As part of their life cycle, neutralophilic bacteria are often exposed to varying environmental stresses, among which fluctuations in pH are the most frequent. In particular, acid environments can be encountered in many situations from fermented food to the gastric compartment of the animal host. Herein, we review the current knowledge of the molecular mechanisms adopted by a range of Gram-positive and Gram-negative bacteria, mostly those affecting human health, for coping with acid stress. Because organic and inorganic acids have deleterious effects on the activity of the biological macromolecules to the point of significantly reducing growth and even threatening their viability, it is not unexpected that neutralophilic bacteria have evolved a number of different protective mechanisms, which provide them with an advantage in otherwise life-threatening conditions. The overall logic of these is to protect the cell from the deleterious effects of a harmful level of protons. Among the most favoured mechanisms are the pumping out of protons, production of ammonia and proton-consuming decarboxylation reactions, as well as modifications of the lipid content in the membrane. Several examples are provided to describe mechanisms adopted to sense the external acidic pH. Particular attention is paid to Escherichia coli extreme acid resistance mechanisms, the activity of which ensure survival and may be directly linked to virulence.

SummaryInducible 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 ␥-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.

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.

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Pyridoxal 5′‐phosphate (PLP), the well‐known active form of vitamin B6, is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N‐terminal helix‐turn‐helix motif that binds DNA and an effector‐binding C‐terminal domain homologous to PLP‐dependent enzymes. Although it is known that PLP acts as an anti‐activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA‐binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B6 vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA‐binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.

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.

One of the most efficient systems of acid resistance in Escherichia coli, the gad system, is based on the coordinated action of two isoforms of glutamate decarboxylase (GadA and GadB) and of a specific glutamate/␥-aminobutyrate antiporter (GadC). The gadA/BC genes, activated in response to acid stress and in stationary phase cells, are subjected to complex circuits of regulation involving 70 , S , cAMP receptor protein, H-NS, EvgAS, TorRS, GadE, GadX, GadW, and YdeO. Herein, we provide evidence that the nucleoid-associated protein H-NS directly functions as repressor of gadA, one of the structural genes, and gadX, a regulatory gene encoding one of the primary activators of the gad system. Band shift and DNase I footprints reveal that H-NS indeed binds to specific sites in the promoter regions of gadA and gadX and represses the transcription of these genes both in an in vitro system and in vivo. Moreover, we show that a maltose-binding protein MalE-GadX fusion is able to stimulate the promoter activity of gadA/BC, thus indicating that GadX is by itself able to up-regulate the gad genes and that a functional competition between H-NS and GadX takes place at the gadA promoter. Altogether, our results indicate that H-NS directly inhibits gadA and gadX transcription and, by controlling the intracellular level of the activator GadX, indirectly affects the expression of the whole gad system.