Chemical and Biochemical Engineering and Institute for Environmental Technology and Industry, Pusan National University)
  • Published : 2005.08.01

Abstract

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the $\beta$-galactosidase activity ($\beta$-GAL) of a recombinant Agrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing $\beta$-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration­dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of $\beta$-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.

Keywords

References

  1. Shirtliff, M. E., J. T. Mader, and A. K. Camper (2002) Molecular interactions in biofilms. Chem. Biol. 9: 859-871 https://doi.org/10.1016/S1074-5521(02)00198-9
  2. Tsukamoto, T., M. Matsukawa, M. Sano, S. Takahashi, H. Hotta, N. Itoh, T. Hirose, and Y. Kumamoto (1999) Biofilm in complicated urinary tract infection. Int. J. Antimicrob. Ag. 11: 233-236 https://doi.org/10.1016/S0924-8579(99)00021-7
  3. Costerton, J. W. (1999) Introduction to biofilm. Int. J. Antimicrob. Ag. 11: 217-221 https://doi.org/10.1016/S0924-8579(99)00018-7
  4. Lee, M., S. H. Hwang, S. D. Ha, J. H. Jang, D. J. Lim, and J. Y. Kong (2004) Rhamnolipid production in batch and fed-batch fermentation using Pseudomonas aeruginosa BYK-2-KCTC 18012P. Biotechnol. Bioprocess Eng. 9: 267-273 https://doi.org/10.1007/BF02942342
  5. Fuqua, C. and E. P. Greenberg (2002) Listening in on bacteria: Acyl-homoserine lactone signaling. Nat. Rev. Mol. Cell. Biol. 3: 685-695 https://doi.org/10.1038/nrm907
  6. Gray, K. M. (1997) Intercellular communication and group behavior in bacteria. Trends. Microbiol. 5: 184-188 https://doi.org/10.1016/S0966-842X(97)01002-0
  7. Chen, X., S. Schauder, N. Potier, A. Van Dorsselaer, I. Pelczer, B. L. Bassler, and F. M. Hughson (2002) Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415: 545-549 https://doi.org/10.1038/415545a
  8. Dunny, G. M. and B. A. Leonard (1997) Cell-cell communication in gram-positive bacteria. Annu. Rev. Microbiol. 51: 527-564 https://doi.org/10.1146/annurev.micro.51.1.527
  9. Zhu, J., J. W. Beaber, M. I. More, C. Fuqua, A. Eberhard, and S. C. Winans (1998) Analogs of the autoinducer 3- oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J. Bacteriol. 180: 5398-5405
  10. Manefield, M., R. de Nys, N. Kumar, R. Read, M. Givskov, P. Steinberg, and S. Kjelleberg (1999) Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein. Microbiol. 145: 283-291 https://doi.org/10.1099/13500872-145-2-283
  11. Brelles-Marino, G. and E. J. Bedmar (2001) Detection, purification and characterization of quorum-sensing signal molecules in plant-associated bacteria. J. Biotechnol. 91: 197-209 https://doi.org/10.1016/S0168-1656(01)00330-3
  12. Teplitski, M., J. B. Robinson, and W. D. Bauer (2000) Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria. Mol. Plant. Microb. In. 13: 637-648 https://doi.org/10.1094/MPMI.2000.13.6.637
  13. Bassler, B. L., M. Wright, and M. R. Silverman (1994) Multiple signaling systems controlling expression of luminescence in Vibrio harveyi: Sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 13: 273-286 https://doi.org/10.1111/j.1365-2958.1994.tb00422.x
  14. Schaefer, A. L., B. L. Hanzelka, A. Eberhard, and E. P. Greenberg (1996) Quorum sensing in Vibrio fischeri: Probing autoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol. 178: 2897-2901 https://doi.org/10.1128/jb.178.10.2897-2901.1996
  15. Blosser, R. S. and K. M. Gray (2000) Extraction of violacein from Chromobacterium violaceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers. J. Microbiol. Meth. 40: 47-55 https://doi.org/10.1016/S0167-7012(99)00136-0
  16. Paggi, R. A., C. B. Martone, C. Fuqua, and R. E. De Castro (2003) Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus. FEMS Microbiol. Lett. 221: 49-52 https://doi.org/10.1016/S0378-1097(03)00174-5
  17. Lee, H. Y., S. H. Choi, and M. B. Gu (2003) Response of bioluminescent bacteria to sixteen azo dyes. Biotechnol. Bioprocess Eng. 8: 101-105 https://doi.org/10.1007/BF02940264
  18. Zhu, J., Y. Chai, Z. Zhong, S. Li, and S. C. Winans (2003) Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: Detection of autoinducers in Mesorhizobium huakii. Appl. Environ. Microb. 69: 6949-6953 https://doi.org/10.1128/AEM.69.11.6949-6953.2003
  19. Malott, R. J. and R. Y. Lo (2002) Studies on the production of quorum-sensing signal molecules in Mannheimia haemolytica A1 and other Pasteurellaceae species. FEMS Microbiol. Lett. 206: 25-30 https://doi.org/10.1111/j.1574-6968.2002.tb10981.x
  20. Ramussen, T. B., M. Hentzer, T. Bjarnsholt, M. Kothe, L. Eberl, and M. Givskov (2003) Eukaryotic interference with bacterial communication: En route to novel antiinfective compounds. Proceeding of the ASM Conferences, Biofilm 2003. November 1-7. Victoria, Canada
  21. Fuqua, C. and S. C. Winans (1996) Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J. Bacteriol. 178: 435-440 https://doi.org/10.1128/jb.178.2.435-440.1996
  22. Manny, A. J., S. Kjelleberg, N. Kumar, R. de Nys, R. W. Read, and P. Steinberg (1997) Reinvestigation of the sulfuric acid-catalyzed cyclisation of brominated 2-alkyllevulinic acids to 3-alkyl-5-methylene-2(5H)-furanones. Tetrahedron 53: 15813-15826 https://doi.org/10.1016/S0040-4020(97)10034-5
  23. McLean, R. J., M. Whiteley, D. J. Stickler, and C. Fuqua (1997) Evidence of autoinducer activity in naturally occurring biofilms. FEMS Microbiol. Lett. 154: 259-263 https://doi.org/10.1111/j.1574-6968.1997.tb12653.x
  24. Stickler, D. J., N. S. Morris, R. J. McLean, and C. Fuqua (1998) Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Appl. Environ. Microb. 64: 3486-3490
  25. Michels, J. J., E. J. Allain, S. A. Borchardt, P. Hu, and W. F. McCoy (2000) Degradation pathway of homoserine lactone bacterial signal molecules by halogen antimicrobials identified by liquid chromatography with photodiode array and mass spectrometric detection. J. Chromatogr. A 898: 153-165 https://doi.org/10.1016/S0021-9673(00)00849-9
  26. Hong, S. P., C. H. Lee, S. K. Kim, H. S. Yun, and J. H. Lee (2004) Mobile phase compositions for ceramide III by normal phase high performance liquid chromatography. Biotechnol. Bioprocess Eng. 9: 47-51 https://doi.org/10.1007/BF02949321
  27. Grouls, R. J. E., E. W. Ackerman, H. H. M. Korsten, L. J. Hellebrekers, and D. D. Breimer (1997) Partition coefficients (n-octanol/water) of N-butyl-p-aminobenzoate and other local anesthetics measured by reversed-phase highperformance liquid chromatography. J. Chromatogr. B 694: 421-425 https://doi.org/10.1016/S0378-4347(97)00207-7
  28. Patrick, G. L. (1997) An Introduction to Medicinal Chemistry. pp. 128-153. Oxford Univ. Press, NY, USA