Cross-Regulation of the Bacillus subtilis glnRA and tnrA Genes Provides Evidence for DNA Binding Site Discrimination by GlnR and TnrA

Abstract

Two Bacillus subtilis transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have the same consensus sequence. Expression of the tnrA gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/ TnrA site which lies immediately upstream of the ؊35 region of the tnrA promoter is required for regulation of tnrA expression by both GlnR and TnrA. Expression of the glnRA operon, which contains two GlnR/TnrA binding sites (glnRAo1 and glnRAo2) in its promoter region, is repressed by both GlnR and TnrA. The glnRAo2 site, which overlaps the ؊35 region of the glnRA promoter, was shown to be required for regulation by both GlnR and TnrA, while the glnRAo1 site which lies upstream of the ؊35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding to tnrA and glnRA promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of 55 nM to the GlnR/TnrA site in the tnrA promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the glnRA promoter region were greater than 3 M. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR–DNA complexes

“…GlnR binds cooperatively to the two operators located in the glnRA promoter ( Fig. 2A) (21,22). A faint intermediately shifted band is observed in the EMSA experiments and is most readily apparent when FBI-GS is present (Fig.…”

“…Although GlnR is the major regulator of glnA expression, the TnrA regulon is comprised primarily of genes required for the utilization of alternative nitrogen sources (3,22). Previous studies have shown that as bacterial growth becomes progressively more nitrogenrestricted, GS expression is derepressed before the pathways for the catabolism of nitrogen-containing compounds are expressed at high levels (8,34,35).…”

“…2 A) (22). To confirm that the GlnR activated by FBI-GS is binding to the glnRA operator sites in vitro, glnRA DNA fragments containing mutations in each operator were analyzed with the EMSA (Fig.…”

“…The glnRA promoter DNA fragments used for the EMSAs have been described (22). The reaction buffer used for initial incubation, serial dilutions, and DNA-binding reactions contained 10 mM Tris (pH 8.0), 150 mM sodium glutamate, 20 mM glutamine, 5 mM MgCl2, 1 mM DTT, 8% (vol/vol) glycerol, 0.05% N-octyl glucoside, 100 g/ml ovalbumin, 10% Ficoll PM400, and 20 M GS protomer.…”

At the Crossroads of Bacterial Metabolism and Virulence Factor Synthesis in Staphylococci

“…GlnR. GlnR is responsive to the concentration of glutamate, glutamine, and ammonium in the growth medium (239). The carbon skeleton of glutamate and glutamine is the TCA cycle intermediate ␣-ketoglutarate.…”