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Notes: 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.

Notes: Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.

Notes: Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σs-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli. The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.

Notes: Salmonella typhimurium is exposed to major shifts in H+ concentration both in its natural and pathogenic environments. The organism undergoes extensive changes in gene expression in response to these pH fluctuations. A current question of regulatory biology is how a change in external pH selectively modulates transcription. We have analysed the expression of one such pH-regulated locus, aniG, and found it is controlled by several additional environmental conditions including osmolarity and oxygen. For factors such as osmolarity and anaerobiosis, an environmentally triggered change in DNA supercoiling has been suggested as a means for controlling gene expression. Thus, environmentally induced changes in DNA topology were explored as a possible common means for establishing the multiple controls on aniG. The involvement of DNA supercoiling in the genetic response of S. typhimurium to external pH has not previously been defined. This report establishes that alkaline environments lower the linking number of reporter plasmids when compared to acidic environments. A consistent pattern was then established whereby conditions or mutations leading to either increased or decreased negative supercoiling were associated with altered expression of aniG. A similar relationship was observed for another environmentally regulated locus, proU. The DNA topology effects on aniG expression were dependent on the presence of EarA, the negative regulator of aniG. These data can be explained by a model in which repressor-operator interactions are very sensitive to changes in operator conformation. These environmentally induced topological influences on operator DNA structure contribute to the magnitude of pH control exerted upon aniG.

Notes: Salmonella typhimurium responds to a variety of environmental stresses by accumulating the alternative sigma factor σS. The repertoire of σS -dependent genes that are subsequently expressed confers tolerance to a variety of potentially lethal conditions including low pH and stationary phase. The mechanism(s) responsible for triggering σS accumulation are of considerable interest, because they help to ensure survival of the organism during encounters with suboptimal environments. Two genes associated with regulating σS levels in S. typhimurium have been identified. The first is clpP, encoding the protease known to be responsible for degrading σS in Escherichia coli. The second is dksA, encoding a protein of unknown function not previously associated with regulating σS levels. As predicted, clpP mutants accumulated large amounts of σS even in log phase. However, dksA mutants failed to accumulate σS in stationary phase and exhibited lower accumulation during acid shock in log phase. DksA appears to be required for the optimal translation of rpoS based upon dksA mutant effects on rpoS transcriptional and translational lacZ fusions. The region of rpoS mRNA between codons 8 and 73 is required to see the effects of dksA mutations. This distinguishes the role of DksA from that of HF-I (hfq ) in rpoS translation, as the HF-I target area occurs well upstream of the rpoS start codon. DksA appears to be involved in the expression of several genes in addition to rpoS based on two-dimensional SDS–PAGE analysis of whole-cell proteins. As a result of their effects on gene expression, mutations in clpP and dksA decreased the virulence of S. typhimurium in mice, consistent with a role for σS in pathogenesis.

Notes: Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro–environment of the bacteria–containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin–Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of ß–galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein–di–ß–D–galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post–infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA actitvity was detected only at early post–infection times (4 h), suggesting that the vacuole may have a mild–acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive ß–galactosidase substrate can provide information about the micro–enviroment within which an intracellular pathogen, such as S. typhimurium, resides.

Notes: The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid. Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor σS encoded by rpoS. Differences between the acid responses of virulent S. typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts. The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon σS for their synthesis. It is predicted that one or more of these ASPs is essential for the sustained system. The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H2O2 and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance. Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance. Consistent with this model, σS proved to be induced by acid shock. Our results also revealed a connection between the transient and sustained ATR systems. Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant. One member of the AtbR regulon, designated atrB, was found to be co-regulated by σS and AtbR. Both regulators had a negative effect on atrB expression. The results suggest AtrB serves as a link between the sustained and transient ATR systems. When σS concentration are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance. Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells.

Notes: 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.

Notes: 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.