Journal of Bacteriology, September 2004, p . 6032-6041, Vol . 186, No . 18

Escherichia coli Glutamate- and Arginine-Dependent Acid Resistance Systems Increase Internal pH and Reverse Transmembrane Potential

Hope Richard and John W . Foster^*

Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama

Received 20 February 2004/ Accepted 22 June 2004

 
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 abouthow these systems protect cells from acid stress . AR2 and AR3are thought to consume intracellular protons through amino aciddecarboxylation . Antiport mechanisms then exchange decarboxylationproducts for new amino acid substrates . This form of protonconsumption could maintain an internal pH [pH_i] conducive tocell survival . The model was tested by estimating the pH_i andtransmembrane potential [] of cells acid stressed at pH 2.5.During acid challenge, glutamate- and arginine-dependent systemselevated pH_i from 3.6 to 4.2 and 4.7, respectively . However,when pH_i was manipulated to 4.0 in the presence or absence ofglutamate, only cultures challenged in the presence of glutamatesurvived, indicating that a physiological parameter aside frompH_i was also important . Measurements of indicated that aminoacid-dependent acid resistance systems help convert membranepotential from an inside negative to inside positive charge,an established acidophile strategy used to survive extreme acidicenvironments . Thus, reversing may be a more important acidresistance strategy than maintaining a specific pH_i value.

 
Enteric organisms that colonize and cause disease in the human intestine must first endure a transient but extreme acid challengein the stomach . The normal human stomach presents an antimicrobialacid environment averaging pH 2, with an emptying time of approximately2 h [53] . As a result, acid-sensitive pathogens like Vibrio cholerae must be ingested in massive numbers [10 to 100 million] to increase the possibility that some will survive and enter the intestine . Other microbes, such as Escherichia coli and Shigella, can colonize or cause disease even when small numbers of cells [10 to 100] are ingested . These resilient microbesare equipped with potent acid resistance systems able to withstandpH 2 challenge for at least 2 h [11, 31, 32, 52] . In fact, E.coli possesses a level of acid resistance rivaling that of thegastric pathogen Helicobacter pylori [39, 45, 50, 59].

It has now been shown that E . coli uses four inducible acid resistance systems to survive extreme acid environments . Acid resistance system 1 [AR1], also referred to as the oxidativeor glucose-repressed system, is acid induced in stationary phase.Its expression requires the alternative sigma factor RpoS andthe cyclic AMP receptor protein CRP [11] . However, the structural components of AR1 as well as the mechanism by which it protects are still unknown . The second AR system, AR2, requires extracellular glutamate to work at pH 2.0 and is induced upon entry into stationary phase or by log-phase growth in acidic minimal medium [10]. Known components of glutamate-dependent acid resistance include two isoforms of glutamate decarboxylase [GadA and GadB] anda putative glutamate:-aminobutyric acid [GABA] antiporter calledGadC [11, 12, 19, 33, 46] . The third system, AR3, is similarto AR2 except that AR3 only protects cells if extracellulararginine is present . AR3 is induced by low pH under anaerobicconditions and has only been demonstrated following growth incomplex media . This arginine-dependent system is composed ofthe acid-inducible arginine decarboxylase AdiA and the AdiCantiporter, which exchanges extracellular arginine for the intracellularend product of decarboxylation, agmatine [11, 15, 22, 31] . Thelast AR system was recently described as lysine dependent andprobably involves the inducible lysine decarboxylase [22].

Although it is clear that these systems protect E . coli during transient exposure to pH 2, how they actually function has been subject to speculation . It is believed that AR2 and AR3 protectthe cell from acid stress by consuming intracellular protonsduring each decarboxylation reaction [11, 14] . The siphoningoff of intracellular protons was proposed to enhance pH homeostasisand allow the cell to maintain an internal pH compatible withviability . This model suggests that a specific internal pH may be crucial for survival during exposure to extreme acid stress. If the cell's internal pH fell below that point, it would succumb. The data obtained in the present study indicate that maintenanceof a specific internal pH may not be paramount to cell survival.Survival may depend on E . coli taking an approach used by acidophiles, which is to reverse the electrical membrane potential [] inthe presence of extreme low pH . Converting membrane potential from negative inside to positive inside can repel protons and mitigate the excess proton motive force [PMF] that can form.

 
Bacterial strains and culture conditions. The strains used in this study included EK227, wild-type K-12;EK592, wild-type MG1655; EK590, clcB clcA [derived from MG1655[24]]; EF333, gadC::Tn10 [3]; EF522, gadA::pRR10 [AP] gadB::Tn10 [3]; and EF996, uncB-C ilv::Tn10 [derived from EK227] . Mediaincluded Luria-Bertani broth [LB] and brain heart infusion [BHI]medium containing 0.4% glucose [LBG and BHIG] . LB broth, whereindicated, was buffered with either 100 mM morpholinepropanesulfonicacid [MOPS; pH 8.0] or morpholineethanesulfonic acid [MES; pH5.0] . For internal pH measurements, these media also contained25 mM sucrose to block nonspecific binding of radiolabeled sucrose[see below] . Acid challenge medium was minimal EG [58] preparedat various pH values [adjusted with HCl] . For the reasons notedabove, EG also contained 25 mM sucrose when used for internalpH measurements . To test whether Na⁺- or K⁺-deficient medium was important, Milli-Q Ultrapure water was prepared at pH 2.5 either with or without 1 mM glutamic acid · HCl . In addition,a Na⁺- and K⁺-deficient medium [M63 K/Na-deficient] containing15 mM [NH₄]₂SO₄, 18 µM FeSO₄ · 7H₂O, 1 mM MgSO₄,and 0.2% glucose was used . All chemicals used were ultrapure[Sigma or Fluka] or Suprapur [EM] . Na⁺ and K⁺ measurements weremade using sodium and potassium ion-specific combination electrodes[Thermo-Orion] . Cultures were grown at 37°C with shakingat 220 rpm . Exponential-phase cultures were grown to an opticaldensity at 600 nm of 0.4 [2 x 10⁸ CFU/ml], while stationary-phasecultures were grown overnight [18 h].

Acid resistance assays. Acid resistance assays were performed as described previously[11] . Briefly, cells were grown overnight in LB MOPS and LBMES for AR1, LBG for AR2, or BHIG for AR3 . LBG was used whenstudying the glutamate-dependent system, but since argininedecarboxylase is not induced well in LBG, BHIG was used to inducethis system . Stationary-phase cultures were diluted 1:1,000into prewarmed pH 2.5 EG medium without amino acid supplementation[for AR1], with 1.6 mM glutamate [for AR2], or with 1.0 mM arginine[AR3] . At various time points, 10-µl aliquots were removedand serially diluted, and 10 µl of each dilution was platedon LB plates . CFU were determined, and percent survival wascalculated relative to time zero.

Internal pH measurements. Internal pH was estimated by measuring the distribution of aweak acid [radiolabeled salicylic acid] across the membrane[4, 8, 23] . Salicylate has been used by us and others as a reliable indicator of internal pH [13, 24, 26, 34, 47] . Control cultureswere grown to log phase [2 x 10⁸ CFU per ml] or stationary phase[10⁹CFU per ml] in LBG containing 25 mM sucrose [final pH attime of assay, 6.9] . Sucrose was added to prevent nonspecificbinding of radiolabeled sucrose, used later for water spacemeasurements . Cultures to test decarboxylase-dependent effectson internal pH were grown overnight to stationary phase in LBGcontaining 25 mM sucrose for AR2 measurements or in BHIG containing25 mM sucrose for AR3 measurements . Cultures were then harvestedby centrifugation and resuspended in pH 7 EG medium containingno additions or in pH 2.5 EG medium with and without 40 mM glutamateor arginine . Final cell density after resuspension was 6 x 10⁹ CFU/ml . Two reactions were required for each assay . A totalof 2,000 to 3,000 dpm of ³H₂O/µl [0.1 µCi/µl]was added to each reaction mixture . Next, 2,000 to 3,000 dpmof [¹⁴C]salicylate [56.5 mCi/mmol] was added to one tube, andthe same amount of [¹⁴C]sucrose [462 mCi/mmol] was added tothe other at time zero . At specific times [30 or 60 min], 5µl was taken from each tube for a direct isotope count,and 100-µl aliquots were centrifuged through equal amounts of dibutylphthalate [50 µl] and silicone oil [50 µl]to separate the supernatant from the cell pellet . The amountof [¹⁴C]sucrose, which does not penetrate the cytoplasmic membrane,was used to determine the extracellular and periplasmic waterspace remaining after centrifugation . Disintegrations per minutefor [¹⁴C]salicylate and ³H₂O were then used to determine the internal pH value by the following equation: pH_i = log{[[A_in]/[A_out]] [10^pKa + 10^pH_out] – 10^pK_a}, where [A] is a measure ofsalicylic acid and the pK_a is 3.0.

measurements. was measured using radiolabeled lipophilic anions or cations[4, 23] . Cells were grown as for internal pH measurements . Log-phaseand stationary-phase cultures were harvested by centrifugationand resuspended in pH 2.5 EG medium containing 40 mM glutamateor 40 mM arginine . Final cell densities were approximately 6x 10⁹ CFU per ml . Each measurement involved two assays, onefor charge distribution and one to determine water space . Thegeneral methodology was similar to that used to estimate internalpH . For charge distribution, 1,200 dpm of [¹⁴C]tetraphenylphosphoniumbromide [TPP⁺; 5 mCi/mmol] or potassium [¹⁴C]thiocyanate [S¹⁴CN^–; 54 mCi/mmol]/µl was added to one tube along with 2,000to 3,000 dpm of ³H₂O . Extracellular and intracellular water spaces were determined as above . Extracellular water space was used to correct for the carryover of extracellular radiolabeled lipophilic ion not removed during centrifugation . At 30 min,5 µl was taken for total direct counts and 100 µlwas centrifuged through dibutylphthalate-silicone oil . The accumulationsof [¹⁴C]TPP⁺ [or S¹⁴CN^–] and ³H₂O in cell pellets wereused to determine [23] by the following equation: = RT/zF· ln[[A_out]/[A_in]], where R is the gas constant [8.28J/K · mol], T is temperature [310.15 K], z is the chargeof the molecule [+ or –], and F is the Faraday constant[96,485 J/V · mol].

Similar assays were done with butanol-treated cells [20% butanol] to determine nonspecific binding, which was subtracted as background from the experimental results . Background counts were no morethan 10% of experimental counts.

Whole-cell decarboxylation and antiport measurements. Transport and conversion of [³H]arginine and [³H]glutamate to[³H]agmatine or [³H]-aminobutyric acid, respectively, and thesubsequent end product efflux were measured using intact andTriton X-100-permeabilized cells . Wild-type and gadA/B and gadCmutant cells were grown for 22 h in 3 ml of BHIG [for argininedecarboxylation] or LBG [for glutamate decarboxylation] . Cellswere harvested by centrifugation, washed twice with EG medium[pH 7.0], and resuspended to 10⁸ cells/ml in 3.0 ml of prewarmedEG medium adjusted to pH 2.5 with HCl or to other pH valuesas indicated . The medium contained 1.0 mM arginine, including4 µCi of [³H]arginine [61 Ci/mmol] per ml or a final concentrationof 0.4 mM glutamate including 22,000 dpm of [³H]glutamate/µl.At timed intervals, 500-µl aliquots of cell-free supernatantswere obtained by filtration [0.45-µm-pore-size filters]and adjusted to pH 7.5 with 5 N NaOH, and 30-µl sampleswere subjected to paper chromatographic separation as describedpreviously [29] . Marked bands were cut and counted for radioactivity.Percent conversion was calculated from total radioactivity oneach strip.

Western blot analysis and cellular location of GadC. Cells were grown overnight in 50 ml of EG pH 7.7 and EG pH 5.5.The cells were harvested by centrifugation at 4,500 x g for10 min [4°C], resuspended in 5 ml of cold 10 mM HEPES buffer[pH 7.4], and passed twice through a French pressure cell [Aminco]at 16,000 lb/in² . Crude extracts were cleared of cell debrisby centrifugation at 4,500 x g for 10 min [two times] . The resultingcleared supernatant was ultracentrifuged at 90,000 x g [4°C]to separate membrane and soluble fractions . Membrane pellets were washed with 2.0 ml of 10 mM HEPES buffer [pH 7.4] to remove residual soluble proteins and resuspended in 300 µl ofthe same buffer . Soluble fractions were also centrifuged at90,000 x g to remove residual membrane . Protein concentrationswere measured using the Bio-Rad protein assay reagent.

Western blot analysis of these fractions was carried out using rabbit anti-GadC antibodies raised against a GadC-specific peptide [N'-CRARSPHYIVMNDKKH] by Genemed Synthesis, Inc . Membrane andsoluble fractions [3 µg of protein] were separated on10% polyacrylamide-sodium dodecyl sulfate gels [30] . Proteins were transferred to Immobilon-P [polyvinylidene difluoride] membranes with a Semiphore transfer cell [Hoefer Scientific]at 100 mA for 2 h . The membranes were blocked with 5% nonfatmilk in Tris-buffered saline [10 mM Tris [pH 8], 150 mM NaCl]containing 0.05% Tween 20 and incubated with rabbit primary[1:8,000] and mouse anti-rabbit secondary [1:8,000] antibodiesfor 1 h at room temperature . The blot was developed with ECLdetection reagents [Amersham Pharmacia Biotech].

 
The role of Cl^– transporters and the F0/F1 proton-translocating ATPase in acid resistance. The goal of this study was to further define how the amino acid-dependentAR systems protect against acid stress . We initially wantedto determine how similar these systems were to the amino acid-independentsystem AR1, whose mechanism is also a mystery . An elegant studyby Iyer et al . previously demonstrated that Cl^– transportersencoded by the clc genes of E . coli were important for AR2 andAR3 function [1, 21] . However, their potential role in system1 was not explored . Consequently, we examined whether the Clctransporters also contributed to AR1.

Wild-type and clcA/B cells were grown to stationary phase inLB MES pH 5.5, an inducing condition for AR1, and then testedfor survival in pH 2.5 minimal medium without amino acid supplementation[Fig . 1A] . Both strains exhibited normal AR1 survival, indicatingthat the Clc Cl^– transporters are not required for AR1.Our results also confirmed that the clc transporters are notessential but are important to both AR2 [Fig . 1B] and AR3 [data not shown] function . These data indicate that a fundamental mechanistic difference exists between the amino acid-dependentand -independent acid resistance mechanisms.

 
Another potential contributor to acid resistance is the F0/F1H⁺-translocating ATPase . The F0/F1 ATPase is a well-establishedmover of protons across the cell membrane . This complex couplesthe energy released as protons move into the cell to the generationof ATP from ADP and P_i [56] . The ATPase can also function inthe opposite direction, hydrolyzing ATP to pump protons outof the cell [28, 60] . Because the basic problem of acid stressis thought to be cytoplasmic acidification, a H⁺ pumping system like the F0/F1 ATPase has the potential to aid in resistanceto extreme acid stress [26] . To explore this possibility, each of the three acid resistance systems was tested for its dependence on the proton-translocating ATPase by comparing the pH 2.5 resistance of an atp mutant strain to that of the wild type . The data presentedin Fig . 2A reveal that the ATPase was important for the protectionprovided by AR1 . However, AR1 was not totally dependent on theATPase, since residual survival was seen in the atp mutant.In contrast, the ATPase was not needed for AR2 or AR3 to functionproperly and did not function as a proton extruder in this situation.[Fig . 2B and C] . These results and the chloride transporterresults revealed fundamental differences between amino acid-dependentand -independent acid resistance mechanisms.

 
These results also addressed another question . Some amino acid decarboxylation reactions in other organisms are coupled toATP synthesis carried out by the F0/F1 proton-translocatingATPase [2, 40, 44, 49] . Knowing that the ATPase is not requiredfor systems 2 and 3 indicates that any effect systems 2 or 3may have on internal pH is most likely due to the glutamateand arginine decarboxylation reactions themselves and not dueto an indirect effect on ATP synthesis.

AR2 and AR3 help acid-stressed cells maintain an elevated internal pH. We then examined if the amino acid-dependent systems [AR2 and AR3] contribute to internal pH homeostasis as predicted . Internal pH measurements were made on cells suspended at various external pH values [Table 1] . Control experiments using pH 7-grown exponential-phasecells gave an internal pH value [pH 7.8] comparable to thatin previous reports [51, 57] . Cells grown at pH 7 to stationaryphase had an internal pH of 7.6.

 
When stationary-phase cells were examined during acid stressat external pH 2.5, differences in internal pH were observeddepending on whether the cells were challenged in the presenceor absence of glutamate or arginine . In the absence of aminoacid supplementation, the internal pH was 3.6 . When glutamateor arginine was added, internal pH rose to pH 4.2 and 4.7, respectively[Table 1] . These results indicate that AR2 and AR3 do elevateinternal pH . However, the internal pH achieved was lower thananticipated . Based on studies with Salmonella, we expected thatinternal pH levels lower than pH 5.5 would be lethal [13, 43].The internal pH gained by the arginine-dependent system was higher than with the glutamate-dependent system, yet survival was somewhat lower . The difference in internal pH attained bythese different systems suggested that maintenance of internalpH is not the only element contributing to survival . AR1 andthe lysine-dependent system were not tested for effects on internalpH because viability could not be maintained above 10% overthe course of the experiments.

Internal pH levels achieved during acid stress correlate to the pH of maximal glutamate or arginine decarboxylation. Since the internal pH measured in acid-resistant cells was lowerthan anticipated, we sought another method to confirm the readings.One approach used various forms of green fluorescent protein,whose fluorescence level changes with pH . The results indicatedthat the internal pH at external pH 2.5 was below the rangeof detection with this method [pH 5] [data not shown] . Thissupported the earlier estimates . We then realized that the reportedpH optima of the decarboxylases [pH 4 and 5 for glutamate andarginine decarboxylases, respectively] seemed remarkably similarto the internal pH measurements . If this correlation held, onewould predict that decarboxylation by intact cells should bemaximal at external pH 2.5, at which internal pH [pH 4 to 5]was near the pH optima of the decarboxylases.

Before determining the pH at which intact cells maximally converted glutamate to GABA, we established that GadC serves as the antiporter for glutamate-dependent acid resistance . Previous work fromour laboratory revealed that AdiC was the antiporter for the arginine-dependent acid resistance system [11, 15, 31] . GadC,however, is only presumed to be the glutamate:GABA antiporter,based on computer sequence analysis . To provide evidence thatGadC is the antiporter, we first established that this proteinwas membrane associated [Fig . 3A] and then demonstrated thatgadC mutants would only decarboxylate glutamic acid if TritonX-100 were used to solubilize the membrane and bypass the transportrequirement [Fig . 3B].

 
We then measured the extracellular pH at which intact cellscarried out maximal transport and conversion of glutamate toGABA [or arginine to agmatine] and compared those values towhat occurred with Triton X-100-permeabilized cells, where internalpH approximates the external pH . Conversion of substrate toproduct was determined using equal amounts of protein from intactand permeabilized cells, equal amounts of substrate, and equalreaction times as described in Materials and Methods . The decarboxylationof glutamate to GABA in permeabilized cells occurred maximallybetween pH 4.4 and 5 [Fig. 4B] . Under similar conditions, maximalconversion of arginine to agmatine took place between pH 5 and5.5 [Fig. 4C] . These values were in agreement with published reports of pH optima using cell extracts [5, 55] . However, thesituation was very different using intact cells, where pH homeostasismechanisms such as amino acid decarboxylation operate to alkalinizeinternal pH relative to external pH . Under these conditions,maximal conversion for both systems occurred around pH 2.5,as predicted [Fig . 4A] [11] . The results suggest that optimaldecarboxylation at external pH 2.5 by intact cells is consistentwith an internal pH between pH 4 and 5 and that the differentinternal pH values generated using glutamate versus argininemight reflect the different pH optima of the two decarboxylases.

 
Of course, for intact cells, the external pH allowing maximal conversion is factorial, reflecting separate pH optima of transport and decarboxylation . Although it has yet not been tested, itis possible that the antiporters only activate at external pH2.5 . Thus, the optimal pH of substrate-to-product conversionby intact cells may only reflect transport . However, once theamino acids enter the cell, it seems unlikely that the decarboxylasescould cause internal pH to rise much above their pH optima.

A specific internal pH value is not required for extreme acid stress survival. Taking into consideration that the internal pH values calculatedfor AR2 and AR3 were different from one another yet both systemsprotected cells from acid stress, an experiment was performed to determine if a specific internal pH was required for survival during acid stress . Cells were acid challenged with and without glutamate, but culture conditions were manipulated such thatthe internal pH of both cultures was equal . If a given pH wereall that was required for survival, then both cultures shouldsurvive equally well . To conduct this experiment, the externalpH values of the two cultures were adjusted independently sothat the internal pH values were equivalent over the time ofthe experiment [Fig . 5A] . When this was done, cells challengedin the presence of glutamate still survived better than cellschallenged without glutamate [Fig . 5B, 60 min] . This resultsupports the hypothesis that achieving a specific internal pHvalue is not the only goal of these acid resistance systems.Since cells challenged at pH 2.5 in the presence of glutamateor arginine still generated a greater pH than cells challengedwithout amino acid, maintenance of pH may be of greater importanceto survival than achieving a specific internal pH value [Fig.5B, inset].

 
Reversal of during survival under extreme acid stress. Clues as to how E . coli handles pH 2 acid stress could comefrom acidophiles, organisms that naturally live and grow underextreme acid stress . Acidophiles use a novel approach to copewith the rigors of low pH . Neutralophiles like E . coli maintainan electrochemical gradient [] with a negative inside chargeas part of generating PMF [16, 25] . However, the of acidophilesgrowing at low pH is reversed, consisting of an inside positivecharge [16, 35] . It has been proposed that this strategy helpsrepel protons and maintain a higher internal pH . In acidophiles,this is thought to be achieved by an array of transporters andion pumps that convert a negative to a positive under extremelyacidic conditions [20, 35, 37].

We hypothesized that E . coli might carry out a similar feat as a result of the decarboxylation and antiport process . Toaddress this possibility, values for E . coli under extremeacid stress conditions were determined . Log-phase cells at pH7 exhibited an expected negative [–86 mV] and a PMF of–140 mV [Table 1] . These values are somewhat lower thanin some other reports [–160 to –180 mV] due to differencesin growth media [LBG and BHIG here, versus minimal media], aeration,and the fact that these cells were fermenting rather than respiring.Stationary-phase cells at pH 7 had a somewhat lower [–52mV] and a PMF of approximately –100 mV . However, under extreme acid stress at pH 2.5, E . coli changed its normally negative to a positive when glutamate or arginine was present[Table 1] . Cells challenged at pH 2.5 for 30 min in the absenceof glutamate or arginine had a near zero . In contrast, theaddition of glutamate or arginine reversed the membrane potential,and became positive inside [+30 for glutamate and +80 for arginine]. Confirmation of this reversal was obtained using TPP⁺, which was excluded from cells when the glutamate decarboxylase system was functioning [data not shown] . The production of a positive in acid-stressed, living E . coli was a surprising finding,but it may explain the survival characteristics of the organism.

There has been controversy over similar studies performed using H . pylori, another neutralophilic organism that can survive extreme acid pH [36, 54] . One report indicated that H . pyloriinverts during extreme acid stress, similar to acidophilesand consistent with our results in E . coli [36] . The second report found that the urease this organism produces will elevate internal pH when external pH is 1.2, but in contrast to acidophiles, they did not find Helicobacter inverted transmembrane electrical potential [54] . In fact, the PMF measured was as high as –254mV . The first study was criticized by the latter one for not taking into account nonspecific binding of radioactive probes. The results presented here with butanol-treated E . coli clearly accounted for nonspecific binding and still measured a positive inside electrical potential.

Neither potassium nor sodium ions are required for glutamate- or arginine-dependent acid resistance mechanisms. Knowing that E . coli reverses its transmembrane potential raisesthe question of the source of the positive charges leading topositive inside . Two obvious candidates would be sodium orpotassium ions, known to contribute to pH homeostasis underother circumstances [23, 41, 42] . To address this question, cultures were grown to stationary phase in EG to induce the glutamate decarboxylase system . Cells were washed several timesin MC buffer [10 mM MgCl₂ and 5 mM CaCl₂] to remove most residualNa⁺ and K⁺ . The washed cells were then added to pH 2.5 water[Milli-Q Ultrapure] containing 1 mM glutamic acid · HClor to M63 media containing different amounts of both ions . Therewere no significant survival differences in pH 2.5 media containingK⁺ concentrations ranging from 10 µM to 100 mM or Na⁺ concentrations ranging from 100 µM to 100 mM . After 2 h at pH 2.5, survival was maintained between 50 and 80% regardless of the Na⁺ or K⁺ concentration [data not shown] . The resultssuggest that neither Na⁺ nor K⁺ ions were required for the systemto work . The low intracellular pH [approximately 4.5] wouldlikely prevent many, if not all, other housekeeping ion movementmechanisms from working efficiently . Thus, the accumulationof the positively charged decarboxylation product most likelyaccounts for the reversal of transmembrane potential.

 
E . coli is a remarkably acid-resistant neutralophilic organism that prefers growth near neutral pH but is able to withstand transient exposures to pH 2 environments for hours . Four systems contribute to this acid resistance . The two most robust systemsuse glutamate and arginine decarboxylases . Transcriptional controls regulating the synthesis of these systems have been heavilystudied; however, the mechanisms by which they provide acidresistance have not been established . The prevailing hypothesishas been that protons entering the cell are consumed by thedecarboxylase reaction via exchange with the amino acid -carboxyl group . The decarboxylated product is returned to the exterior by antiport in exchange for more substrate [38] . The ultimate goal of this cycle would be to raise internal pH to a level that protects sensitive cell constituents . Surprisingly, Na⁺:H⁺ and potassium:H⁺ antiport systems, thought to be important for pH homeostasis under pH conditions more suited for growth, appeared unimportant for survival under extreme acid stress [6, 7, 9,27, 61] . The results of this work indicate that the amino acid-dependentAR systems increase internal pH and reverse transmembrane potential.Whether one feature is more important than the other is notknown.

The CO₂ that evolved as a result of decarboxylation did not appear to contribute to pH homeostasis at this acid extreme. Carbonic anhydrase essentially adds water, not free protons,to CO₂ to make carbonic acid [H₂CO₃]; carbonic acid can then dissociate to HCO₃^– and H⁺ . Because the pK_a of this reactionis 6.5, an internal pH that is estimated to be between 4.2 and4.7 will prevent this dissociation [18] . Another reason carbonicanhydrase probably does not contribute to pH homeostasis atthis extreme is that the protein has an alkaline pH optimum,making it unlikely that the enzymatic formation of carbonic acid will occur at pH 4 to 5 . However, the system might contribute to pH homeostasis in the vicinity of the pK_a of carbonic acid.

Although glutamate and arginine decarboxylases were shown toraise internal pH at external pH 2.5, the cytoplasm remainedremarkably acidic [pH 4.2 to 4.7] . Ordinarily, E . coli prefersto keep internal pH slightly alkaline [pH 7.8] during growth.This surprising finding was supported by two other results.First, besides using the radiolabel distribution assay, effortsto use green fluorescent protein derivatives for internal pHmeasurements indicated that the internal pH during pH 2.5 acidstress was below pH 5, the limit of detection by this method.Second, if internal pH were considerably higher than our measurementsindicated, then other amino acid decarboxylases with higherpH optima, such as ornithine decarboxylase [pH optimum 7.0],might be expected to function as effective acid resistance systems.However, ornithine decarboxylase does not protect cells at pH2.5, a finding we attribute to the high pH optimum limitingenzymatic function at the acidic internal pH reported here [3, 22] . The situation with lysine decarboxylase [CadA] furthersupports the hypothesis . This enzyme has a pH optimum of pH 5.7, which is more acidic than ornithine decarboxylase but less acidic than the arginine or glutamate enzymes [48] . Consistentwith its intermediate pH optimum, there is a lysine-dependentacid resistance mechanism, but it is much less effective thanthe glutamate or arginine systems [22].

A major advance in our understanding of acid resistance camewith the discovery that the chloride transport proteins of E.coli [ClcA and ClcB] are important [but not essential] to acidresistance [21] . In their discussion, Iyer et al . proposed that at pH 2.5, protons cross the membrane and enter the cell inthe form of uncharged HCl molecules that dissociate intracellularlyinto H⁺ and Cl^– . At the high KCl concentration used in their experiments [40 mM], that is certainly possible . The intracellular protons are then consumed by decarboxylation, and the excessCl^– [or other anions] is thought to be removed via theClc chloride transporters, which are thought to be channels.They proposed that these "channels" would provide an electricalshunt to prevent excessive inner membrane polarization predictedto occur, in the case of the arginine-dependent system, duringthe exchange of the intracellular decarboxylation product agmatine[+2] for the extracellular substrate arginine [+1] . In theirmodel, excessive charge, negative inside, would build in theabsence of the Clc channels . Cl^– exit through the channelswould prevent the cell interior from becoming too negativelycharged . Although not explicitly stated, their model impliedthat when all the systems are functioning, the resulting wouldstill be negative inside when external pH is 2.5 . The discovery that the Clc products are actually H⁺:Cl^– antiporterssuggests that the original model of Iyer et al . requires revision[1, 21].

We have carried out the internal pH and measurements neededto test the model, and we found that E . coli does not hyperpolarizebut reverses membrane potential during acid stress, mimickingthe strategy of an acidophile . We propose that the reversalof transmembrane charge by AR2 and AR3 is the direct result of proton influx combined with the production of positively charged decarboxylation products [GABA or agmatine] inside thecell . At low Cl^– ion concentrations [no added KCl], itis generally perceived that protons, under pH 2.5 acidic conditions, enter the cell without an associated chloride ion [Fig . 6]. If true, then proton intrusion alone would add considerablyto the positive charges inside the cell . For instance, whenthe external pH is 2.5, internal pH moves from roughly 7.5 to4.5, meaning the number of protons inside the cell [startingat 10 protons per cell at pH 7.5] increases 1,000-fold [to approximately10,000 protons per cell] . This may be enough to dissipate thenormally negative interior charge of stationary-phase cells.Movement of about 10,000 H⁺ ions across the membrane can changethe calculated by 60 mV [17] . Of course, more than 10,000 protons must enter the cell to lower pH, because many protons becomebuffered by cell constituents, which can also add to the positivecharge.

 
At steady state in the presence of glutamate or arginine, additional protons that enter are effectively consumed by the decarboxylation reaction, which keeps internal pH near 4.5 . This consumptionremoves the proton from pH consideration, but the associatedpositive charge remains in the end product [GABA or agmatine].Although the charge is eventually removed by what is probablyelectrogenic antiport, there must always be a pool of end productin the cell to continuously drive antiport . Thus, the intracellularpool of agmatine at any one instant could be as high as halfof the original intracellular arginine pool [approximately 5to 10 mM] . This scenario is consistent with our findings.

The precise role of the Clc antiporters in this system remains unclear . Eliminating the antiporters clearly impairs acid resistance, whether high-Cl^– or low-Cl^– media are used [15] [Fig . 1B] . It would make sense if these antiporters allowedentry of Cl^– ions to counter the positive inside producedas a result of proton influx and decarboxylation, so that whenthe inside charge is positive, the Clc antiporters could allow entry of negative charges and prevent membrane hyperpolarization in the direction opposite to that predicted by Iyer et al . [21]. This would also allow excess H⁺ ions to be expelled from the cytoplasm in exchange for Cl^–.

Calculations by Iyer et al . established the conversion rateof glutamate to GABA to be approximately three times that ofarginine to agmatine within minutes of exposure to pH 2.5 [10⁴ versus 3 x 10³ molecules per min per cell, respectively] [21].This does not contradict the differences we observed in the measurements, where the arginine system generated 2.7 timesmore positive charge than glutamate . The apparent contradictioncan be resolved by considering the different ionizable groupson glutamate and arginine.

The -carboxyl groups of both glutamate and arginine have pK_avalues of 2.1 and, thus, they will be less than 50% protonatedat external pH 2.5 . Upon entering what is probably a less-acidicGadC antiporter channel, we predict the remaining H⁺ will bereleased to the periplasm, and so the group on both amino acidswould enter as COO^– . However, glutamate, but not arginine,also has a side chain carboxyl group with a dissociation constantof 4.3 . If the pH of the mouth of the antiporter is near externalpH 2.5 [pH 3 for instance], then this carboxyl group will bemostly protonated as it enters the cell . Once inside the cytoplasm,about half of those side chain protons will dissociate at thehigher internal pH [4.2] and then be consumed again during decarboxylationto form GABA . The result would be a futile proton cycle as wellas an electroneutral conversion . Thus, the higher reported conversionof glutamate to GABA would not generate as much internal positivecharge as the slower arginine system.

How might the cell recover from an inverted ? In our model,as acid stress is reduced [i.e., as external pH becomes lessacidic] proton intrusion stops, but the decarboxylases continue to remove internal protons and allow internal pH to rise . The excess positive charges leading to the inverted are now inthe form of decarboxylation products that can be removed bythe antiporters . In the case of arginine and agmatine, a +2 is extruded in exchange for a +1 charge . As long as no moreH⁺ flows into the cell, this exchange combined with the importof anions by the Clc antiporters [or other means] are predictedto restore a negative inside electrical potential . Finally,normal homeostatic mechanisms take over, and the cell can resumegrowing.

How might an inside positive membrane potential aid survivalat extreme acid pH? One hypothesis is that converting membranepotential from negative inside to positive inside may be a wayto repel protons . While it is true that internal pH falls to4.2 to 4.7, the drop would be more severe without the repellingforce of a positive inside charge . However, in the experimentwhere internal pH was maintained at 4.0 with and without glutamate,the cells with glutamate survived better, arguing that the primaryrole of a positive would not be to repel protons to changeinternal pH . Alternatively, a positive might mitigate excessPMF that can form when pH is large.

An internal pH of 4.5 and a positive inside membrane potential would be disastrous to growing E . coli cells . We propose these conditions pose only minor problems to a cell under extremeacid stress . At external pH 2.5, the internal pH of the cellis so low that very few metabolic reactions can even take place.For instance, protein synthesis is undetectable under theseconditions, and the cells clearly are not growing . As a resultof metabolic stasis, the cell could also survive a relativelyshort period of reversed transmembrane potential . The overallresult may actually protect the cell from inadvertent, self-inflicteddamage.

In sum, the results presented indicate that even though E . coli cannot grow under acidophilic pH conditions, the organism may have learned to traverse the gastric acid barrier by adoptingpart of the acidophile survival strategy . The decarboxylasesystems may protect against severe acid stress in two ways.Consuming protons by decarboxylation would produce a less acidicinternal pH and generate an inside positive potential that couldhelp repel protons and/or prevent excessive PMF.

 
This work was supported by National Institutes of Health award R01-GM61147.

We thank Patricia Couling for help in preparing the manuscript.

 
* Corresponding author . Mailing address: Department of Microbiology and Immunology, University of South Alabama College of Medicine, 307 University Blvd., Mobile, AL 36688 . Phone: [251] 460-6323 . Fax: [251] 460-7931 . E-mail: fosterj@sungcg.usouthal.edu .

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