Angela Tramonti

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.

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.

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.

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.

Allosteric Communication of Tryptophan Synthase FUNCTIONAL AND REGULATORY PROPERTIES OF THE βS178P MUTANT

The α2β2 tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the βS178P mutant. Ser-178 is located at the end of helix 6 of the β subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of βSer-178 is hydrogen bonded to Gly-181 of loop 6 of the α subunit only when α subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the βS178P mutation suggests that the hydrogen bond involving αGly-181 is disrupted as a result of localized structural perturbations. The ratio of α to β subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight α−β complex. Both the activity of the α subunit and the inhibitory effect of the α subunit ligands indole-3-acetylglycine andd,l-α-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the β subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine andd,l-α-glycerol-3-phosphate do not affect the β activity. Accordingly, the distribution of l-serine intermediates at the β-site, dominated by the α-aminoacrylate, is only slightly influenced by α subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the α-aminoacrylate tautomer absorbing at 450 nm. Crystals of the βS178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the α and β activity in the α2β2 complex with respect to the isolated subunits results from interactions that involve residues different from βSer-178 and (ii) βSer-178 is a critical residue in ligand-triggered signals between α and β active sites.

Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii

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.

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

ABSTRACT In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.

Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.

The critical structural role of a highly conserved histidine residue in group II amino acid decarboxylases

Glutamate decarboxylase is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme, belonging to the subset of PLP‐dependent decarboxylases classified as group II. Site‐directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding. This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases. A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241.