1887

Abstract

The spores of have characteristic properties and consist of complex structures including various types of proteins. To perform comprehensive analysis of the protein composition of the spores, the proteins extracted from the spore were analysed by a combination of one-dimensional PAGE and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using Turboquest SEQUEST software interfaced with the DNA sequence database of . A total of 154 proteins were identified, and 69 of them were novel. The remaining 85 proteins have been previously reported as sporulation-specific proteins or as proteins that are synthesized in vegetative cells. The expression pattern of each gene deduced to encode novel spore proteins was analysed using a series of strains carrying a reporter gene. The results revealed that the expression of 26 genes was dependent on sporulation-specific sigma factors, namely σ, σ, σ and σ. In this study, it is demonstrated that the combination of the techniques of SDS-PAGE and LC-MS/MS, with the mutant library of , is an effective tool for the analysis of complicated cellular structures.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-12-3971
2002-12-01
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/12/1483971a.html?itemId=/content/journal/micro/10.1099/00221287-148-12-3971&mimeType=html&fmt=ahah

References

  1. Aronson, A. I. & Fitz-James, P. ( 1976; ). Structure and morphogenesis of the bacterial spore coat. Bacteriol Rev 40, 360-402.
    [Google Scholar]
  2. Bagyan, I., Setlow, B. & Setlow, P. ( 1998; ). New small, acid-soluble proteins unique to spores of Bacillus subtilis: identification of the coding genes and regulation and function of two of these genes. J Bacteriol 180, 6704-6712.
    [Google Scholar]
  3. Bourne, N., Fitz-James, P. C. & Aronson, A. I. ( 1991; ). Structural and germination defects of Bacillus subtilis spores with altered contents of a spore coat protein. J Bacteriol 173, 6618-6625.
    [Google Scholar]
  4. Buchanan, C. E. & Ling, M.-L. ( 1992; ). Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis. J Bacteriol 174, 1717-1725.
    [Google Scholar]
  5. Cutting, S. M. & Vander Horn, P. B. ( 1990; ). Genetic analysis. In Molecular Biological Methods for Bacillus , pp. 27-64. Edited by C. R. Harwood & S. M. Cutting. Chichester:Wiley.
  6. Deuerling, E., Paeslack, B. & Schumann, W. ( 1995; ). The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift. J Bacteriol 177, 4105-4112.
    [Google Scholar]
  7. Driks, A. ( 1999; ). Bacillus subtilis spore coat. Microbiol Mol Biol Rev 63, 1-20.
    [Google Scholar]
  8. Driks, A. ( 2001; ). Proteins of the spore core and coat. In Bacillus subtilis and its Closest Relatives from Genes to Cells , pp. 527-535. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC:American Society for Microbiology.
  9. Fawcett, P., Eichenberger, P., Losick, R. & Youngman, P. ( 2000; ). The transcriptional profile of early to middle sporulation in Bacillus subtilis. Proc Natl Acad Sci USA 97, 8063-8068.[CrossRef]
    [Google Scholar]
  10. Francesconi, S. C., MacAlister, T. J., Setlow, B. & Setlow, P. ( 1988; ). Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis. J Bacteriol 170, 5963-5967.
    [Google Scholar]
  11. Goldman, R. C. & Tipper, D. J. ( 1978; ). Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component. J Bacteriol 135, 1091-1106.
    [Google Scholar]
  12. Gould, G. W. ( 1983; ). Mechanisms of resistance and dormancy. In The Bacterial Spores , pp. 173-210. Edited by A. Hurst & G. W. Gould. London:Academic Press.
  13. Helmann, J. D. & Moran, C. P.Jr ( 2001; ). RNA polymerase and sigma factors. In Bacillus subtilis and its Closest Relatives from Genes to Cells , pp. 527-535. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC:American Society for Microbiology.
  14. Henner, D. J. ( 1990; ). Inducible expression of regulatory genes in Bacillus subtilis. Methods Enzymol 185, 223-228.
    [Google Scholar]
  15. Henriques, A. O & Moran, C. P.Jr ( 2000; ). Structure and assembly of the bacterial endospore coat. Methods 20, 95-110.[CrossRef]
    [Google Scholar]
  16. Henriques, A. O., Beall, B. W., Roland, K. & Moran, C. P.Jr ( 1995; ). Characterization of cotJ, a σE-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores. J Bacteriol 177, 3394-3406.
    [Google Scholar]
  17. Henriques, A. O., Beall, B. W. & Moran, C. P.Jr ( 1997; ). CotM of Bacillus subtilis, a member of the α-crystallin family of stress proteins, is induced during development and participates in spore outer coat formation. J Bacteriol 179, 1887-1897.
    [Google Scholar]
  18. Henriques, A. O., Melsen, L. R. & Moran, C. P.Jr ( 1998; ). Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. J Bacteriol 180, 2285-2291.
    [Google Scholar]
  19. Hirose, I., Sano, K., Shioda, I., Kumano, M., Nakamura, K. & Yamane, K. ( 2000; ). Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study. Microbiology 146, 65-75.
    [Google Scholar]
  20. Igo, M. M. & Losick, R. ( 1986; ). Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis. J Mol Biol 191, 615-624.[CrossRef]
    [Google Scholar]
  21. Kobayashi, K., Hashiguchi, K., Yokozeki, K. & Yamanaka, S. ( 1998; ). Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli. Biosci Biotechnol Biochem 62, 1109-1114.[CrossRef]
    [Google Scholar]
  22. Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors ( 1997; ). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[CrossRef]
    [Google Scholar]
  23. LaVallie, E. R. & Stahl, M. L. ( 1989; ). Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation. J Bacteriol 171, 3085-3094.
    [Google Scholar]
  24. Moriyama, R., Fukuoka, H., Miyata, S., Kudoh, S., Hattori, A., Kozuka, S., Yasuda, Y., Tochikubo, K. & Makino, S. ( 1999; ). Expression of a germination-specific amidase, SleB, of bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores. J Bacteriol 181, 2373-2378.
    [Google Scholar]
  25. Paidhungat, M. & Setlow, P. ( 2001; ). Spore germination and outgrowth. In Bacillus subtilis and its Closest Relatives from Genes To Cells , pp. 537-548. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC:American Society for Microbiology.
  26. Piggot, P. J. & Losick, R. ( 2001; ). Sporulation genes and intercompartmental regulation. In Bacillus subtilis and its Closest Relatives from Genes To Cells , pp. 483-517. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC:American Society for Microbiology.
  27. Rufo, G. A.Jr, Sullivan, B. J., Sloma, A. & Pero, J. ( 1990; ). Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis. J Bacteriol 172, 1019-1023.
    [Google Scholar]
  28. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  29. Schuch, R. & Piggot, P. J. ( 1994; ). The dacFspoIIA operon of Bacillus subtilis, encoding σF, is autoregulated. J Bacteriol 176, 4104-4110.
    [Google Scholar]
  30. Serrano, M., Zilhao, R., Ricca, E., Ozin, A. J., Moran, C. P.Jr & Henriqous, A. O. ( 1999; ). A Bacillus subtilis secreted protein with a role in endospore coat assembly and function. J Bacteriol 181, 3632-3643.
    [Google Scholar]
  31. Seyler, R. W.Jr, Henriques, A. O., Ozin, A. J. & Moran, C. P.Jr ( 1997; ). Assembly and interactions of cotJ-encoded proteins, constituents of the inner layers of the Bacillus subtilis spore coat. Mol Microbiol 25, 955-966.[CrossRef]
    [Google Scholar]
  32. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. ( 1996; ). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850-858.[CrossRef]
    [Google Scholar]
  33. Simpson, E. B., Hancock, T. W. & Buchanan, C. E. ( 1994; ). Transcriptional control of dacB, which encodes a major sporulation-specific penicillin-binding protein. J Bacteriol 176, 7767-7769.
    [Google Scholar]
  34. Sloma, A., Rudolph, C. F., Rufo, G. A.Jr, Sullivan, B. J., Theriault, K. A., Ally, D. & Pero, J. ( 1990; ). Gene encoding a novel extracellular metalloprotease in Bacillus subtilis. J Bacteriol 172, 1024-1029.
    [Google Scholar]
  35. Stragier, P. & Losick, R. ( 1996; ). Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet 30, 297-341.[CrossRef]
    [Google Scholar]
  36. Takamatsu, H., Hiraoka, T., Kodama, T., Koide, H., Kozuka, S., Tochikubo, K. & Watabe, K. ( 1998; ). Cloning of a novel gene yrbB, encoding a protein located in the spore integument of Bacillus subtilis. FEMS Microbiol Lett 166, 361-367.[CrossRef]
    [Google Scholar]
  37. Takamatsu, H., Kodama, T., Imamura, A., Asai, K., Kobayashi, K., Nakayama, T., Ogasawara, N. & Watabe, K. ( 2000; ). The Bacillus subtilis yabG gene is transcribed by SigK RNA polymerase during sporulation, and yabG mutant spores have altered coat protein composition. J Bacteriol 182, 1883-1888.[CrossRef]
    [Google Scholar]
  38. Vagner, V., Dervyn, E. & Ehrlich, S. D. ( 1998; ). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144, 3097-3104.[CrossRef]
    [Google Scholar]
  39. Valbuzzi, A., Ferrari, E. & Albertini, A. M. ( 1999; ). A novel member of the subtilisin-like protease family from Bacillus subtilis. Microbiology 145, 3121-3127.
    [Google Scholar]
  40. Wu, J.-J., Schuch, R. & Piggot, P. J. ( 1992; ). Characterization of a Bacillus subtilis sporulation operon that includes genes for an RNA polymerase σ factor and for a putative dd-carboxypeptidase. J Bacteriol 174, 4885-4892.
    [Google Scholar]
  41. Yates, J. R.3rd, Eng, J. K., McCormack, A. L. & Schieltz, D. ( 1995; ). Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem 67, 1426-1436.[CrossRef]
    [Google Scholar]
  42. Zhang, J., Fitz-James, P. C. & Aronson, A. I. ( 1993; ). Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis. J Bacteriol 175, 3757-3766.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-12-3971
Loading
/content/journal/micro/10.1099/00221287-148-12-3971
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