莢膜多醣體 (CPS)已知是創傷弧菌感染的重要毒立因子之ㄧ，並且與該菌對小鼠的致命性感染極為相關。創傷弧菌的莢膜多醣體是由酸性的多醣體所構成，在分類上被歸屬於第一群莢膜多醣體。先前我們實驗室藉由比較性基因體雜交以及南方墨點法分析有莢膜菌株和無莢膜菌株在基因上的差異，發現了創傷弧菌YJ016基因體中ORF VV0337到ORF VV0366這段區域裏的基因與創傷弧菌合成莢膜多醣體有關。在本研究中我首先以柏克萊果蠅基因體計劃之神經網路絡啟動子預測軟體進行預測，找到4個可能的啟動子在這段區域之中。另外藉由其他的生物資訊分析軟體找到了Shine-Dalgarno (SD)序列、兩段Rho-independent的終止子，以及一個39 bp稱為JUMPStart的高度保留於多醣體基因群前端的DNA序列。接著，經由反轉錄聚合酶連鎖反應(RT-PCR)發現不管是培養在30℃或是37℃的情況下，這段區域中的29個ORF都會表現。從使用來自相鄰的ORF上的引子進行RT-PCR的結果推測此區域可能包含ORF VV0337~VV0345和ORF VV0347~VV0364兩個操縱子和 ORF VV0365以及ORF VV0366兩個單獨的基因。由ORF VV0349、ORF VV0350、ORF VV0352以及ORF VV0353的註解我們推測它們可能參與在不曾被報導與第一群多醣體共同表現的細胞外多醣體colanic acid的合成。我們將其中的ORF VV0349以對偶基因交換的方式刪除，發現此突變株的菌落變成半透明樣，且對人類血清殺菌反應極具敏感性，顯示了ORF VV0349確實與創傷弧菌莢膜多醣體的合成有關。另一方面，將預測能產生核苷酸去氫酶的ORF VV0365進行刪除，發現此突變株的菌落型態為不透光，且其對人類血清殺菌能力的抗性與野生株YJ016相當，顯示ORF VV0365與創傷弧菌莢膜多醣體的合成無關。我們的研究結果顯示，ORF VV0337到ORF VV0366這個區域確實與創傷弧菌莢膜多醣體的合成相關，而非參與在colanic acid的合成。

The capsular polysaccharides (CPS) has been shown to be a primary virulence factor of Vibrio vulnificus and correlate with bacterial lethality in mice. CPS of V. vulnificus is composed of acidic polysaccharides, and therefore is classified as group 1. Our laboratory has previously found a region from ORF VV0337 to ORF VV0366 that may be associated with CPS synthesis in the genome of strain YJ016 by comparative genomic hybridization and Southern analysis. In this study, four promoters were predicted in this region by Neural Network Promoter Prediction (NNPP) of Berkeley Drosophila Genome Project. The Shine-Dalgarno sequences, two Rho-independent terminators, and a conserved 39 bp sequence, the JUMPStart sequence, which is involved in the production of various polysaccharides, were also predicted by bioinformatic analyses. The expression of twenty-nine ORFs in this region has been tested by RT-PCR and all were shown to be expressed at both 37℃ and 30℃. Two operons (VV0337~VV0345 and VV0347~VV0364) and two single genes (VV0365 and VV0366) were suggested based on the results of RT-PCR with the primers derived from adjacent ORFs. ORF VV0349, ORF VV0350, ORF VV0352, and ORF VV0353 were annotated as genes involved in the synthesis of an exopolysaccharide, colonic acid, which has not been reported to be coexpressed with the group 1 CPS. One of them, ORF VV0349 was deleted in V. vulnificus, and the VV0349 mutant became translucent and sensitive to human serum bactericidal effect, indicating that this ORF is involved in the synthesis of CPS. On the other hand, ORF VV0365, which may encode nucleotide sugar dehydrogenase, was found to be not required for CPS synthesis, since the VV0365 mutant was opaque in colonial morphology and as resistant to human serum as the parental strain YJ016. Our data demonstrate that the genes in this region are indeed involved in the synthesis of CPS, but not the colonic acid.

1. Amako, K., K. Okada, and S. Miake. 1984. Evidence for the presence of a capsule in Vibrio vulnificus. J Gen Microbiol 130:2741-3.
2. Amaro, C., and E. G. Biosca. 1996. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl Environ Microbiol 62:1454-7.
3. Amaro, C., E. G. Biosca, B. Fouz, E. Alcaide, and C. Esteve. 1995. Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels. Appl Environ Microbiol 61:1133-7.
4. Amaro, C., E. G. Biosca, B. Fouz, and E. Garay. 1992. Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains of Vibrio vulnificus biotypes 1 and 2 by silver staining and immunoblotting. Curr Microbiol 25:99-104.
5. Amaro, C., E. G. Biosca, B. Fouz, A. E. Toranzo, and E. Garay. 1994. Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice. Infect Immun 62:759-63.
6. Bailey, M. J., Hughes, C., and Koronakis, V. 1997. RfaH and the ops element, components of a novel system controlling bacterial transcription elongation. Mol Microbiol 26:845-851.
7. Bisharat, N., V. Agmon, R. Finkelstein, R. Raz, G. Ben-Dror, L. Lerner, S. Soboh, R. Colodner, D. N. Cameron, D. L. Wykstra, D. L. Swerdlow, and J. J. Farmer, 3rd. 1999. Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Israel Vibrio Study Group. Lancet 354:1421-4.
8. Bullen, J. J., P. B. Spalding, C. G. Ward, and J. M. Gutteridge. 1991. Hemochromatosis, iron and septicemia caused by Vibrio vulnificus. Arch Intern Med 151:1606-9.
9. Bush, C. A., P. Patel, S. Gunawardena, J. Powell, A. Joseph, J. A. Johnson, and J. G. Morris. 1997. Classification of Vibrio vulnificus strains by the carbohydrate composition of their capsular polysaccharides. Anal Biochem 250:186-95.
10. Chen, C. Y., K. M. Wu, Y. C. Chang, C. H. Chang, H. C. Tsai, T. L. Liao, Y. M. Liu, H. J. Chen, A. B. Shen, J. C. Li, T. L. Su, C. P. Shao, C. T. Lee, L. I. Hor, and S. F. Tsai. 2003. Comparative genome analysis of Vibrio vulnificus, a marine pathogen. Genome Res 13:2577-87.
11. Chuang, Y. C., C. Y. Yuan, C. Y. Liu, C. K. Lan, and A. H. Huang. 1992. Vibrio vulnificus infection in Taiwan: report of 28 cases and review of clinical manifestations and treatment. Clin Infect Dis 15:271-6.
12. Chuang, Y. P., C. T. Fang, S. Y. Lai, S. C. Chang, and J. T. Wang. 2006. Genetic determinants of capsular serotype K1 of Klebsiella pneumoniae causing primary pyogenic liver abscess. Journal of Infectious Diseases 193:645–54.
13. Devi, S. J., U. Hayat, C.E. Frasch, A.S. Kreger, and J.G. Morris, Jr. . 1995. Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model. Infect. Immun. 63:2906-2911.
14. Donnenberg, M. S. 1991. Construction of an eae deletion mutant of enterophathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59:4310-7.
15. Gray, L. D., and A. S. Kreger. 1989. Detection of Vibrio vulnificus cytolysin in V. vulnificus-infected mice. Toxicon 27:439-64.
16. Gray, L. D. a. A. S. K. 1985. Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus. Infect. Immun. 48:62-72.
17. Hayat, U., G. P. Reddy, C. A. Bush, J. A. Johnson, A. C. Wright, and J. G. Morris, Jr. 1993. Capsular types of Vibrio vulnificus: an analysis of strains from clinical and environmental sources. J Infect Dis 168:758-62.
18. Hollis, D. G., R. E. Weaver, C. N. Baker, and C. Thornsberry. 1976. Halophilic Vibrio species isolated from blood cultures. J Clin Microbiol 3:425-31.
19. Hsueh, P. R., C. Y. Lin, H. J. Tang, H. C. Lee, J. W. Liu,, and a. Y. C. C. Y. C. Liu. 2004. Vibrio vulnificus in Taiwan. Emerging Infectious Diseases 10:1363-1368.
20. Jeong, H. S., K. C. Jeong, H. K. Choi, K. J. Park, K. H. Lee, J. H. Rhee, and S. H. Choi. 2001. Differential expression of Vibrio vulnificus elastase gene in a growth phase-dependent manner by two different types of promoters. J Biol Chem 276:13875-80.
21. Kang, M. K., E.C. Jhee, B.S. Koo, J.Y. Yang, B.H. Park, J.S. Kim, H.W. Rho, H.R. Kim, and J.W. Park. 2002. Induction of nitric oxide synthase expression by Vibrio vulnificus cytolysin. Biochem. Biophys. Res. Commun. 290:1090-1095.
22. Kim, H. R., H. W. Rho, M. H. Jeong, J. W. Park, J. S. Kim, B. H. Park, U. H. Kim, and S. D. Park. 1993. Hemolytic mechanism of cytolysin produced from V. vulnificus. Life Sci 53:571-7.
23. Kreger, A., and D. Lockwood. 1981. Detection of extracellular toxin(s) produced by Vibrio vulnificus. Infect Immun 33:583-90.
24. Linkous, D. A., and J. D. Oliver. 1999. Pathogenesis of Vibrio vulnificus. FEMS Microbiol Lett 174:207-14.
25. Litwin, C. M., T. W. Rayback, and J. Skinner. 1996. Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect Immun 64:2834-8.
26. Maeda, H., T. Akaike, Y. Sakata, and K. Maruo. 1993. Role of bradykinin in microbial infection: enhancement of septicemia by microbial proteases and kinin. Agents Actions Suppl 42:159-65.
27. Maria, C. L., M. K. Jones, and A. C. Wright. 2006. Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon. J Bacteriol 188:1987-98.
28. Maruo, K., T. Akaike, T. Ono, and H. Maeda. 1998. Involvement of bradykinin generation in intravascular dissemination of Vibrio vulnificus and prevention of invasion by a bradykinin antagonist. Infect Immun 66:866-9.
29. Miyoshi, N. 1987. Purification and characterization of Vibrio vulnificus protease. Microbiol Immunol 31:13-25.
30. Muench, K. H. 1989. Hemochromatosis and infection: alcohol and iron, oysters and sepsis. Am J Med 87:40N-43N.
31. O'Neill, K. R., S. H. Jones, and D. J. Grimes. 1992. Seasonal incidence of Vibrio vulnificus in the Great Bay estuary of New Hampshire and Maine. Appl Environ Microbiol 58:3257-62.
32. Osaka, K., M. Komatsuzaki, H. Takahashi, S. Sakano, and N. Okabe. 2004. Vibrio vulnificus septicaemia in Japan: an estimated number of infections and physicians' knowledge of the syndrome. Epidemiol. Infect. 132:993-996.
33. 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 M06-24. J Bacteriol 174:2620-30.
34. Shao, C. P., and L. I. Hor. 2000. Metalloprotease is not essential for Vibrio vulnificus virulence in mice. Infect Immun 68:3569-73.
35. Shinoda, S., M. Kobayashi, H. Yamada, S. Yoshida, M. Ogawa, and Y. Mizuguchi. 1987. Inhibitory effect of capsular antigen of Vibrio vulnificus on bactericidal activity of human serum. Microbiol. Immunol. 31:393-401.
36. Simpson, L. M., and J. D. Oliver. 1993. Regulation of proteolytic activity of Vibrio vulnificus by iron-containing compounds. Microb Pathog 14:249-52.
37. Simpson, L. M., and J. D. Oliver. 1983. Siderophore production by Vibrio vulnificus. Infect Immun 41:644-9.
38. Simpson, L. M., V.K. White, S.F. Zane, and J.D. Oliver. 1987. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect. Immun. 55:269-272.
39. Snook, C. F., P. A. Tipton, and L. J. Beamer. 2003. Crystal structure of GDP-mannose dehydrogenase: a key enzyme of alginate biosynthesis in P. aeruginosa. Biochemistry 42:4658-4668.
40. Strom, M. S., and R. N. Paranjpye. 2000. Epidemiology and pathogenesis of Vibrio vulnificus. Microbes Infect 2:177-88.
41. Subramani, S. 1992. Targeting of proteins into the peroxisomal matrix. J Membr Biol 125:99-106.
42. Whitfield, C. 2006. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu. Rev. Biochem. 75:39–68.
43. Whitfield, C., and I. S. Roberts. 1999. Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol Microbiol 31:1307-19.
44. Wright, A. C., J.L. Powell, M.K. Tanner, L.A. Ensor, A.B. Karpas, J.G. Morris, Jr., and M.B. Sztein. 1999. Differential expression of Vibrio vulnificus capsular polysaccharide. Infect. Immun. 67:2250-2257.
45. Wright, A. C., J. L. Powell, J. B. Kaper, and J. G. Morris, Jr. 2001. Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus. Infect Immun 69:6893-901.
46. Wright, A. C., L. M. Simpson, J. D. Oliver, and J. G. Morris, Jr. 1990. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect Immun 58:1769-73.
47. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-19.
48. Yoshida, S., M. Ogawa, and Y. Mizuguchi. 1985. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect. Immun. 47:446-451.
49. Zuppardo, A. B., and R. J. Siebeling. 1998. An epimerase gene essential for capsule synthesis in Vibrio vulnificus. Infect Immun 66:2601-6.
50. 王瑞烽. 2004. 創傷弧菌參與莢膜多醣體合成之基因群的比較基因體學分析. 成功大學微生物暨免疫學研究所碩士論文.
51. 陳銘祥. 2006. 創傷弧菌中誘導式表現系統之建立. 成功大學微生物暨免疫學研究所碩士論文.