Mutational analysis of the TnrA-binding sites in the Bacillus subtilis nrgAB and gabP promoter regions

Abstract

Transcription of the Bacillus subtilis nrgAB promoter is activated during nitrogen-limited growth by the TnrA protein. A common inverted repeat, TGTNAN7TNACA (TnrA site), is centered 49 to 51 bp upstream of the transcriptional start sites for the TnrA-regulated nrgAB, gabP P2, and nas promoters. Oligonucleotide-directed mutagenesis of the nrgAB promoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of nrgAB expression. Mutations that alter the relative distance between the two half-sites of the nrgAB TnrA site abolish nitrogen regulation of nrgAB expression. Spacer mutations that change the relative distance between the TnrA site and -35 region of the nrgAB promoter reveal that activation of nrgAB expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of the conserved nucleotides in the gabP P2 TnrA site showed that this sequence is also required for nitrogen-regulated gabP P2 expression. The TnrA protein, expressed in an overproducing Escherichia coli strain, had a 625-fold-higher affinity for the wild-type nrgAB promoter DNA than for a mutated nrgAB promoter DNA fragment that is unable to activate nrgAB expression in vivo. These results indicate that the proposed TnrA site functions as the binding site for the TnrA protein. TnrA was found to activate nrgAB expression during late exponential growth in nutrient sporulation medium containing glucose, suggesting that cells become nitrogen limited during growth in this medium.

FIG. 1

Alignment of nitrogen-regulated promoters. Nucleotides corresponding to the conserved upstream inverted repeat are boxed. The −35 region of the nrgAB promoter is overlined.

FIG. 2

Retardation analysis of wild-type and mutant nrgAB promoter regions by TnrA. A 164-bp nrgAB promoter DNA fragment containing the B. subtilis wild-type TnrA-binding site (lanes 1 to 6) and the mutant TnrA-binding site from NRG416-42G (lanes 7 to 12) was used in the binding reactions. Total amounts of protein from TnrA overexpression extracts present in the reaction mixtures: lanes 1 and 7, 0 μg; lanes 2 and 8, 8 ng; lanes 3 and 9, 40 ng; lanes 4 and 10, 200 ng; lanes 5 and 11, 1.0 μg; lanes 6 and 12, 5.0 μg.

FIG. 3

Growth and β-galactosidase expression from the (nrgA-lacZ)407 fusion in wild-type and tnrA cells during growth in either NSM or NSM containing 1% glucose. Samples were removed periodically, and β-galactosidase activity was assayed in cell extracts. Data from a typical experiment are shown. Triangles, SF407 [(nrgA-lacZ)407] cells grown in NSM plus glucose; squares, SF407T [(nrgA-lacZ)407 tnrA62] cells grown in NSM plus glucose; circles, SF407 [(nrgA-lacZ)407] cells grown in NSM. Open symbols, Klett units; closed symbols, β-galactosidase specific activity.