GadE (YhiE) activates glutamate decarboxylase-dependent acid resistance in Escherichia coli K-12

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:gamma-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.     

Carbon dioxide increases acid resistance in Escherichia coli

AIMS: To investigate how carbon dioxide affects the acid resistance of Escherichia coli. METHODS AND RESULTS: Escherichia coli W3110 was grown in minimal EG medium at pH 7.5, and cells were adapted at pH 5.5 at 37 degrees C with and without supply of carbon dioxide and nitrogen gases. The number of colonies grown on LB medium was measured after cells were challenged in minimal EG medium of pH 2.5 at 37 degrees C under various conditions. When carbon dioxide was supplied at both the acid adaptation and challenge stages, 94% of cells survived after the acid challenge for 1 h, while the survival rates were 50 and 67% when nitrogen gas and glutamate were supplied respectively. After the acid challenge for 3 h, the survival rate observed with the carbon dioxide gas supply was again 2.5-fold higher than those with the nitrogen gas supply. CONCLUSION: Carbon dioxide was shown to participate in the maintenance of high viability under acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information for research into bacterial pathogenesis, fermentation and food preservation.     

Differential inactivation of glucose- and glutamate dependent acid resistance of Escherichia coli TMW 2.497 by high-pressure treatments

The inactivation by 200–400 MPa and post-pressure survival at acid conditions of E. coli TMW 2.497 was characterized by the measurement of intracellular pH (pH_in), viable cell counts, glutamate (Glu) and arginine (Arg) consumption, and the influence of mild adaptation to mild acid stress prior to pressure treatment. Glutamate and arginine did not affect viable cell counts or the pH_in during pressure application but improved the ability to maintain a high pH_in after pressure treatment. In pH 4.0 buffer without arg and glu, a 3 log reduction of cell counts occurred after 24 h of incubation, whereas little or no loss of viability was observed after 24 h incubation in the presence of glu and arg. During post-pressure incubation at pH 4.0, 10 mM glutamate were metabolized but only 2 mM arginine were used, indicating that glutamate rather than arginine was responsible for the protective effect on pH_in and survival. In conclusion, the pressure induced, irreversible loss of the transmembrane ΔpH correlates to cell death and glu stabilizes the pH_in of E. coli during post-pressure incubation.     

Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli

The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach. Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine alpha-decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm. The arginine system afforded significant protection to wild-type E. coli cells in our acid shock experiments. The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC. Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine. Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli. Evidence for a lysine-dependent XAR system in E. coli is also presented. The protection by lysine, however, is milder than that by arginine.     

Polyamines and regulation of ornithine biosynthesis in Escherichia coli.

The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.     

A glutamate-dependent acid resistance gene in Escherichia coli.

Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 isolates screened, 3 Tet(r) strains were identified as extremely acid sensitive (<0.1% survival at pH 2.5 for 2 h). Flanking sequences of the Tn10 inserts were amplified by inverse PCR. The sequences encoded a hydrophobic partial peptide of 88 residues. A random-primer-generated probe hybridized to Kohara clones 279 and 280 at 32 min (33.7 min on the revised genomic map EcoMap7) near gadB (encoding glutamate decarboxylase). The gene was designated xasA for extreme acid sensitive. xasA::Tn10 strains grown at pH 7 to 8 showed 100-fold-less survival in acid than the parent strain. Growth in mild acid (pH 5 to 6) restored acid resistance; anaerobiosis was not required, as it is for acid resistance in rpoS strains. xasA::Tn10 eliminated enhancement of acid resistance by glutamic acid. xasA was found to be a homolog of gadC recently sequenced in Shigella flexneri, in which it appears to encode a permease for the decarboxylated product of GadB. These results suggest that GadC (XasA) participates in a glutamate decarboxylase alkalinization cycle to protect E. coli from cytoplasmic acidification. The role of the glutamate cycle is particularly important for cultures grown at neutral pH before exposure to extreme acid.     

Regulatory network of acid resistance genes in Escherichia coli

Overexpression of the response regulator EvgA confers an acid-resistant phenotype to exponentially growing Escherichia coli. This acid resistance is partially abolished by deletion of ydeP, yhiE or ydeO, genes induced by EvgA overexpression. Microarray analysis identified two classes of operons (genes). The first class contains seven operons induced by EvgA overexpression in the absence of ydeO, an AraC/XylS regulator gene. The second class contains 12 operons induced by YdeO overexpression. Operons in the second class were induced by EvgA overexpression only in the presence of ydeO. EvgA is likely to directly upregulate operons in the first class, and indirectly upregulate operons in the second class via YdeO. Analysis using the motif-finding program alignace identified an 18 bp inverted repeat motif in six upstream regions of all seven operons directly regulated by EvgA. Gel mobility shift assays showed the specific binding of EvgA to the six sequences. Introduction of mutations into the inverted repeats upstream of ydeP and b1500-ydeO resulted in reduction in EvgA-induced ydeP and ydeO expression and acid resistance. These results suggest that EvgA binds to the inverted repeats and upregulates the downstream genes. Overexpression of YdeP, YdeO and YhiE conferred acid resistance to exponentially growing cells, whereas GadX overexpression did not. Microarray analysis also identified several GadX-activated genes. Several genes induced by overexpression of YdeO and GadX overlapped; however, yhiE was induced only by YdeO. The acid resistance induced by YdeO overexpression was abolished by deletion of yhiE, gadC, slp-yhiF, hdeA or hdeD, genes induced by YdeO overexpression, suggesting that several genes orchestrate YdeO-induced acid resistance. We propose a model of the regulatory network of the acid resistance genes.     

Mechanisms of acid resistance in enterohemorrhagic Escherichia coli.

Enterohemorrhagic strains of Escherichia coli must pass through the acidic gastric barrier to cause gastrointestinal disease. Taking into account the apparent low infectious dose of enterohemorrhagic E. coli, 11 O157:H7 strains and 4 commensal strains of E. coli were tested for their abilities to survive extreme acid exposures (pH 3). Three previously characterized acid resistance systems were tested. These included an acid-induced oxidative system, an acid-induced arginine-dependent system, and a glutamate-dependent system. When challenged at pH 2.0, the arginine-dependent system provided more protection in the EHEC strains than in commensal strains. However, the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. Because E. coli must also endure acid stress imposed by the presence of weak acids in intestinal contents at a pH less acidic than that of the stomach, the ability of specific acid resistance systems to protect against weak acids was examined. The arginine- and glutamate-dependent systems were both effective in protecting E. coli against the bactericidal effects of a variety of weak acids. The acids tested include benzoic acid (20 mM; pH 4.0) and a volatile fatty acid cocktail composed of acetic, propionic, and butyric acids at levels approximating those present in the intestine. The oxidative system was much less effective. Several genetic aspects of E. coli acid resistance were also characterized. The alternate sigma factor RpoS was shown to be required for oxidative acid resistance but was only partially involved with the arginine- and glutamate-dependent acid resistance systems. The arginine decarboxylase system (including adi and its regulators cysB and adiY) was responsible for arginine-dependent acid resistance. The results suggest that several acid resistance systems potentially contribute to the survival of pathogenic E. coli in the different acid stress environments of the stomach (pH 1 to 3) and the intestine (pH 4.5 to 7 with high concentrations of volatile fatty acids). Of particular importance to the food industry was the finding that once induced, the acid resistance systems will remain active for prolonged periods of cold storage at 4 degrees C.     

Polyamines decrease Escherichia coli outer membrane permeability.

The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels. We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis. In both cases, inhibition was observed. Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation. The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine. Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder. These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins. These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.     

Polyamines reduce paraquat-induced soxS and its regulon expression in Escherichia coli

Polyamines, ubiquitous polycationic compounds, are involved in many cellular responses and relieve paraquat-induced cytotoxicity inEscherichia coli. We constructed a newE. coli mutant strain, JIL528, which is deficient in the biosynthesis of both putrescine and spermidine, to examine the physiological role of polyamines under oxidative stress caused by paraquat. Putrescine and spermidine downregulate the expression ofsoxS induced by paraquat in a concentration-dependent manner. The product of SoxS is a key regulator governing cellular responses against oxidative stress inE. coli. The downregulation ofsoxS expression by polyamines was not shown in thesoxR mutant background. Glucose-6-phosphate dehydrogenase (G6PDH; encoded byzwf) and manganese-containing superoxide dismutase (Mn-SOD; encoded bysodA) activities induced by paraquat were decreased by exogenous polyamines. The induction of thezwf expression by paraquat was also decreased by exogenous polyamines. The polyamine-deficient mutant strain JIL528 showed a highersoxS expression than its parent polyamine-proficient wild type BW1157, on exogenous supplementation of paraquat concentrations below 1 mol/L. While the growth rate of the mutant was decreased,soxS expression was increased in a concentration-dependent manner above 0.01 mol/L of paraquat. In contrast, growth inhibition of the mutant by paraquat was relieved, andsoxS was no longer induced by exogenous putrescine (1 mmol/L). In conclusion, polyamines protect against paraquat-induced toxicity but downregulatesoxS expression, suggesting that the protective role of polyamines against oxidative damage induced by paraquat results insoxS downregulation.     

Chaperone Hsp31 contributes to acid resistance in stationary-phase Escherichia coli

Hsp31, the product of the sigmaS - and sigmaD -dependent hchA gene, is a heat-inducible chaperone implicated in the management of protein misfolding at high temperatures. We show here that Hsp31 plays an important role in the acid resistance of starved Escherichia coli but that it has little influence on oxidative-stress survival.     

Escherichia coli acid resistance: tales of an amateur acidophile

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.     

Variation in acid resistance among shiga toxin-producing clones of pathogenic Escherichia coli

Pathogenic strains of Escherichia coli, such as E. coli O157:H7, have a low infectious dose and an ability to survive in acidic foods. These bacteria have evolved at least three distinct mechanisms of acid resistance (AR), including two amino acid decarboxylase-dependent systems (arginine and glutamate) and a glucose catabolite-repressed system. We quantified the survival rates for each AR mechanism separately in clinical isolates representing three groups of Shiga toxin-producing E. coli (STEC) clones (O157:H7, O26:H11/O111:H8, and O121:H19) and six commensal strains from ECOR group A. Members of the STEC clones were not significantly more acid resistant than the commensal strains when analyzed using any individual AR mechanism. The glutamate system provided the best protection in a highly acidic environment for all groups of isolates (<0.1 log reduction in CFU/ml per hour at pH 2.0). Under these conditions, there was notable variation in survival rates among the 30 O157:H7 strains, which depended in part on Mg(2+) concentration. The arginine system provided better protection at pH 2.5, with a range of 0.03 to 0.41 log reduction per hour, compared to the oxidative system, with a range of 0.13 to 0.64 log reduction per hour. The average survival rate for the O157:H7 clonal group was significantly less than that of the other STEC clones in the glutamate and arginine systems and significantly less than that of the O26/O111 clone in the oxidative system, indicating that this clonal group is not exceptionally acid resistant with these specific mechanisms.