Abstract
We describe a general, in vivo method for identifying Bacillus subtilis genes controlled by specific, nonessential regulatory factors. We establish the use of this approach by identifying, isolating, and characterizing a gene dependent on sigma B, an alternate transcription factor which is found early in stationary phase but which is not essential for sporulation. The method relies on two features: (i) a plate transformation technique to introduce a null mutation into the regulatory gene of interest and (ii) random transcriptional fusions to a reporter gene to monitor gene expression in the presence and absence of a functional regulatory product. We applied this genetic approach to isolate genes comprising the sigma B regulon. We screened a random Tn917lacZ library for fusions that required an intact sigma B structural gene (sigB) for greatest expression, converting the library strains from wild-type sigB+ to sigB delta::cat directly on plates selective for chloramphenicol resistance. We isolated one such fusion, csbA::Tn917lacZ (csb for controlled by sigma B), which mapped between hisA and degSU on the B. subtilis chromosome. We cloned the region surrounding the insertion, identified the csbA reading frame containing the transposon, and found that this frame encoded a predicted 76-residue product which was extremely hydrophobic and highly basic. Primer extension and promoter activity experiments identified a sigma B-dependent promoter 83 bp upstream of the csbA coding sequence. A weaker, tandem, sigma A-like promoter was likewise identified 28 bp upstream of csbA. The csbA fusion was maximally expressed during early stationary phase in cells grown in Luria broth containing 5% glucose and 0.2% glutamine. This timing of expression and medium dependence were very similar to those for ctc, the only other recognized gene dependent on sigma B.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Atkinson M. R., Fisher S. H. Identification of genes and gene products whose expression is activated during nitrogen-limited growth in Bacillus subtilis. J Bacteriol. 1991 Jan;173(1):23–27. doi: 10.1128/jb.173.1.23-27.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Binnie C., Lampe M., Losick R. Gene encoding the sigma 37 species of RNA polymerase sigma factor from Bacillus subtilis. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5943–5947. doi: 10.1073/pnas.83.16.5943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boylan S. A., Suh J. W., Thomas S. M., Price C. W. Gene encoding the alpha core subunit of Bacillus subtilis RNA polymerase is cotranscribed with the genes for initiation factor 1 and ribosomal proteins B, S13, S11, and L17. J Bacteriol. 1989 May;171(5):2553–2562. doi: 10.1128/jb.171.5.2553-2562.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheo D. L., Bayles K. W., Yasbin R. E. Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis. J Bacteriol. 1991 Mar;173(5):1696–1703. doi: 10.1128/jb.173.5.1696-1703.1991. [DOI] [PMC free article] [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]
- Dubnau D., Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol. 1971 Mar 14;56(2):209–221. doi: 10.1016/0022-2836(71)90460-8. [DOI] [PubMed] [Google Scholar]
- Dubnau E., Weir J., Nair G., Carter L., 3rd, Moran C., Jr, Smith I. Bacillus sporulation gene spo0H codes for sigma 30 (sigma H). J Bacteriol. 1988 Mar;170(3):1054–1062. doi: 10.1128/jb.170.3.1054-1062.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duncan M. L., Kalman S. S., Thomas S. M., Price C. W. Gene encoding the 37,000-dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus, and cryptic function. J Bacteriol. 1987 Feb;169(2):771–778. doi: 10.1128/jb.169.2.771-778.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ferrari F. A., Trach K., Hoch J. A. Sequence analysis of the spo0B locus reveals a polycistronic transcription unit. J Bacteriol. 1985 Feb;161(2):556–562. doi: 10.1128/jb.161.2.556-562.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibson M. M., Ellis E. M., Graeme-Cook K. A., Higgins C. F. OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium. Mol Gen Genet. 1987 Apr;207(1):120–129. doi: 10.1007/BF00331499. [DOI] [PubMed] [Google Scholar]
- Hahn J., Albano M., Dubnau D. Isolation and characterization of Tn917lac-generated competence mutants of Bacillus subtilis. J Bacteriol. 1987 Jul;169(7):3104–3109. doi: 10.1128/jb.169.7.3104-3109.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heijne G. The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J. 1986 Nov;5(11):3021–3027. doi: 10.1002/j.1460-2075.1986.tb04601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
- Helmann J. D., Márquez L. M., Chamberlin M. J. Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene. J Bacteriol. 1988 Apr;170(4):1568–1574. doi: 10.1128/jb.170.4.1568-1574.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Igo M. M., Losick R. Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis. J Mol Biol. 1986 Oct 20;191(4):615–624. doi: 10.1016/0022-2836(86)90449-3. [DOI] [PubMed] [Google Scholar]
- Igo M., Lampe M., Ray C., Schafer W., Moran C. P., Jr, Losick R. Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis. J Bacteriol. 1987 Aug;169(8):3464–3469. doi: 10.1128/jb.169.8.3464-3469.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jaacks K. J., Healy J., Losick R., Grossman A. D. Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis. J Bacteriol. 1989 Aug;171(8):4121–4129. doi: 10.1128/jb.171.8.4121-4129.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kalman S., Duncan M. L., Thomas S. M., Price C. W. Similar organization of the sigB and spoIIA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase. J Bacteriol. 1990 Oct;172(10):5575–5585. doi: 10.1128/jb.172.10.5575-5585.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kleckner N., Roth J., Botstein D. Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics. J Mol Biol. 1977 Oct 15;116(1):125–159. doi: 10.1016/0022-2836(77)90123-1. [DOI] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- Leighton T. New types of RNA polymerase mutations causing temperature-sensitive sporulation in bacillus subtilis. J Biol Chem. 1977 Jan 10;252(1):268–272. [PubMed] [Google Scholar]
- Lemaux P. G., Herendeen S. L., Bloch P. L., Neidhardt F. C. Transient rates of synthesis of individual polypeptides in E. coli following temperature shifts. Cell. 1978 Mar;13(3):427–434. doi: 10.1016/0092-8674(78)90317-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Love P. E., Lyle M. J., Yasbin R. E. DNA-damage-inducible (din) loci are transcriptionally activated in competent Bacillus subtilis. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6201–6205. doi: 10.1073/pnas.82.18.6201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moran C. P., Jr, Johnson W. C., Losick R. Close contacts between sigma 37-RNA polymerase and a Bacillus subtilis chromosomal promoter. J Mol Biol. 1982 Dec 15;162(3):709–713. doi: 10.1016/0022-2836(82)90399-0. [DOI] [PubMed] [Google Scholar]
- Moran C. P., Jr, Lang N., Losick R. Nucleotide sequence of a Bacillus subtilis promoter recognized by Bacillus subtilis RNA polymerase containing sigma 37. Nucleic Acids Res. 1981 Nov 25;9(22):5979–5990. doi: 10.1093/nar/9.22.5979. [DOI] [PMC free article] [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]
- O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
- Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
- Piggot P. J., Curtis C. A., de Lencastre H. Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis. J Gen Microbiol. 1984 Aug;130(8):2123–2136. doi: 10.1099/00221287-130-8-2123. [DOI] [PubMed] [Google Scholar]
- Price C. W., Doi R. H. Genetic mapping of rpoD implicates the major sigma factor of Bacillus subtilis RNA polymerase in sporulation initiation. Mol Gen Genet. 1985;201(1):88–95. doi: 10.1007/BF00397991. [DOI] [PubMed] [Google Scholar]
- Ray C., Hay R. E., Carter H. L., Moran C. P., Jr Mutations that affect utilization of a promoter in stationary-phase Bacillus subtilis. J Bacteriol. 1985 Aug;163(2):610–614. doi: 10.1128/jb.163.2.610-614.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
- Sancar A., Rupp W. D. A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region. Cell. 1983 May;33(1):249–260. doi: 10.1016/0092-8674(83)90354-9. [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]
- Schmidt R., Margolis P., Duncan L., Coppolecchia R., Moran C. P., Jr, Losick R. Control of developmental transcription factor sigma F by sporulation regulatory proteins SpoIIAA and SpoIIAB in Bacillus subtilis. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9221–9225. doi: 10.1073/pnas.87.23.9221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stragier P., Losick R. Cascades of sigma factors revisited. Mol Microbiol. 1990 Nov;4(11):1801–1806. doi: 10.1111/j.1365-2958.1990.tb02028.x. [DOI] [PubMed] [Google Scholar]
- Tatti K. M., Moran C. P., Jr Promoter recognition by sigma-37 RNA polymerase from Bacillus subtilis. J Mol Biol. 1984 May 25;175(3):285–297. doi: 10.1016/0022-2836(84)90349-8. [DOI] [PubMed] [Google Scholar]
- Wanner B. L., McSharry R. Phosphate-controlled gene expression in Escherichia coli K12 using Mudl-directed lacZ fusions. J Mol Biol. 1982 Jul 5;158(3):347–363. doi: 10.1016/0022-2836(82)90202-9. [DOI] [PubMed] [Google Scholar]
- Weir J., Dubnau E., Ramakrishna N., Smith I. Bacillus subtilis spo0H gene. J Bacteriol. 1984 Feb;157(2):405–412. doi: 10.1128/jb.157.2.405-412.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamori T., Ito K., Nakamura Y., Yura T. Transient regulation of protein synthesis in Escherichia coli upon shift-up of growth temperature. J Bacteriol. 1978 Jun;134(3):1133–1140. doi: 10.1128/jb.134.3.1133-1140.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]