Mutational analysis of the TnrA-binding sites in the Bacillus subtilis nrgAB and gabP promoter regions.
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.
Wray
L V
LV
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Zalieckas
J M
JM
Ferson
A E
AE
Fisher
S H
SH
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D003470:Culture Media; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics*; D027361:Organic Anion Transporters; D051922:PII Nitrogen Regulatory Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012097:Repressor Proteins; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C085245:NrgA protein, Bacillus subtilis; C085246:NrgB protein, Bacillus subtilis; D027361:Organic Anion Transporters; D051922:PII Nitrogen Regulatory Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C101458:ScgR protein, Bacillus subtilis; D014157:Transcription Factors; C513601:gabP protein, E coli