John W. Foster

Escherichia colihas an ability, rare among theEnterobacteriaceae, to survive extreme acid stress under various host (e.g., human stomach) and nonhost (e.g., apple cider) conditions. Previous microarray studies have exposed a cluster of 12 genes at 79 centisomes collectively called an acid fitness island (AFI). Four AFI genes,gadA,gadX,gadW, andgadE, were already known to be involved in an acid resistance system that consumes an intracellular proton through the decarboxylation of glutamic acid. However, roles for the other eight AFI gene products were either unknown or subject to conflicting findings. Two new aspects of acid resistance are described that require participation of five of the remaining eight AFI genes. YhiF (a putative regulatory protein), lipoprotein Slp, and the periplasmic chaperone HdeA protectedE. colifrom organic acid metabolites produced during fermentation once the external pH was reduced to pH 2.5. HdeA appears to handle protein damage caused when protonated organic acids diffuse into the cell and dissociate, thereby decreasing internal pH. In contrast, YhiF- and Slp-dependent systems appear to counter the effects of the organic acids themselves, specifically succinate, lactate, and formate, but not acetate. A second phenomenon was defined by two other AFI genes,yhiDandhdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>10⁸CFU per ml). Density-dependent acid resistance does not require any demonstrable secreted factor and may involve cell contact-dependent activation. These findings further define the complex physiology ofE. coliacid resistance.

Escherichia coliprefers growth in neutral pH environments but can withstand extremely acidic conditions (pH 2) for long periods. Of the fourE. colisystems that contribute to acid resistance, one, the glutamate-dependent system, is remarkable in its efficacy and regulatory complexity. The resistance mechanism involves the intracellular consumption of protons by the glutamate decarboxylase isozymes GadA and GadB. The antiporter GadC then exports the product, γ-aminobutyric acid, in exchange for fresh glutamate. A microarray study using overexpressed regulators uncoveredevgASandydeOas potential regulators ofgadE, now known to encode the essential activator of thegadAandgadBCgenes. Examination ofevgAandydeOunder normal expression conditions revealed that their products do activategadEexpression but only under specific conditions. They were important during exponential growth in acidified minimal medium containing glucose but were unnecessary forgadEexpression in stationary-phase cells grown in complex medium. The response regulator EvgA activatesgadEdirectly and indirectly via induction of the AraC-like regulatorydeO. Evidence obtained usinggadE-lacZoperon fusions also revealed that GadE was autoinduced. Electrophoretic mobility shift assays indicated that EvgA, YdeO, and GadE bind to different regions upstream ofgadE, indicating they all act directly at thegadEpromoter. Since GadE controls the expression of numerous genes besidesgadAandgadBC, the relevance of these regulatory circuits extends beyond acid resistance.

Gastrointestinal pathogens are faced with an extremely acidic environment. Within moments, a pathogen such as Escherichia coli O157:H7 can move from the nurturing pH 7 environment of a hamburger to the harsh pH 2 milieu of the stomach. Surprisingly, certain microorganisms that grow at neutral pH have elegantly regulated systems that enable survival during excursions into acidic environments. The best-characterized acid-resistance system is found in E. coli.

Summary: Escherichia coli survives pH 2 acid stress at a level rivalling Helicobacter pylori. Of the three E. coli acid resistance systems involved, the one most efficient and most studied uses isozymes of glutamate decarboxylase (GadA/GadB) to consume intracellular protons, and a glutamate:γ‐amino butyric acid (GABA) anti‐porter (GadC) to expel GABA in exchange for extracellular glutamate. Because acid resistance is a critical factor in resisting stomach acidity, mechanisms that control this system are extremely important. Here we show that an Era‐like, molecular switch GTPase called TrmE regulates glutamate‐dependent acid resistance. Western blot analysis revealed a TrmE‐dependent, glucose‐induced system and a TrmE‐independent, glucose‐repressed pathway. Gene fusion studies indicated that the TrmE requirement for GadA/B production takes place at both the transcriptional and translational levels. TrmE controls GAD transcription by affecting the expression of GadE, the essential activator of the gadA and gadBC genes. TrmE most probably controls gadE expression indirectly by influencing the synthesis or activity of an unknown regulator that binds the gadE control region. Translational control of GAD production by TrmE appears to be more direct, affecting synthesis of the decarboxylase and the anti‐porter proteins. TrmE GTPase activity was critical for both the transcriptional and translational effects. Thus, TrmE is part of an increasingly complex control network designed to integrate diverse physiological signals and forecast future exposures to extreme acid. The significance of this network extends beyond acid resistance as the target of this control, GadE, regulates numerous genes in addition to gadA/BC.

Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pH _i ) conducive to cell survival. The model was tested by estimating the pH _i and transmembrane potential (ΔΨ) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pH _i from 3.6 to 4.2 and 4.7, respectively. However, when pH _i was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pH _i was also important. Measurements of ΔΨ indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing ΔΨ may be a more important acid resistance strategy than maintaining a specific pH _i value.

Extreme acid resistance is a remarkable property of virulent and avirulent Escherichia coli. The ability to resist environments in which the pH is 2.5 and below is predicted to contribute significantly to the survival of E. coli during passage through the gastric acid barrier. One acid resistance system imports glutamate from acidic environments and uses it as a proton sink during an intracellular decarboxylation reaction. Transcription of the genes encoding the glutamate decarboxylases and the substrate-product antiporter required for this system is induced under a variety of conditions, including the stationary phase and a low pH. Acid induction during log-phase growth in minimal medium appears to occur through multiple pathways. We recently demonstrated that GadE, the essential activator of the genes, was itself acid induced. In this report we present evidence that there is a regulatory loop involving cross-repression of two AraC-like regulators, GadX and GadW, that can either assist or interfere with GadE activation of the gad decarboxylase and antiporter genes, depending on the culture conditions. Balancing cross-repression appears to be dependent on cAMP and the cAMP regulator protein (CRP). The control loop involves the GadX protein repressing the expression of gadW and the GadW protein repressing or inhibiting RpoS, which is the alternative sigma factor that drives transcription of gadX. CRP and cAMP appear to influence GadX-GadW cross-repression from outside the loop by inhibiting production of RpoS. We found that GadW represses the decarboxylase genes in minimal medium and that growth under acidic conditions lowers the intracellular cAMP levels. These results indicate that CRP and cAMP can mediate pH control over gadX expression and, indirectly, expression of the decarboxylase genes. Mutational or physiological lowering of cAMP levels increases the level of RpoS and thereby increases the production of GadX. Higher GadX levels, in turn, repress gadW and contribute to induction of the gad decarboxylase genes. The presence of multiple pH control pathways governing expression of this acid resistance system is thought to reflect different environmental routes to a low pH.

Summary: Commensal and pathogenic strains of Escherichia coli possess three inducible acid resistance systems that collaboratively protect cells against acid stress to pH 2 or below. The most effective system requires glutamate in the acid challenge media and relies on two glutamate decarboxylases (GadA and B) combined with a putative glutamate:γ‐aminobutyric acid antiporter (GadC). A complex network of regulators mediates induction of this system in response to various media, pH and growth phase signals. We report that the LuxR‐like regulator GadE (formerly YhiE) is required for expression of gadA and gadBC regardless of media or growth conditions. This protein binds directly to the 20 bp GAD box sequence found in the control regions of both loci. Two previously identified AraC‐like regulators, GadX and GadW, are only needed for gadA/BC expression under some circumstances. Overexpression of GadX or GadW will not overcome a need for GadE. However, overexpression of GadE can supplant a requirement for GadX and W. Data provided also indicate that GadX and GadE can simultaneously bind the area around the GAD box region and probably form a complex. The gadA, gadBC and gadE genes are all induced by low pH in exponential phase cells grown in minimal glucose media. The acid induction of gadA/BC results primarily from the acid induction of gadE. Constitutive expression of GadE removes most pH control over the glutamate decarboxylase and antiporter genes. The small amount of remaining pH control is governed by GadX and W. The finding that gadE mutations also diminish the effectiveness of the other two acid resistance systems suggests that GadE influences the expression of additional acid resistance components. The number of regulatory proteins (five), sigma factors (two) and regulatory feedback loops focused on gadA/BC expression make this one of the most intensively regulated systems in E. coli.

To survive in extremely acidic conditions, Escherichia coli has evolved three adaptive acid resistance strategies thought to maintain internal pH. While the mechanism behind acid resistance system 1 remains enigmatic, systems 2 and 3 are known to require external glutamate (system 2) and arginine (system 3) to function. These latter systems employ specific amino acid decarboxylases and putative antiporters that exchange the extracellular amino acid substrate for the intracellular by-product of decarboxylation. Although GadC is the predicted antiporter for system 2, the antiporter specific for arginine/agmatine exchange has not been identified. A computer-based homology search revealed that the yjdE (now called adiC ) gene product shared an overall amino acid identity of 22% with GadC. A series of adiC mutants isolated by random mutagenesis and by targeted deletion were shown to be defective in arginine-dependent acid resistance. This defect was restored upon introduction of an adiC ⁺ -containing plasmid. An adiC mutant proved incapable of exchanging extracellular arginine for intracellular agmatine but maintained wild-type levels of arginine decarboxylase protein and activity. Western blot analysis indicated AdiC is an integral membrane protein. These data indicate that the arginine-to-agmatine conversion defect of adiC mutants was at the level of transport. The adi gene region was shown to be organized into two transcriptional units, adiAY and adiC , which are coordinately regulated but independently transcribed. The data also illustrate that the AdiA decarboxylase:AdiC antiporter system is designed to function only at acid levels sufficient to harm the cell.

An important feature ofEscherichia colipathogenesis is an ability to withstand extremely acidic environments of pH 2 or lower. This acid resistance property contributes to the low infectious dose of pathogenicE. colispecies. One very efficientE. coliacid resistance system encompasses two isoforms of glutamate decarboxylase (gadAandgadB) and a putative glutamate:γ-amino butyric acid (GABA) antiporter (gadC). The system is subject to complex controls that vary with growth media, growth phase, and growth pH. Previous work has revealed that the system is controlled by two sigma factors, two negative regulators (cyclic AMP receptor protein [CRP] and H-NS), and an AraC-like regulator called GadX. Earlier evidence suggested that the GadX protein acts both as a positive and negative regulator of thegadAandgadBCgenes depending on environmental conditions. New data clarify this finding, revealing a collaborative regulation between GadX and another AraC-like regulator called GadW (previously YhiW). GadX and GadW are DNA binding proteins that form homodimers in vivo and are 42% homologous to each other. GadX activates expression ofgadAandgadBCat any pH, while GadW inhibits GadX-dependent activation. Regulation ofgadAandgadBCby either regulator requires an upstream, 20-bp GAD box sequence. Northern blot analysis further indicates that GadW represses expression ofgadX. The results suggest a control circuit whereby GadW interacts with both thegadAandgadXpromoters. GadW clearly repressesgadXand, in situations where GadX is missing, activatesgadAandgadBC.GadX, however, activates onlygadAandgadBCexpression. CRP also repressesgadXexpression. It does this primarily by repressing production of sigma S, the sigma factor responsible forgadXexpression. In fact, the acid induction ofgadAandgadBCobserved when rich-medium cultures enter stationary phase corresponds to the acid induction of sigma S production. These complex control circuits impose tight rein over expression of thegadAandgadBCsystem yet provide flexibility for inducing acid resistance under many conditions that presage acid stress.

Summary: Salmonella enterica serovar Typhimurium periodically experiences acid stress in a variety of host and non‐host environments. An encounter with non‐lethal acid stress (pH > 4) induces an assortment of physiological changes, called the acid tolerance response (ATR), that helps the cell to tolerate extreme low pH (pH 3). These physiological changes differ in log phase and stationary phase cells and are controlled by different regulatory proteins. OmpR is an acid‐induced response regulator critical to the stationary phase ATR but not to the log phase ATR. As OmpR also controls the expression of the acid‐induced viru‐lence operon ssrAB, acid shock induction of ompR was examined to gain insight into how Salmonella links virulence with survival at extreme acid pH. The results indicate that acid pH induces ompR from a promoter different from that used for basal expression. Transcription from this promoter is repressed by the histone‐like protein H‐NS and requires OmpR‐P for induction. The classic sensor kinase EnvZ and acetyl phosphate collaborate to produce the optimum level of OmpR‐P needed for autoinduction. Although OmpR‐P is required for acid‐induced expression of ompR in wild‐type cells, OmpR is not needed for ompR transcription in the absence of H‐NS. Thus, the role of OmpR‐P in autoinduction is to help to counteract repression by H‐NS. This evidence, combined with the finding that relaxing DNA supercoiling with novobiocin also increased ompR transcription, suggests that acid stress induces ompR by altering local DNA topology, not by changing the phosphorylation status of OmpR.