臺灣博碩士論文加值系統

溶藻弧菌是一種重要的伺機性病原體，主要於人體及養殖水產引起弧菌症。弧菌屬細菌能自我形成具保護功能之生物膜，使其病原體更易入侵和感染生物體，而生物膜的存在被認為可能與慢性疾病的產生和疾病的大流行有關。胺醯組胺酸雙胜肽酶(PepD, EC 3.4.13.3)為胜肽酶家族M20中的一員，被研究認為影響細菌生物膜的形成。過去對於細菌中胺醯組胺酸雙胜肽酶的研究很少，只針對其序列和部分生化特性進行探討，並無其生理角色或活性區胺基酸相關之研究。本論文從溶藻弧菌ATCC 17749中首次發現胺醯組胺酸雙胜肽酶之基因，並對其進行序列分析、生化特性及活性區胺基酸之研究。此基因之開放讀碼區(ORF)序列共有1473個鹼基對，可轉譯出一條長490個胺基酸的蛋白質，計算其分子量約為53.6 kDa。此胺基酸序列和其他弧菌屬之PepD蛋白質序列比對有非常高之相似度，同時與大腸桿菌及鼠傷寒桿菌相似度達63%。將溶藻弧菌pepD基因殖入pET-28a(+)質體中，表現N端帶有His-tag之重組蛋白，並利用Ni-NTA親和層析管柱純化之。純化出的蛋白質可水解雙胜肽L-carnosine及其他特定Xaa-His雙胜肽，但無水解三胜肽之活性。經酵素動力學研究，溶藻弧菌PepD蛋白對雙胜肽L-carnosine之Km與kcat值分別為5.38 mM與0.132 s-1。研究發現溶藻弧菌PepD重組蛋白於pH 7.4及37℃環境下有最佳活性。此酵素活性可被金屬螯合劑EDTA及雙胜肽類似物抑制劑bestatin所抑制，推測溶藻弧菌PepD蛋白為金屬雙胜肽酶。經序列分析預測溶藻弧菌PepD蛋白上胺基酸位置His80、Asp82、Asp119、Glu149、Glu150、Asp173及His461為活性區胺基酸。將其分別定點突變為H80A、D82A、D119A、E149A、E150A、D173A及H461A後，突變蛋白皆失去原有之活性。此外，以PepV蛋白結晶結構為模板做出溶藻弧菌PepD蛋白之同源模擬，顯示出相同之活性區胺基酸。因此，根據本論文實驗結果將首次提出胺醯組胺酸雙胜肽酶活性區胺基酸之分佈情形與其可能扮演之功能。

Vibrio alginolyticus is one of the important opportunistic pathogens causing vibriosis in aquacultured species and human. Vibrio spp. are examples to form a stable protective biofilm that might facilitate the transmission of pathogens. Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3), a member of peptidase family M20, was considered to be involved in bacterial biofilm formation. The researches on bacterial PepD were less known and only investigated genetically and biochemically. A newly defined aminoacylhistidine dipeptidase from Vibrio alginolyticus ATCC 17749 was characterized via the determination of the corresponding gene sequence, biochemical properties and identification of the active site residues. The cloned fragment contained an ORF of 1473 bp, encoding a 490 amino acid residues protein with a calculated molecular weight of 53.6 kDa. The deduced amino acid sequence shared high sequence identity with PepD from various Vibrio spp. and both 63% from Escherichia coli and Salmonella typhimurium. The pepD gene of V. alginolyticus was cloned into the pET-28a(+) expression vector and expressed as a (His)6-PepD fusion protein. Following the Ni-NTA chromatographic purification, the purified enzyme displays catalytic activity on digestion of an unusual dipeptide L-carnosine (β-Ala-L-His) with Km 5.38 mM and kcat 0.132 s-1 and other Xaa-His dipeptides, but not histidine-containing tripeptides. Expressed PepD was observed with optimal activity at pH 7.4 and 37℃. The enzymatic activity was inhibited by a dipeptide analogue inhibitor bestatin and metal-chelating agent EDTA, indicating PepD as a metallo-dipeptidase. Sequence analysis revealed that His80, Asp82, Asp119, Glu149, Glu150, Asp173, and His461 are probable the active site residues of V. alginolyticus PepD. The results derived from site-directed mutagenesis showed that the mutants of H80A, D82A, D119A, E149A, E150A, D173A, and H461A lose their full activities as compared with wild-type PepD. The homology model of V. alginolyticus PepD obtained on the basis of L. delbrueckii PepV structure exhibits the similar active site pocket as predicted. Therefore, the results obtained from this study present, for the first time, the investigation of the putative active site residues of bacterial aminoacylhistidine dipeptidase and their possible roles in catalysis.

Table of Contents
Abstract (Chinese) I
Abstract (English) III
Acknowledgement V
Keywords VI
Abbreviations VII
Table of Contents X
List of Figures XIII
List of Tables XV

Chapter 1 Introduction 1
1.1 Vibrio species 1
1.2 Vibrio alginolyticus 3
1.3 Biofilm 5
1.4 Proteolytic Enzymes 8
1.4.1 Metallopeptidase 10
1.4.2 Peptidase family M20 11
1.4.3 Aminoacylhistidine dipeptidase 12
1.4.4 Peptidase V 13
1.4.5 Carnosinase 15
1.4.6 Other Carnosine-Hydrolyzing Enzymes 17
1.5 Research Goal 18

Chapter 2 Materials and Methods 19
2.1 Bacterial strains, plasmids, animal, and cell 19
2.2 Chemicals and Reagents 19
2.3 Kits 21
2.4 Equipments 22
2.5 Solutions 23
2.6 Identification of V. alginolyticus pepD Gene Sequence 27
2.6.1 Primer design for V. alginolyticus pepD identification 27
2.6.2 Extraction of V. alginolyticus genomic DNA 27
2.6.3 PCR amplification of pepD from V. alginolyticus 27
2.6.4 DNA sequencing of V. alginolyticus pepD 28
2.6.5 Sequence analysis of V. alginolyticus pepD 29
2.7 Cloning and Expression of V. alginolyticus pepD 30
2.7.1 Construction of E. coli expression vector containing pepD gene 30
2.7.2 Expression of V. alginolyticus pepD gene in E. coli 31
2.8 Purification of Expressed V. alginolyticus PepD 32
2.8.1 Purification of PepD by affinity chromatography 32
2.8.2 Protein concentration determination 33
2.8.3 SDS-PAGE and Native-PAGE analysis 33
2.9 Characterization of Expressed V. alginolyticus PepD 35
2.9.1 Enzymatic Activity Assay of PepD 35
2.9.2 pH optimum 35
2.9.3 Temperature optimum and thermostability 36
2.9.4 Substrate specificity 36
2.9.5 Inhibitor profile 36
2.9.6 Enzyme kinetics 37
2.10 Mutagenesis Analysis of V. alginolyticus pepD 37
2.10.1 Site-directed mutagenesis on V. alginolyticus pepD 37
2.10.2 Circular dichroism (CD) spectroscopy 38

Chapter 3 Results 40
3.1 V. alginolyticus pepD Gene Sequence 40
3.2 Expression and Purification of V. alginolyticus PepD in E. coli 42
3.3 Effects of pH and Temperature on Activity of PepD 44
3.4 Effects of Inhibitors on Activity of PepD 46
3.5 Substrate Specificity 49
3.6 Enzyme Kinetics 50
3.7 Site-directed Mutagenesis Analysis of V. alginolyticus PepD 51
3.8. Structural Features of PepD 56
Chapter 4 Discussion and Conclusion 58
Chapter 5 Future Work 64
Chapter 6 Reference 65
Appendix 1 75
Appendix 2 77
Appendix 3 78
Appendix 4 80
4.1 Production of PepD Monoclonal Antibody 80
4.1.1 Immunization of mice 80
4.1.2 Preparation of myeloma cells 80
4.1.3 Fusion of myeloma cells with spleen cells 81
4.1.4 Screening of hybridoma clone by ELISA method 82
4.1.5 Limiting dilution of hybridoma clones 83
4.1.6 Ascites production in mice 83
4.1.7 Western blot analysis 83
Appendix 5 85
Appendix 6 86
Appendix 7 87
Appendix 8 92







List of Figures
Fig. 1. Scanning Electron Microscopy (SEM) of V. alginolyticus intermediary morphologies. 1
Fig. 2. Model of the development of a mature biofilm from planktonic cells. 6
Fig. 3. An enzymatic reaction catalyzed by aminoacylhistidine dipeptidase. 12
Fig. 4. (A) Ribbon diagrams of the crystal structure of PepV and (B) Stereo view of zinc-binding residues of PepV. 14
Fig. 5. Common histidine-containing dipeptides present in mammals. 15
Fig. 6. Flowchart of expression vector pET-28a(+)-pepD construction. 31
Fig. 7. Formation of a Schiff base by L-histidine and OPA. 35
Fig. 8. Nucleotide sequences and predicted amino acid sequences of V. alginolyticus pepD gene. 41
Fig. 9. SDS-PAGE and western blot analysis of purified PepD. 43
Fig. 10. pH optimum of PepD. 44
Fig. 11. Thermostability of PepD. 45
Fig. 12. Temperature optimum of PepD. 45
Fig. 13. Effects of benzamidine (A), EDTA (B), bestatin (C), and NEM (D) at various concentrations on PepD activity. 48
Fig. 14. Substrate specificity of PepD for Xaa-His dipeptides and histidine-containing tripeptides. 49
Fig. 15. (A) Michaelis-Menten plot for PepD catalyzed the hydrolysis of L-carnosine in 50 mM Tris-HCl, pH 7.4 at 37℃. (B) Lineweaver-Burk plot calculated from the respective Michaelis-Menten plot. 50
Fig. 16. Comparison of the amino acid sequences of V. alginolyticus PepD and L. delbrueckii PepV. 52
Fig. 17. SDS-PAGE of purified PepD wild-type and mutant proteins. 53
Fig. 18. Native PAGE of purified PepD wild-type and mutant proteins. 53
Fig. 19. Enzymatic activities of PepD WT and mutants on L-carnosine. 54
Fig. 20. The CD spectra of V. alginolyticus PepD wild-type and mutant proteins. 55
Fig. 21. Three-dimensional ribbon of the crystal structure of PepV (left) and the generated PepD model based on PepV (right). 56
Fig. 22. Stereo view of PepD (light gray) superimposed with the active site of PepV (blue). 57

















List of Tables
Table 1. Association of Vibrio spp. with different clinical syndromes. 2
Table 2. Classification of peptidases. 8
Table 3. Reaction conditions and cycling parameters for the PCR reaction. 28
Table 4. Reaction conditions of pCR®2.1-TOPO-pepD digestion and pET-28a(+)-pepD ligation. 30
Table 5. Solutions and volumes for preparing SDS-PAGE and Native-PAGE separating gel and stacking gel. 34

Chapter 6 Reference
1. Albertini, M. C., Accorsi, A., Teodori, L., Pierfelici, L., Uquccioni, F., Rocchi, M. B., Burattini, S., and Citterio, B. 2006. Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination. Cytometry A. 69, 260-265.
2. Alexander, J. B. and Ingram, G. A. 1992. Noncellular nonspecific defence mechanism of fish. Annu. Rev. Fish Dis. 2, 249-279.
3. Badger, J., Sauder, J. M., Adams, J. M., Antonysamy, S., Bain, K., Bergseid, M. G., Buchanan, S. G., Buchanan, M. D., Batiyenko, Y., Christopher, J. A., Emtage, S., Eroshkina, A., Feil, I., Furlong, E. B., Gajiwala, K. S., Gao, X., He, D., Hendle, J., Huber, A., Hoda, K., Kearins, P., Kissinger, C., Laubert, B., Lewis, H. A., Lin, J., Loomis, K., Lorimer, D., Louie, G., Maletic, M., Marsh, C. D., Miller, I., Molinari, J., Muller-Diechmann, H. J., Newman, J. M., Noland, B. W., Pagarigan, B., Park, F., Peat, T. S., Post, K. W., Radojicic, S., Ramos, A., Romero, R., Rutter, M. E., Sanderson, W. E., Schwinn, K. D., Tresser, J., Winhoven, J., Wright, T. A., Wu, L., Xu, J., and Harris, T. J. 2005. Structural analysis of a set of proteins resulting from a bacterial genomics project. Proteins. 60, 787-796.
4. Bando, K., Shimotsuji, T., Toyoshima, H., Hayashi, C., Miyai, K. 1984. Fluorometric assay of human serum carnosinase activity in normal children, adults and patients with myopathy. Ann. Clin. Biochem. 21, 510-514.
5. Baross, J. and Liston, J. 1970. Occurrence of Vibrio parahaemolyticus and related hemolytic vibrios in marine environments of Washington State. Appl. Microbiol. 20, 179-186.
6. Bersani, C., Bianchi, M. A., Cantoni, C., and Baretta, G. 1980. Carnosinase: its presence in bacteria. Arch. Vet. Ital. 31 (suppl. 1-2), 1-3.
7. Biagini, A., and Puigserver, A. 2001. Sequence analysis of the aminoacylase-1 family. A new proposed signature for metalloexopeptidases. Comp. Biochem. Physiol. B. 128, 469-481.
8. Blake, P. A., Weaver, R. E., and Hollis, D. G. 1980. Diseases of humans (other than cholera) caused by vibrios. Annu. Rev. Microbiol. 34, 341-367.
9. Boldyrev, A. A. 2000. Problems and perspectives in studying the biological role of carnosine. Biochemistry (Mosc.). 65, 751-756.
10. Brombacher, E., Dorel, C., Zehnder, A. J., and Landini, P. 2003. The curli biosynthesis regulator CsgD co-ordinates the expression of both positive and negative determinants for biofilm formation in Escherichia coli. Microbioloy. 149, 2847-2857.
11. Butterworth, R. J., Wassif, W. S., Sherwood, R. A., Gerges, A., Poyser, K. H., Garthwaite, J., Peters, T. J., and Bath, P. M. 1996. Serum neuron-specific enolase, carnosinase, and their ratio in acute stroke. An enzymatic test for predicting outcome？ Stroke. 27, 2064-2068.
12. Chevrier, B., Schalk, C., D’Orchymont, H., Rondeau, J. M., Moras, D., and Tarnus, C. 1994. Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family. Structure. 2, 283-291.
13. Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., and Marrie, T. J. 1987. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41, 435-464.
14. Costerton, J. W., Ellis, B., Lam, K., Johnson, F., and Khoury, A. E. 1994. Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria. Antimicrob. Agents. Chemother. 38, 2803-2809.
15. Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., and Lappin-Scott, H. M. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49, 711-745.
16. Csámpai, A., Kutlán, D., Tóth, F., and Molnár-Perl, I. 2004. o-Phthaldialdehyde derivatization of histidine: stoichiometry, stability and reaction mechanism. J. Chromatogr. A. 1031, 67-78.
17. Davies, D. G. and Geesey, G. G. 1995. Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture. Appl. Environ. Microbiol. 61, 860-867.
18. Decker, E. A., Livisay, S. A., and Zhou, S. 2000. A re-evaluation of the antioxidant activity of purified carnosine. Biochemistry (Mosc.). 65, 766-770.
19. Desmarais, W., Bienvenue, D. L., Bzymek, K. P., Petsko, G. A., Ringe, D., and Holz, R. C. 2006. The high-resolution structures of the neutral and the low pH crystals of aminopeptidase from Aeromonas proteolytica. J. Biol. Inorg. Chem. 11, 398-408.
20. Finlay, B. B. and Falkow, S. 1997. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136-169.
21. Fuqua, W. C., Winans, S. C., and Greenberg, E. P. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. Annu. Rev. Microbiol. 50, 727-751.
22. Gahrn-Hansen, B. and Hornstrup, M. K. 1994. Extraintestinal infections caused by Vibrio parahaemolyticus and Vibrio alginolyticus at the country of Funen 1987-1992. Ugeskr. Laeg. 156, 5279-5282.
23. Gilboa, R., Greenblatt, H. M., Perach, M., Spungin-Bialik, A., Lessel, U., Wohlfahrt, G., Schomburg, D., Blumberg, S., and Shoham, G. 2000. Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 Å resolution. Acta Crystallogr. D. Biol. Crystallogr. 56, 551-558.
24. Goarant, C., Merien, F., Berthe, F., Mermoud, I., and Perolat, P. 1999. Arbitrarily primed PCR to type Vibrio spp. pathogenic for shrimp. Appl. Environ. Microbiol. 65, 1145-1151.
25. Greenblatt, H. M., Almog, O., Maras, B., Spungin-Bialik, A., Barra, D., Blumberg, S., and Shoham, G. 1997. Streptomyces griseus aminopeptidase: X-ray crystallographic structure at 1.75 Å resolution. J. Mol. Biol. 265, 620-636.
26. Hakansson, K. and Miller, C. G. 2002. Structure of peptidase T from Salmonella typhimurium. Eur. J. Biochem. 269, 443-450.
27. Hall-Stoodley, L. and Stoodley, P. 2005. Biofilm formation and dispersal and the transmission of human pathogens. Trends Microbiol. 13, 7-10.
28. Hellendorn, M. A., Franke-Fayard, B. M., Mierau, I., Venema, G., and Kok, J. 1997. Cloning and analysis of the PepV dipeptidase gene of Lactococcus lactis MG1363. J. Bacteriol. 179, 3410-3415.
29. Hlady, W. G. and Klontz, K. C. 1996. The epidemiology of Vibrio infections in Florida, 1981-1993. J. Infect. Dis. 173, 1176-1183.
30. Hoyle, B. D. and Costerton, W. J. 1991. Bacterial resistance to antibiotics: the role of biofilms. Prog. Drug. Res. 37, 91-105.
31. Jackson, M. C., Kucera, C. M., and Lenney, J. F. 1991. Purification and properties of human serum carnosinase. Clin. Chim. Acta. 196, 193-205.
32. Jackson, M. C. and Lenney, J. F. 1996. The distribution of carnosine and related dipeptides in rat and human tissues. Inflamm. Res. 45: 132-135.
33. Janda, J. M., Powers, C., Bryant, R. G., and Abbott, S. L. 1988. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin. Microbiol. Rev. 1, 245-267.
34. Jozic, D., Bourenkow, G., Bartunik, H., Scholze, H., Dive, V., Henrich, B., Huber, R., Bode, W., and Maskos, K. 2002. Crystal structure of the dinuclear zinc aminopeptidase PepV from Lactobacillus delbrueckii unravels its preference for dipeptides. Structure. 10, 1097-1106.
35. Klein, J., Henrich, B., and Plapp, R. 1986. Cloning and expression of the pepD gene of Escherichia coli. J. Gen. Microbiol. 132, 2337-2343.
36. Kirsh, M., Dembinski, D. R., Hartman, P. E., and Miller, C. G. 1978. Salmonella typhimurium peptidase active on carnosine. J. Bacteriol. 134, 361-374.
37. Komeda, H. and Asano, Y. 2005. A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for β-alanyl dipeptides. FEBS J. 272, 3075-3084.
38. Lawrence, J. R., Korber, D. R., Hoyle, B. D., Costerton, J. W., and Caldwell, D. E. 1991. Optical sectioning of microbial biofilms. J. Bacteriol. 173, 6558-6567.
39. Lee, K. K. 1995. Pathogenesis studies on Vibrio alginolyticus in the grouper, Epinephelus malabaricus Bloch et Schneider. Microb. Pathogen. 19, 39-48.
40. Lee, K. K., Yu, S. R., Chen, F. R., Yang, T. Z., and Liu, P. C. 1996. Virulence of Vibrio alginolyticus isolated from diseased tiger prawn Penaeus monodon. Curr. Microbiol. 32, 229-231.
41. Lenney, J. F., George, R. P., Weiss, A. M., Kucera, C. M., Chan, P., and Rinzler, G. S. 1982. Human serum carnosinase: characterization, distinction from cellular carnosinase, and activation by cadmium. Clin. Chim. Acta. 123, 221-231.
42. Lenney, J. F., Peppers, S. C., Kucera-Orallo, C. M., and George, R. P. 1985. Characterization of human tissue carnosinase. Biochem. J. 228, 653-660.
43. Levine, W. C. and Griffin, P. M. 1993. Vibrio infections on the Gulf Coast: results of first year of regional surveillance. J. Infect. Dis. 167, 479-483.
44. Licciano, M., Stabili, L., and Giangrande, A. 2005. Clearance rates of Sabella spallanzanii and Branchiomma luctuosum (Annelida: Polychaeta) on a pure culture of Vibrio alginolyticus. Water Res. 39, 4375-4384.
45. Lindner, H. A., Alary, A., Boju, L. I., Sulea, T., and Ménard, R. 2005. Roles of dimerization domain residues in binding and catalysis by aminoacylase-1. Biochemistry. 44, 15645-15651.
46. Marano, N. N., Daniels, N. A., Easton, A. N., McShan, A., Ray, B., Wells, J. G., Griffin, P. M., and Angulo, F. J. 2000. A survey of stool culturing practices for vibrio species at clinical laboratories in Gulf Coast states. J. Clin. Microbiol. 38, 2267-2270.
47. Matsiota-Bernard, P. and Nauciel, C. 1993. Vibrio alginolyticus wound infection after exposure to sea water in an air crash. Eur. J. Clin. Microbiol. Infect. Dis. 12, 474-475.
48. Mead, P., Slutsker, L., Dietz, V., McCaig, L., Bresee, J., Shapiro, C., Griffin, P. M., and Tauxe, R. V. 1999. Food-related illness and disease in the United States. Emerg. Infect. Dis. 5, 1-20.
49. Miller, C. G. and Schwartz, G. 1978. Peptidase-deficient mutants of Escherichia coli. J. Bacteriol. 135, 603-611.
50. Miller, M. B. and Bassler, B. L. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55, 165-199.
51. Miyamoto, Y., Nakamura, K., and Takizawa, K. 1961. Pathogenic halophiles. Proposals of a new genus “Oceanomonas” and of the amended species names. Jpn. J. Microbiol. 5, 477-486.
52. Molitoris, E., Joseph, S. W., Krichevsky, M. I., Sindhuhardja, W., and Colwell, R. R. 1985. Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia. Appl. Environ. Microbiol. 50, 1388-1394.
53. Morris, J. G. Jr. and Black, R. E. 1985. Cholera and other vibrioses in the United States. N. Engl. J. Med. 312, 343-350.
54. Murphey, W. H., Lindmark, D. G., and Mosovich, L. 1973. Serum carnosinase deficiency concomitant with mental retardation. Pediatr. Res. 7, 601-606.
55. Olsen, A., Jonsson, A., and Normark, S. 1989. Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli. Nature. 338, 652-655.
56. Opal, S. M. and Saxon, J. R. 1986. Intracranial infection by Vibrio alginolyticus following injury in salt water. J. Clin. Microbiol. 23, 373-374.
57. O’Toole, G., Kaplan, H. B., and Kolter, R. 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54, 49-79.
58. Perry, T. L., Hansen, S., Tischler, B., Bunting, R., and Berry, K. 1976. Carnosinaemia: a new metabolic disorder associated with neurological disease and mental defect. N. Engl. J. Med. 277, 1219-1227.
59. Petroff, O. A., Hyder, F., Rothman, D. L., and Mattson, R. H. 2001. Homocarnosine and seizure control in juvenile myoclonic epilepsy and complex partial seizures. Neurology. 56, 709-715.
60. Pratt, L. A. and Kolter, R. 1998. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol. Microbiol. 30, 285-293.
61. Prigent-Combaret, C., Vidal, O., Dorel, C., and Lejeune, P. 1999. Abiotic surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli. J. Bacteriol. 181, 5993-6002.
62. Rao, M. B., Tanksale, A. M., Ghatge, M. S., and Deshpande, V. V. 1998. Molecular and biotechnological of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597-635.
63. Rawlings, N. D. and Barrett, A. J. 1993. Evolutionary families of peptidases. Biochem. J. 290, 205-218.
64. Rawlings, N. D. and Barrett, A. J. 1995. Evolutionary families of metallopeptidases. Meth. Enzymol. 248, 183-228.
65. Rawlings, N. D., Morton, F. R., and Barret, A. J. 2006. MEROPS: the peptidase database. Nucleic Acids Res. 34, D270-272.
66. Reina Prieto, J. and Heravs Palazon, J. 1993. Otitis media due to Vibrio alginolyticus: the risks of the Mediterranean sea. An. Esp. Pediatr. 39, 361-363.
67. Rippey, S. R. 1994. Infectious diseases associated with molluscan shellfish consumption. Clin. Microbiol. Rev. 7, 419-425.
68. Rose, J. B., Epstein, P. R., Lipp, E. K., Sherman, B. H., Bernard, S. M., and Patz, J. A. 2001. Climate variability and change in the United States: potential impacts on water- and foodborne diseases caused by microbiologic agents. Environ. Health Perspect. 109 (Suppl. 2), 211-221.
69. Rowsell, S., Pauptit, R. A., Tucker, A. D., Melton, R. G., Blow, D. M., and Brick, P. 1997. Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy. Structure. 5, 337-347.
70. Russo, S. and Baumann, U. 2004. Crystal structure of a dodecameric tetrahedral shaped aminopeptidase. J. Biol. Chem. 279, 51275-51281.
71. Sakazaki R. 1968. Proposal of Vibrio alginolyticus for the biotype 2 of Vibrio parahaemolyticus. Jpn. J. Med. Sci. Biol. 21, 359-362.
72. Schembri, M. A., Kjaergaard, K., and Klemm, P. 2003. Global gene expression in Escherichia coli biofilms. Mol. Microbiol. 48, 253-267.
73. Schmidt U., Chmel, H., Cobbs, C. 1979. Vibrio alginolyticus infections in humans. J. Clin. Microbiol. 10, 666-668.
74. Schroeder, U., Henrich, B., Fink, J., and Plapp, R. 1994. PepD of Escherichia coli K-12, a metallopeptidase of low substrate specificity. FEMS Microbiol. Lett. 123, 153-160.
75. Seidler, N. W. 2000. Carnosine prevents the glycation-induced changes in electrophoretic mobility of aspartate aminotransferase. J. Biochem. Mol. Toxicol. 14, 215-220.
76. Stewart, P. S. 2001. Multicellular resistance: biofilms. Trends Microbiol. 9, 34-39.
77. Suda, H., Aoyagi, T., Takeuchi, T., and Umezawa, H. 1976. Inhibition of aminopeptidase B and leucine aminopeptidase by bestatin and its stereoisomer. Arch. Biochem. Biophys. 177, 196-200.
78. Teufel, M., Saudek, V., Ledig, J. P., Bernhardt, A., Boularand, S., Carreau, A., Cairns, N. J., Carter, C., Cowley, D. J., Duverger, D., Ganzhorn, A. J., Guenet, C., Heintzelmann, B., Laucher, V., Sauvage, C., and Smirnova, T. 2003. Sequence identification and characterization of human carnosinase and a closely related nonspecific dipeptidase. J. Biol. Chem. 278, 6521-6531.
79. Tucker, A. D., Rowsell, S., Melton, R. G., and Pauptit, R. A. 1996. A new crystal form of carboxypeptidase G2 from Pseudomonas sp. strain RS-16 which is more amenable to structure determination. Acta Crystallogr. D. 52, 890-892.
80. van der Drift, C. and Ketelaars, H. C. 1974. Carnosinase: its presence in Pseudomonas aeruginosa. Antonie Van Leeuwenhoek. 40, 377-384.
81. Vaughan-Jones, R. D., Spitzer, K. W., and Swietach, P. 2006. Spatial aspects of intracellular pH regulation in heart muscle. Prog. Biophys. Mol. Biol. 90, 207-224.
82. Vidal, O., Longin, R., Prigent-Combaret, C., Dorel, C., Hooreman, M., and Lejeune, P. 1998. Isolation of an Escherichia coli K-12 mutant strain able to form biofilm on inert surfaces: involvement of a new ompR allele that increases curli expression. J. Bacteriol. 180, 2442-2449.
83. Vongerichten, K. F., Klein, J. R., Matern, H., and Plapp, R. 1994. Cloning and nucleotide sequence analysis of pepV, a carnosinase gene from Lactobacillus delbrueckii subsp. lactis DSM 7290, and partial characterization of the enzyme. Microbiology. 140, 2591-2600.
84. Walker, N. D., McEwan, N. R., and Wallace, R. J. 2005. A pepD-like peptidase from the ruminal bacterium, Prevotella albensis. FEMS Microbiol. Lett. 243, 399-404.
85. Wassif, W. S., Sherwood, R. A., Amir, A., Idowu, B., Summers, B., Leigh, N., and Peters, T. J. 1994. Serum carnosinase activities in central nervous system disorders. Clin. Chim. Acta. 225, 57-64.
86. Weiner, R., Langille, S., and Quintero, E. 1995. Structure, function and immunochemistry of bacterial exopolysaccharides. J. Ind. Microbiol. 15, 339-346.
87. Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R., Teitzel, G. M., Lory, S., and Greenberg, E. P. 2001. Gene expression in Pseudomonas aeruginosa biofilms. Nature. 413, 860-864.
88. Yiallouros, I., Kappelhoff, R., Schilling, O., Wegmann, F., Helms, M. W., Auge, A., Brachtendorf, G., Berkhoff, E. G., Beermann, B., Hinz, H. J., Konig, S., Peter-Katalinic, J., Stocker, W. 2002. Activation mechanism of pro-astacin: role of the pro-peptide, tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing. J. Mol. Biol. 234, 237-246.
89. Zen-Yoji, H., Le Clair, R. A., Ota, K., and Montague, T. S. 1973. Comparison of Vibrio parahaemolyticus cultures isolated in the United States with those isolated in Japan. J. Infect. Dis. 127, 237-241.
90. Zhu, J. and Mekalanos, J. J. 2003. Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae. Dev. Cell. 5, 647-656.