Mechanisms of Transcription Activation Exerted by GadX and GadW at the gadA and gadBC Gene Promoters of the Glutamate-Based Acid Resistance System in Escherichia coli

Abstract

In Escherichia coli the gad system protects the cell from the extreme acid stress encountered during transit through the host stomach. The structural genes gadA, gadB, and gadC encode two glutamate decarboxylase isoforms and a glutamate/gamma-aminobutyrate (GABA) antiporter, respectively. Glutamate decarboxylation involves both proton consumption and production of GABA, a neutral compound which is finally exported via the GadC antiporter. Regulation of gadA and gadBC transcription is very complex, involving several circuits controlling expression under different growth phase, medium, and pH conditions. In this study we found that the AraC-like activators GadX and GadW share the same 44-bp binding sites in the gadA and gadBC regulatory regions. The common binding sites are centered at 110.5 bp and 220.5 bp upstream of the transcriptional start points of the gadA and gadBC genes, respectively. At the gadA promoter this regulatory element overlaps one of the binding sites of the repressor H-NS. The DNA of the gadBC promoter has an intrinsic bend which is centered at position -121. These findings, combined with transcriptional regulation studies, may account for the two different mechanisms of transcriptional activation by GadX and GadW at the two promoters studied. We speculate that while at the gadA promoter GadX and GadW activate transcription by displacing H-NS via an antirepressor mechanism, at the gadBC promoter the mechanism of activation involves looping of the DNA sequence between the promoter and the activator binding site.