JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 5129-5135 Vol. 173, No. 160021-9193/91/165129-07$02.00/0Copyright ©3 1991, American Society for Microbiology

Inducible pH Homeostasis and the Acid Tolerance Responseof Salmonella typhimuriumJOHN W. FOSTER* AND HOLLY K. HALL

Department of Microbiology and Immunology, College of Medicine,University of South Alabama, Mobile, Alabama 36688

Received 22 March 1991/Accepted 3 June 1991

The acid tolerance response (ATR) is an adaptive system triggered at external pH (pH.) values of 5.5 to 6.0that will protect cells from more severe acid stress (J. Foster and H. Hall, J. Bacteriol. 172:771-778, 1990).Correlations between the internal pH (pHi) of adapted versus unadapted cells at pH. of 3.3 indicate that theATR system produces an inducible pH-homeostatic function. This function serves to maintain the pH1 above 5to 5.5. Below this range, cells rapidly lose viability. Development of this pH homeostasis mechanism wassensitive to protein synthesis inhibitors and operated only to augment the pHi at pH. values below 4. Incontrast, classical constitutive pH homeostasis was insensitive to protein synthesis inhibitors and was efficientonly at pH. values above 4. Physiological studies indicated an important role for the Mg2+-dependentproton-translocating ATPase in affording ATR-associated survival during exposure to severe acid challenges.Along with being acid intolerant, cells deficient in this ATPase did not exhibit inducible pH homeostasis. Wespeculate that adaptive acid tolerance is important to Salmonella species in surviving acid encounters in boththe environment and the infected host.

pH homeostasis is the process whereby a cell maintains arelatively constant intracellular pH (pHi) over a broad rangeof external (pH.) values. The basis for this phenomenon isthe apparent modulation of primary cellular proton pumps aswell as potassium/proton and sodium/proton antiport sys-tems (4, 18, 21). This process appears subject primarily toallosteric control (such as the pH set point for pump activa-tion) since, as we will show, pH homeostasis functionsnormally in the presence of protein synthesis inhibitors. Asfar as genetic control, the system can be referred to asconstitutive. However, a superimposing genetic responsewhich can further protect a cell from acid stress has beendiscovered in Salmonella typhimurium (10) and Escherichiacoli (12, 13). The process, referred to as the acid toleranceresponse (ATR), is triggered in Salmonella species at pHvalues between 6.0 and 5.5, but protects cells against muchstronger acid (pH 3.0 to 4.0) when nonadaptive pH homeo-stasis normally fails. The ATR system includes at least 18proteins as determined by two-dimensional polyacrylamidegel electrophoresis (PAGE) analysis (10, 26). Recently, thefur gene product (ferric uptake regulator) has been impli-cated as contributing a key regulatory function. Mutations inthis locus confer Atr- and acid-sensitive phenotypes as wellas prevent the expression of several acid-regulated genes(11). In addition, a role in the ATR for a functional H+-translocating ATPase, the product of the atp operon (for-merly unc), was suggested from genetic studies.One can envision a variety of mechanisms that could

protect the cell from extreme acid stress. These includeincreases in internal buffering capacity or proton extrusionrate, as well as a decreased membrane proton conductance.As an alternative, the cell could prevent and/or repair aciddamage as a result of lower pHi. We have studied each ofthese possibilities and have further examined survivors ofstrong acid stress in an effort to ascertain what has allowedthem to survive.

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains and cultural conditions. The bacterialstrains used were all derivatives of S. typhimurium LT2 andare listed in Table 1. Culture media included E mediumsupplemented with 0.4% glucose (27) and LB medium (6).Chloramphenicol, 2,4-dinitrophenol (DNP), and carboxylcyanide m-chlorophenylhydrazone (CCCP) were added asindicated for each experiment. N,N'-Dicyclohexylcarbodi-imide (DCCD) was used at 5 mM. This concentration wasused because Salmonella species are relatively resistant tothis compound, presumably owing to poor outer membranepermeability.Measurement of ATR. The procedure for observing the

adaptive ATR was presented in detail by Foster and Hall(10). Briefly, cultures were grown with shaking in minimal Eglucose broth under semiaerobic conditions to 108 cells perml (3 ml of medium in a culture tube [13 mm by 100 mm]).The medium pH was adjusted to 5.8 with HCI, and theculture was allowed to adapt for one doubling (approximate-ly 60 to 70 min). Unadapted cultures were grown directly to2 x 108 cells per ml at pH 7.7. At that point, the pH of bothcultures was adjusted to 3.30 and further incubated for 90min. Viable counts were taken at 0 and 90 min by dilution infresh minimal E medium and plating on LB agar. Thepercent viability was measured as follows: (CFU at 90min/CFU at time zero) x 100.

Genetic procedures. Transductions were performed withphage P22 as described earlier (1, 2, 25). Plasmid extractionsand other DNA manipulations were outlined previously (9).Measurement of pH,. The method for measuring the pH,

involved the distribution of radiolabeled weak acids or basesacross the cellular membrane. The procedure was derivedfrom those of Booth et al. (5) and Atkinson and Winkler (3).To determine intracellular water space, we grew cells inminimal medium containing 25 mM sucrose. At mid-logphase, 3 ml of cells was harvested and resuspended into 200,d of culture supernatant. 3H20 and ["4C]sucrose were eachadded to 3,000 dpm/,ul, a 5-,ul sample was removed for totalcounts, and the remainder was incubated at 37°C for 10 min

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TABLE 1. S. typhimurium strains used in this study

Strain Relevant genotype Source oror phenotype reference

JF316 pyrD95 zad::TnJOJF1638 aniG::Mu dJ AearA324 9JF1819 atr-J 10JF1892 atr-J atp-102::TnJO 10JF1930 atr-12 (ace) Acid survivorJF1949 atr-12 atp-102::TnJO JF1930 x SF342JF1955 icd-1 aniG::Mu dJ AearA324 Acid survivorJF1969 icd-1 aniG::Mu dJ AearA324/

pFW75(icd')JF1971 icd-6 Acid survivorJF1952 Aatp-1IpHF72(atp')JF1953 aniG::Mu dJ AearA atp-102::TnJO JF1638 x SF342JF2278 Ura- Acid survivorJF2280 atr-14 Acid survivorJF2283 pyrA Acid survivorJF2276 icd-8 Acid survivorSF342 atp-102::TnlO G. Ames

(reaction mix A). Following incubation, duplicate samples(100 RI) were centrifuged through 50 ,u of dibutyl phthalateand 50 [lI of silicon oil. The tubes were frozen at -70°C, andthe cell pellet was sliced from the tube. The pellet was placedfor 5 min in a minivial containing 100 p,l of 1% sodiumdodecyl sulfate (SDS). Scintillation fluid was added, and theseries of vials were counted in an LKB 1219 scintillationcounter. The total water per cell pellet was calculated as3H20 disintegrations per minute of the pellet divided by thetotal 3H20 per microliter of reaction mix. Extracellular H20was equal to the disintegrations per minute of [14C]sucrose inthe pellet divided by the total ['4C]sucrose per microliter ofreaction mix. Intracellular H20 was equal to total H20minus extracellular H20.An almost identical procedure was used to determine the

distribution of weak acids (['4C]benzoic acid or ['4C]salicylicacid) or base (['4C]methylamine). The difference with thismethod is that the reaction mix (200 pll) contained 3,000 dpmeach of 3H20 and ['4C]benzoic acid or methylamine per ,u.The formula used to calculate pH, was

total acid concentration inK 10pH o opK

pH, = log [total acid concentration out(l)p

Radiolabeled methylamine, benzoic acid, and salicylic acidwere used to measure pH, at pH. levels of 7.7, 4.0 to 7.0, and3.3 to 4.5, respectively. pH, measurements were madewithin 20 min of a pHo shift.Measurement of acid-induced damage to f8-galactosidase.

Cells constitutively synthesizing P-galactosidase were ad-justed to the pH indicated in Fig. 3A and solubilized by theSDS-chloroform procedure of Miller (20). At the timesindicated, the solution was neutralized and ,-galactosidaseactivity was measured.

RESULTSThe ATR augments pH homeostasis. The fundamental

question we must address is how adaptation to mild acidifi-cation protects the cell from subsequent extreme acid expo-sure at pH, 3.3. One possibility is that pH homeostasismechanisms are enhanced during adaptation, thereby allow-ing the cell to maintain the pH, in the viable range for a

longer period during severe acid stress. If this model iscorrect, one would expect to observe measurable pH, differ-

01.5_\CL~~~~~~~~~~~~~~~~a

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Exp ralp0t aDl~~~~~~~~~~~~~~~~~~~~~~~~(

0~~~~~~~~~~~8 7 6 5 4 3

External pHFIG. 1. Effects of pHi on the pHwof unadapted cells. Cells were

grown to 2 x 108 cells per ml in E medium plus glucose (pH 7.7), andthe medium pH was adjusted to the values indicated. After 20 mn ofequilibration, thepHl values were measured as described in Mate-rials and Methods. The accuracy of each measurement, done intriplicate, was ±0o.1 unit.

ences when comparing adapted with unadapted cells inacidic environments. To test this theory, we first monitoredpHi and ApH of unadapted cells at various pH. values byusing radiolabeled weak acids (benzoic acid [pK = 4.2] andsalicylic acid [pK = 3.0]) and a weak base (methylamine [pK= 10.16]) as outlined in Materials and Methods. pH1 valuesobtained for acid-shifted cells are somewhat lower thanpreviously reported by Hickey and Hirshfield (14) becausethe cultures were grown under the semiaerobic conditionsbest suited to ATR development. As shown in Fig. 1, the pH,decreased from 7.8 to 5.8 when the pH. was shifted from 7.5to 4.0. Concomitantly, the difference between pHo and pH1(ApH) increased from 0.3 to 1.8. We have recently shownthat unadapted cells that are shifted to pH. 4.0 retainviability at or near 100% for several hours (8). However,once pHo falls below 4.0, cells progressively lose viability asdetermined by viable plate count (8) and by vital stainingwith propidium iodide or acridine orange (data not shown).The reason for this loss of viability became evident when pH,was measured within 15 min of a shift to pH 3.3. During thisshift the pH, fell dramatically to 4.4 and ApH dropped from1.8 to approximately 1.0 (Fig. 1). Cell viability remainedabove 80% during this short time frame, indicating that thedecrease in pH, was not due to cell death.

If the process of adaptation affects pH homeostasis mech-anisms or buffering capacity, an increased (more alkaline)pH, should be evident when comparing adapted with un-adapted cells, at least under low-pH stress. Initial compari-sons between adapted and unadapted cells made at variouspHo values ranging from 7.5 to 4.0 revealed no differences inpH, (Table 2). However, a dramatic difference in pH, wasobserved when comparing adapted and unadapted cells atpHo 3.3. Within minutes, the pH, of unadapted cell culturesfell 0.5 to 0.9 pH unit lower than that of adapted cells (Table2). The data support the hypothesis that adaptation enablesthe cells to better maintain pH,. The ability of adapted cellsto maintain a less acidic pH, could explain the tolerance toexternal acid conferred by the ATR. This adaptive pH,maintenance most probably reflects an increase in pH ho-meostasis capability. Although there are alternative possi-

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INDUCIBLE pH HOMEOSTASIS 5131

TABLE 2. pHi of S. typhimuriuma

pHi" of:pH.

Unadapted cells Adapted cells

7.5 7.8 ± 0.15.8 7.1 ± 0.1 7.1 ± 0.15.0 6.4 ± 0.1 6.6 ± 0.14.5 6.1 ± 0.1 6.0 ± 0.14.0 5.8 0.1 5.8 0.13.3 4.4 ± 0.2 5.3 ± 0.1

a Radiolabeled benzoic acid assay or salicylic acid assay. Assayed at 15 minafter shift.

b Values are mean t range of variation for three experiments.

bilities, they are unlikely and will be addressed below. In ourmodel, the ATR system enhances pH homeostasis capacityonly below (not above) pH. 4.0.

Protein synthesis is required for ATR-enhanced homeosta-sis. The pH homeostasis mechanisms engaged above pHo 4function normally even in the absence of protein synthesis,as shown in Table 3. Unadapted cells shifted to pHo 4.2maintained a pHi of 5.9 in either the presence or absence ofchloramphenicol. We will refer to this form of pH homeo-stasis as constitutive. However, although pH homeostasisabove pHo 4.0 did not require de novo protein synthesis,ATR survival below pHo 4 was shown previously to requirede novo protein synthesis (10). Therefore, to establish a linkbetween inducible pH homeostasis capability and ATRsurvival, it was necessary to demonstrate that, contrary towhat was observed with constitutive pH homeostasis, denovo protein synthesis was required for inducible homeosta-sis. The data in Table 3 provide that evidence. At pHo 3.3,adapted cells maintained a 0.5 pH unit increase in pHi whencompared with unadapted cells. However, the addition ofchloramphenicol 15 min prior to adaptation eliminated pHienhancement as well as survival. This observation mustreflect either an additional pH homeostasis system inducedduring adaptation or a new protein(s) that protects preexist-ing homeostasis mechanisms. Previous studies with two-dimensional PAGE analyses have shown that the productionof 12 proteins is enhanced during adaptation. One or more ofthese proteins could be involved in the process describedhere.

Lethal pHi. The next logical question was to determine theapproximate pHi at which protein damage could be demon-strated and at which cells begin to lose viability. We exam-ined the question of lethal pH1 by using the uncouplingagents DNP and CCCP. DNP and CCCP are protonophoresthat will tend to equilibrate pH1 and pHo. Therefore, one canestimate the lethal pH, by adding these protonophores to

TABLE 3. Effect of protein synthesis on pH homeostasis

Without chloramphenicol With chloramphenicola

pHi" % Viability' pH, % Viability

4.2 5.9 + 0.1 103 5.9 + 0.1 953.3 (unadapted) 4.6 ± 0.1 0.06 NDd ND3.3 (adapted) 5.1 ± 0.1 82 4.6 + 0.1 <0.001

a Chloramphenicol (60 i.g/ml) was added 15 min prior to adaptation or pHshift if unadapted.

b pH, was measured at 25 min after the shift.c Viability was measured 90 min after shift to pH, 3.3.d ND, not determined.

7

2c 6.5

L 6

E 5.5

204 5C

4.5

4

100

-b 10

S 0.1

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pHo = 5.0 A

0 200 400 600 800Dinitrophenol (uM)F01-0 B

DNP 0 400 400 0 100 200 400pHo 7.7 7.7 6.0 5.1 5.1 5.1 5.1pH 7.8 7.7 6.0 6.5 5.8 5.4 5.1

FIG. 2. Effect ofDNP on pH, and viability. (A) Cells were grownin minimal E medium plus glucose (pH 7.7) to an optical density at600 nm of 0.4. Subsequently, the pH was adjusted to 5.0 anddifferent amounts of DNP were added to parallel cultures. The pH,was measured at 30 min as described in Materials and Methods. (B)The effect of manipulating pHi on viability is shown. DNP wasadded to cultures suspended in medium buffered to the indicatedpHo. Viability was measured 120 min after addition of DNP.

cells suspended in medium at pH 5.0, a pH. that is ordinarilyinnocuous to the cell. Correlations made between measuredpH, and viability will indicate the pHi at which viabilitydeclines. As shown in Fig. 2A, the addition of increasingamounts of DNP to cells suspended in pH 5.0 mediumresulted in a concomitant decrease in pH,. Allowing DNP toact for longer periods did not cause greater changes in pHibeyond what is shown in the figure. Subsequent measure-ments of viability after removing DNP revealed that celldeath occurred at pH, 5.4 and below (Fig. 2B). As a control,DNP at 400 ,uM did not cause a loss of viability when addedat pH. 6 or above (Fig. 2B). Similar results were alsoobtained with CCCP (data not shown).We next examined the effect of pH on P-galactosidase

activity to confirm that this level of pHi can indeed damageproteins. Using permeabilized cells, we first determined that3-galactosidase did not undergo significant irreversible acid

denaturation until pH 5.5, at which point 30% of its activitywas destroyed over 60 min (Fig. 3A). At pH 5.0, 90% of theactivity was destroyed within the same time frame (Fig. 3A).We took advantage of this fact to correlate protein damageand loss of viability in an ATR experiment with strain JF1638(Fig. 3B and C). JF1638 constitutively synthesizes P-galac-tosidase. Figure 3B reflects survival based upon viablecounts, whereas Fig. 3C indicates the amount of P-galactosi-dase activity remaining in the same culture. The percentagesof 3-galactosidase and surviving viable cells were almostidentical over the 90-min exposure period to pH. 3.3.Consequently, P-galactosidase activity can be used to mon-itor pHi-induced protein damage. In addition, the data con-firm that the pHi at which protein damage becomes evidentmost probably is between 5.0 and 5.5 since, in vitro, a-ga-lactosidase begins to denature between pH 5.5 and 5.0. Invivo, as the pHi decreased below 5.5, P-galactosidase activ-ity began to be irreversibly destroyed. Unadapted cellswhich were unable to maintain a pHi between 5 and 5.5

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0.001JF1638 iod icd/pFW75 atr-1 atr-12

FIG. 4. ATR mutants. (A) Complementation of the Atr- (acid-sensitive) phenotype of atp::TnJO (second pair of bars) by clonedSalmonella atp+ operon (pHF72). Survival data at pH 3.3 are givenat 90 min. Symbols: L, unadapted cells; El, adapted cells. (B)Complementation of the Atr(Con) (constitutive acid-tolerant) phe-notype of an icd mutant (second pair of bars) (JF1955) by cloned icd(third pair of bars) from S. typhimurium (pFW75). The fourth andfifth pairs of bars illustrate the constitutive acid tolerant phenotypesof JF1819 (atr-1) and JF1930 (atr-12 [ace]).

FIG. 3. Correlation between viability and the measurement ofacid-damaged protein. (A) Cells (JF1638) constitutively producing,-galactosidase were suspended (37°C) at the pH indicated and thenpermeabilized to equilibrate pHi and pH. with a chloroform-SDSmixture described by Miller (19). ,-Galactosidase activity wasmonitored at 0, 30, and 60 min as indicated by readjusting the pH to7 prior to assay. (B and C) JF1638 was adapted at pH 5.8 orremained unadapted at pH 7.7. Following acid challenge (pH 3.3),viability (panel B) and active P-galactosidase (panel C) were mea-sured at 0, 45, and 90 min after challenge.

succumbed more quickly to the deleterious effects of acidpHi, as evidenced by a more rapid denaturation of ,-galac-tosidase. These data support the protonophore data indicat-ing that pHi levels below 5.5 are lethal and will damageinternal proteins.pH 5.8 adaptation will not prevent acid-induced death

caused by artificial acidification of the cytoplasm by protono-phores. The evidence above indicated that adaptation en-hanced the ability to maintain pHi. However, it was consid-ered possible that the ATR system also providedmechanisms for repair or prevention of acid damage. Thiswas suggested by Goodson and Rowbury (13) in their workon acid habituation of E. coli. To test this hypothesis inSalmonella spp., adapted and unadapted cells were adjustedto pHo 5.1 and then DNP or CCCP was added to 400 or 33,uM, respectively. These concentrations will almost equili-brate pH1 with pH.. If acid damage is either prevented or

repaired by adapted cells, viability should be improved over

unadapted cells after the protonophore is removed. How-ever, no significant difference in viability could be demon-strated by this method (data not shown). Neither was thereany difference in pH1, which was 5.3. Therefore, during pH5.8 adaptation no internal system capable of repairing aciddamage appears to be induced. The primary purpose of the

preshock ATR system, then, is to maintain pH1 and, in sodoing, to retain viability.

Screening for spontaneous acid-tolerant mutants. To beginto understand acid tolerance, we screened survivors oflong-term acid treatment to identify constitutively acid-resistant mutants. Previous use of this strategy resulted inthe discovery of one mutation, designated atr-J (strainJF1819), that resulted in the acid-resistant phenotype. Amore detailed analysis of acid survivors was undertaken forthis current search. Unadapted cells (JF1638 or LT2) grownto 2 x 108 cells per ml were exposed to pH 3.3 until only 100to 1,000 cells per ml survived. These were plated onto LBmedium for further analysis. Surprisingly, 18% of the survi-vors screened proved to be auxotrophs. Of these, 44%required glutamate, 31% required uracil, and 25% requiredsomething else (methionine, proline, arginine, purines, orundetermined amino acids). All of the glutamate-requiringmutations tested mapped at 25 min on the S. typhimuriumlinkage map (cotransducible with phoP [22]) and were foundto be deficient in isocitrate dehydrogenase (data not shown).Another acid-tolerant mutant, atr-12, was discovered and

found to require acetate for maximum growth. Cotransduc-tion with zad: :TnJO placed the mutation at 3 min, suggestingthe involvement of the aceEF lpd operon and hence pyru-vate dehydrogenase. Repair of this locus also restored anormal ATR. It is unclear why a deficiency in pyruvatedehydrogenase would lead to acid tolerance.

icd mutations increase internal buffer capacity. The data inFig. 4B illustrate that the icd mutants isolated by thistechnique were constitutively acid tolerant when comparedwith the icd' parental strain. Furthermore, a cloned icd'locus from Salmonella spp. not only complemented theglutamate auxotrophy, but also reestablished a normal ATRresponse. Why are icd mutants acid tolerant? Lakshmi and

A

LT2 unc::TnlO unc::TnOlOpHF72

C Unadapted Adapted

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TABLE 4. pHi of ATR mutants"

pHi at pHo of:Strain Genotype 3.3 3.3

7.5 4-4 (unadapted) (adapted)

LT2 7.8 6.1 4.4 5.1SF342 atp 7.9 5.9 4.7 4.5JF1819 atr-J 7.8 6.3 5.7 NDbJF1930 atr-12 (ace) 7.8 6.2 5.5 NDJF1791 icd 7.8 6.6 5.5 ND

a pHi was measured as indicated in Materials and Methods by usingradiolabeled weak acid or base distributions. The range of variation was 0.1pH unit.

b ND, not determined.

Helling (17) reported that icd mutants accumulate enormousamounts of intracellular citrate and isocitrate (40 to 50 mM).Since the pK1 for these acids is 6.4, they could conceivablybuffer the pHi toward this value, preventing it from droppingbelow the critical 5.5 point vital for survival. Measurementsof pHi confirmed this (Table 4). At a pH. of 4.4, the pHi ofan icd mutant was 0.5 unit higher (pHi 6.6) than that of itsicd' parent (pH1 6.1), whereas little difference was noted atpHo 7.5 (pHi 7.8). A more dramatic difference was noted atpHo 3.3, at which the pHi of unadapted icd cells was 1.1units more basic than that of unadapted LT2 cells. In accordwith the accumulation of citrate as the means of acidresistance, secondary mutations in gitA (citrate synthase)prevented the accumulation of citrate in an icd mutant andsimultaneously eliminated the acid tolerance phenotype.Even though it would seem to be an attractive ATR

mechanism, isocitrate dehydrogenase does not appear to bea major component of the adaptive ATR. This was suggestedby the fact that the activity of this enzyme did not changeduring adaptation (data not shown).The effect of icd mutations on acid tolerance has also

allowed us to address a fundamental question of acid-induced death. We have presumed that death results frominternal acid damage caused by a lowered pHi and that theATR system prevents the pHi from reaching these lethallevels. An alternative scenario would be that external acidcould damage a cell surface component(s) essential for cellviability. As a subsequent, indirect consequence of celldeath, the pHi of the nonviable cell would decrease. In thiscase, lowering of the pH; would occur secondarily to death.The isolation of acid-tolerant icd mutants that possess anelevated internal buffer capacity provides elegant proof thatacid-induced death is directly related to lowered pH,. In-creased internal buffer capacity would not prevent externalacid damage to the cell.

atr-i and atr-12 (ace) mutations affect ATR pH homeostasiscapability. Both the atr-J and atr-12 (ace) mutations result ina constitutive acid tolerance phenotype (Fig. 4B). To deter-mine whether this may be due to increased buffer capacity,as was the case for icd mutants, or to an effect upon ATR pHhomeostasis, we measured the pHi values of both mutants atseveral different medium pHs. The results (Table 4) indicatethat even when unadapted, both atr(Con) mutants havedramatically improved pH homeostasis capability at pH 3.3when compared with LT2. Equally dramatic was the factthat, as opposed to the icd mutants, no significant differenceswere observed above pH 4 for either the atr-J or atr-12 (ace)mutant. This indicates that these mutations specificallyenhance the ability of the cell to handle severe acid stress inthe same range affected by the ATR and do not represent a

100

10 K/

01

0.01

0.001-

LT2 JF2280 JF2276 JF1 930Phenotype WT Ura- lcd- Ace-Adapt (pH5.8) - + - - + - - + - +Starvation No No Carbon No No Ura No No No No Ace

FIG. 5. Effect of starvation on acid tolerance. Cells were eitherprocessed for adaptation as indicated in Materials and Methods orstarved for a given nutrient as indicated in the figure. Carbon anduracil starvation was conducted at 0.02% and 5 ,ug/ml, respectively.Where indicated, acetate was added to 0.04%. Survival values arethose following 2 h of pH 3.3 acid treatment.

general increase in buffer capacity as was the case for icdmutants.Does starvation provide protection from acid-induced

death? Although it is clear that the majority of auxotrophs(icd) survived acid stress as a result of increased buffercapacity, why were so many other auxotrophic mutationsassociated with acid survival? An obvious possibility is thatstarvation for a given nutrient might elicit some degree ofacid tolerance and thus provide a selective advantage. Wehave tested this theory by using several of the spontaneousmutants selected in this study. For example, carbon starva-tion of LT2 and uracil starvation of JF2278 did provide sometolerance to severe acid that was not evident when glucoseor uracil was plentiful (Fig. 5). However, the degree of acidprotection for carbon or uracil starvation was only a fractionof that afforded by pH 5.8 adaptation. It is reasonable toassume that survival of many of the auxotrophs was due tostarvation-induced cross-protection. However, it is particu-larly interesting that the atr-12 (ace) mutant was acid toler-ant whether or not it was starved for acetate. The indicationis that the acid tolerance of this mutant must be due to aneffect upon the ATR and not simply to starvation-inducedcross-protection.The Mg2+-dependent proton-translocating ATPase is re-

quired for adaptive acid tolerance. The atp operon codes forthe FoF1 proton-translocating ATPase required for oxidativesynthesis of ATP and for the generation of proton motiveforce under anaerobic conditions. The Fo subunits are intrin-sic membrane proteins that form a pore through whichprotons can pass. The F1 sector (ATPase) binds to Fo andprotrudes into the cytoplasm. Protons passing into the cellthrough Fo will generate ATP via F1, whereas exiting pro-tons will expend an ATP. Thus, FoF1 can pump protons outof the cell and conceivably raise the pHi. It was shownpreviously that a TnJO insertion into the atp (formerly unc)operon conferred upon the cell an extreme acid-sensitivephenotype and an inability to undergo adaptive acid toler-ance. The obvious question of whether the atp::TnlO inser-tion affects pH homeostasis is addressed in Table 4. Al-though the loss of ATPase did not influence pH homeostasisat pHo 7.5 or 4.4, the atp::TnJO insertion clearly prevented

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100

o-O

10

0.1

0.001I v/I IX77 VI-17ALT2 LT2 LT2 atp::Tn10

pH 3.3 3.3 4.2 3.3DgCDD + + +FIG. 6. Effect of DCCD on the development of acid tolerance.

Cells were processed for adaptation as indicated in Materials andMethods. The challenge pH used for each set of experiments isshown below the figure. Where indicated, 5 mM DCCD was added15 min prior to adjusting the culture to the challenge pH. SF342 was

the atp::TnJO strain used. Symbols: C, unadapted cells; 1, adaptedcells.

ATR-enhanced homeostasis below pH. 4 (e.g., pH 3.3).Whether this implies a requirement for ATP or a directinvolvement of the ATPase as a proton pump is unresolved.The atp operon from Salmonella species was cloned andsubsequently shown to complement the succinate-negativephenotype that is characteristic of atp mutants, as well as toreestablish the ability to develop acid tolerance (Fig. 4).On the basis of the results obtained with the atp mutant, a

biochemical approach was used to gain further insight intothe role of the ATPase. DCCD is known to inhibit ATPaseactivity by covalently modifying subunit c of the intrinsicmembrane sector Fo (24). DCCD prevents proton transloca-tion through the Fo pore, thereby inhibiting the synthesis ofATP by the F1 sector (for a review, see reference 23). Wereasoned that if the H+-translocating ATPase was importantto acid tolerance, DCCD should prevent the development ofacid tolerance, presumably by interfering with H+ translo-cation out of the cell. The results of these experiments areshown in Fig. 6. The second pair of bars shows that DCCDclearly prevented acid tolerance when added 45 min afteradaptation of cells to pH 5.8 (15 min prior to the shift to pH3.3). However, no loss in viability occurred when DCCDwas added to cells shifted to pH 4.2 (third pair of bars). Thissuggests that constitutive pH homeostasis does not require

the ATPase but that this pump is required for induciblehomeostasis.

Since the location of the TnJO insertion in the atp operon

is unknown, it remains possible that the mutant is making an

Fo without F1. This could lead to a proton leak. However, itseems unlikely that the atp mutant is acid sensitive as a

result of a leaky F0. If some Fo pore is made, the addition ofDCCD to the atp::TnJO strain should prevent proton move-

ment through the pore into the cell and thus afford some

protection against acid. This was not the case. The atp::TnJOmutant remained exquisitely sensitive to acid in the presence

of DCCD (Fig. 6).

DISCUSSION

The data presented in this communication add to ourknowledge of low-pH stress and the adaptive ATR. First, wehave demonstrated that acid-induced death is the directresult of lowered pHi. Loss of viability is not due to externaldamage to the cell. This conclusion is based on the fact thatone can prevent acid-induced death by increasing the inter-nal buffer capacity (e.g., accumulation of citrate by an icdmutant). Increased internal buffering will not prevent exter-nal acid damage. Our second finding is that acid damagecausing inviability occurs when the pH1 decreases below 5.5and rapidly accumulates when the pH1 drops below 5.0. Thiswas demonstrated by artifically lowering the pH1 via proto-nophores as well as by measuring damage to internal 3-ga-lactosidase. Third, the reason why adapted cells survivestrong-acid conditions better than unadapted cells is due toan enhanced ability to maintain the pH1 above 5.0. The pH1of adapted cells was maintained 0.5 to 0.9 unit more alkalinethan that of unadapted cells. Evidence that the ATR systemprovides the mechanism(s) for augmenting pH, homeostasiswas found in that adaptive enhancement of pHi required denovo protein synthesis during adaptation at pH 5.8. Normalconstitutive pH homeostasis that occurred down to pH. 4did not depend upon the synthesis of new proteins.

Several lines of evidence implicate the proton-translocat-ing ATPase as an important component of acid tolerance.Mutants lacking the ATPase (atp::TnJO) are acid sensitive,as are atp+ cells in the presence of DCCD. Measurements ofpH1 also indicate that atp::TnJO mutants cannot adaptivelyenhance pH1 as do wild-type cells. The fact that DCCDimparts an ATR- phenotype on wild-type cells suggests thatthe ATPase might serve as a proton pump to extrude protonsduring the ATR. Alternatively, DCCD might prevent thesynthesis of ATP needed to power alternate homeostasisevents. Proton-translocating ATPase activity from otherorganisms has been shown to increase in response to adecrease in cytoplasmic pH (16). It is not yet clear whethera similar phenomenon may occur here. One possibility isthat a protein is induced during adaptation that interacts withpreexisting H+-translocating ATPase complexes, renderingthem more active or more stable under extremely acidicconditions.During this study, additional acid-resistant mutants have

been found. Some of them (auxotrophs) can be explained bya partial cross-protection to acid as a result of starvation fora specific nutrient. There is evidence that starvation forcarbon source can lead to general protection against otherstress conditions such as heat shock (19). A possible reasonfor this is that starvation has been shown to induce thechaparonin class of stress proteins (e.g., DnaK and GroE),which are thought to renature improperly folded, denaturedproteins. Thus, starvation could contribute to increasedsurvival at low pH if these same chaparonins can refoldacid-denatured proteins.

In contrast to the auxotrophs noted above, other acid-resistant mutants such as atr-J and atr-12 may more directlyinfluence the ATR system. Although we cannot yet unam-biguously attribute the acid tolerance of the atr-J and atr-12(ace) mutants to an increased ATR, it is noteworthy thatboth mutants exhibited elevated pH1 at pH. 3.3 but not atpH0 4.4. This was unlike the obvious buffering effect causedby the icd mutations, when pH1 was higher at both pH.values. This evidence supports a link between these muta-tions and the ATR system. Previously we reported that theatr-J mutation affected the synthesis of several ATR poly-

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INDUCIBLE pH HOMEOSTASIS 5135

peptides (10). These new mutants will be examined in asimilar manner.

In addition to what has been reported here, we haverecently implicated the ferric uptake regulator (fur) as a

major contributor to the regulation of the ATR (11). Muta-tions in the fur locus impart an Atr-, acid-sensitive pheno-type, deregulate the production of eight ATR proteins, andeliminate the expression of several acid-regulated genes. furmutations were also shown to prevent the development ofthe inducible pHi homeostasis mechanism described here.The role of Fur in this system appears to be independent ofiron, since the ATR is unaffected by iron availability. Wehave suggested that Fur may sense changes in pHi andregulate a set of low-pH-inducible genes, including membersof the ATR system.From the evidence obtained so far, a two-phase working

model can be envisoned to explain how a cell copes with theeventuality of low-pH stress. The first phase of protection,preshock, occurs as the environmental pH approaches 5.8,when the cell will induce the ATR-associated pH homeosta-sis system. This system will operate at pHo values below 4.0.During severe acid stress, this mechanism will maintain thepHi near 5.5 and minimize acid denaturation of internalproteins. The second phase of this protective model involvesthe acid shock proteins induced once the pHo drops tobetween pH 5 and pH 3. Acid shock is a response distinctfrom the ATR. A completely different set of proteins areinduced during acid shock. We and others have found thatthis shift also induces several of the heat shock proteinsclassified as chaparonins (8, 15). We predict that theseproteins are important both in preventing acid denaturationand in refolding denatured proteins once the cells are re-lieved of severe acid stress. The ,B-galactosidase experi-ments do provide evidence that protein denaturation willoccur at the low pHi values measured during severe acidstress (Fig. 3).Thus, the accumulated data indicate that the adaptive

ATR is a complex system designed to shield the cell fromexcessive H+ ion concentrations through a form of induciblepH homeostasis. The potential contribution of this system,as well as other aspects of pH-regulated gene expression, tothe pathogenesis of Salmonella species is formidable (1,8-11, 14, 15, 25). During its life cycle, this neutrophilicorganism encounters a variety of acidic environments, in-cluding pond water, stomach acid, and colon contents.Furthermore, as intracellular parasites, Salmonella speciesare exposed to low-pH conditions in the phagosomes andphagolysosomes of macrophages (7). Considerable work willbe required to reveal the functional details of this responseand its role in virulence.

ACKNOWLEDGMENTS

We thank Z. Aliabadi, H. Winkler, D. Wood, and M. Spector forhelpful discussions and critical reading of the manuscript. We arealso indebted to R. Thompson for her careful preparation of themanuscript.

This research was supported by National Science Foundationgrant DCB-89-04839.

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