Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Oct;177(19):5686–5695. doi: 10.1128/jb.177.19.5686-5695.1995

Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression.

B R Belitsky 1, P J Janssen 1, A L Sonenshein 1
PMCID: PMC177381  PMID: 7559359

Abstract

The Bacillus subtilis gltAB genes, coding for the two subunits of glutamate synthase, are transcribed divergently from the gltC gene, encoding a LysR-type transcriptional activator of gltAB. The predicted gltA and gltC transcription start sites are separated by 51 to 52 bp. A 15-bp, consensus binding site (Box I) for LysR-type proteins was found centered at position -64 with respect to the gltA transcription start. This site was shown by mutational analysis to be required both for GltC-mediated activation of gltA and for autorepression of gltC. Box II, which is similar to Box I, is centered 22 bp downstream of Box I and overlaps the -35 region of the gltA promoter. Box II was found to be essential for activation of gltA but not for gltC autoregulation. Introduction of approximately one additional helical turn of DNA between Box I and Box II enhanced gltA expression 7- to 40-fold under nonactivating conditions and about 2-fold under activating conditions. Expression of gltA was dramatically decreased when the distance between Box I and Box II was altered by a nonintegral number of helical turns of DNA. gltC autorepression was abolished by most of the inserts between Box I and Box II but was augmented by adding one helical turn.

Full Text

The Full Text of this article is available as a PDF (405.1 KB).

Selected References

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

  1. Bartowsky E., Normark S. Interactions of wild-type and mutant AmpR of Citrobacter freundii with target DNA. Mol Microbiol. 1993 Nov;10(3):555–565. doi: 10.1111/j.1365-2958.1993.tb00927.x. [DOI] [PubMed] [Google Scholar]
  2. Belitsky B. R., Sonenshein A. L. Mutations in GltC that increase Bacillus subtilis gltA expression. J Bacteriol. 1995 Oct;177(19):5696–5700. doi: 10.1128/jb.177.19.5696-5700.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bender R. A., Snyder P. M., Bueno R., Quinto M., Magasanik B. Nitrogen regulation system of Klebsiella aerogenes: the nac gene. J Bacteriol. 1983 Oct;156(1):444–446. doi: 10.1128/jb.156.1.444-446.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohannon D. E., Rosenkrantz M. S., Sonenshein A. L. Regulation of Bacillus subtilis glutamate synthase genes by the nitrogen source. J Bacteriol. 1985 Sep;163(3):957–964. doi: 10.1128/jb.163.3.957-964.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bohannon D. E., Sonenshein A. L. Positive regulation of glutamate biosynthesis in Bacillus subtilis. J Bacteriol. 1989 Sep;171(9):4718–4727. doi: 10.1128/jb.171.9.4718-4727.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Byerly K. A., Urbanowski M. L., Stauffer G. V. The metR binding site in the Salmonella typhimurium metH gene: DNA sequence constraints on activation. J Bacteriol. 1991 Jun;173(11):3547–3553. doi: 10.1128/jb.173.11.3547-3553.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Ernsting B. R., Denninger J. W., Blumenthal R. M., Matthews R. G. Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein? J Bacteriol. 1993 Nov;175(22):7160–7169. doi: 10.1128/jb.175.22.7160-7169.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fisher R. F., Long S. R. Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA. J Mol Biol. 1993 Oct 5;233(3):336–348. doi: 10.1006/jmbi.1993.1515. [DOI] [PubMed] [Google Scholar]
  12. Fouet A., Sonenshein A. L. A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis. J Bacteriol. 1990 Feb;172(2):835–844. doi: 10.1128/jb.172.2.835-844.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gao J., Gussin G. N. Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa. EMBO J. 1991 Dec;10(13):4137–4144. doi: 10.1002/j.1460-2075.1991.tb04991.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Glaser P., Kunst F., Arnaud M., Coudart M. P., Gonzales W., Hullo M. F., Ionescu M., Lubochinsky B., Marcelino L., Moszer I. Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees. Mol Microbiol. 1993 Oct;10(2):371–384. [PubMed] [Google Scholar]
  15. Goethals K., Van Montagu M., Holsters M. Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1646–1650. doi: 10.1073/pnas.89.5.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henikoff S., Haughn G. W., Calvo J. M., Wallace J. C. A large family of bacterial activator proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6602–6606. doi: 10.1073/pnas.85.18.6602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Itaya M., Kondo K., Tanaka T. A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. Nucleic Acids Res. 1989 Jun 12;17(11):4410–4410. doi: 10.1093/nar/17.11.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jefferson R. A., Burgess S. M., Hirsh D. beta-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8447–8451. doi: 10.1073/pnas.83.22.8447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jin S., Sonenshein A. L. Transcriptional regulation of Bacillus subtilis citrate synthase genes. J Bacteriol. 1994 Aug;176(15):4680–4690. doi: 10.1128/jb.176.15.4680-4690.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karow M. L., Glaser P., Piggot P. J. Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2012–2016. doi: 10.1073/pnas.92.6.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  22. LeDeaux J. R., Grossman A. D. Isolation and characterization of kinC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinases KinA and KinB in Bacillus subtilis. J Bacteriol. 1995 Jan;177(1):166–175. doi: 10.1128/jb.177.1.166-175.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ogasawara N., Nakai S., Yoshikawa H. Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res. 1994;1(1):1–14. doi: 10.1093/dnares/1.1.1. [DOI] [PubMed] [Google Scholar]
  24. Oliver G., Gosset G., Sanchez-Pescador R., Lozoya E., Ku L. M., Flores N., Becerril B., Valle F., Bolivar F. Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12. Gene. 1987;60(1):1–11. doi: 10.1016/0378-1119(87)90207-1. [DOI] [PubMed] [Google Scholar]
  25. Parsek M. R., Ye R. W., Pun P., Chakrabarty A. M. Critical nucleotides in the interaction of a LysR-type regulator with its target promoter region. catBC promoter activation by CatR. J Biol Chem. 1994 Apr 15;269(15):11279–11284. [PubMed] [Google Scholar]
  26. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [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. Schell M. A. Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol. 1993;47:597–626. doi: 10.1146/annurev.mi.47.100193.003121. [DOI] [PubMed] [Google Scholar]
  29. Schell M. A., Poser E. F. Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. J Bacteriol. 1989 Feb;171(2):837–846. doi: 10.1128/jb.171.2.837-846.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schneider K., Beck C. F. Promoter-probe vectors for the analysis of divergently arranged promoters. Gene. 1986;42(1):37–48. doi: 10.1016/0378-1119(86)90148-4. [DOI] [PubMed] [Google Scholar]
  31. Schwacha A., Bender R. A. The nac (nitrogen assimilation control) gene from Klebsiella aerogenes. J Bacteriol. 1993 Apr;175(7):2107–2115. doi: 10.1128/jb.175.7.2107-2115.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Stanssens P., Opsomer C., McKeown Y. M., Kramer W., Zabeau M., Fritz H. J. Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 1989 Jun 26;17(12):4441–4454. doi: 10.1093/nar/17.12.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Toledano M. B., Kullik I., Trinh F., Baird P. T., Schneider T. D., Storz G. Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell. 1994 Sep 9;78(5):897–909. doi: 10.1016/s0092-8674(94)90702-1. [DOI] [PubMed] [Google Scholar]
  35. Urbanowski M. L., Stauffer G. V. Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium. J Bacteriol. 1989 Oct;171(10):5620–5629. doi: 10.1128/jb.171.10.5620-5629.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang L., Helmann J. D., Winans S. C. The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite. Cell. 1992 May 15;69(4):659–667. doi: 10.1016/0092-8674(92)90229-6. [DOI] [PubMed] [Google Scholar]
  37. Wang S. P., Stacey G. Studies of the Bradyrhizobium japonicum nodD1 promoter: a repeated structure for the nod box. J Bacteriol. 1991 Jun;173(11):3356–3365. doi: 10.1128/jb.173.11.3356-3365.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wu W. F., Urbanowski M. L., Stauffer G. V. Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium. J Bacteriol. 1995 Apr;177(7):1834–1839. doi: 10.1128/jb.177.7.1834-1839.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Zhou C., Abaigar L., Jong A. Y. A protocol for using T7 DNA polymerase in oligonucleotide site-directed mutagenesis. Biotechniques. 1990 May;8(5):503–503. [PubMed] [Google Scholar]

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

RESOURCES