Nonribosomal Peptide Synthase is Responsible for the Biosynthesis of Siderophore in Vibrio vulnificus MO6-24/O

  • Kim, In-Hwang (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Shim, Jung-Im (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Lee, Ko-Eun (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Hwang, Won (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Kim, Ik-Jung (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Choi, Sang-Ho (Department of Agricultural and Biotechnology and Center for Agricultural Biomaterials, Seoul National University) ;
  • Kim, Kun-Soo (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University)
  • Published : 2008.01.31

Abstract

Vibrio vulnificus produces siderophores, low-molecular-weight iron-chelating compounds, to obtain iron under conditions of iron deprivation. To identify genes associated with the biosynthesis of siderophore in V. vulnificus MO6-24/O, we screened clones with mini-Tn5 random insertions for those showing decreased production of siderophore. Among 6,000 clones screened, nine such clones were selected. These clones contain the transposon inserted in VV2_0830 (GenBank accession number) that is a homolog of a nonribosomal peptide synthase (NRPS). There is an another NRPS module, VV2_0831, 49-bp upstream to VV2_0830. We named these two genes vvs (Vibrio vulnificus siderophore synthase) A and B, respectively. Mutation of either vvsA or vvsB showed a decreased production of siderophore. The expression of an NRPS-lux fusion was negatively modulated by the presence of iron, and the regulation was dependent on Fur (ferric uptake regulator). However, the expression of the NRPS genes was still not fully derepressed in the iron-rich condition, even in furnull mutant cells, suggesting that some other unknown factors are involved in the regulation of the genes. We also demonstrated that the NRPS genes are important for virulence of the pathogen in a mice model.

Keywords

References

  1. Aisen, P., A. Leibman, and C. L. Sia. 1972. Molecular weight and subunit structure of hagfish transferrin. Biochemistry 11: 3461-3464 https://doi.org/10.1021/bi00768a021
  2. Biosca, E. G., B. Fouz, E. Alcaide, and C. Amaro. 1996. Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2. Appl. Environ. Microbiol. 62: 928-935
  3. Butterton, J. R., M. H. Choi, P. I. Watnick, P. A. Carroll, and S. B. Calderwood. 2000. Vibrio cholerae VibF is required for vibriobactin synthesis and is a member of the family of nonribosomal peptide synthases. J. Bacteriol. 182: 1731-1738 https://doi.org/10.1128/JB.182.6.1731-1738.2000
  4. Butterton, J. R., J. A. Stoebner, S. M. Payne, and S. B. Calderwood. 1992. Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J. Bacteriol. 174: 3729-3738 https://doi.org/10.1128/jb.174.11.3729-3738.1992
  5. Choi, H. K., N. Y. Park, D. Kim, H. J. Chung, S. Ryu, and S. H. Choi. 2002. Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus. J. Biol. Chem. 277: 47292-47299 https://doi.org/10.1074/jbc.M206893200
  6. Clark, V. L. and P. M. Bavoil. 1994. Bacterial pathogenesis. Methods Enzymol. 235: 313-372
  7. Crosa, J. H. 1997. Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria. Microbiol. Mol. Biol. Rev. 61: 319-336
  8. Crosa, J. H. and C. T. Walsh. 2002. Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol. Mol. Biol. Rev. 66: 223-249 https://doi.org/10.1128/MMBR.66.2.223-249.2002
  9. De Lorenzo, V., M. Herrero, U. Jakubzik, and K. N. Timmis. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J. Bacteriol. 172: 6568-6572 https://doi.org/10.1128/jb.172.11.6568-6572.1990
  10. Dessaux, Y., J. Tempe, and S. K. Farrand. 1987. Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955. Mol. Gen. Genet. 208: 301-308 https://doi.org/10.1007/BF00330457
  11. Escolar, L., J. Perez-Matin, and V. de Lorenzo. 1999. Opening the iron box: Transcriptional metalloregulation by the Fur protein. J. Bacteriol. 181: 6223-6229
  12. Griffiths, G. L., S. P. Sigel, S. M. Payne, and J. B. Neilands. 1984. Vibriobactin, a siderophore from Vibrio cholerae. J. Biol. Chem. 259: 383-385
  13. Herrero, M., V. de Lorenzo, and K. N. Timmis. 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J. Bacteriol. 172: 6557-6567 https://doi.org/10.1128/jb.172.11.6557-6567.1990
  14. Johnston, J. M., S. F. Becker, and L. M. McFarland. 1985. Vibrio vulnificus. Man and the sea. JAMA 253: 2850-2853 https://doi.org/10.1001/jama.253.19.2850
  15. Klebba, P. E., J. M. Rutz, J. Liu, and C. K. Murphy. 1993. Mechanisms of TonB-catalyzed iron transport through the enteric bacterial cell envelop. J. Bioenerg. Biomembr. 25: 603-611
  16. Lankford, C. E. 1973. Bacterial assimilation of iron. Crit. Rev. Microbiol. 2: 273-331 https://doi.org/10.3109/10408417309108388
  17. Lee, H. J., K. J. Park, A. Y. Lee, S. G. Park, B. C. Park, K. H. Lee, and S. J. Park. 2003. Regulation of fur expression by RpoS and fur in Vibrio vulnificus. J. Bacteriol. 185: 5891-5896 https://doi.org/10.1128/JB.185.19.5891-5896.2003
  18. Lee, J. H., J.-E. Rhee, U. Park, H.-M. Ju, B. C. Lee, T. S. Kim, H. S. Jeong, and S. H. Choi. 2007. Identification and functional analysis of Vibrio vulnificus SmcR, a novel global regulator. J. Microbiol. Biotechnol. 17: 325-334
  19. Lee, K.-E., J.-S. Bang, C.-H. Baek, D.-K. Park, W. Hwang, and K.-S. Kim. 2007. IVET-based identification of virulence factors in Vibrio vulnificus MO6-24/O. J. Microbiol. Biotechnol. 17: 234-243
  20. Lieu, H.-Y., H.-S. Song, S.-N. Yang, J.-H. Kim, H.-J. Kim, Y.-D. Park, C.-S. Park, and H.-Y. Kim. 2006. Identification of proteins affected by iron in Saccharomyces cerevisiae using proteome analysis J. Microbiol. Biotechnol. 16: 946-951
  21. Litwin, C. M., T. W. Rayback, and J. Skinner. 1996. Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect. Immun. 64: 2834-2838
  22. Lorenzo, M. D., S. Popperlaars, M. Stork, M. Nagasawa, M. E. Tolmasky, and J. H. Crosa. 2004. A nonribosomal peptide synthase with a novel domain organization is essential for siderophore biosynthesis in Vibrio anguillarum. J. Bacteriol. 186: 7327-7336 https://doi.org/10.1128/JB.186.21.7327-7336.2004
  23. McIntosh, M. A., C. L. Pickett, S. S. Chenault, and C. F. Earhart. 1978. Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins. Biochem. Biophys. Res. Commun. 81: 1106-1112 https://doi.org/10.1016/0006-291X(78)91250-0
  24. 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
  25. O'Brien, I. G. and F. Gibson. 1970. The structure of enterochelin and related 2,3-dihydroxy-N-benzoylserine conjugates from Escherichia coli. Biochim. Biophys. Acta 215: 393-402 https://doi.org/10.1016/0304-4165(70)90038-3
  26. Okujo, N., M. Saito, S. Yamamoto, T. Yoshida, S. Miyoshi, and S. Shinoda. 1994. Structure of vulnibactin, a new polyaminecontaining siderophore from Vibrio vulnificus. Biometals 7: 109-116
  27. Panina, E. M., A. A. Mironov, and M. S. Gelfand. 2001. Comparative analysis of FUR regulons in gamma-proteobacteria. Nucleic Acids Res. 29: 5195-5206 https://doi.org/10.1093/nar/29.24.5195
  28. Reddy, G. P., U. Hayat, C. Abeygunawardana, C. Fox, A. C. Wright, D. R. Maneval Jr., C. A. Bush, and J. G. Morris Jr. 1992. Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus MO6-24. J. Bacteriol. 174: 2620-2630 https://doi.org/10.1128/jb.174.8.2620-2630.1992
  29. Sambrook, J., E. F. Fritsh, and T. maniastis. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Sping Harbor Laboratory Press, Cold Spring Harbor, N.Y
  30. Simpson, L. M. and J. D. Oliver. 1983. Siderophore production by Vibrio vulnificus. Infect. Immun. 41: 644-649
  31. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram negative bacteria. Bio/Technology 1: 784-791 https://doi.org/10.1038/nbt1183-784
  32. Watnick, P. I., J. R. Butterton, and S. B. Calderwood. 1998. The interaction of the Vibrio cholerae transcription factors Fur and IrgB with the overlapping promoters of two virulence genes, irgA and irgB. Gene 209: 65-70
  33. Webster, A. C. and Litwin, C. M. 2000. Cloning and characterization of vuuA, a gene encoding the Vibrio vulnificus ferric vulnibactin receptor. Infect. Immun. 68: 526-534 https://doi.org/10.1128/IAI.68.2.526-534.2000
  34. Weinberg, E. D. 1974. Iron and susceptibility to infectious disease. Science 184: 952-956 https://doi.org/10.1126/science.184.4140.952
  35. Weinberg, E. D. 1978. Iron and infection. Microbiol. Rev. 42: 45-66
  36. Wright, A. C., L. M. Simpson, and J. D. Oliver. 1981. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect. Immun. 34: 503-507
  37. Wyckoff, E. E., J. A. Stoebner, and K. E. Reed. 1997. Cloning of a Vibrio cholerae vibriobactin gene cluster: Identification of genes required for early steps in siderophore biosynthesis. J. Bacteriol. 179: 7055-7962 https://doi.org/10.1128/jb.179.22.7055-7062.1997