1887

Microbiology Society logo

Volume 134, Issue 8

Molecular Cloning and Genetic Analysis of the Determinant for Gamma-Lysin, a Two-component Toxin of Free

Abstract

The -lysin determinant of strain Smith 5R has been cloned in phage and plasmid vectors in . Genetic evidence is presented which demonstrates that -lysin requires the co-operative action of two polypeptides expressed by the closely linked and genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of -lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of -lysin, but not by anti--, anti-- or anti--lysin senlm. Subcloning and transposon Tn5 mutagenesis identified a 3·5 kb region which was necessary for -lysin expression in . Two genes ( and ) were mapped and their polyoeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of -lysin purified from culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of ) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the culture supernate containing component B of -lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the and genes are closely linked in the chromosome of several strains of .

  • Received:
  • Revised:
  • Published Online:
© Society for General Microbiology 1988
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-134-8-2179
1988-08-01
2024-04-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/134/8/mic-134-8-2179.html?itemId=/content/journal/micro/10.1099/00221287-134-8-2179&mimeType=html&fmt=ahah

References

  1. Amann E., Brosius J. 1985; ‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli.. Gene 40:183–190
     [Google Scholar]
  2. Anderson J.C. 1977; Experimental staphylococcal mastitis in the mouse: the induction of chronic mastitis and its response to antibiotic therapy.. Journal of Comparative Pathology 87:611–621
     [Google Scholar]
  3. Anderson J.C. 1983; Veterinary aspects of staphylococci.. In Staphylococci and Staphylococcal Infections 28 pp. 193–237 Easmon C.S.F., Adlam C. Edited by London: Academic Press;
     [Google Scholar]
  4. Appleyard R.K. 1954; Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12.. Genetics 39:440–452
     [Google Scholar]
  5. Berg D.E., Weiss A., Crossland L. 1980; Polarity of Tn5 insertion mutations in Escherichia coli.. Journal of Bacteriology 142:439–446
     [Google Scholar]
  6. Bolivar F., Rodriguez R.L., Greene P.J., Betlach M.V., Heynecker H.L., Boyer H.W., Crosa J.H., Falkow S. 1977; Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.. Gene 2:95–113
     [Google Scholar]
  7. Burnette W.N. 1981; 'Western blotting': electrophoretic transfer of protein from sodium dodecyl sulphate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.. Analytical Biochemistry 112:195–203
     [Google Scholar]
  8. Clyne M., De azavedo J., Arbuthnott J. 1987; Production of gamma haemolysin by toxic shock syndrome associated strains of Staphylococcus aureus.. Journal of Clinical Microbiology 26:535–539
     [Google Scholar]
  9. Coleman D.C., Foster T.J. 1981; Analysis of the reduction in expression of tetracycline resistance by transposon Tn 10 in the multicopy state.. Molecular and General Genetics 182:171–177
     [Google Scholar]
  10. Dougan G., Kehoe M. 1984; The minicell system as a method for studying expression from plasmid DNA.. Methods in Microbiology 17:233–258
     [Google Scholar]
  11. Dougan G., Sherratt D. 1977; The transposon Tn/ as a probe for studying ColEl structure and function.. Molecular and General Genetics 151:151–160
     [Google Scholar]
  12. Easmon C.S.F., Adlam C. 1983 editor Staphylococci and Staphylococcal Infections 1 and 2 London: Academic Press;
     [Google Scholar]
  13. Fackrell H.B., Wiseman G.M. 1976a; Production and purification of the gamma haemolysin of Staphylococcus aureus 'Smith 5R'.. Journal of General Microbiology 92:1–10
     [Google Scholar]
  14. Fackrell H.B., Wiseman G.M. 1976b; Properties of the gamma haemolysin of Staphylococcus aureus ‘Smith 5R’.. Journal of General Microbiology 92:11–24
     [Google Scholar]
  15. Guyonnet F., Plommet M. 1970; Hemolysine gamma de Staphylococcus aureus: purification et proprietes.. Annales de I’Institut Pasteur 118:19–33
     [Google Scholar]
  16. Guyonnet F., Plommet M., Bouillane C. 1968; Purification de l’hemolysine gamma de Staphylococcus aureus.. Comptes rendus hebdomadaires des seances de I’Academie des sciences 267:1181–1182
     [Google Scholar]
  17. Helfman D.M., Fermamisco J.R., Fiddes J.C., Thomas G.P., Hughes S.H. 1983; Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library.. Proceedings of the National Academy of Sciences of the United States of America 80:31–35
     [Google Scholar]
  18. Jonsson P., Lindberg M., Haraldsson I., Wadstrom T. 1985; Virulence of Staphylococcus aureus in the mouse mastitis model: studies of alpha haemolysin, coagulase and protein A as possible virulence determinants with protoplast fusion and gene cloning.. Infection and Immunity 49:765–769
     [Google Scholar]
  19. Laemmli U.K. 1970; Cleavage of the structural protein during the assembly of the head of bacteriophage T4.. Nature; London: 227680–685
     [Google Scholar]
  20. Loenen W.A.M., Brammar W.J. 1980; A bacteriophage vector for cloning large restriction fragments made with several restriction enzymes.. Gene 10:249–259
     [Google Scholar]
  21. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  22. Mccartney C., Arbuthnott J.P. 1978; Mode of action of membrane-damaging toxins produced by staphylococci.. In Bacterial Toxins and Cell Membranes pp. 89–122 Jeljaszewicz J., Wadstrom T. Edited by London: Academic Press;
     [Google Scholar]
  23. Miller J.H. 1972 Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  24. Mollby R., Wadstrom T. 1971; Separation of gamma hemolysin from Staphylococcus aureus Smith 5R.. Infection and Immunity 3:633–635
     [Google Scholar]
  25. Novick R.P. 1963; Properties of a cryptic high- frequency transducing phage in Staphylococcus aureus.. Virology 33:155–166
     [Google Scholar]
  26. O’REILLY M., Deazavedo J.C.S., Kennedy S., Foster T.J. 1986; Inactivation of the alpha- haemolysin of Staphylococcus aureus 8325-4 by site- directed mutagenesis and studies on expression of its haemolysins.. Microbial Pathogenesis 1:125–138
     [Google Scholar]
  27. O’TOOLE P.W., Foster T.J. 1986a; Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus.. Microbial Pathogenesis 1:583–594
     [Google Scholar]
  28. O’TOOLE P.W., Foster T.J. 1986b; Epidermolytic toxin serotype B of Staphylococcus aureus is plasmid-encoded.. FEMS Microbiology Letters 36:311–314
     [Google Scholar]
  29. Ranneli D.M., Jones C.L., Johns M.B., Mussey G.J., Khan S.A. 1985; Molecular cloning of staphylococcal enterotoxin B gene in Escherichia coli and Staphylococcus aureus.. Proceedings of the National Academy of Sciences of the United States of America 82:5850–5854
     [Google Scholar]
  30. Rigby P.W.J., Diekman M., Rhodes C., Berg P. 1977; Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase.. Journal of Molecular Biology 113:237–251
     [Google Scholar]
  31. Smith M.L., Price S.A. 1938; Staphylococcus gamma haemolysin.. Journal of Pathology and Bacteriology 47:379–393
     [Google Scholar]
  32. Southern E.M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis.. Journal of Molecular Biology 98:503–517
     [Google Scholar]
  33. Sparling P.F. 1983; Applications of genetics to studies of bacterial virulence.. Philosophical Transactions of the Royal Society B303:199–207
     [Google Scholar]
  34. Taylor A.G., Bernheimer A.W. 1974; Further characterization of staphylococcal gamma-hemolysin.. Infection and Immunity 10:54–59
     [Google Scholar]
  35. Taylor A.G., Plommet M. 1973; Anti-gamma haemolysin as a diagnostic test in staphylococcal osteomyelitis.. Journal of Clinical Pathology 26:409–412
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-134-8-2179
Loading
Molecular Cloning and Genetic Analysis of the Determinant for Gamma-Lysin, a Two-component Toxin of Staphylococcus aureus
Microbiology 134, 2179 (1988); https://doi.org/10.1099/00221287-134-8-2179
/content/journal/micro/10.1099/00221287-134-8-2179
/content/journal/micro/10.1099/00221287-134-8-2179
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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