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. 1994 Mar;176(5):1348–1358. doi: 10.1128/jb.176.5.1348-1358.1994

Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis.

F M Hulett 1, J Lee 1, L Shi 1, G Sun 1, R Chesnut 1, E Sharkova 1, M F Duggan 1, N Kapp 1
PMCID: PMC205199  PMID: 8113174

Abstract

Bacillus subtilis has an alkaline phosphatase (APase) gene family composed of at least four genes. All members of this gene family are expressed postexponentially, either in response to phosphate starvation or sporulation induction or, in some cases, in response to both. The phoA gene (formerly called phoAIV) and the phoB gene (formerly called phoAIII) products have both been isolated from phosphate-starved cells, and a mutation in either gene decreased the total APase expressed under phosphate starvation conditions. Data presented here show that a phoA phoB double mutant reduced APase production during phosphate starvation by 98%, indicating that these two genes are responsible for most of the APase activity during phosphate-limited growth. The promoter for phoA was cloned and used, with the phoB promoter, to examine phosphate regulation in B. subtilis. phoA-lacZ reporter gene assays showed that the expression of the phoA gene commences as the culture enters stationary phase as a result of limiting phosphate concentrations in the growth medium, thereby mimicking the pattern of total APase expression. Induction persists for approximately 2 h and is then turned off. phoA is transcribed from a single promoter which initiates transcription 19 bp before the translation initiation codon. PhoP and PhoR are members of the two-component signal transduction system believed to regulate gene expression in response to limiting phosphate. The expression of phoA or phoB in response to phosphate starvation was equally dependent on PhoP and PhoR for induction. lacZ-promoter fusions showed that both phoA and phoB were hyperinduced, or failed to turn off induction after 2 h, in a spo0A strain of B. subtilis. Mutations in genes which are required for phosphorylation of Spo0A, spo0B and spo0F, also resulted in phoA and phoB hyperinduction, suggesting that phosphorylation of Spo0A is required for the repression of both APases in wild-type strains. The hyperinduction of either APase gene in a spo0A strain was dependent on PhoP and PhoR. Analysis of a phoP-lacZ promoter fusion showed that the phoPR operon is hyperinduced in a spo0A mutant strain, suggesting that Spo0A approximately P represses APases by repressing phoPR transcription. We propose a model for PHO regulation in B. subtilis whereby the phoPR operon is transcribed in response to limiting phosphate concentration, resulting in activation of the PHO regulon transcription, including transcription of phoA and phoB. When the phosphate response fails to overcome the nutrient deficiency, signals for phosphorylation of Spo0A result in production of Spo0A approximately P, which represses transcription of phoPR, thereby repressing synthesis of the PHO regulon.

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Selected References

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  1. Albano M., Hahn J., Dubnau D. Expression of competence genes in Bacillus subtilis. J Bacteriol. 1987 Jul;169(7):3110–3117. doi: 10.1128/jb.169.7.3110-3117.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bookstein C., Edwards C. W., Kapp N. V., Hulett F. M. The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis. J Bacteriol. 1990 Jul;172(7):3730–3737. doi: 10.1128/jb.172.7.3730-3737.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burbulys D., Trach K. A., Hoch J. A. Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell. 1991 Feb 8;64(3):545–552. doi: 10.1016/0092-8674(91)90238-t. [DOI] [PubMed] [Google Scholar]
  4. Chesnut R. S., Bookstein C., Hulett F. M. Separate promoters direct expression of phoAIII, a member of the Bacillus subtilis alkaline phosphatase multigene family, during phosphate starvation and sporulation. Mol Microbiol. 1991 Sep;5(9):2181–2190. doi: 10.1111/j.1365-2958.1991.tb02148.x. [DOI] [PubMed] [Google Scholar]
  5. Chibazakura T., Kawamura F., Takahashi H. Differential regulation of spo0A transcription in Bacillus subtilis: glucose represses promoter switching at the initiation of sporulation. J Bacteriol. 1991 Apr;173(8):2625–2632. doi: 10.1128/jb.173.8.2625-2632.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dubnau D. Genetic competence in Bacillus subtilis. Microbiol Rev. 1991 Sep;55(3):395–424. doi: 10.1128/mr.55.3.395-424.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferrari E., Henner D. J., Perego M., Hoch J. A. Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. J Bacteriol. 1988 Jan;170(1):289–295. doi: 10.1128/jb.170.1.289-295.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ferrari E., Howard S. M., Hoch J. A. Effect of stage 0 sporulation mutations on subtilisin expression. J Bacteriol. 1986 Apr;166(1):173–179. doi: 10.1128/jb.166.1.173-179.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilman M. Z., Chamberlin M. J. Developmental and genetic regulation of Bacillus subtilis genes transcribed by sigma 28-RNA polymerase. Cell. 1983 Nov;35(1):285–293. doi: 10.1016/0092-8674(83)90231-3. [DOI] [PubMed] [Google Scholar]
  10. Hulett F. M., Bookstein C., Jensen K. Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis. J Bacteriol. 1990 Feb;172(2):735–740. doi: 10.1128/jb.172.2.735-740.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hulett F. M., Jensen K. Critical roles of spo0A and spo0H in vegetative alkaline phosphatase production in Bacillus subtilis. J Bacteriol. 1988 Aug;170(8):3765–3768. doi: 10.1128/jb.170.8.3765-3768.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hulett F. M., Kim E. E., Bookstein C., Kapp N. V., Edwards C. W., Wyckoff H. W. Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure. J Biol Chem. 1991 Jan 15;266(2):1077–1084. [PubMed] [Google Scholar]
  13. Jensen K. K., Sharkova E., Duggan M. F., Qi Y., Koide A., Hoch J. A., Hulett F. M. Bacillus subtilis transcription regulator, Spo0A, decreases alkaline phosphatase levels induced by phosphate starvation. J Bacteriol. 1993 Jun;175(12):3749–3756. doi: 10.1128/jb.175.12.3749-3756.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kapp N. V., Edwards C. W., Chesnut R. S., Hulett F. M. The Bacillus subtilis phoAIV gene: effects of in vitro inactivation on total alkaline phosphatase production. Gene. 1990 Nov 30;96(1):95–100. doi: 10.1016/0378-1119(90)90346-s. [DOI] [PubMed] [Google Scholar]
  15. Lee J. W., Hulett F. M. Nucleotide sequence of the phoP gene encoding PhoP, the response regulator of the phosphate regulon of Bacillus subtilis. Nucleic Acids Res. 1992 Nov 11;20(21):5848–5848. doi: 10.1093/nar/20.21.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Makino K., Shinagawa H., Amemura M., Kawamoto T., Yamada M., Nakata A. Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins. J Mol Biol. 1989 Dec 5;210(3):551–559. doi: 10.1016/0022-2836(89)90131-9. [DOI] [PubMed] [Google Scholar]
  17. Makino K., Shinagawa H., Amemura M., Kimura S., Nakata A., Ishihama A. Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro. J Mol Biol. 1988 Sep 5;203(1):85–95. doi: 10.1016/0022-2836(88)90093-9. [DOI] [PubMed] [Google Scholar]
  18. Marahiel M. A., Zuber P., Czekay G., Losick R. Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis. J Bacteriol. 1987 May;169(5):2215–2222. doi: 10.1128/jb.169.5.2215-2222.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miki T., Minami Z., Ikeda Y. The genetics of alkaline phosphatase formation in Bacillus subtilis. Genetics. 1965 Nov;52(5):1093–1100. doi: 10.1093/genetics/52.5.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mueller J. P., Bukusoglu G., Sonenshein A. L. Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system. J Bacteriol. 1992 Jul;174(13):4361–4373. doi: 10.1128/jb.174.13.4361-4373.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mueller J. P., Sonenshein A. L. Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression. J Bacteriol. 1992 Jul;174(13):4374–4383. doi: 10.1128/jb.174.13.4374-4383.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Palva A., Vigren G., Simonen M., Rintala H., Laamanen P. Nucleotide sequence of the tetracycline resistance gene of pBC16 from Bacillus cereus. Nucleic Acids Res. 1990 Mar 25;18(6):1635–1635. doi: 10.1093/nar/18.6.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Perego M., Wu J. J., Spiegelman G. B., Hoch J. A. Mutational dissociation of the positive and negative regulatory properties of the Spo0A sporulation transcription factor of Bacillus subtilis. Gene. 1991 Apr;100:207–212. doi: 10.1016/0378-1119(91)90368-l. [DOI] [PubMed] [Google Scholar]
  24. Piggot P. J., Buxton R. S. Bacteriophage PBSX-induced deletion mutants of Bacillus subtilis 168 constitutive for alkaline phosphatase. J Gen Microbiol. 1982 Apr;128(4):663–669. doi: 10.1099/00221287-128-4-663. [DOI] [PubMed] [Google Scholar]
  25. Predich M., Nair G., Smith I. Bacillus subtilis early sporulation genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase containing sigma H. J Bacteriol. 1992 May;174(9):2771–2778. doi: 10.1128/jb.174.9.2771-2778.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robertson J. B., Gocht M., Marahiel M. A., Zuber P. AbrB, a regulator of gene expression in Bacillus, interacts with the transcription initiation regions of a sporulation gene and an antibiotic biosynthesis gene. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8457–8461. doi: 10.1073/pnas.86.21.8457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Seki T., Yoshikawa H., Takahashi H., Saito H. Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis. J Bacteriol. 1987 Jul;169(7):2913–2916. doi: 10.1128/jb.169.7.2913-2916.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seki T., Yoshikawa H., Takahashi H., Saito H. Nucleotide sequence of the Bacillus subtilis phoR gene. J Bacteriol. 1988 Dec;170(12):5935–5938. doi: 10.1128/jb.170.12.5935-5938.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shimotsu H., Henner D. J. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene. 1986;43(1-2):85–94. doi: 10.1016/0378-1119(86)90011-9. [DOI] [PubMed] [Google Scholar]
  31. Spiegelman G., Van Hoy B., Perego M., Day J., Trach K., Hoch J. A. Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci. J Bacteriol. 1990 Sep;172(9):5011–5019. doi: 10.1128/jb.172.9.5011-5019.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Strauch M. A., Spiegelman G. B., Perego M., Johnson W. C., Burbulys D., Hoch J. A. The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J. 1989 May;8(5):1615–1621. doi: 10.1002/j.1460-2075.1989.tb03546.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Strauch M., Webb V., Spiegelman G., Hoch J. A. The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1801–1805. doi: 10.1073/pnas.87.5.1801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vellanoweth R. L., Rabinowitz J. C. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Mol Microbiol. 1992 May;6(9):1105–1114. doi: 10.1111/j.1365-2958.1992.tb01548.x. [DOI] [PubMed] [Google Scholar]
  35. Wanner B. L. Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? J Bacteriol. 1992 Apr;174(7):2053–2058. doi: 10.1128/jb.174.7.2053-2058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wanner B. L., Wilmes-Riesenberg M. R. Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli. J Bacteriol. 1992 Apr;174(7):2124–2130. doi: 10.1128/jb.174.7.2124-2130.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weir J., Predich M., Dubnau E., Nair G., Smith I. Regulation of spo0H, a gene coding for the Bacillus subtilis sigma H factor. J Bacteriol. 1991 Jan;173(2):521–529. doi: 10.1128/jb.173.2.521-529.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yamada M., Makino K., Amemura M., Shinagawa H., Nakata A. Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes. J Bacteriol. 1989 Oct;171(10):5601–5606. doi: 10.1128/jb.171.10.5601-5606.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. von Heijne G., Abrahmsén L. Species-specific variation in signal peptide design. Implications for protein secretion in foreign hosts. FEBS Lett. 1989 Feb 27;244(2):439–446. doi: 10.1016/0014-5793(89)80579-4. [DOI] [PubMed] [Google Scholar]

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