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1.  Control of Acid Resistance in Escherichia coli 
Journal of Bacteriology  1999;181(11):3525-3535.
Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor ςS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be ςS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the ςS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.
PMCID: PMC93821  PMID: 10348866
2.  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.
PMCID: PMC168100  PMID: 8795195
3.  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 Mg2+ 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.
PMCID: PMC1087542  PMID: 15870339
4.  Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. 
Journal of Bacteriology  1995;177(14):4097-4104.
Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.
PMCID: PMC177142  PMID: 7608084
5.  Escherichia coli Glutamate- and Arginine-Dependent Acid Resistance Systems Increase Internal pH and Reverse Transmembrane Potential 
Journal of Bacteriology  2004;186(18):6032-6041.
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 (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (ΔΨ) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi 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 pHi 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 pHi value.
PMCID: PMC515135  PMID: 15342572
6.  Acid Resistance Systems Required for Survival of Escherichia coli O157:H7 in the Bovine Gastrointestinal Tract and in Apple Cider Are Different 
Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider. This property is thought to contribute to the low infectious dose of the organism. Three acid resistance (AR) systems are expressed in stationary-phase cells. AR system 1 is σS dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively. In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract. Wild-type and mutant E. coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves. Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider. Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH. Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells. AR system 3-deficient cells survived well in both apple cider and calves. Taken together, these results indicate that E. coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered.
PMCID: PMC492388  PMID: 15294816
7.  Purification of Synechocystis sp. Strain PCC6308 Cyanophycin Synthetase and Its Characterization with Respect to Substrate and Primer Specificity 
Synechocystis sp. strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli. The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 ± 30 kDa and consists of identical subunits of 85 ± 5 kDa. The Km values for arginine (49 μM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates. During in vitro cyanophycin synthesis, 1.3 ± 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP. The optima for the enzyme-catalyzed reactions were pH 8.2 and 50°C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), β-hydroxy aspartic acid (35 and 37%), aspartic acid β-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture. In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), d-aspartic acid (37 and 30%), l-glutamic acid (13 and 5%), and ornithine (23 and 11%). On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid β-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [3H]lysine (15% of maximum activity) and [3H]canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas l-glutamic acid was not incorporated. No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine). Various samples of chemically synthesized poly-α,β-d,l-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-α-l-aspartic acid was almost inactive.
PMCID: PMC92852  PMID: 11319097
8.  YjdE (AdiC) Is the Arginine:Agmatine Antiporter Essential for Arginine-Dependent Acid Resistance in Escherichia coli 
Journal of Bacteriology  2003;185(15):4402-4409.
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.
PMCID: PMC165756  PMID: 12867448
9.  A Requirement of TolC and MDR Efflux Pumps for Acid Adaptation and GadAB Induction in Escherichia coli 
PLoS ONE  2011;6(4):e18960.
The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H+ transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown.
Methods and Principal Findings
TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 105-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5–6.0, but not at pH 6.5–8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5.
TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach.
PMCID: PMC3082540  PMID: 21541325
10.  Outer and Inner Membrane Proteins Compose an Arginine-Agmatine Exchange System in Chlamydophila pneumoniae▿ † 
Journal of Bacteriology  2008;190(22):7431-7440.
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.
PMCID: PMC2576674  PMID: 18790867
11.  Products of the Escherichia coli Acid Fitness Island Attenuate Metabolite Stress at Extremely Low pH and Mediate a Cell Density-Dependent Acid Resistance▿  
Journal of Bacteriology  2007;189(7):2759-2768.
Escherichia coli has an ability, rare among the Enterobacteriaceae, 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, and gadE, 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 protected E. coli from 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, yhiD and hdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>108 CFU 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 of E. coli acid resistance.
PMCID: PMC1855797  PMID: 17259322
12.  Role of rpoS in Acid Resistance and Fecal Shedding of Escherichia coli O157:H7 
Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator ςS and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 101 to 104 CFU, we found the wild-type strain in feces of mice given lower doses (102 versus 103 CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 104 CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P ≤ 0.05). Thus, ςS appears to play a role in E. coli O157:H7 passage in mice and shedding from calves, possibly by inducing expression of the glucose-repressed RpoS-dependent AR determinant and thus increasing resistance to gastrointestinal stress. These findings may provide clues for future efforts aimed at reducing or eliminating this pathogen from cattle herds.
PMCID: PMC91873  PMID: 10653728
13.  Arginine-Agmatine Antiporter in Extreme Acid Resistance in Escherichia coli 
Journal of Bacteriology  2003;185(22):6556-6561.
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 α-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.
PMCID: PMC262112  PMID: 14594828
14.  Low-pH Rescue of Acid-Sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-Regulated Arginine Decarboxylase System 
Journal of Bacteriology  2013;195(13):3062-3072.
For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the ParaBAD or PrhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter PrhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter ParaBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ΔaroD and ΔphoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.
PMCID: PMC3697538  PMID: 23645603
15.  pH-Dependent Expression of Periplasmic Proteins and Amino Acid Catabolism in Escherichia coli 
Journal of Bacteriology  2002;184(15):4246-4258.
Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.
PMCID: PMC135203  PMID: 12107143
16.  pH-Dependent Modulation of Cyclic AMP Levels and GadW-Dependent Repression of RpoS Affect Synthesis of the GadX Regulator and Escherichia coli Acid Resistance 
Journal of Bacteriology  2003;185(23):6852-6859.
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.
PMCID: PMC262709  PMID: 14617649
17.  Characterization of the Escherichia coli O157:H7 Sakai GadE Regulon▿ †  
Journal of Bacteriology  2008;191(6):1868-1877.
Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of the glutamate decarboxylase (GAD) system, the most efficient acid resistance (AR) mechanism in E. coli. The full contribution of GadE to the AR and virulence of E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles of the mutant with that of the wild type in the exponential and stationary phases of growth. Inactivation of gadE significantly altered the expression of 60 genes independently of the growth phase and of 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly downregulated the expression of gadA, gadB, and gadC and of many acid fitness island genes. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Inactivation of ler in the ΔgadE strain reversed the effect of gadE deletion on LEE expression, indicating that Ler is necessary for LEE repression by GadE. GadE is also involved in downregulation of LEE expression under conditions of moderately acidic pH. Characterization of AR of the ΔgadE strain revealed that GadE is indispensable for a functional GAD system and for survival of E. coli O157:H7 in a simulated gastric environment. Altogether, these data indicate that GadE is critical for the AR of E. coli O157:H7 and that it plays an important role in virulence by downregulating expression of LEE.
PMCID: PMC2648353  PMID: 19114477
18.  Arginine-Dependent Acid Resistance in Salmonella enterica Serovar Typhimurium 
Journal of Bacteriology  2006;188(15):5650-5653.
Salmonella enterica serovar Typhimurium does not survive a pH 2.5 acid challenge under conditions similar to those used for Escherichia coli (J. W. Foster, Nat. Rev. Microbiol. 2:898-907, 2004). Here, we provide evidence that S. enterica serovar Typhimurium can display arginine-dependent acid resistance (AR) provided the cells are grown under anoxic conditions and not under the microaerobic conditions used for assessment of AR in E. coli. The role of the arginine decarboxylase pathway in Salmonella AR was shown by the loss of AR in mutants lacking adiA, which encodes arginine decarboxylase; adiC, which encodes the arginine-agmatine antiporter; or adiY, which encodes an AraC-like regulator. Transcription of adiA and adiC was found to be dependent on AdiY, anaerobiosis, and acidic pH.
PMCID: PMC1540025  PMID: 16855258
19.  Characterization of an Escherichia coli O157:H7 Plasmid O157 Deletion Mutant and Its Survival and Persistence in Cattle▿  
Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic ΔpO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the ΔpO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the ΔpO157 mutant. The ΔpO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the ΔpO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the ΔpO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed ∼50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.
PMCID: PMC1855633  PMID: 17277224
20.  The Lysine Decarboxylase CadA Protects Escherichia coli Starved of Phosphate against Fermentation Acids▿ †  
Journal of Bacteriology  2007;189(6):2249-2261.
Conflicting results have been reported for the rate and extent of cell death during a prolonged stationary phase. It is shown here that the viability of wild-type cells (MG1655) could decrease ≥108-fold between days 1 and 14 and between days 1 and 6 of incubation under aerobic and anaerobic phosphate (Pi) starvation conditions, respectively, whereas the cell viability decreased moderately under ammonium and glucose starvation conditions. Several lines of evidence indicated that the loss of viability of Pi-starved cells resulted primarily from the catabolism of glucose into organic acids through pyruvate oxidase (PoxB) and pyruvate-formate lyase (PflB) under aerobic and anaerobic conditions, respectively. Weak organic acids that are excreted into the medium can reenter the cell and dissociate into protons and anions, thereby triggering cell death. However, Pi-starved cells were efficiently protected by the activity of the inducible GadABC glutamate-dependent acid resistance system. Glutamate decarboxylation consumes one proton, which contributes to the internal pH homeostasis, and removes one intracellular negative charge, which might compensate for the accumulated weak acid anions. Unexpectedly, the tolerance of Pi-starved cells to fermentation acids was markedly increased as a result of the activity of the inducible CadBA lysine-dependent acid resistance system that consumes one proton and produces the diamine cadaverine. CadA plays a key role in the defense of Salmonella at pH 3 but was thought to be ineffective in Escherichia coli since the protection of E. coli challenged at pH 2.5 by lysine is much weaker than the protection by glutamate. CadA activity was favored in Pi-starved cells probably because weak organic acids slowly reenter cells fermenting glucose. Since the environmental conditions that trigger the death of Pi-starved cells are strikingly similar to the conditions that are thought to prevail in the human colon (i.e., a combination of low levels of Pi and oxygen and high levels of carbohydrates, inducing the microbiota to excrete high levels of organic acids), it is tempting to speculate that E. coli can survive in the gut because of the activity of the GadABC and CadBA glutamate- and lysine-dependent acid resistance systems.
PMCID: PMC1899392  PMID: 17209032
21.  Functional Heterogeneity of RpoS in Stress Tolerance of Enterohemorrhagic Escherichia coli Strains 
The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher β-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.
PMCID: PMC1489321  PMID: 16820496
22.  Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase. 
Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.
PMCID: PMC168131  PMID: 8795225
23.  Functional Genomics Enables Identification of Genes of the Arginine Transaminase Pathway in Pseudomonas aeruginosa▿  
Journal of Bacteriology  2007;189(11):3945-3953.
Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of the metabolic versatility of this organism. To identify genes involved in arginine catabolism, we have employed DNA microarrays to analyze the transcriptional profiles of this organism in response to l-arginine. While most of the genes involved in arginine uptake, regulation, and metabolism have been identified as members of the ArgR (arginine-responsive regulatory protein) regulon in our previous study, they did not include any genes of the arginine dehydrogenase (ADH) pathway. In this study, 18 putative transcriptional units of 38 genes, including the two known genes of the ADH pathway, kauB and gbuA, were found to be inducible by exogenous l-arginine in the absence of ArgR. To identify the missing genes that encode enzymes for the initial steps of the ADH pathway, the potential physiological functions of those candidate genes in arginine utilization were studied by growth phenotype analysis of knockout mutants. Expression of these genes was induced by l-arginine in an aruF mutant strain devoid of a functional arginine succinyltransferase pathway, the major route of arginine utilization. Disruption of dadA, a putative catabolic alanine dehydrogenase-encoding gene, in the aruF mutant produced no growth on l-arginine, suggesting the involvement of l-alanine in arginine catabolism. This hypothesis was further supported by the detection of an l-arginine-inducible arginine:pyruvate transaminase activity in the aruF mutant. Knockout of aruH and aruI, which encode an arginine:pyruvate transaminase and a 2-ketoarginine decarboxylase in an operon, also abolished the ability of the aruF mutant to grow on l-arginine. The results of high-performance liquid chromatography analysis demonstrated consumption of 2-ketoarginine and suggested that generation of 4-guanidinobutyraldehyde occurred in the aruF mutant but not in the aruF aruI mutant. These results led us to propose the arginine transaminase pathway that removes the α-amino group of l-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the arginine transaminase pathway. This work depicted a wider network of arginine metabolism than we previously recognized.
PMCID: PMC1913404  PMID: 17416670
24.  Acid- and Base-Induced Proteins during Aerobic and Anaerobic Growth of Escherichia coli Revealed by Two-Dimensional Gel Electrophoresis 
Journal of Bacteriology  1999;181(7):2209-2216.
Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.
PMCID: PMC93635  PMID: 10094700
25.  Sigma Factor N, Liaison to an ntrC and rpoS Dependent Regulatory Pathway Controlling Acid Resistance and the LEE in Enterohemorrhagic Escherichia coli 
PLoS ONE  2012;7(9):e46288.
Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σN), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σN-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σS) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σS was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σN allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σS stability, gadE, ler or GDAR, indicating a dependence on transcription from a σN promoter(s) and not RNAP competition for the phenotype. Among a library of σN enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σS stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σN regulate GDAR and LEE expression through downregulation of σS at the post-translational level; likely by altering σS stability or activity. The regulatory interplay between NtrC, other EBPs, and σN–σS, represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.
PMCID: PMC3459932  PMID: 23029465

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