1887

Abstract

A genomic library constructed from isolate MT444 DNA in the plasmid vector pBR328 was screened using host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 C and found to contain a 31 kb dlll fragment of inserted DNA. This fragment was present in seven isolates of which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of and failed to identify any additional proteins compared to control containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for . The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 557. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designated Neither protease nor lecithinase activities were detectable in recombinants expressing gene Haemolytic activity was observed from 6 C to 37 C for erythrocytes from a number of mammalian species and also from fish. Gene was expressed in as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of cultured under conditions of iron-sufficiency or iron-restriction. The results indicate that the availability of iron modulates the expression of the gene.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-141-6-1331
1995-06-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/141/6/mic-141-6-1331.html?itemId=/content/journal/micro/10.1099/13500872-141-6-1331&mimeType=html&fmt=ahah

References

  1. Auld D.S., Vallee B.L. 1990; Zinc coordination, function and structure of zinc enzymes and other proteins.. Biochemistry 29:5647–5659
    [Google Scholar]
  2. Bandín I., Santos Y., Bruno D.W., Raynard R.S., Toranzo A.E., Barja J.L. 1991; Lack of biological activities in the extracellular products of Kenibacterium salmoninarum. . Can J Fish Aquat Sei 48:421–425
    [Google Scholar]
  3. Banner C.R., Rohovec J.S., Fryer J.L. 1991; A new value for mol percent guanine + cytosine of DNA for the salmonid fish pathogen Kenibacterium salmoninarum. . FEMS Microbiol Lett 79:57–60
    [Google Scholar]
  4. Bradford M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.. Anal Biochem 72:248–254
    [Google Scholar]
  5. Bruno D.W. 1988; The relationship between auto-agglutination, cell surface hydrophobicity and virulence of the fish pathogen Renibacterium salmoninarum. . FEMS Microbioi Lett 51:135–140
    [Google Scholar]
  6. Bruno D.W. 1990; Presence of saline extractable protein associated with virulent strains of the fish pathogen Kenibacterium salmoninarum. . Bull Eur Assoc Fish Pathol 10:8–10
    [Google Scholar]
  7. Bruno D.W., Munro A.L.S. 1986; Haematological assessment of rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L, infected with Kenibacterium salmoninarum. . J Fish Dis 9:195–204
    [Google Scholar]
  8. Daly J.G., Stevenson R.M.W. 1990; Characterization of the Kenibacterium salmoninarum haemagglutinin.. J Gen Microbiol 136:949–953
    [Google Scholar]
  9. Domann E., Leimeister-Wächter M., Goebel W., Chakraborty T. 1991; Molecular cloning, sequencing and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene.. Infect Immun 59:65–72
    [Google Scholar]
  10. Döring G., Obernesser H.-J., Botzenhart K. 1981; Extracellular toxins of Pseudomonas aeruginosa II. Effect of two proteases on human immunoglobulins IgG, IgA and secretory IgA.. Zentralbl Bakteriol Mikrobiol Hyg 1 Abt Orig A 249:89–98
    [Google Scholar]
  11. Dougan G., Kehoe M. 1984; The minicell system as a method for studying expression from plasmid DNA.. Methods Microbiol 17:233–258
    [Google Scholar]
  12. Evelyn T.P.T. 1993; Bacterial kidney disease - BKD . In Bacterial Diseases of Fish pp. 177–195 Inglis V., Roberts R.J., Bromage N.R. Edited by Oxford: Blackwell;
    [Google Scholar]
  13. Evenden A.J., Gilpin M.L., Gilpin M.L., Munn C.B. 1990; The cloning and expression of a gene encoding haemolytic activity from the fish pathogen R.salmoninarum. . FEMS Microbiol Eett 71:31–34
    [Google Scholar]
  14. Evenden A.J., Grayson T.H., Gilpin M.L., Munn C.B. 1993; Renibacterium salmoninarum and bacterial kidney disease - the unfinished jigsaw.. Annu Rev Fish Dis 3:87–104
    [Google Scholar]
  15. Finlay B.B., Falkow S. 1989; Common themes in microbial pathogenicity.. Microbiol Rev 53:210–230
    [Google Scholar]
  16. Getchell R.G., Rohovec J.S., Fryer J.L. 1985; Comparison of Renibacterium salmoninarum isolates by antigenic analysis.. Fish Pathol 20:149–150
    [Google Scholar]
  17. Goodfellow M., Embley T.M., Austin B. 1985; Numerical taxonomy and emended description of Renibacterium salmoninarum. . J Gen Microbiol 131:2739–2752
    [Google Scholar]
  18. Grayson T.H. 1993 Molecular cloning and characterisation of potential vaccine antigens from Renibacterium salmoninarum. PhD thesis University of Plymouth, UK.:
    [Google Scholar]
  19. Griffiths S.G., Lynch W.H. 1991; Instability of the major soluble antigen produced by Renibacterium salmoninarum. . J Fish Dis 14:55–66
    [Google Scholar]
  20. Guan C., Li P., Riggs P.D., Inouye H. 1987; Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein.. Gene 67:21–30
    [Google Scholar]
  21. Heck L.W., Morihara K., McRae W.B., Miller E.i. 1986; Specific cleavage of human type III and IV collagens by Pseudomonas aeruginosa elastase.. Infect Immun 51:115–118
    [Google Scholar]
  22. Hong Y., Ghebrehiwet B. 1992; Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components Clq and C31.. Clin Immunol Immunopathol 62:133–138
    [Google Scholar]
  23. Jongeneel C.V., Bouvier J., Bairoch A. 1989; A unique signature identifies a family of zinc-dependent metallopeptidases.. FEBS Lett 242:211–214
    [Google Scholar]
  24. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 222:680–685
    [Google Scholar]
  25. Maina C.V., Riggs P.D., Grandea A.G. III Slatko B.E., Moran L.S., Tagliamonte J.A., McReynolds L.A., Guan G. 1988; A vector to express and purify foreign proteins in Escherichia coli by fusion to, and separation from, maltose-binding protein.. Gene 74:365–373
    [Google Scholar]
  26. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning-, a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Mengaud J., Geoffroy C., Cossart P. 1991; Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases.. Infect Immun 59:1043–1049
    [Google Scholar]
  28. Platt T. 1986; Transcription termination and regulation of gene expression.. Annu Rev Biochem 55:339–372
    [Google Scholar]
  29. Rockey D.D., Turaga P.S.D., Wiens G.D., Cook B.A., Kaattari S.L. 1991; Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell- surface protein.. Can J Microbiol 37:758–763
    [Google Scholar]
  30. Simonen M., Palva I. 1993; Protein secretion in Bacillus species.. Microbiol Rev 57:109–137
    [Google Scholar]
  31. Southern E.M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis.. J Mol Biol 98:503–517
    [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.. Proc Natl Acad Sci USA 764350–4354
    [Google Scholar]
  33. Turaga P., Wiens G., Kaattari S. 1987; Bacterial kidney disease: the potential role of soluble protein antigen(s).. J Fish Biol Suppl A 31:191–194
    [Google Scholar]
  34. Vallee B.L, Auld D.S. 1989; Short and long spacer sequences and other structural features of zinc binding sites in zinc enzymes.. FEBS Lett 257:138–140
    [Google Scholar]
  35. Wiens G.D., Kaattari S.L. 1991; Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum. . Infect Immun 59:631–637
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-141-6-1331
Loading
/content/journal/micro/10.1099/13500872-141-6-1331
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error