Abstract
Two novel Bacillus subtilis genes that regulate the production of several extracellular enzymes were clones and characterized. These two genes are organized as part of an operon. When cloned in a multicopy plasmid, the first gene (tenA, transcription enhancement) stimulates alkaline protease production at the transcriptional level. The second gene (tenI) exerts an opposite effect to reduce alkaline protease production. The production of neutral protease, levansucrase, and alkaline protease can be stimulated up to 11- to 55-fold. Thus, tenA is a new member of the deg (regulatory genes for degradative enzymes) family in B. subtilis. A functional degS product is required to observe the stimulatory effect from tenA. Between the promoter and the ribosome-binding site of tenA, there exists a terminatorlike structure. Deletion of this structure doubles the expression of tenA. Neither tenA nor tenI is essential for cell growth and the production of extracellular enzymes. However, inactivation of these genes causes a delay in sporulation. This operon is located close to tre on the genetic linkage map. The overall organization of this operon and its relationship with other known regulatory factors in the deg family are discussed.
Full text
PDF![46](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/47b0c5f6a681/jbacter00091-0068.png)
![47](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/735f0686aae5/jbacter00091-0069.png)
![48](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/c3a555c5da7d/jbacter00091-0070.png)
![49](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/5134a7869bd4/jbacter00091-0071.png)
![50](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/4d39e901ea2d/jbacter00091-0072.png)
![51](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/c93e71838257/jbacter00091-0073.png)
![52](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/03238fec8a7d/jbacter00091-0074.png)
![53](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/a1867960907e/jbacter00091-0075.png)
![54](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fd22/207154/51c5bb91d3f2/jbacter00091-0076.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- Amster-Choder O., Houman F., Wright A. Protein phosphorylation regulates transcription of the beta-glucoside utilization operon in E. coli. Cell. 1989 Sep 8;58(5):847–855. doi: 10.1016/0092-8674(89)90937-9. [DOI] [PubMed] [Google Scholar]
- Burnett T. J., Shankweiler G. W., Hageman J. H. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation. J Bacteriol. 1986 Jan;165(1):139–145. doi: 10.1128/jb.165.1.139-145.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
- Danna K. J. Determination of fragment order through partial digests and multiple enzyme digests. Methods Enzymol. 1980;65(1):449–467. doi: 10.1016/s0076-6879(80)65055-1. [DOI] [PubMed] [Google Scholar]
- Dedonder R. A., Lepesant J. A., Lepesant-Kejzlarová J., Billault A., Steinmetz M., Kunst F. Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl Environ Microbiol. 1977 Apr;33(4):989–993. doi: 10.1128/aem.33.4.989-993.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henner D. J., Yang M., Ferrari E. Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems. J Bacteriol. 1988 Nov;170(11):5102–5109. doi: 10.1128/jb.170.11.5102-5109.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoch J. A., Barat M., Anagnostopoulos C. Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J Bacteriol. 1967 Jun;93(6):1925–1937. doi: 10.1128/jb.93.6.1925-1937.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunst F., Debarbouille M., Msadek T., Young M., Mauel C., Karamata D., Klier A., Rapoport G., Dedonder R. Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems. J Bacteriol. 1988 Nov;170(11):5093–5101. doi: 10.1128/jb.170.11.5093-5101.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunst F., Pascal M., Lepesant-Kejzlarova J., Lepesant J. A., Billault A., Dedonder R. Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168. Biochimie. 1974;56(11-12):1481–1489. doi: 10.1016/s0300-9084(75)80270-7. [DOI] [PubMed] [Google Scholar]
- Lepesant J. A., Kunst F., Lepesant-Kejzlarová J., Dedonder R. Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg. Mol Gen Genet. 1972;118(2):135–160. doi: 10.1007/BF00267084. [DOI] [PubMed] [Google Scholar]
- Mahadevan S., Reynolds A. E., Wright A. Positive and negative regulation of the bgl operon in Escherichia coli. J Bacteriol. 1987 Jun;169(6):2570–2578. doi: 10.1128/jb.169.6.2570-2578.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
- Msadek T., Kunst F., Henner D., Klier A., Rapoport G., Dedonder R. Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU. J Bacteriol. 1990 Feb;172(2):824–834. doi: 10.1128/jb.172.2.824-834.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ochi K., Kandala J. C., Freese E. Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation. J Biol Chem. 1981 Jul 10;256(13):6866–6875. [PubMed] [Google Scholar]
- Pascal M., Kunst F., Lepesant J. A., Dedonder R. Characterization of two sucrase activities in Bacillus subtilis Marburg. Biochimie. 1971;53(10):1059–1066. doi: 10.1016/s0300-9084(71)80193-1. [DOI] [PubMed] [Google Scholar]
- Priest F. G. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev. 1977 Sep;41(3):711–753. doi: 10.1128/br.41.3.711-753.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Projan S. J., Carleton S., Novick R. P. Determination of plasmid copy number by fluorescence densitometry. Plasmid. 1983 Mar;9(2):182–190. doi: 10.1016/0147-619x(83)90019-7. [DOI] [PubMed] [Google Scholar]
- 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]
- Schnetz K., Toloczyki C., Rak B. Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes. J Bacteriol. 1987 Jun;169(6):2579–2590. doi: 10.1128/jb.169.6.2579-2590.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
- Shimotsu H., Henner D. J. Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes. J Bacteriol. 1986 Oct;168(1):380–388. doi: 10.1128/jb.168.1.380-388.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stahl M. L., Ferrari E. Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation. J Bacteriol. 1984 May;158(2):411–418. doi: 10.1128/jb.158.2.411-418.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steinmetz M., Le Coq D., Aymerich S., Gonzy-Tréboul G., Gay P. The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet. 1985;200(2):220–228. doi: 10.1007/BF00425427. [DOI] [PubMed] [Google Scholar]
- Takagi M., Takada H., Imanaka T. Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT. J Bacteriol. 1990 Jan;172(1):411–418. doi: 10.1128/jb.172.1.411-418.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Wang L. F., Doi R. H. Complex character of senS, a novel gene regulating expression of extracellular-protein genes of Bacillus subtilis. J Bacteriol. 1990 Apr;172(4):1939–1947. doi: 10.1128/jb.172.4.1939-1947.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang L. F., Doi R. H. Promoter switching during development and the termination site of the sigma 43 operon of Bacillus subtilis. Mol Gen Genet. 1987 Apr;207(1):114–119. doi: 10.1007/BF00331498. [DOI] [PubMed] [Google Scholar]
- Wong S. L. Development of an inducible and enhancible expression and secretion system in Bacillus subtilis. Gene. 1989 Nov 30;83(2):215–223. doi: 10.1016/0378-1119(89)90107-8. [DOI] [PubMed] [Google Scholar]
- Wong S. L., Price C. W., Goldfarb D. S., Doi R. H. The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1184–1188. doi: 10.1073/pnas.81.4.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Wu X. C., Nathoo S., Pang A. S., Carne T., Wong S. L. Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis. J Biol Chem. 1990 Apr 25;265(12):6845–6850. [PubMed] [Google Scholar]
- Yang M. Y., Ferrari E., Henner D. J. Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. J Bacteriol. 1984 Oct;160(1):15–21. doi: 10.1128/jb.160.1.15-21.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]