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. 1990 Aug;87(16):6238–6242. doi: 10.1073/pnas.87.16.6238

Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis.

M J Weickert 1, G H Chambliss 1
PMCID: PMC54508  PMID: 2117276

Abstract

Catabolite repression of the Bacillus subtilis alpha-amylase gene (amyE) involves an operator sequence located just downstream of the promoter (amyR), overlapping the transcription start site. Oligonucleotide site-directed mutagenesis of this sequence identified bases required for catabolite repression. Two mutations increased both the 2-fold symmetry of the operator and the repression ratio. Although many mutations reduced the repression ratio 3- to 11-fold, some also caused a 2-fold or greater increase in amylase production. Others caused hyperproduction without affecting catabolite repression. Homologous sequences in other catabolite-repressed B. subtilis promoters suggest a common regulatory site may be involved in catabolite repression.

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Selected References

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

  1. Adhya S., Garges S. How cyclic AMP and its receptor protein act in Escherichia coli. Cell. 1982 Jun;29(2):287–289. doi: 10.1016/0092-8674(82)90145-3. [DOI] [PubMed] [Google Scholar]
  2. Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
  3. Ebright R. H., Cossart P., Gicquel-Sanzey B., Beckwith J. Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7274–7278. doi: 10.1073/pnas.81.23.7274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ebright R. H., Cossart P., Gicquel-Sanzey B., Beckwith J. Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli. Nature. 1984 Sep 20;311(5983):232–235. doi: 10.1038/311232a0. [DOI] [PubMed] [Google Scholar]
  5. Fisher S. H., Magasanik B. 2-Ketoglutarate and the regulation of aconitase and histidase formation in Bacillus subtilis. J Bacteriol. 1984 Apr;158(1):379–382. doi: 10.1128/jb.158.1.379-382.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Freese E., Klofat W., Galliers E. Commitment to sporulation and induction of glucose-phosphoenolpyruvate-transferase. Biochim Biophys Acta. 1970 Nov 24;222(2):265–289. doi: 10.1016/0304-4165(70)90115-7. [DOI] [PubMed] [Google Scholar]
  7. Fujita Y., Fujita T. Identification and nucleotide sequence of the promoter region of the Bacillus subtilis gluconate operon. Nucleic Acids Res. 1986 Feb 11;14(3):1237–1252. doi: 10.1093/nar/14.3.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fujita Y., Fujita T., Miwa Y., Nihashi J., Aratani Y. Organization and transcription of the gluconate operon, gnt, of Bacillus subtilis. J Biol Chem. 1986 Oct 15;261(29):13744–13753. [PubMed] [Google Scholar]
  9. Fujita Y., Miwa Y. Identification of an operator sequence for the Bacillus subtilis gnt operon. J Biol Chem. 1989 Mar 5;264(7):4201–4206. [PubMed] [Google Scholar]
  10. Helmann J. D., Wang Y., Mahler I., Walsh C. T. Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons. J Bacteriol. 1989 Jan;171(1):222–229. doi: 10.1128/jb.171.1.222-229.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henkin T. M., Sonenshein A. L. Mutations of the Escherichia coli lacUV5 promoter resulting in increased expression in Bacillus subtilis. Mol Gen Genet. 1987 Oct;209(3):467–474. doi: 10.1007/BF00331151. [DOI] [PubMed] [Google Scholar]
  12. Kammerer W., Deuschle U., Gentz R., Bujard H. Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process. EMBO J. 1986 Nov;5(11):2995–3000. doi: 10.1002/j.1460-2075.1986.tb04597.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kawazu T., Nakanishi Y., Uozumi N., Sasaki T., Yamagata H., Tsukagoshi N., Udaka S. Cloning and nucleotide sequence of the gene coding for enzymatically active fragments of the Bacillus polymyxa beta-amylase. J Bacteriol. 1987 Apr;169(4):1564–1570. doi: 10.1128/jb.169.4.1564-1570.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laoide B. M., Chambliss G. H., McConnell D. J. Bacillus licheniformis alpha-amylase gene, amyL, is subject to promoter-independent catabolite repression in Bacillus subtilis. J Bacteriol. 1989 May;171(5):2435–2442. doi: 10.1128/jb.171.5.2435-2442.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lehtovaara P., Ulmanen I., Palva I. In vivo transcription initiation and termination sites of an alpha-amylase gene from Bacillus amyloliquefaciens cloned in Bacillus subtilis. Gene. 1984 Oct;30(1-3):11–16. doi: 10.1016/0378-1119(84)90099-4. [DOI] [PubMed] [Google Scholar]
  16. MAGASANIK B. Catabolite repression. Cold Spring Harb Symp Quant Biol. 1961;26:249–256. doi: 10.1101/sqb.1961.026.01.031. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Martin I., Debarbouille M., Klier A., Rapoport G. Induction and metabolite regulation of levanase synthesis in Bacillus subtilis. J Bacteriol. 1989 Apr;171(4):1885–1892. doi: 10.1128/jb.171.4.1885-1892.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Melin L., Magnusson K., Rutberg L. Identification of the promoter of the Bacillus subtilis sdh operon. J Bacteriol. 1987 Jul;169(7):3232–3236. doi: 10.1128/jb.169.7.3232-3236.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Melin L., Rutberg L., von Gabain A. Transcriptional and posttranscriptional control of the Bacillus subtilis succinate dehydrogenase operon. J Bacteriol. 1989 Apr;171(4):2110–2115. doi: 10.1128/jb.171.4.2110-2115.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Murphy N., McConnell D. J., Cantwell B. A. The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme beta-glucanase. Nucleic Acids Res. 1984 Jul 11;12(13):5355–5367. doi: 10.1093/nar/12.13.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nakajima R., Imanaka T., Aiba S. Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene. J Bacteriol. 1985 Jul;163(1):401–406. doi: 10.1128/jb.163.1.401-406.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nicholson W. L., Chambliss G. H. Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis. J Bacteriol. 1985 Mar;161(3):875–881. doi: 10.1128/jb.161.3.875-881.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nicholson W. L., Park Y. K., Henkin T. M., Won M., Weickert M. J., Gaskell J. A., Chambliss G. H. Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence. J Mol Biol. 1987 Dec 20;198(4):609–618. doi: 10.1016/0022-2836(87)90204-x. [DOI] [PubMed] [Google Scholar]
  25. Oda M., Sugishita A., Furukawa K. Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon. J Bacteriol. 1988 Jul;170(7):3199–3205. doi: 10.1128/jb.170.7.3199-3205.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  27. Rosenkrantz M. S., Dingman D. W., Sonenshein A. L. Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine. J Bacteriol. 1985 Oct;164(1):155–164. doi: 10.1128/jb.164.1.155-164.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sadler J. R., Sasmor H., Betz J. L. A perfectly symmetric lac operator binds the lac repressor very tightly. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6785–6789. doi: 10.1073/pnas.80.22.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Schleif R. DNA binding by proteins. Science. 1988 Sep 2;241(4870):1182–1187. doi: 10.1126/science.2842864. [DOI] [PubMed] [Google Scholar]
  31. Setlow B., Setlow P. Levels of cyclic GMP in dormant, germinated, and outgrowing spores and growing and sporulating cells of Bacillus megaterium. J Bacteriol. 1978 Oct;136(1):433–436. doi: 10.1128/jb.136.1.433-436.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Setlow P. Inability of detect cyclic AMP in vegetative or sporulating cells or dormant spores of Bacillus megaterium. Biochem Biophys Res Commun. 1973 May 15;52(2):365–372. doi: 10.1016/0006-291x(73)90720-1. [DOI] [PubMed] [Google Scholar]
  33. Sloma A., Ally A., Ally D., Pero J. Gene encoding a minor extracellular protease in Bacillus subtilis. J Bacteriol. 1988 Dec;170(12):5557–5563. doi: 10.1128/jb.170.12.5557-5563.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Straney D. C., Straney S. B., Crothers D. M. Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex. J Mol Biol. 1989 Mar 5;206(1):41–57. doi: 10.1016/0022-2836(89)90522-6. [DOI] [PubMed] [Google Scholar]
  35. Takeda Y., Ohlendorf D. H., Anderson W. F., Matthews B. W. DNA-binding proteins. Science. 1983 Sep 9;221(4615):1020–1026. doi: 10.1126/science.6308768. [DOI] [PubMed] [Google Scholar]
  36. Takkinen K., Pettersson R. F., Kalkkinen N., Palva I., Söderlund H., Käriäinen L. Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene. J Biol Chem. 1983 Jan 25;258(2):1007–1013. [PubMed] [Google Scholar]
  37. Uozumi N., Sakurai K., Sasaki T., Takekawa S., Yamagata H., Tsukagoshi N., Udaka S. A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa. J Bacteriol. 1989 Jan;171(1):375–382. doi: 10.1128/jb.171.1.375-382.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vasantha N., Thompson L. D., Rhodes C., Banner C., Nagle J., Filpula D. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J Bacteriol. 1984 Sep;159(3):811–819. doi: 10.1128/jb.159.3.811-819.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Weickert M. J., Chambliss G. H. Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene. J Bacteriol. 1989 Jul;171(7):3656–3666. doi: 10.1128/jb.171.7.3656-3666.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wittman V., Wong H. C. Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators. J Bacteriol. 1988 Jul;170(7):3206–3212. doi: 10.1128/jb.170.7.3206-3212.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Yamashita S., Kawamura F., Yoshikawa H., Takahashi H., Kobayashi Y., Saito H. Dissection of the expression signals of the spoA gene of Bacillus subtilis: glucose represses sporulation-specific expression. J Gen Microbiol. 1989 May;135(5):1335–1345. doi: 10.1099/00221287-135-5-1335. [DOI] [PubMed] [Google Scholar]
  43. Yang M., Galizzi A., Henner D. Nucleotide sequence of the amylase gene from Bacillus subtilis. Nucleic Acids Res. 1983 Jan 25;11(2):237–249. doi: 10.1093/nar/11.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]

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