Neutralophilic bacteria, especially those affecting human health, possess mechanisms that enhance growth in moderate acid (pH 4–6) and enable survival in extrem

Inducible bacterial amino acid decarboxylases are expressed at the end of active cell division to counteract acidification of the extracellular environment during fermentative growth. It has been proposed that acid resistance in some enteric bacteria strictly relies on a glutamic acid‐dependent system. The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB. The gadC gene, located downstream of gadB, has been proposed to encode a putative antiporter implicated in the export of γ‐aminobutyrate, the glutamic acid decarboxylation product. In the present work, we provide in vivo evidence that gadC is co‐transcribed with gadB and that the functional glutamic acid‐dependent system requires the activities of both GadA/B and GadC. We also found that expression of gad genes is positively regulated by acidic shock, salt stress and stationary growth phase. Mutations in hns, the gene for the histone‐like protein H‐NS, cause derepressed expression of the gad genes, whereas the rpoS mutation abrogates gad transcription even in the hns background. According to our results, the master regulators H‐NS and RpoS are hierarchically involved in the transcriptional control of gad expression: H‐NS prevents gad expression during the exponential growth whereas the alternative sigma factor RpoS relieves H‐NS repression during the stationary phase, directly or indirectly accounting for transcription of gad genes.

TheEscherichia colichromosome contains two distantly located genes,gadAandgadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H⁺and producing γ-aminobutyric acid. This compound is exported via the protein product of thegadCgene, which is cotranscribed withgadB. Here we demonstrate that transcription of bothgadAandgadBCis positively controlled bygadX, a gene downstream ofgadA, encoding a transcriptional regulator belonging to the AraC/XylS family. ThegadXpromoter encompasses the 67-bp region preceding thegadXtranscription start site and contains both RpoD and RpoS putative recognition sites. Transcription ofgadXoccurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of thegadstructural genes. However, P_T5lacO-controlledgadXexpression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when thegadsystem is normally repressed. Autoregulation of the wholegadsystem is inferred by the positive effect of GadX on thegadApromoter andgadAXcotranscription. Transcription ofgadXis derepressed in anhnsmutant and strongly reduced in bothrpoSandhns rpoSmutants, consistent with the expression profile ofgadstructural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX bindsgadAandgadBCpromoters at different sites and with different binding affinities.

Summary: Escherichia 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/XylS‐like 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 post‐transcriptional 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 5′ 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 5′‐WANDNCTDWTWKTRAYATWAWMATG KCTGATNTTTWYNTYAK‐3′, is also present in the regulatory region of the slp‐yhiF, hdeAB and gadE‐mtdEF 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/γ-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.