The Escherichia coli chromosome contains two distantly located genes, gadA and gadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H ؉ and producing ␥-aminobutyric acid. This compound is exported via the protein product of the gadC gene, which is cotranscribed with gadB. Here we demonstrate that transcription of both gadA and gadBC is positively controlled by gadX, a gene downstream of gadA, encoding a transcriptional regulator belonging to the AraC/XylS family. The gadX promoter encompasses the 67-bp region preceding the gadX transcription start site and contains both RpoD and RpoS putative recognition sites. Transcription of gadX occurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of the gad structural genes. However, P T5 lacO-controlled gadX expression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when the gad system is normally repressed. Autoregulation of the whole gad system is inferred by the positive effect of GadX on the gadA promoter and gadAX cotranscription. Transcription of gadX is derepressed in an hns mutant and strongly reduced in both rpoS and hns rpoS mutants, consistent with the expression profile of gad structural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX binds gadA and gadBC promoters at different sites and with different binding affinities.

SummaryEscherichia coli has the remarkable ability to resist severe acid stress for several hours. With the notable exception of the gadBC operon, the most important genes involved in acid resistance are present within the acid fitness island (AFI), a 15 kb H-NS-repressed and RpoS-controlled genome region. The AraC/XylSlike transcriptional regulators GadX and GadW are also encoded within this region. In this article, we show that gadW transcription occurs from two native promoters, which are affected by the transcription of the divergently transcribed and GadX-dependent gadY small RNA, and from the gadX promoter. The gadXW dicistronic transcript is subjected to posttranscriptional processing in which GadY is involved. In contrast, gadW transcription negatively affects gadY transcription. By aligning the GadX/GadW binding site on the gadY promoter with the GadX/ GadW binding sites previously identified in the gadA and gadBC 5Ј regulatory regions, we generated a 42 bp GadX/GadW consensus sequence. DNase I footprinting analyses confirmed that a 42 bp GadX/ GadW binding site, which matched the consensus sequence 5Ј-WANDNCTDWTWKTRAYATWAWMATG KCTGATNTTTWYNTYAK-3Ј, is also present in the regulatory region of the slp-yhiF, hdeAB and gadEmtdEF operons, all of which belong to the AFI. The presence of five GadX/GadW-specific binding sites in the AFI suggests that GadX and GadW may act as H-NS counter-silencers.

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/␥-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.