1887

Abstract

A secreted phosphodiesterase/alkaline phosphatase, APaseD, was purified from a culture of JH646MS. Its phosphodiesterase activity was reminiscent of an APase isolated and characterized previously. Immunoassay and N-terminal sequencing showed the two proteins to be identical. Using the first 20 amino acids of the mature protein, a BLAST search of GenBank was used to find an homologous sequence. An exact match was found but in a putative non-coding region. It was hypothesized that there was a base pair deletion in the gene. A DNA fragment internal to the coding region was generated by PCR using template DNA from a strain which produced APaseD. The PCR fragment was cloned and used to interrupt the gene. Western blot analysis of the parent and the mutated strains showed that APaseD was missing in the mutant. Resequencing of the gene revealed a larger ORF encoding a protein similar in size to the 49 kDa APaseD estimated by SDS-PAGE. The promoter was then cloned, sequenced and used in promoter fusions which showed that the gene was phosphate-starvation-induced and dependent on PhoP and PhoR for expression.

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1996-08-01
2021-07-30
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References

  1. Albano M., Hahn J., Dubnau D. 1987; Expression of competence genes in Bacillus subtilis . J Bacteriol 169:3110–3117
    [Google Scholar]
  2. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410
    [Google Scholar]
  3. Archibald A.R., Hancock I.C., Hatwood C. R. 1993; Cell wall structure, synthesis, and turnover. In Bacillus subtilis and Other Gram-positive Bacteria : Biochemistry, Physiology and Molecular Genetics pp. 381–410 Sonenshein A.L., Hoch J.A., Losick R. Edited by Washington, DC :: American Society for Microbiology.;
    [Google Scholar]
  4. Bookstein C., Edwards C.W., Kapp N.V., Hulett F.M. 1990; The Bacillus subtilis 168 alkaline phosphatase III gene; impact of a phoAIII mutation on total alkaline phosphatase synthesis. J Bacteriol 172:3730–3737
    [Google Scholar]
  5. Burbulys D., Trach K.A., Hoch J.A. 1991; Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64:545–552
    [Google Scholar]
  6. Cutting S.M., Vander Horn P.B. 1990; Genetic analysis. In Molecular Biological Methods for Bacillus pp. 27–74 Harwood C.R., Cutting S.M. Edited by New York:: John Wiley.;
    [Google Scholar]
  7. Dubnau D. 1991; Genetic competence in Bacillus subtilis . Microbiol Rev 55:395–424
    [Google Scholar]
  8. Ferrari E., Howard S., Hoch J.A. 1986; Effect of stage 0 sporulation mutants on subtilisin expression. J Bacteriol 166:173–179
    [Google Scholar]
  9. Ferrari E., Henner D., Perego M., Hoch J.A. 1988; Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. J Bacteriol 170:289–295
    [Google Scholar]
  10. Gilman M., Chamberlin M. 1993; Developmental and genetic regulation of Bacillus subtilis genes transcribed by sigma-28 RNA containing polymerase. Cell 35:285–293
    [Google Scholar]
  11. Grant W.D. 1979; Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis . J Bacteriol 137:35–43
    [Google Scholar]
  12. Hanahan D. 1985; Techniques for transformation of E. coli . In DNA Cloning II: A Practical Approach pp. 109–135 Glover D.M. Edited by Washington, DC:: IRL Press.;
    [Google Scholar]
  13. Hulett F.M. 1993; Regulation of phosphorus metabolism. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics pp. 229–235 Sonenshein A.L., Hoch J.A., Losick R. Edited by Washington, DC:: American Society for Microbiology.;
    [Google Scholar]
  14. Hulett F.M. 1996; The signal transduction network for Pho regulation in Bacillus subtilis . Mol Microbiol 19:933–939
    [Google Scholar]
  15. Hulett F.M., Jensen K. 1988; Critical roles of spo0H and spoOH in vegetative alkaline phosphatase production in Bacillus subtilis . J Bacteriol 170:3765–3768
    [Google Scholar]
  16. Hulett F.M., Bookstein C., Jensen K. 1990; Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis . J Bacteriol 172:735–740
    [Google Scholar]
  17. Hulett F.M., Kim E.E., Bookstein C., Kapp N.V., Edwards C.W., Wyckoff H.W. 1991; Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase threedimensional structure. Biol Chem 226:1077–1084
    [Google Scholar]
  18. Hulett F. M., Sun G., Liu W. 1994a; The Pho regulon of Bacillus subtilis is regulated by sequential action of two genetic switches. In Phosphate in Microorganisms: Cellular and Molecular Biology pp. 50–54 Torriani-Gorini A., Yagil E., Silver S. Edited by Washington, DC:: American Society for Microbiology.;
    [Google Scholar]
  19. Hulett F. M., Lee J., Shi L., Sun G., Chesnut R., Sharkova E., Duggan M. F., Kapp N. 1994b; Sequential action of two-component genetic switches regulates the Pho regulon in Bacillus subtilis . J Bacteriol 176:1348–1358
    [Google Scholar]
  20. Jensen K.K., Sharkova E., Duggan M.F., Qi Y., Koide A., Hoch J.A., Hulett F.M. 1993; Bacillus subtilis transcription regulator, Spo0A, decreases alkaline phosphatase levels induced by phosphate starvation. J Bacteriol 175:3749–3756
    [Google Scholar]
  21. Kusser W., Fiedler F. 1982; Purification, Mr-value and subunit structure of a teichoic acid hydrolase from Bacillus subtilis . FEBS Lett 149:67–70
    [Google Scholar]
  22. Kusser W., Fiedler F. 1983; Teichoicase from Bacillus subtilis Marburg. J Bacteriol 155:302–310
    [Google Scholar]
  23. Lampen J.O., Wang W., Mezes P.S.F., Yang Y.Q. 1984; Lactamases of bacilli: nature and processing. In Genetics and Biotechnology of Bacilli pp. 129–140 Ganesan A.T., Hoch J.A. Edited by New York:: Academic Press.;
    [Google Scholar]
  24. Le Hegarat J.C., Anagnostopoulos C. 1973; Purification, subunit structure and properties of two repressible phospho- hydrolases of Bacillus subtilis . Eur J Biochem 39:525–539
    [Google Scholar]
  25. Mauël C., Young M., Karamata D. 1991; Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units. J Gen Microbiol 137:929–941
    [Google Scholar]
  26. Marahiel M.A., Zuber P., Czekay G., Losick R. 1987; Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis . J Bacteriol 169:2215–2222
    [Google Scholar]
  27. Matsudaira P. 1987; Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
    [Google Scholar]
  28. Msadek T., Kunst F., Rapoport G. 1993; Two-component regulatory systems. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics pp. 729–745 Sonenshein A.L., Hoch J.A., Losick R. Edited by Washington, DC:: American Society for Microbiology.;
    [Google Scholar]
  29. Nagarajan V. 1993; Protein secretion. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics pp. 713–726 Sonenshein A.L., Hoch J.A., Losick R. Edited by Washington, DC:: American Society for Microbiology.;
    [Google Scholar]
  30. Ogasawara N., Fujita Y., Kobayashi Y., Sadaie Y., Tanaka T., Takahashi H., Yamane K. 1995; Systematic sequencing of the Bacillus subtilis genome: progress report of the Japanese group. Microbiology 141:257–259
    [Google Scholar]
  31. Ogawa K., Akagawa E., Nakamura K., Yamane K. 1995; Determination of a 21 548 bp nucleotide sequence around the 24° region of the Bacillus subtilis chromosome. Microbiology 141:269–275
    [Google Scholar]
  32. Soldo B., Lazarevic V., Pagni M., Mauël C., Karamata D. 1995; The 8th international conference on Bacilli . p. 77:
    [Google Scholar]
  33. Sun G., Sharkova E., Chesnut R., Birkey S., Duggan M.F., Sorokin A., Pujic P., Ehrlich S.D., Hulett F.M. 1996; Regulators of aerobic and anaerobic respiration in Bacillus subtilis . J Bacteriol 178:1374–1385
    [Google Scholar]
  34. Vasantha N., Thompson L.D., Rhodes C., Banner C., Nagle J., Filpula D. 1984; Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequences and mature protein. J Bacteriol 159:811–819
    [Google Scholar]
  35. Von Heijne G., Abrahmsen L. 1989; Species-specific variation in signal peptide design. FEBS Lett 244:439–446
    [Google Scholar]
  36. Yamane K., Maruo B. 1978a; Purification and characterization of extracellular soluble and membrane-bound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis . J Bacteriol 134:100–107
    [Google Scholar]
  37. Yamane K., Maruo B. 1978b; Alkaline phosphatase possessing alkaline phosphodiesterase activity and other phosphodiesterases in Bacillus subtilis . J Bacteriol 134:108–114
    [Google Scholar]
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