Daniela De Biase

The response to stationary-phase stress conditions in Escherichia coli : role and regulation of the glutamic acid decarboxylase system

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.

Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase

Glutamate decarboxylase is a vitamin B6‐dependent enzyme, which catalyses the decarboxylation of glutamate to γ‐aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH‐dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure‐based site‐directed mutagenesis that the triple helix bundle formed by the N‐termini of the protein at acidic pH is the major determinant for this behaviour.

Functional Characterization and Regulation of gadX, a Gene Encoding an AraC/XylS-Like Transcriptional Activator of the Escherichia coli Glutamic Acid Decarboxylase System

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 gamma-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.

Structures of {Gamma}-Aminobutyric Acid (Gaba) Aminotransferase, a Pyridoxal 5'-Phosphate, and [2Fe-2S] Cluster-Containing Enzyme, Complexed with {Gamma}-Ethynyl-Gaba and with the Antiepilepsy Drug Vigabatrin

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, γ-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-Å resolution) and in complex with vigabatrin as well as with the close analogue γ-ethynyl-GABA (to 2.3 and 2.8 Å, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5′-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.

GadX/GadW‐dependent regulation of the Escherichia coli acid fitness island: transcriptional control at the gadY–gadW divergent promoters and identification of four novel 42 bp GadX/GadW‐specific binding sites

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.

Structures of GABA aminotransferase, a pyridoxal 5'-phosphate and [2Fe-2S] cluster containing enzyme, complexed with -EthynylGABA and with the antiepilepsy drug vigabatrin

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, γ-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-Å resolution) and in complex with vigabatrin as well as with the close analogue γ-ethynyl-GABA (to 2.3 and 2.8 Å, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5′-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.

Coping with low pH: molecular strategies in neutralophilic bacteria

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.

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

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.

Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon

For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH < 2.5) encountered during transit through the host stomach. The glutamate‐dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP‐dependent enzyme which performs a proton‐consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamatein/γ‐aminobutyrateout (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH‐dependent intracellular activation of E. coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non‐pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co‐regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA‐enriched foods possess health‐promoting properties.

Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB

Escherichia coli and other enterobacteria exploit the H+‐consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10‐fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N‐terminal 14 residues, involved in the formation of two triple‐helix bundles, were deleted by mutagenesis. X‐ray structures, obtained in the presence of the substrate analogue acetate, identified halide‐binding sites at the base of each N‐terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5′‐phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl− exchange activities of ClC‐ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.