Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Sep;176(18):5673–5680. doi: 10.1128/jb.176.18.5673-5680.1994

Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis.

M Ogura 1, M Kawata-Mukai 1, M Itaya 1, K Takio 1, T Tanaka 1
PMCID: PMC196770  PMID: 8083159

Abstract

Bacillus subtilis secretes extracellular proteases whose production is positively regulated by a two-component regulatory system, DegS-DegU, and other regulatory factors including DegR. To identify an additional regulatory gene(s) for exoprotease production, we performed a shotgun cloning in the cell carrying multiple copies of degR and found a transformant producing large amounts of the exoproteases. The plasmid in this transformant, pLC1, showed a synergistic effect with multiple copies of degR on the production of the extracellular proteases, and it required degS for its enhancing effect. The DNA region responsible for the enhancement contained the proB gene, as shown by restriction analyses and sequence determination. The proB gene encoding gamma-glutamyl kinase was followed by the proA gene encoding glutamyl-gamma-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon. pLC1 contained the complete proB gene and a part of proA lacking the proA C-terminal region. It was also found that proB on the chromosome showed a synergistic effect with multiple copies of degR. We consider on the basis of these results that the metabolic intermediate, gamma-glutamyl phosphate, would transmit a signal to DegS, resulting in a higher level of phosphorylated DegU. Possible involvement of DegR in this process is discussed.

Full text

PDF
5673

Images in this article

5676Image
on p.5676

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aiba H., Mizuno T., Mizushima S. Transfer of phosphoryl group between two regulatory proteins involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli. J Biol Chem. 1989 May 25;264(15):8563–8567. [PubMed] [Google Scholar]
  2. Amory A., Kunst F., Aubert E., Klier A., Rapoport G. Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis. J Bacteriol. 1987 Jan;169(1):324–333. doi: 10.1128/jb.169.1.324-333.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baich A. Proline synthesis in Escherichia coli. A proline-inhibitable glutamic acid kinase. Biochim Biophys Acta. 1969 Dec 30;192(3):462–467. doi: 10.1016/0304-4165(69)90395-x. [DOI] [PubMed] [Google Scholar]
  4. Buxton R. S. Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage. J Gen Virol. 1980 Feb;46(2):427–437. doi: 10.1099/0022-1317-46-2-427. [DOI] [PubMed] [Google Scholar]
  5. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  6. Dahl M. K., Msadek T., Kunst F., Rapoport G. Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase. J Bacteriol. 1991 Apr;173(8):2539–2547. doi: 10.1128/jb.173.8.2539-2547.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahl M. K., Msadek T., Kunst F., Rapoport G. The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis. J Biol Chem. 1992 Jul 15;267(20):14509–14514. [PubMed] [Google Scholar]
  8. Deutch A. H., Rushlow K. E., Smith C. J. Analysis of the Escherichia coli proBA locus by DNA and protein sequencing. Nucleic Acids Res. 1984 Aug 10;12(15):6337–6355. doi: 10.1093/nar/12.15.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feng J., Atkinson M. R., McCleary W., Stock J. B., Wanner B. L., Ninfa A. J. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J Bacteriol. 1992 Oct;174(19):6061–6070. doi: 10.1128/jb.174.19.6061-6070.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Forst S., Delgado J., Inouye M. Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6052–6056. doi: 10.1073/pnas.86.16.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gaur N. K., Dubnau E., Smith I. Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies. J Bacteriol. 1986 Nov;168(2):860–869. doi: 10.1128/jb.168.2.860-869.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hayzer D. J., Leisinger T. The gene-enzyme relationships of proline biosynthesis in Escherichia coli. J Gen Microbiol. 1980 Jun;118(2):287–293. doi: 10.1099/00221287-118-2-287. [DOI] [PubMed] [Google Scholar]
  14. Hess J. F., Oosawa K., Kaplan N., Simon M. I. Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell. 1988 Apr 8;53(1):79–87. doi: 10.1016/0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  15. Hess J. F., Oosawa K., Matsumura P., Simon M. I. Protein phosphorylation is involved in bacterial chemotaxis. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7609–7613. doi: 10.1073/pnas.84.21.7609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Honjo M., Nakayama A., Fukazawa K., Kawamura K., Ando K., Hori M., Furutani Y. A novel Bacillus subtilis gene involved in negative control of sporulation and degradative-enzyme production. J Bacteriol. 1990 Apr;172(4):1783–1790. doi: 10.1128/jb.172.4.1783-1790.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Igo M. M., Ninfa A. J., Silhavy T. J. A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation. Genes Dev. 1989 May;3(5):598–605. doi: 10.1101/gad.3.5.598. [DOI] [PubMed] [Google Scholar]
  18. Inuzuka M., Miyano H., Tomoeda M. Specific action of 4-nitropyridine 1-oxide on Excherichia coli K-12 pro+ strains leading to the isolation of proline-requiring mutants: isolation and characterization of pro-mutants. Antimicrob Agents Chemother. 1976 Aug;10(2):325–332. doi: 10.1128/aac.10.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Itaya M. Construction of a novel tetracycline resistance gene cassette useful as a marker on the Bacillus subtilis chromosome. Biosci Biotechnol Biochem. 1992 Apr;56(4):685–686. doi: 10.1271/bbb.56.685. [DOI] [PubMed] [Google Scholar]
  20. Itaya M., Tanaka T. Complete physical map of the Bacillus subtilis 168 chromosome constructed by a gene-directed mutagenesis method. J Mol Biol. 1991 Aug 5;220(3):631–648. doi: 10.1016/0022-2836(91)90106-g. [DOI] [PubMed] [Google Scholar]
  21. Kawamura F., Doi R. H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 Oct;160(1):442–444. doi: 10.1128/jb.160.1.442-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keener J., Kustu S. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4976–4980. doi: 10.1073/pnas.85.14.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee T. Y., Makino K., Shinagawa H., Nakata A. Overproduction of acetate kinase activates the phosphate regulon in the absence of the phoR and phoM functions in Escherichia coli. J Bacteriol. 1990 May;172(5):2245–2249. doi: 10.1128/jb.172.5.2245-2249.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  25. Lukat G. S., McCleary W. R., Stock A. M., Stock J. B. Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):718–722. doi: 10.1073/pnas.89.2.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McLaughlin J. R., Murray C. L., Rabinowitz J. C. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283–11291. [PubMed] [Google Scholar]
  27. Mukai K., Kawata-Mukai M., Tanaka T. Stabilization of phosphorylated Bacillus subtilis DegU by DegR. J Bacteriol. 1992 Dec;174(24):7954–7962. doi: 10.1128/jb.174.24.7954-7962.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nagami Y., Tanaka T. Molecular cloning and nucleotide sequence of a DNA fragment from Bacillus natto that enhances production of extracellular proteases and levansucrase in Bacillus subtilis. J Bacteriol. 1986 Apr;166(1):20–28. doi: 10.1128/jb.166.1.20-28.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ninfa A. J., Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909–5913. doi: 10.1073/pnas.83.16.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pang A. S., Nathoo S., Wong S. L. Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis. J Bacteriol. 1991 Jan;173(1):46–54. doi: 10.1128/jb.173.1.46-54.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parkinson J. S. Signal transduction schemes of bacteria. Cell. 1993 Jun 4;73(5):857–871. doi: 10.1016/0092-8674(93)90267-t. [DOI] [PubMed] [Google Scholar]
  32. Perego M., Hoch J. A. Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis. J Bacteriol. 1988 Jun;170(6):2560–2567. doi: 10.1128/jb.170.6.2560-2567.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Piggot P. J., Hoch J. A. Revised genetic linkage map of Bacillus subtilis. Microbiol Rev. 1985 Jun;49(2):158–179. doi: 10.1128/mr.49.2.158-179.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schaeffer P., Millet J., Aubert J. P. Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A. 1965 Sep;54(3):704–711. doi: 10.1073/pnas.54.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith C. J., Deutch A. H., Rushlow K. E. Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis. J Bacteriol. 1984 Feb;157(2):545–551. doi: 10.1128/jb.157.2.545-551.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tanaka T., Kawata-Mukai M. Stabilization of the phosphorylated form of Bacillus Subtilis DegU caused by degU9 mutation. FEMS Microbiol Lett. 1994 Jan 1;115(1):93–96. doi: 10.1111/j.1574-6968.1994.tb06620.x. [DOI] [PubMed] [Google Scholar]
  37. Tanaka T., Kawata M. Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases. J Bacteriol. 1988 Aug;170(8):3593–3600. doi: 10.1128/jb.170.8.3593-3600.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tanaka T., Kawata M., Mukai K. Altered phosphorylation of Bacillus subtilis DegU caused by single amino acid changes in DegS. J Bacteriol. 1991 Sep;173(17):5507–5515. doi: 10.1128/jb.173.17.5507-5515.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tanaka T., Kawata M., Nagami Y., Uchiyama H. prtR enhances the mRNA level of the Bacillus subtilis extracellular proteases. J Bacteriol. 1987 Jul;169(7):3044–3050. doi: 10.1128/jb.169.7.3044-3050.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Ward J. B., Jr, Zahler S. A. Genetic studies of leucine biosynthesis in Bacillus subtilis. J Bacteriol. 1973 Nov;116(2):719–726. doi: 10.1128/jb.116.2.719-726.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weiss V., Magasanik B. Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8919–8923. doi: 10.1073/pnas.85.23.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wong S. L., Wang L. F., Doi R. H. Cloning and nucleotide sequence of senN, a novel 'Bacillus natto' (B. subtilis) gene that regulates expression of extracellular protein genes. J Gen Microbiol. 1988 Dec;134(12):3269–3276. doi: 10.1099/00221287-134-12-3269. [DOI] [PubMed] [Google Scholar]
  44. Wylie D., Stock A., Wong C. Y., Stock J. Sensory transduction in bacterial chemotaxis involves phosphotransfer between Che proteins. Biochem Biophys Res Commun. 1988 Mar 15;151(2):891–896. doi: 10.1016/s0006-291x(88)80365-6. [DOI] [PubMed] [Google Scholar]
  45. Yang M., Ferrari E., Chen E., Henner D. J. Identification of the pleiotropic sacQ gene of Bacillus subtilis. J Bacteriol. 1986 Apr;166(1):113–119. doi: 10.1128/jb.166.1.113-119.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yang M., Shimotsu H., Ferrari E., Henner D. J. Characterization and mapping of the Bacillus subtilis prtR gene. J Bacteriol. 1987 Jan;169(1):434–437. doi: 10.1128/jb.169.1.434-437.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  48. Zuber P., Losick R. Use of a lacZ fusion to study the role of the spoO genes of Bacillus subtilis in developmental regulation. Cell. 1983 Nov;35(1):275–283. doi: 10.1016/0092-8674(83)90230-1. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES