1887

Abstract

SUMMARY: The CWLA lytic amidase of 168 was purified and antisera raised against the purified protein. No expression of could be demonstrated under any conditions by the use of the antisera and fusion analysis. Two lytic enzymes of apparent molecular masses 34 and 30 kDa (as measured by renaturing SDS-PAGE) were found to be mitomycin C-inducible, the larger of which corresponds to a protein immunologically related to CWLA. Both of these inducible lysins were found to be encoded by prophage PBSX. Prophage SPβ was shown by renaturing SDS-PAGE to produce a 43 kDa lytic enzyme unrelated immunologically to CWLA. The smaller of the two PBSX enzymes was purified and found to be an -acetylmuramyl--alanine amidase of 32 kDa (as measured by SDS-PAGE and Coomassie blue staining) which cross-reacts only weakly with the anti-CWLA sera. The potential origin of and its possible relationship to the other phage lytic enzymes are discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-12-3177
1993-12-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/12/mic-139-12-3177.html?itemId=/content/journal/micro/10.1099/00221287-139-12-3177&mimeType=html&fmt=ahah

References

  1. Anagnostopoulos C., Spizizen J. 1961; Requirements for transformation in Bacillus subtilis. Journal of Bacteriology 81:741–746
    [Google Scholar]
  2. Burnette W.M. 1981; Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmod-ified nitrocellulose and radiographic detection with antibody and radioiodinated protein-A. Analytical Biochemistry 112:195–203
    [Google Scholar]
  3. Buxton R.S. 1976; Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX. Journal of Virology 20:22–28
    [Google Scholar]
  4. Buxton R.S. 1980; Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage. Journal of General Virology 46:427–437
    [Google Scholar]
  5. Foster S.J. 1991; Cloning, expression, sequence analysis and biochemical characterization of an autolytic amidase of B. subtilis 168 trpC2. Journal of General Microbiology 137:1987–1998
    [Google Scholar]
  6. Foster S.J. 1992; Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing gel electrophoresis. Journal of Bacteriology 174:464–470
    [Google Scholar]
  7. Ghuysen J.-M., Tippr D.J., Strominger J.L. 1966; Enzymes that degrade bacterial cell walls. Methods in Enzymology 8:685–699
    [Google Scholar]
  8. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166:557–580
    [Google Scholar]
  9. Hemphill H.E. 1990; Bacteriophages. In Molecular Biological Methods for Bacillus pp. 451–191 Harwood C.R., Cutting S.M. Edited by Chichester: Wiley & Sons.;
    [Google Scholar]
  10. Herbold D.R., Glaser L. 1975; Bacillus subtilis N-acetylmuramic acid l-alanine amidase. Journal of Biological Chemistry 250:1676–1682
    [Google Scholar]
  11. Kemp E.H., Sammons R.L., Moir A., Sun D., Setlow P. 1991; Analysis of transcriptional control of the gerD spore germination gene of Bacillus subtilis 168. Journal of Bacteriology 173:4646–4652
    [Google Scholar]
  12. Kuroda A., Sekiguchi J. 1990; Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene. Journal of General Microbiology 136:2209–2216
    [Google Scholar]
  13. Kuroda A., Sekiguchi J. 1991; Molecular cloning and sequencing of a major Bacillus subtilis autolysin gene. Journal of Bacteriology 173:7304–7312
    [Google Scholar]
  14. Kuroda A., Imazeki M., Sekiguchi J. 1991; Purification and characterization of a cell wall hydrolase encoded by the cwlA gene of Bacillus subtilis. FEMS Microbiology Letters 81:9–14
    [Google Scholar]
  15. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature: London; 227680–685
    [Google Scholar]
  16. Lazarevic V., Margot P., Soldo B., Karamata D. 1992; Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetyl muramoyl-l-alanine amidase and its modifier. Journal of General Microbiology 138:1949–1961
    [Google Scholar]
  17. Lipsky R.H., Rosenthal R., Zahler S.A. 1981; Defective specialized SPβ transducing bacteriophages of Bacillus subtilis that carry the sup-3 or sup-44 gene. Journal of Bacteriology 148:1012–1015
    [Google Scholar]
  18. Margot P. 1992 Genetique des autolysines de Bacillus subtilis. PhD thesis University of Lausanne Switzerland.:
    [Google Scholar]
  19. Margot P., Karamata D. 1992; Identification of the structural genes for N-acetylmuramoyl-l-alanine amidase and its modifier in Bacillus subtilis 168: inactivation of these genes by insertional mutagenesis has no effect on growth or ceil separation. Molecular and General Genetics 232:359–366
    [Google Scholar]
  20. Margot P., Roten C.-A.H., Karamata D. 1991; N- acetylmuramoyl-l-alanine amidase assay based on specific radioactive labelling of muropeptide l-alanine: quantification of the enzyme activity in the autolysin deficient Bacillus subtilis 168 ,flaD strain. Analytical Biochemistry 198:15–18
    [Google Scholar]
  21. Mauel C., Karamata D. 1984; Characterization of proteins induced by mitomycin C treatment of Bacillus subtilis. Journal of Virology 49:806–812
    [Google Scholar]
  22. Piggot P.J. 1989; Revised genetic map of Bacillus subtilis 168. In Regulation of Procaryotic Development pp. 1–41 Smith I., Slepecky R.A., Setlow P. Edited by Washington, DC: American Society for Microbiology.;
    [Google Scholar]
  23. Rogers H.J., Taylor C., Rayter S., Ward J.B. 1984; Purification and properties of an autolytic endo-β-glucosaminidase and the N-acetylmuramyl-l-alanine amidase from Bacillus subtilis strain 168. Journal of General Microbiology 130:2395–2402
    [Google Scholar]
  24. Rosenthal R., Toye P.A., Korman R.Z., Zahler S.A. 1979; The prophage of SPβ c2dcitKl, a defective specialized transducing phage of Bacillus subtilis. Genetics 92:721–739
    [Google Scholar]
  25. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  26. Thurm P., Garro A.J. 1975; Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX. Journal of Virology 16:184–191
    [Google Scholar]
  27. Ward J.B., Curtis C.A.M., Taylor C., Buxton R.S. 1982; Purification and characterization of two phage PBSX-induced lytic enzymes of Bacillus subtilis168: an N-acetylmuramoyl-l-alanine amidase and an N-acetylmuramidase. Journal of General Microbiology 128:1171–1178
    [Google Scholar]
  28. Warner F.D., Kitos G.A., Romano M.P., Hemphill H.E. 1977; Characterization of SPβ a temperate bacteriophage from Bacillus subtilis168M. Canadian Journal of Microbiology 23:45–51
    [Google Scholar]
  29. Wood H.E., Dawson M.T., Devine K.M., Mcconnell D.J. 1990; Characterization of PBSX, a defective prophage of Bacillus subtilis. Journal of Bacteriology 172:2667–2674
    [Google Scholar]
  30. Youngman P. 1990; Use of transposons and integrational vectors for mutagenesis and construction of gene fusions in Bacillus species. In Molecular Biological Methods for Bacillus pp. 221–266 Harwood S.R., Cutting S.M. Edited by Chichester: Wiley & Sons.;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-12-3177
Loading
/content/journal/micro/10.1099/00221287-139-12-3177
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