創傷弧菌是一種嗜鹽性革蘭氏陰性弧菌，會造成人體嚴重的傷口感染及致死性的敗血症。此菌能分泌一種稱之為Vvn (Vibrio vulnificus nuclease)的核酸脢至細胞間質。其特性為耐熱、並且可以水解DNA及RNA。我們實驗室曾利用任意致突變法分離到9株在不同胺基酸殘基發生突變而導致核酸脢活性喪失的突變株。其突變點分別為Arg212Cys, Asp37Glu, Ala31Pro, Arg110Cys, Cys149Gly, Tyr35Phe, Gly98Val, Thr173Pro及Val202Gly。這些點突變都是位於具高相似度的細菌性核酸脢之高度保留區。根據Bordo及Argos之研究結果，我們推測其中四株含有安全的突變，亦即胺基酸的改變可能不會影響蛋白質的結構。其它的突變株則含有不安全的突變，亦即胺基酸的改變可能會影響蛋白質的結構。本研究中，利用特定點突變的方法，將含有不安全突變的突變株，轉變成含有安全突變的突變株。其中兩個突變株，Gly98Ala及Arg212Lys，其核酸脢的活性轉為陽性，顯示此兩位置的胺基酸並未參與核酸脢之活性。接著將其餘七個含安全突變且喪失核酸脢活性的Vvn突變蛋白 (Asp37Glu, Ala31Pro, Arg110Cys, Cys149Gly, Tyr35Phe, Thr173Pro及Val202Ala)純化出來，並分析其蛋白質二級結構以及與DNA結合的能力。經由旋光光度分析儀分析發現所有突變蛋白與野生型的二級結構沒有太大的差異，同時在electrophoretic mobility shift assays試驗中，我們發現這些Vvn突變株均能與DNA結合。再對照Vvn以X-ray crystallographyn解出的立體結構，發現這些發生突變的胺基酸並非位於催化區域，而是位於催化區域的周圍，我們推測這些位置上胺基酸的改變可能間接影響Vvn的活性。

Vibrio vulnificus is a halophilic, gram-negative bacterium associated with serious wound infections and fatal septicemia in human. This organism produces a periplasmic nuclease, Vibrio vulnificus nuclease (Vvn), which is thermostable and capable of digesting both DNA and RNA. Previously, we have isolated nine nuclease-negative mutants, including Arg212Cys, Asp37Glu, Ala31Pro, Arg110Cys, Cys149Gly, Tyr35Phe, Gly98Val, Thr173Pro and Val202Gly by random mutagenesis. All of these mutations have affected the amino acid residues located in the regions highly conserved in members of this nuclease subfamily. Four of the mutations belong to the “safe” mutations which may not affect the protein structure, while the others belong to the “unsafe” mutations which may affect the protein structure according to Bordo and Argos. In this study, I used site-directed mutagenesis to alter the “unsafe” mutations to “safe” ones. Two of the five Vvn mutants generated by site-directed mutagenesis, Gly98Ala and Arg212Lys, were found to be nuclease-positive, suggesting that these amino acid residues were not involved in the nuclease activity of Vvn. The other mutants, Arg110Lys, Cys149Al and Val202Ala, were still nuclease-negative and were subjected to further analysis. The Vvn mutants containing the “safe” mutations, Ala31Pro, Tyr35Phe, Asp37Glu, Thr173Pro, Arg110Cys, Cys149Ala and Val202Ala, were then fused with a His-tag at the C-terminus, and the recombinant Vvn mutant proteins were purified and examined for their secondary structure and DNA binding ability. The circular dichroism (CD) analysis results demonstrated that there was no obvious difference in the secondary structure between the Vvn mutants and the wild type Vvn. Our electrophoretic mobility shift assays results showed that these Vvn mutants still bind DNA. The locations of the altered amino acid residues in these Vvn mutants were all found to be outside the catalytic region defined by the X-ray crystallography data, suggesting that the mutations may indirectly affect the hydrolysis ability of Vvn.

1. Adams R. L., J. T. The Biochemistry of the Nucleic Acids, 10th Ed., Chapman and Hall. New York. (1986)
2. Amaro C., Biosca E. G. Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice. Infect. Immun. 62. 759. (1994)
3. Amaro C., Biosca E. G. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl. Environ. Microbiol. 62. 1454. (1996)
4. Arias C. R., Pualte M. J. Genetic relatedness among environmental, clinical, and diseased-eel Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR. Appl. Enviro. Microbiol. 64. 3403. (1998)
5. Austin B., Austin D. A. Bacterial fish pathogens: disease of farmed and wild fish. 3rd rev. New York: Springer. (1999)
6. Bisharat N., Agmon V. Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Lancet 354. 1421. (1999)
7. Boivin R., Bellemare G. A novel approach to the rapid isolation and nucleotide sequencing of genomic clones. Genet. Anal. Tech. Appl. 8. 181. (1991)
8. Bordo D., Argos P. Suggestions for “Safe” residue substitutions in site-directed mutagenesis. J. Mol. Biol. 217. 721. (1991)
9. Brennt C. E., Wright A. C. Growth of Vibrio vulnificus in serum from alcoholics: association with high transferrin iron saturation. J. Infect. Dis. 164. 1030. (1991)
10. Buchrieser C., Gangar V. V. Multiple Vibrio vulnificus strains in oysters as demonstrated by clamped homogeneous electric field gel electrophoresis. Appl. Environ. Microbiol. 61. 1163. (1995)
11. Bullen J. J., Spalding P. B. Hemochromatosis, iron and septicemia caused by Vibrio vulnificus. Arch. Intern. Med. 151. 1606. (1991)
12. Cantinieaux B., Boelaert J. R. Nutrophils from patients with secondary haemosiderosis contain excessive amounts of autotoxic iron. Eur. J. Haematol. 51. 161. (1993)
13. Chan T. Y., Chow D. P. Vibrio vulnificus septicemia in a patient with liver cirrhosis. Southeast. Asian J. Tropl. Med. Public Health. 25. 215. (1994)
14. Chen H. M., Dimagno T. J. The effect of Glu75 of staphylococcal nuclease on enzyme activity, protein stability and protein unfolding. Proteins 261. 599. (1999)
15. Chuang Y. C., Young C. D. Vibrio vulnificus infection. Scan J. Infect. Dis. 21. 721. (1989)
16. Chuang Y. C., Yuan C. Y. Vibrio vulnificus infection in Taiwan: report of 28 cases and review of clinical manifestations and treatment. Clin. Infect. Dis. 15. 271. (1992)
17. Dalsgaard A., Frimodt-Moller N. Clinical manifestations and molecular epidemiology of Vibrio vulnificus infections in Denmark. Eur. J. Clin. Microbiol. Infect. Dis. 15. 227. (1996)
18. Dayal H. H., Trieff N. M. Preventing Vibrio vulnificus infections: who should bear responsibility ? Am. J. Prev. Med. 9. 191. (1993)
19. Enzlin J. H., Scharer O. D. The active site of the DNA repair endonuclease XPF-ERCC1 forms a highly conserved nuclease motif. EMBO J. 21. 2045. (2002)
20. Focareta T., Manning P. A. Distinguishing between the extracellular DNase of Vibrio vulnificus and development of a transformation system. Mol. Microbiol. 5. 2547. (1991)
21. Foccareta T., Manning P. A. Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization of the deoxyribonuclease (DNase) together with its periplasmic localization in Escherichia coli K-12. Gene 53. 31. (1987)
22. Friedhoff P., Kolmes B. Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis. Nucleic Acids Res. 24. 2632. (1996)
23. Gander R. M., Larocco M. T. Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus. J. Clin. Microbiol. 27. 1015. (1989)
24. Gerlt J. A., Acuqa M. D. Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of staphylococcal nuclease. Biochem. 29. 3677. (1990)
25. Gray L. D., Kreer A. S. Mouse skin damage caused by cytolysin from Vibrio vulnificus and by V. vulnificus infection. J. Infect. Dis. 155. 236. (1987)
26. Gray L. D., Kreger A. S. Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus. Infect. Immun. 48. 62. (1985)
27. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166. 557. (1983)

28. Hase C. C., Finkelstein R. A. Comparison of the Vibrio cholerae Hemagglutinin/protease and the Pseudomonas aeruginosa elastase. Infect. Immun. 58. 4011. (1990)
29. Hase C. C., Finkestein R. A. Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain. J. Bacteriol. 173. 3311. (1991)
30. Hlady W. G. Vibrio infections associated with raw oyster consumption in Florida, 1981-1994. J. Food Prot. 60. 353. (1997)
31. Hoffmann T. J., Nelson B. Vibrio vulnificus septicemia. Arch. Inte. Med. 148. 1825. (1988)
32. Hollis D. G., Weaver R. E. Vibrio species isolated from blood cultures. J. Clin. Microbiol. 3. 425. (1976)
33. Hor L. I., Chang L. K. Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection. Microbiol. Immunol. 44. 871. (2000)
34. Hor L. I., Chang T. T. Survival of Vibrio vulnificus in whole blood from patients with chronic liver diseases: association with phagocytosis by neutrophils and serum ferritin levels. J. Infect. Dis. 179. 275. (1999)
35. Jakcson J. K., Murphree R. L. Evidence that mortality from Vibrio vulnificus infection results from single strains among heterogeneous populations in shell-fish. J. Clin. Microbiol. 35. 2098. (1997)
36. Johnson D. E., Calia F. M. Resistance of Vibrio vulnificus to serum bactericidal and opsonizing factors: relation to virulence in suckling mice and humans. J. Infect. Dis. 150. 413. (1984)
37. Kaspar C. W., Tamplin M. L. Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish. Appl. Environ. Microiol. 59. 2425. (1993)
38. Klontz K. C., Lieb S. Syndromes of Vibrio vulnificus infections. Clinical and epidemiologic features in Florida cases, 1981-1987. Ann. Intern. Med. 109. 318. (1988)
39. Koenig K. L., Mueller J. Vibrio vulnificus. Hazard on the half shell. West. J. Med. 155. 400. (1991)
40. Kook H., Lee S. E. Vibrio vulnificus hemolysin dilates rat thoracic aorta by activating guanylate cyclase. Life Sci. 59. 236. (1996)
41. Kook H., Rhee J. H. Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin. Eur. J. Pharmacol. 365. 267. (1999)
42. Ku W. Y., Yuan H. S. The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis. Nucleic Acids Res. 30. 1670. (2002)
43. Leung K. W., Liaw Y. C. Significance of local electrostatic interactions in staphylococcal nuclease studied by site-directed mutagenesis. J. Biol. Chem. 276. 46039 (2001)
44. Light J., Lerner R. A. Random mutagenesis of staphylococcal nuclease and phage display selection. Bioorg. Med. Chem. 3. 955. (1995)
45. Linder K., Oliver J. D. Membrane fatty acid and virulence change in the viable but nonculturable state of Vibrio vulnificus. Appl. Envion. Microbiol. 55. 2837. (1989)
46. Loll P. J., Lattman E. E. Active site mutant Glu 43 Asp in staphylococcal nuclease displays nonlocal structural changes. Biochem. 29. 6866. (1990)
47. Moris-Jr J. G., Black R. E. Cholera and other vibrioses in the United States. N. Engl. J. Med. 312. 343. (1985)
48. Mark S. S., Rohinee N. Epidemiology and pathogenesis of Vibrio vulnificus. Microbe. Infect. 2. 177. (2000)
49. Martinez J. L., Delgado-Irbarren A. Mechanisms of iron acquisition and bacterial virulence. FEMS Microbiol. Rev. 6. 45. (1990)
50. Meidler R., Morad I. Detection of anticodon nuclease residues involved in tRNALys cleavage specificity. J. Mol. Biol. 287. 499. (1999)
51. Miyoshi N., Shimizu C. Purification and characterization of Vibrio vulnificus protease. Microbiol. Immunol. 31. 13. (1987)
52. Moulard M., Condemine G. Characterization of the nucM gene coding for a nuclease of the phytopathogenic bacteria Erwinia chrysanthemi. Mol. Microbiol. 8. 685. (1993)
53. Moulard M., Condemine G. Purification and characterization of the nuclease NucM of Erwinia chrysanthemi. Biochim. Biophys. Acta. 1262. 133. (1995)
54. Muench K. H. Hemochromatosis and infection: alcohol and iron, oysters and sepsis. Am. J. Med. 87. 40. (1989)
55. Nguyen D. M., Schleif R. F. Isolation and physical characterization of random insertions in Staphylococcal nuclease. J. Mol. Biol. 282. 751. (1998)
56. Oliver J. D., Hite F. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl. Environ. Microbiol. 61. 2624. (1995)
57. Otto B. R., Verweij-Van Vught A. M. Transferrin and heme-compounds as iron sources for pathogenic bacteria. Crit. Rev. Microbiol. 18. 217. (1992)
58. Powell J. L., Wright A. C. Release of tumor necrosis factor alpha in response to Vibrio vulnificus capsular polysaccharide in in vivo and in vitro models,. Infect. Immun. 65. 3713. (1997)
59. Shao C. P., Hor L. I. Isolation and Characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin. Infect. Immun. 69. 5943. (2001)
60. Shao C. P., Wu S. I. Cloning and characterization of a periplasmic nuclease of Vibrio vulnificus and its role in preventing uptake of foreign DNA. Appl. Environ. Microiol. 67. 82. (2001)
61. Shao C. P., Hor L. I. Metalloprotease is not essential for Vibrio vulnificus virulence in mice. Infect. Immun. 68. 3569. (2000)
62. Shapiro R. L., Altekruse S. The role of Gulf Coast oysters harvested in warmer months in Vibrio vulnificus infections in the United States, 1988-1996. J. Infect. Dis. 178. 752. (1998)
63. Shevchik V. E., Condemine G. Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide is isomerase activity. EMBO J. 13. 2007. (1994)
64. Tamplin M. L., Specter S. Differential complement activation and susceptibility to human serum bactericidal action by Vibrio species. Infect. Immun. 42. 1187. (1983)
65. Tamplin M. L., Specter S. Vibrio vulnificus resists phagocytosis in the absence of serum opsonins. Infect. Immun. 49. 715. (1985)
66. Tefany F. J., Lee S. Iron overload and Vibrio vulnificus septicemia. Au. J. Dermatol. 31. 27. (1990)
67. Warnock E. W. 3d., Mac-Math T. L. Primary Vibrio vulnificus septicemia. J. Emerg. Med. 11. 153. (1993)
68. Weichart D., Kjelleberg S. Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus. Microbiol. 142. 845. (1996)
69. Weichart D., McDougald D. In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus. Appl. Environ. Microbiol. 63. 2754. (1997)
70. Winkler U. Mutants of Serratia marcescens defective or superactive in the release of a nuclease. Mol. Gen. Genetic. 11187. (1968)
71. Witholt B., Boekout M. An efficient procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal. Biochem. 74. 160. (1976)
72. Wright A. C., Simpson L. M. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect. Immun. 58. 1769. (1990)
73. Yoshida S., Ogawa M. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect. Immun. 47. 446. (1985)
74. Zuppardo A. B., Sitbeling R. J. An epimerase gene essential for capsule synthesis in Vibrio vulnificus. Infect. Immun. 66. 2601. (1998)
75. 蔡幸紋，以任意致突變技術鑑定創傷弧菌核酸分解脢中參與酵素活性之胺基酸殘基，90年6月