Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Identification and Functional Analysis of Vibrio vulnificus SmcR, a Novel Global Regulator

  • Lee, Jeojng-Hyun (Department of Food Science and Technology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University) ;
  • Rhee, Jee-Eun (Department of Food Science and Technology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University) ;
  • Park, U-Ryung (Department of Food Science and Technology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University) ;
  • Ju, Hyun-Mok (Department of Food Science and Technology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University) ;
  • Lee, Byung-Cheol (School of Life Sciences and Biotechnology, Korea University) ;
  • Kim, Tae-Sung (School of Life Sciences and Biotechnology, Korea University) ;
  • Jeong, Hye-Sook (Laboratory of Enteric Infections, Department of Microbiology, National Institute of Health Korea) ;
  • Choi, Sang-Ho (Department of Food Science and Technology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University)
  • Published : 2007.02.28
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Abstract

Recently, quorum sensing has been implicated as an important global regulator controlling the production of numerous virulence factors such as capsular polysaccharides in bacterial pathogens. The nucleotide and deduced amino acid sequences of smcR, a homolog of V. harveyi luxR identified from V. vulnificus ATCC29307, were analyzed. The amino acid sequence of SmcR from V. vulnificus was 72 to 92% similar to those of LuxR homologs from Vibrio spp. Functions of SmcR were assessed by the construction of an isogenic mutant, whose smcR gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of smcR resulted in a significant alteration in biofilm formation, in type of colony morphology, and in motility. When compared with the wild-type, the smcR mutant exhibited reduced survival under adverse conditions, such as acidic pH and hyperosmotic stress. The smcR mutant exhibited decreased cytotoxic activity toward INT 407 cells in vitro. Furthermore, the intraperitoneal $LD_{50}$ of the smcR mutant was approximately $10^2$ times higher than that of parental wild-type. Therefore, it appears that SmcR is a novel global regulator, controlling numerous genes contributing to the pathogenesis as well as survival of V. vulnificus.

Keywords

References

  1. Chen, X., S. Schauder, N. Polter, 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
  2. Croxatto, A., V. J. Chalker, J. Lauritz, J. Jass, A. Hardman, P. Williams, M. Camara, and D. L. Milton. 2002. VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum. J. Bacteriol. 184: 1617-1629 https://doi.org/10.1128/JB.184.6.1617-1629.2002
  3. de Kevit, T. R. and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 68: 4839-4849 https://doi.org/10.1128/IAI.68.9.4839-4849.2000
  4. Donnenberg, M. S. and J. B. Kaper. 1991. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59: 4310-4317
  5. Fuqua, W. C., S. C. Winans, and E. P. Greenberg. 1994. Quorum sensing in bacteria: The LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176: 269-275 https://doi.org/10.1128/jb.176.2.269-275.1994
  6. Greenberg, E. P., J. W. Hastings, and S. Ulitzur. 1979. Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria. Arch. Microbiol. 120: 87-91 https://doi.org/10.1007/BF00409093
  7. Jeong, H. S., L. H. Lee, K. H. Lee, S. J. Park, and S. H. Choi. 2003. SmcR and cyclic AMP receptor protein coactivate Vibrio vulnificus vvpE encoding elastase through the RpoSdependent promoter in a synergistic manner. J. Biol. Chem. 276: 45072-45081
  8. Jeong, K. C., H. S. Jeong, J. H. Rhee, S. E. Lee, S. S. Chung, A. M. Starks, G. M. Escudero, P. A. Gulig, and S. H. Choi. 2000. Construction and phenotypic evaluation of Vibrio vulnificus vvpE mutant for elastolytic protease. Infect. Immun. 68: 5096-5160 https://doi.org/10.1128/IAI.68.9.5096-5106.2000
  9. Jobling, M. and R. K. Holmes. 1997. Characterization of hapR, a positive regulator of the Vibrio cholerae HA/ protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol. Microbiol. 26: 1023-1034 https://doi.org/10.1046/j.1365-2958.1997.6402011.x
  10. Ju, H. M., I. G. Hwang, G. J. Woo, T. S. Kim, and S. H. Choi. 2005. Identification of the Vibrio vulnificus fexA gene and evaluation of its influence on virulence. J. Microbiol. Biotechnol. 15: 1337-1345
  11. Keen, N. T., S. Tamaki, D. Kobayashi, and D. Trollinger. 1988. Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70: 191-197 https://doi.org/10.1016/0378-1119(88)90117-5
  12. Kim S. Y., S. E. Lee, Y. R. Kim, C. M. Kim, R. Y. Ryu, H. E. Choy, S. S. Chung, and J. H. Rhee. 2003. Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensing system. Mol. Microbiol. 48: 1647-1664 https://doi.org/10.1046/j.1365-2958.2003.03536.x
  13. Kovacikova, G. and K. Skorupski. 2002. Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter. Mol. Microbiol. 46: 1135-1147 https://doi.org/10.1046/j.1365-2958.2002.03229.x
  14. Lee, B. C., S. H. Choi, and T. S. Kim. 2004. Application of sulforhodamine B assay for determining cytotoxicity of Vibrio vulnificus against human intestinal cells. J. Microbiol. Biotechnol. 14: 350-355
  15. Lee, J. H., N. Y. Park, S. J. Park, and S. H. Choi. 2003. Identification and characterization of the Vibrio vulnificus phosphomannomutase gene. J. Microbiol. Biotechnol. 13: 149-154
  16. Linkous, D. A. and J. D. Oliver. 1999. Pathogenesis of Vibrio vulnificus. FEMS Microbiol. Lett. 174: 207-214 https://doi.org/10.1111/j.1574-6968.1999.tb13570.x
  17. McCarter, L. L. 1998. OpaR, a homolog of Vibrio harveyi LuxR, controls opacity of Vibrio parahaemolyticus. J. Bacteriol. 180: 3166-3173
  18. McDougald, D., S. A. Rice, and S. Kjelleberg. 2000. The marine pathogen Vibrio vulnificus encodes a putative homologue of the Vibrio harveyi regulatory gene, luxR: A genetic and phylogenetic comparison. Gene 248: 213-221 https://doi.org/10.1016/S0378-1119(00)00117-7
  19. McDougald, D., S. A. Rice, and S. Kjelleberg. 2001. SmcRdependent regulation of adaptive phenotypes in Vibrio vulnificus. J. Bacteriol. 183: 758-762 https://doi.org/10.1128/JB.183.2.758-762.2001
  20. Mekalanos, J. J. 1992. Environmental signals controlling expression of virulence determinants in bacteria. J. Bacteriol. 174: 1-7 https://doi.org/10.1128/jb.174.1.1-7.1992
  21. Miller, M. B. and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199 https://doi.org/10.1146/annurev.micro.55.1.165
  22. Miller, J. F., J. J. Mekalanos, and S. Falkow. 1989. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science 243: 916-922 https://doi.org/10.1126/science.2537530
  23. Miller, V. L. and J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: Osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170: 2575-2583 https://doi.org/10.1128/jb.170.6.2575-2583.1988
  24. Nesper, J., C. M. Lauriano, K. E. Klose, D. Kapfhammer, A. Kraib, and J. Reidl. 2001. Characterization of Vibrio cholerae O1 El Tor galU and galE mutants: Influence on lipopolysaccharide structure, colonization, and biofilm formation. Infect. Immun. 69: 435-445 https://doi.org/10.1128/IAI.69.1.435-445.2001
  25. Oka, A., H. Sugisaki, and M. Takanami. 1981. Nucleotide sequence of the kanamycin resistance transposon Tn903. J. Mol. Biol. 147: 217-226 https://doi.org/10.1016/0022-2836(81)90438-1
  26. Park, N. A., J. H. Lee, B. C. Lee, T. S. Kim, and S. H. Choi. 2006. Identification and characterization of the wbpO gene essential for lipopolysaccharide synthesis in Vibrio vulnificus. J. Microbiol. Biotechnol. 16: 808-816
  27. Reed, L. J. and H. Muench. 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27: 439-497
  28. Rhee, J. E., J. H. Rhee, P. Y. Ryu, and S. H. Choi. 2002. Identification of the cadBA operon from Vibrio vulnificus and its influence on survival to acid stress. FEMS Microbiol. Lett. 208: 245-251 https://doi.org/10.1111/j.1574-6968.2002.tb11089.x
  29. Rhee, J. E., H. M. Ju, U. Park, B. C. Park, and S. H. Choi. 2004. Identification of the Vibrio vulnificus cadC and evaluation of its role in acid tolerance. J. Microbiol. Biotechnol. 14: 1093-1098
  30. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  31. Schauder, S. and B. L. Bassler. 2001. The language of bacteria. Genes Dev. 15: 1468-1480 https://doi.org/10.1101/gad.899601
  32. Schauder, S., K. Showkat, M. G. Surrette, and B. B. Bassler. 2001. The LuxS family of bacterial autoinducers: Biosynthesis of a novel quorum-sensing signal molecule. Mol. Microbiol. 41: 463-476 https://doi.org/10.1046/j.1365-2958.2001.02532.x
  33. Shao, C. P. and L. I. Hor. 2001. Regulation of metalloprotease gene expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue. J. Bacteriol. 183: 1369-1375 https://doi.org/10.1128/JB.183.4.1369-1375.2001
  34. Strom, M. and R. N. Paranjpye. 2000. Epidemiology and pathogenesis of Vibrio vulnificus. Microbes Infect. 2: 177- 188 https://doi.org/10.1016/S1286-4579(00)00270-7
  35. Williams, P., M. Camara, A. Hardman, S. Swift, D. Milton, V. J. Hope, K. Winzer, B. Middleton, D. I. Pritchard, and B. W. Bycroft. 2000. Quorum sensing and the populationdependent control of virulence. Philos. Trans. R. Soc. Lond. B 355: 667-680