Identification of DNA sequences involved in regulating Bacillus subtilis glnRA expression by the nitrogen source

J Mol Biol. 1991 Jul 20;220(2):241-53. doi: 10.1016/0022-2836(91)90010-4.


The DNA binding protein, GlnR, encoded by glnR, is believed to be directly responsible for regulating glnRA expression in Bacillus subtilis. Identification of cis-acting loci involved in glnRA control is the focus of this study. Analysis of glnRA-lacZ transcriptional fusions harboring deletions extending into the promoter region demonstrated that sequences upstream from position -35, relative to the transcription start-point, were necessary for nitrogen source regulation. These sequences included a 21 base-pair (bp) element, from positions -40 to -60, having 2-fold symmetry; the element shares homology to certain binding sites utilized by proteins having the alpha-helix-turn-alpha-helix motif, of which GlnR is a member. Involvement of this element in regulation was examined by using synthetic DNA fragments containing the promoter and upstream sequences driving lacZ expression. Fragments extending from positions -63 to -8 and from positions -52 to -8 yielded full and partial regulation, respectively. Regulation from a fragment containing a 5 bp insertion between positions -36 and -37 was impaired. A T.A to A.T transversion mutation at position -41 did not have any detectable effect on regulation, whereas a T.A to C.G transition mutation at the same site resulted in constitutive expression. Using a gel electrophoresis mobility shift assay, it was found that purified GlnR bound to a glnRA restriction fragment that extended from positions -104 to +83; binding was abolished after digestion with HinfI, which cleaves between positions -52 and -48. Furthermore, HinfI digestion was inhibited by the presence of GlnR. Thus, the GlnR binding site extends from the vicinity of position -35 upstream to position -63. We suggest that the glnRA operator is the 21 bp sequence lying within this region.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacillus subtilis / genetics*
  • Bacillus subtilis / metabolism
  • Bacterial Proteins / genetics*
  • Base Sequence
  • Chromosome Deletion
  • DNA-Binding Proteins / metabolism
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial*
  • Genotype
  • Glutamate-Ammonia Ligase / genetics*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nitrogen / metabolism
  • Oligonucleotide Probes
  • Plasmids
  • Promoter Regions, Genetic
  • Protein Conformation
  • Restriction Mapping
  • Sequence Homology, Nucleic Acid
  • Transcription, Genetic


  • Bacterial Proteins
  • DNA-Binding Proteins
  • Oligonucleotide Probes
  • Glutamate-Ammonia Ligase
  • Nitrogen