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

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.