Acid Resistance System Research Articles

Indole enhances acid resistance in Escherichia coli

As a stationary-phase signal, indole is secreted in large quantities by Escherichia coli on enriched media and has been shown to control several genes; however, its impact on acid resistance remains to be studied in detail. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed that indole increases the expression of the glutamine decarboxylase system that includes genes such as gadA, gadB, and gadC genes with no effect on the expression of other acid resistance systems such as arginine decarboxylase ( adiA) and lysine decarboxylase ( cadA, cadB, cadC, and ldcC). Indole also induces yhiE ( gadE) that encodes the regulator required for expression of gadA, gadB, and gadC. These results suggest that indole enhances the survival of E. coli under acidic conditions by increasing the expression of acid resistance genes of the glutamine decarboxylase system, thus increasing its acid resistance.

Acid stress response in Escherichia coli: mechanism of regulation of gadA transcription by RcsB and GadE

Escherichia coli can survive extreme acid stress for several hours. The most efficient acid resistance system is based on glutamate decarboxylation by the GadA and GadB decarboxylases and the import of glutamate via the GadC membrane protein. The expression of the corresponding genes is controlled by GadE, the central activator of glutamate-dependent acid resistance (GDAR). We have previously shown by genetic approaches that as well as GadE, the response regulator of the Rcs system, RcsB is absolutely required for control of gadA/BC transcription. In the presence of GadE, basal activity of RcsB stimulates the expression of gadA/BC, whereas activation of RcsB leads to general repression of the gad genes. We report here the results of various in vitro assays that show RcsB to regulate by direct binding to the gadA promoter region. Furthermore, activation of gadA transcription requires a GAD box and binding of an RcsB/GadE heterodimer. In addition, we have identified an RcsB box, which lies just upstream of the −10 element of gadA promoter and is involved in repression of this operon.

E. coli K-12 and EHEC Genes Regulated by SdiA

Background Escherichia and Salmonella encode SdiA, a transcription factor of the LuxR family that regulates genes in response to N-acyl homoserine lactones (AHLs) produced by other species of bacteria. E. coli genes that change expression in the presence of plasmid-encoded sdiA have been identified by several labs. However, many of these genes were identified by overexpressing sdiA on a plasmid and have not been tested for a response to sdiA produced from its natural position in the chromosome or for a response to AHL.Methodology/Principal FindingsWe determined that two important loci reported to respond to plasmid-based sdiA, ftsQAZ and acrAB, do not respond to sdiA expressed from its natural position in the chromosome or to AHLs. To identify genes that are regulated by chromosomal sdiA and/or AHLs, we screened 10,000 random transposon-based luciferase fusions in E. coli K-12 and a further 10,000 in E. coli O157:H7 for a response to AHL and then tested these genes for sdiA-dependence. We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA. Gene regulation by sdiA of E. coli is only partially dependent upon AHL.Conclusions/SignificanceThe genes of E. coli that respond to plasmid-based expression of sdiA are largely different than those that respond to chromosomal sdiA and/or AHL. This has significant implications for determining the true function of AHL detection by E. coli.

Substrate recognition and transport behavior analyses of amino acid antiporter with coarse-grained models

The L-arginine (Arg)/agmatine (Agm) antiporter AdiC is a vital transport protein of the arginine-dependent extreme acid resistance system of enteric bacteria. Recently, both substrate-free and Arg-bound structures of AdiC were determined by X-ray crystallography. In this article, the two different proteins were investigated with three simple models. Gaussian network model provided the information of conformational changes. It is found that Arg binding induces structural rearrangement in the extracellular domain, and transmembrane helix 6 (TM6) has the most pronounced trend of conformational changes. The moving directions of fluctuation regions were further ascertained by using anisotropy elastic network model and cross-correlation analysis. Interestingly, the two substrate-binding sites hypothesis of AdiC was confirmed directly by molecular docking. Furthermore, the binding preferences of these two sites were explained from the aspects of electrostatic complementarity and geometric matching. These simple coarse-grained analyses can be used as a general and quick method for the mechanism studies of transport proteins.

Interference of the CadC regulator in the arginine-dependent acid resistance system of Shigella and enteroinvasive E. coli

A typical pathoadaptive mutation of Shigella and enteroinvasive Escherichia coli (EIEC) is the inactivation of the cad locus which comprises the genes necessary for lysine decarboxylation, an enzyme involved in pH homoeostasis. In E. coli, the cadBA operon, encoding lysine decarboxylase (CadA) and a lysine cadaverine antiporter (CadB), is submitted to the control of CadC, a positive activator whose gene maps upstream the operon, and is transcribed independently from the same strand. CadC is an integral inner membrane protein which acts both, as signal sensor and as transcriptional regulator responding to the low pH and lysine signals. Analysis of the molecular rearrangements responsible for the loss of lysine decarboxylase activity in Shigella and EIEC has revealed that the inactivation of the cadC gene is a common feature. The 3 major adaptive acid resistance (AR) systems – AR1, AR2, and AR3 – are known to be activated at low pH by Shigella and E. coli, allowing them to withstand extremely acid conditions. In this study, evaluating the survival of S. flexneri, S. sonnei, and EIEC strains complemented with a functional cadC gene and challenged at low pH, we present evidence that CadC negatively regulates the expression of the arginine-dependent adaptive acid-resistance system (AR3), encoded by the adi locus while it has no effect on the expression of AR1 and AR2 systems. Moreover, since our results indicate that in enteroinvasive strains the presence of CadC reduces the expression of the arginine decarboxylase encoding gene adiA, it is possible to hypothesize that the loss of functionality of lysine decarboxylase is counterbalanced by a higher expression of the adi system, and that CadC, besides specifically affecting the regulation of the cadBA operon, is also relevant to other systems responding to low pH.

Comparative analysis of acid resistance in Listeria monocytogenes and Salmonella enterica strains before and after exposure to poultry decontaminants. Role of the glutamate decarboxylase (GAD) system

Data on the ability of chemical poultry decontaminants to induce an acid stress response in pathogenic bacteria are lacking. This study was undertaken in order to compare the survival rates in acid broths of Listeria monocytogenes and Salmonella enterica strains, both exposed to and not exposed to decontaminants. The contribution of the glutamate decarboxylase (GAD) acid resistance system to the survival of bacteria in acid media was also examined. Four strains ( L. monocytogenes serovar 1/2, L. monocytogenes serovar 4b, S. enterica serotype Typhymurium and S. enterica serotype Enteritidis) were tested before (control) and after exposure to trisodium phosphate, acidified sodium chlorite, citric acid, chlorine dioxide and peroxyacids (strains were repeatedly passed through media containing increasing concentrations of a compound). Stationary-phase cells (10 8 cfu/ml) were inoculated into tryptic soy broth (TSB) acidified with citric acid (pH 2.7 and 5.0) with or without glutamate (10 mM) added, and incubated at 37 °C for 15 min. Survival percentages (calculated from viable colonies) varied from 2.47 ± 0.67% to 91.93 ± 5.83%. L. monocytogenes cells previously exposed to acid decontaminants (citric acid and peroxyacids) showed, when placed in acid TSB, a higher ( P < 0.05) percentage of survival (average 38.80 ± 30.52%) than control and pre-exposed to non-acidic decontaminants strains (22.82 ± 23.80%). Similar ( P > 0.05) survival percentages were observed in previously exposed to different decontaminants and control Salmonella strains. The GAD acid resistance system did not apparently play any role in the survival of L. monocytogenes or S. enterica at a low pH. This study demonstrates for the first time that prior exposure to acidic poultry decontaminants increases the percentage of survival of L. monocytogenes exposed to severe acid stress. These results have important implications for the meat industry when considering which decontaminant treatment to adopt.

Metabolic and transcriptional response of recombinant Escherichia coli to elevated dissolved carbon dioxide concentrations

The effect of dissolved carbon dioxide (dCO(2)) concentration on the stoichiometric and kinetic constants and by-product accumulation was determined for Escherichia coli cells producing recombinant green fluorescent protein (GFP). Constant dCO(2), in the range of 20-300 mbar, was maintained during batch cultures by manipulating the inlet gas composition. As dCO(2) increased, specific growth rate (micro) decreased, and acetate accumulation and the time for onset of GFP production increased. Maximum biomass yield on glucose and GFP concentration were affected for dCO(2) above 70 and 150 mbar, respectively. Expression analysis of 16 representative genes showed that E. coli can respond at the transcriptional level upon exposure to increasing dCO(2), and revealed possible mechanisms responsible for the detrimental effects of high dCO(2). Genes studied included those involved in decarboxylation reactions (aceF, icdA, lpdA, sucA, sucB), genes from pathways of production and consumption of acetate (ackA, poxB, acs, aceA, fadR), genes from gluconeogenic and anaplerotic metabolism (pckA, ppc), genes from the acid resistance (AR) systems (adiA, gadA, gadC), and the heterologous gene (gfp). The transcription levels of tricarboxylic acid (TCA) cycle genes (icdA, sucA, sucB) and glyoxylate shunt (aceA) decreased as dCO(2) increased, whereas fadR (that codes for a negative regulator of the glyoxylate operon) and poxB (that codes for PoxB which is involved in acetate production from pyruvate) were up-regulated as dCO(2) increased up to 150 mbar. Furthermore, transcription levels of genes from the AR systems increased as dCO(2) increased up to 150 mbar, indicating that elevated dCO(2) triggers an acid stress response in E. coli cells. Altogether, such results suggest that the increased acetate accumulation and reduction in mu, biomass yield and maximum GFP concentration under high dCO(2) resulted from a lower carbon flux to TCA cycle, the concomitant accumulation of acetyl-CoA or pyruvate, and the acidification of the cytoplasm.

Escherichia coli O157 : H7 glutamate- and arginine-dependent acid-resistance systems protect against oxidative stress during extreme acid challenge

Micro-organisms may simultaneously encounter multiple stresses in their environment. To investigate the protection that several known Escherichia coli O157 : H7 acid-resistance systems might provide against both oxidative and acid stress, the addition of diamide, a membrane-permeable thiol-specific oxidizing agent, or hydrogen peroxide were used concurrent with acid challenge at pH 2.5 to determine bacterial survival. The addition of either diamide or hydrogen peroxide decreased bacterial survival in a dose-dependent manner for E. coli O157 : H7 during challenge at pH 2.5 following overnight growth in LB MES pH 5.5 (acid-resistance system 1, AR1). In contrast, the presence of either glutamate or arginine during challenge provided significant protection against diamide- and hydrogen peroxide-induced oxidative stress during pH 2.5 acid challenge. Oxidative stress protection during acid challenge required gadC and adiA for the glutamate- (AR2) and arginine- (AR3) dependent acid-resistance systems, respectively. In addition, maximal protection against oxidative stress in the presence of glutamate required a low external pH (pH 2.5), since pH 5.5 did not protect. This study demonstrates that the glutamate- and arginine-dependent acid-resistance systems of E. coli O157 : H7 can simultaneously protect against oxidative stress during extreme acid challenge.

A 750 bp sensory integration region directs global control of the Escherichia coli GadE acid resistance regulator

Escherichia coli survives pH 2 environments through an acid resistance (AR) system regulated by the transcriptional activator GadE. Numerous proteins control gadE at an upstream, conserved, 798 bp intergenic region. We show this region produces three transcripts starting at -124 (T1), -324/-317 (T2) and -566 (T3) bp from the gadE start codon. Transcriptional lacZ fusions to gadE promoter regions revealed P1 and P3 were active while P2 alone was not. However, pairing P3 with P2 activated P2 and increased expression 20-fold above P3 alone. The fusions were transferred to Salmonella, which lacks this AR system, and plasmid-borne E. coli-specific regulators EvgA, YdeO, GadE and GadX were introduced. Data revealed that YdeO and GadX activate P3, P2 and P3P2, while GadE autoactivates P1 and represses P3 and P3P2. The developing model indicates that different signals activate YdeO, GadX, or an MnmE-dependent regulator, which stimulate gadE transcription from the P3 and P2 promoters. Once made, GadE activates P1 and represses P3 and P2. The P1 region also enables efficient downstream transcription and translation of the P3 or P2 transcripts. Evidence indicates the entire 750 bp sensory integration locus is necessary for a versatile response.

Projection Structure of a Member of the Amino Acid/Polyamine/Organocation Transporter Superfamily

The L-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15-17% to eukaryotic and human APC transporters. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport L-arginine. Purified detergent-solubilized AdiC particles were dimeric. Reconstitution experiments yielded two-dimensional crystals of AdiC-W293L diffracting beyond 6 angstroms resolution from which we determined the projection structure at 6.5 angstroms resolution. The projection map showed 10-12 density peaks per monomer and suggested mainly tilted helices with the exception of one distinct perpendicular membrane spanning alpha-helix. Comparison of AdiC-W293L with the projection map of the oxalate/formate antiporter from Oxalobacter formigenes, a member from the major facilitator superfamily, indicated different structures. Thus, two-dimensional crystals of AdiC-W293L yielded the first detailed view of a transport protein from the APC superfamily at sub-nanometer resolution.

Outer and Inner Membrane Proteins Compose an Arginine-Agmatine Exchange System inChlamydophila pneumoniae

Most chlamydial strains have a pyruvoyl-dependent decarboxylase protein that converts L-arginine to agmatine. However, chlamydiae do not produce arginine, so they must import it from their host. Chlamydophila pneumoniae has a gene cluster encoding a putative outer membrane porin (CPn1033 or aaxA), an arginine decarboxylase (CPn1032 or aaxB), and a putative cytoplasmic membrane transporter (CPn1031 or aaxC). The aaxC gene was expressed in Escherichia coli producing an integral cytoplasmic membrane protein that catalyzed the exchange of L-arginine for agmatine. Expression of the aaxA gene produced an outer membrane protein that enhanced the arginine uptake and decarboxylation activity of cells coexpressing aaxB and aaxC. This chlamydial arginine/agmatine exchange system complemented an E. coli mutant missing the native arginine-dependent acid resistance system. These cells survived extreme acid shock in the presence of L-arginine. Biochemical and evolutionary analysis showed the aaxABC genes evolved convergently with the enteric arginine degradation system, and they could have a different physiological role in chlamydial cells. The chlamydial system uniquely includes an outer membrane porin, and it is most active at a higher pH from 3 to 5. The chlamydial AaxC transporter was resistant to cadaverine, L-lysine and L-ornithine, which inhibit the E. coli AdiC antiporter.

Examination of Stress and Virulence Gene Expression inEscherichia coliO157:H7 Using Targeted Microarray Analysis

Escherichia coli O157:H7 poses a threat to humans through food- and water-borne transmission. To investigate how environmental stresses affect the Escherichia coli O157:H7 transcriptome, we designed a targeted microarray consisting of stress response and virulence genes (n = 125) to analyze the impact of acidified (pH 3.5), cold (7.5 degrees C), and fresh tryptic soy broth (TSB) (37 degrees C) on E. coli O157:H7 stress response and virulence gene expression. Nutrient replenishment with fresh TSB resulted in 72 differentially expressed genes (> or = 1.5-fold change; p < 0.05), with 65 induced. All queried global and specific stress regulators were affected, as were 12 virulence genes. Cold-shocked cells displayed 17 differentially expressed genes, with 10 being induced. Induction of rpoS, members of the sigma(H) regulon (clpB, dnaK, ftsH), and acid resistance (AR) genes (gadA, gadX) was observed. Porin transcript (ompC, ompF) and gapA and tufA ancillary genes were repressed. Acid shock resulted in 24 differentially expressed genes, with 21 induced. No induction of any stationary phase AR system was observed, though acid-coping mechanisms were recruited, including mar and phoB induction, and repression of ompC and ompF. Stress regulators were induced, including relA, soxS, rpoE, and rpoH. The microarray data were validated by quantitative real-time polymerase chain reaction. Exposure to sublethal stress events led to the induction of diverse stress response networks. In the food chain, sublethal events may render cells increasingly resistant to future stresses, potentially leading to increased survival.

A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis

Acidification of the phagosome is considered to be a major mechanism used by macrophages against bacteria, including Mycobacterium tuberculosis (Mtb). Mtb blocks phagosome acidification, but interferon-gamma (IFN-gamma) restores acidification and confers antimycobacterial activity. Nonetheless, it remains unclear whether acid kills Mtb, whether the intrabacterial pH of any pathogen falls when it is in the phagosome and whether acid resistance is required for mycobacterial virulence. In vitro at pH 4.5, Mtb survived in a simple buffer and maintained intrabacterial pH. Therefore, Mtb resists phagolysosomal concentrations of acid. Mtb also maintained its intrabacterial pH and survived when phagocytosed by IFN-gamma-activated macrophages. We used transposon mutagenesis to identify genes responsible for Mtb's acid resistance. A strain disrupted in Rv3671c, a previously uncharacterized gene encoding a membrane-associated protein, was sensitive to acid and failed to maintain intrabacterial pH in acid in vitro and in activated macrophages. Growth of the mutant was also severely attenuated in mice. Thus, Mtb is able to resist acid, owing in large part to Rv3671c, and this resistance is essential for virulence. Disruption of Mtb's acid resistance and intrabacterial pH maintenance systems is an attractive target for chemotherapy.

Fresh-cut lettuce in modified atmosphere packages stored at improper temperatures supports enterohemorrhagic E. coli isolates to survive gastric acid challenge.

Incidences of foodborne outbreaks involving enterohemorrhagic Escherichia coli strains with mutations in a key regulatory gene, rpoS, have been reported. Incentives, if any, for losing this regulatory function are not clear since the RpoS regulator is required for the expression of several environmental stress tolerance genes. RpoS also positively regulates 2 of the 3 acid-resistance systems of E. coli under aerobic growth conditions and enables the pathogen to survive gastric acid challenge. We selected 7 enterohemorrhagic E. coli isolates, 6 of which are known to carry defective rpoS gene, and then analyzed resistance to synthetic gastric juice after the strains were inoculated on fresh-cut lettuce and stored under modified atmosphere packaging (MAP) conditions. Subatmospheric oxygen partial pressures in MAP enabled all 6 rpoS-defective isolates to induce acid resistance over the 8-d storage period if the temperature was >or= 15 degrees C. No acid resistance was induced for MAP-stored lettuce left at temperatures <or= 10 degrees C or for lettuce packed and stored under aerobic conditions. The data underscore the impending danger of abusive storage temperatures especially with regard to the application of MAP to extend the shelf life of fresh produce. The results also highlight the biological significance of having multiple acid-resistance pathways and the complex regulatory network of enterohemorrhagic E. coli strains.

Inducible acid tolerance mechanisms in enteric bacteria.

Enteric micro-organisms have developed several inducible mechanisms for surviving transient periods of extreme acid stress. Salmonella typhimurium possesses an acid tolerance response (ATR) induced in minimal medium by short exposures to mild acid stress. More than 50 acid shock proteins (ASPs) are induced during adaptation. Eight ASPs are regulated by the major iron regulatory protein, Fur, in an unusual iron-independent manner. The two-component regulator, PhoP, is an autoinduced ASP that controls the induction of three additional ASPs. The stress sigma factor sigma S is an ASP that regulates induction of eight ASPs. Acid induction of sigma S is due to its decreased proteolytic turnover via the ClpXP protease in conjunction with the two-component-type response regulator MviA (RssB in Escherichia coli). Mutations in any of these three regulators leads to a defective ATR. Repair of pH stress-induced DNA damage appears to require the Ada protein (O6-methylguanine methyltransferase) since an ada mutant is both acid and alkaline sensitive. In contrast to S. typhimurium, E. coli and Shigella have acid resistance systems induced in complex media that include a glucose-repressed system protective at pH 2.5 without amino acid supplementation, a glutamate decarboxylase system that requires glutamate and an arginine decarboxylase system unique to E. coli.

Sodium regulates Escherichia coli acid resistance, and influences GadX- and GadW-dependent activation of gadE

Enteric bacteria must survive the extreme acid of the stomach (pH 2 or less) before entering the intestine where they can colonize and cause disease. Escherichia coli is superior to most other Enterobacteriaceae in surviving pH 2 acid stress because it has four known acid-resistance systems, the most studied of which depends on glutamic acid. Glutamate-dependent acid resistance requires glutamate decarboxylase isozymes GadA and GadB, as well as a glutamate/gamma-aminobutyric acid antiporter encoded by gadC. The regulatory protein GadE is the essential activator of the gadA and gadBC genes. The transcription of gadE, however, is controlled by numerous proteins. Two of these proteins, GadX and GadW, are AraC-family regulators whose sensory input signals are not known. Since Na(+) and K(+) play important roles in pH homeostasis, the contribution of these ions toward the regulation of this acid-resistance system was examined. The results indicated that a decrease in Na(+), but not K(+), concentration coincided with diminished acid resistance, and decreased expression of the gadE, gadA and gadBC genes. However, Na(+)-dependent regulation of these genes dissipated in the absence of GadX and GadW. Since Na(+) levels did not regulate gadX or gadW transcription, it is proposed that GadX and GadW sense intracellular Na(+) concentration or some consequence of altered Na(+) levels.

Characterization of an Acid-Dependent Arginine Decarboxylase Enzyme from Chlamydophila pneumoniae

Genome sequences from members of the Chlamydiales encode diverged homologs of a pyruvoyl-dependent arginine decarboxylase enzyme that nonpathogenic euryarchaea use in polyamine biosynthesis. The Chlamydiales lack subsequent genes required for polyamine biosynthesis and probably obtain polyamines from their host cells. To identify the function of this protein, the CPn1032 homolog from the respiratory pathogen Chlamydophila pneumoniae was heterologously expressed and purified. This protein self-cleaved to form a reactive pyruvoyl group, and the subunits assembled into a thermostable (alphabeta)(3) complex. The mature enzyme specifically catalyzed the decarboxylation of L-arginine, with an unusually low pH optimum of 3.4. The CPn1032 gene complemented a mutation in the Escherichia coli adiA gene, which encodes a pyridoxal 5'-phosphate-dependent arginine decarboxylase, restoring arginine-dependent acid resistance. Acting together with a putative arginine-agmatine antiporter, the CPn1032 homologs may have evolved convergently to form an arginine-dependent acid resistance system. These genes are the first evidence that obligately intracellular chlamydiae may encounter acidic conditions. Alternatively, this system could reduce the host cell arginine concentration and produce inhibitors of nitric oxide synthase.

The Escherichia coli AraC-family regulators GadX and GadW activate gadE, the central activator of glutamate-dependent acid resistance

Escherichia coli can survive pH 2 acid stress by using several acid resistance systems. The most efficient of these employs glutamate decarboxylase (GadA/GadB) to consume protons, and an antiporter (GadC) to exchange the intracellular decarboxylation product for external glutamic acid. Expression of the essential transcriptional activator of this system, GadE, is controlled by several regulators in a hierarchical fashion. In this study, two additional activators have been identified. The AraC-family regulators GadX and GadW, previously found to activate gadA/BC in vitro, are now shown in vivo to directly activate gadE expression, which, in turn, activates the gadA/BC genes. In vivo results using E. coli and Salmonella enterica show that these regulators actually have little direct effect on gadA and gadBC promoters. The numerous gadE induction pathways converge on a 798 bp control region situated upstream of the gadE promoter region. Deletions of this control region exposed the region between -798 and -360 nt (relative to the translational start) to be required for maximum gadE-lacZ expression in Luria-Bertani (LB) medium and to be the primary focus of GadX and GadW control. The GadE protein itself, which binds to three GAD box sequences present between -233 and -42 nt, helped activate GadE expression in LB, but only when the -798 to -360 region was absent. These regulatory regions and proteins appear to integrate a variety of physiological signals that forecast a need for GadE-dependent gene expression and acid resistance.

The acid-resistance pathways of Shigella flexneri 2457T

The stationary-phase acid-resistance pathways of Shigella flexneri 2457T have not previously been studied. The two acid-resistance systems, the glutamate-dependent acid-resistance (GDAR) and the oxidative pathways, reported elsewhere for Escherichia coli and S. flexneri 3136, were both detected in S. flexneri 2457T. However, S. flexneri 2457T cells grown overnight under fermentative conditions and acid-shocked in minimal media in the absence of glutamate, an acid test often described as a negative control for both pathways, were capable of surviving acid challenge. It is possible that this resistance is due to the oxidative pathway operating in a non-glucose-repressible manner, or to a novel pathway present in S. flexneri 2457T. The construction of gadB and gadC mutants ruled out any contribution by the GDAR pathway, whilst further characterizing the GDAR properties of S. flexneri 2457T. Interestingly, study of the role of rpoS in the oxidative pathway and the unusual acid-resistance phenotype revealed that the frameshift present in the 2457T rpoS gene results in expression of a truncated RpoS protein, which may be reduced in activity and is not essential for the acid-resistance phenotype of S. flexneri 2457T.

The CrdRS (HP1365-HP1364) Two-Component System Is Not Involved in pH-Responsive Gene Regulation in the Helicobacter pylori Strains 26695 and G27

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease. However, urease-independent mechanisms are likely to contribute to acid adaptation. pH-responsive gene regulation in this organism is mediated by a two-component system (HP0166-HP0165) designated ArsRS and the metal-dependent regulators NikR and Fur. Recently, it was reported that another two-component system termed CrdRS (HP1365-HP1364) is required for pH-responsive regulation of the major acid-resistance systems in the H. pylori strain J99. By the analysis of crdRS null mutants of the H. pylori strains 26695 and G27, we show that low pH induction of both the urease and the amidase genes occurs in the absence of crdRS in these strains, suggesting substantial strain-specific differences in the regulation of a major virulence determinant.