of Salmonella typhimurium

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1991, p Vol. 173, No /91/ $02.00/0 Copyright , American Society for Microbiology Inducible ph Homeostasis and the Acid Tolerance Response of Salmonella typhimurium JOHN W. FOSTER* AND HOLLY K. HALL Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile, Alabama 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 6.0 that will protect cells from more severe acid stress (J. Foster and H. Hall, J. Bacteriol. 172: , 1990). Correlations between the internal ph (phi) of adapted versus unadapted cells at ph. of 3.3 indicate that the ATR system produces an inducible ph-homeostatic function. This function serves to maintain the ph1 above 5 to 5.5. Below this range, cells rapidly lose viability. Development of this ph homeostasis mechanism was sensitive to protein synthesis inhibitors and operated only to augment the phi at ph. values below 4. In contrast, classical constitutive ph homeostasis was insensitive to protein synthesis inhibitors and was efficient only at ph. values above 4. Physiological studies indicated an important role for the Mg2+-dependent proton-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. We speculate that adaptive acid tolerance is important to Salmonella species in surviving acid encounters in both the environment and the infected host. ph homeostasis is the process whereby a cell maintains a relatively constant intracellular ph (phi) over a broad range of external (ph.) values. The basis for this phenomenon is the apparent modulation of primary cellular proton pumps as well as potassium/proton and sodium/proton antiport systems (4, 18, 21). This process appears subject primarily to allosteric control (such as the ph set point for pump activation) since, as we will show, ph homeostasis functions normally in the presence of protein synthesis inhibitors. As far as genetic control, the system can be referred to as constitutive. However, a superimposing genetic response which can further protect a cell from acid stress has been discovered in Salmonella typhimurium () and Escherichia coli (12, 13). The process, referred to as the acid tolerance response (ATR), is triggered in Salmonella species at ph values between 6.0 and 5.5, but protects cells against much stronger acid (ph 3.0 to 4.0) when nonadaptive ph homeostasis normally fails. The ATR system includes at least 18 proteins as determined by two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis (, 26). Recently, the fur gene product (ferric uptake regulator) has been implicated as contributing a key regulatory function. Mutations in this locus confer Atr- and acid-sensitive phenotypes as well as 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 (formerly unc), was suggested from genetic studies. One can envision a variety of mechanisms that could protect the cell from extreme acid stress. These include increases in internal buffering capacity or proton extrusion rate, as well as a decreased membrane proton conductance. As an alternative, the cell could prevent and/or repair acid damage as a result of lower phi. We have studied each of these possibilities and have further examined survivors of strong acid stress in an effort to ascertain what has allowed them to survive. * Corresponding author MATERIALS AND METHODS Bacterial strains and cultural conditions. The bacterial strains used were all derivatives of S. typhimurium LT2 and are listed in Table 1. Culture media included E medium supplemented with 0.4% glucose (27) and LB medium (6). Chloramphenicol, 2,4-dinitrophenol (DNP), and carboxyl cyanide m-chlorophenylhydrazone (CCCP) were added as indicated for each experiment. N,N'-Dicyclohexylcarbodiimide (DCCD) was used at 5 mm. This concentration was used because Salmonella species are relatively resistant to this compound, presumably owing to poor outer membrane permeability. Measurement of ATR. The procedure for observing the adaptive ATR was presented in detail by Foster and Hall (). Briefly, cultures were grown with shaking in minimal E glucose broth under semiaerobic conditions to 8 cells per ml (3 ml of medium in a culture tube [13 mm by mm]). The medium ph was adjusted to 5.8 with HCI, and the culture was allowed to adapt for one doubling (approximately 60 to 70 min). Unadapted cultures were grown directly to 2 x 8 cells per ml at ph 7.7. At that point, the ph of both cultures was adjusted to 3.30 and further incubated for 90 min. Viable counts were taken at 0 and 90 min by dilution in fresh minimal E medium and plating on LB agar. The percent viability was measured as follows: (CFU at 90 min/cfu at time zero) x. Genetic procedures. Transductions were performed with phage P22 as described earlier (1, 2, 25). Plasmid extractions and other DNA manipulations were outlined previously (9). Measurement of ph,. The method for measuring the ph, involved the distribution of radiolabeled weak acids or bases across the cellular membrane. The procedure was derived from those of Booth et al. (5) and Atkinson and Winkler (3). To determine intracellular water space, we grew cells in minimal medium containing 25 mm sucrose. At mid-log phase, 3 ml of cells was harvested and resuspended into 200,d of culture supernatant. 3H20 and ["4C]sucrose were each added to 3,000 dpm/,ul, a 5-,ul sample was removed for total counts, and the remainder was incubated at 37 C for min

2 5130 FOSTER AND HALL TABLE 1. S. typhimurium strains used in this study Strain Relevant or phenotype genotype Source reference or JF316 pyrd95 zad::tnjo JF1638 anig::mu dj AearA324 9 JF1819 atr-j JF1892 atr-j atp-2::tnjo JF1930 atr-12 (ace) Acid survivor JF1949 atr-12 atp-2::tnjo JF1930 x SF342 JF1955 icd-1 anig::mu dj AearA324 Acid survivor JF1969 icd-1 anig::mu dj AearA324/ pfw75(icd') JF1971 icd-6 Acid survivor JF1952 Aatp-1IpHF72(atp') JF1953 anig::mu dj AearA atp-2::tnjo JF1638 x SF342 JF2278 Ura- Acid survivor JF2280 atr-14 Acid survivor JF2283 pyra Acid survivor JF2276 icd-8 Acid survivor SF342 atp-2::tnlo G. Ames (reaction mix A). Following incubation, duplicate samples ( RI) were centrifuged through 50,u of dibutyl phthalate and 50 [li of silicon oil. The tubes were frozen at -70 C, and the cell pellet was sliced from the tube. The pellet was placed for 5 min in a minivial containing p,l of 1% sodium dodecyl sulfate (SDS). Scintillation fluid was added, and the series of vials were counted in an LKB 1219 scintillation counter. The total water per cell pellet was calculated as 3H20 disintegrations per minute of the pellet divided by the total 3H20 per microliter of reaction mix. Extracellular H20 was equal to the disintegrations per minute of [14C]sucrose in the pellet divided by the total ['4C]sucrose per microliter of reaction mix. Intracellular H20 was equal to total H20 minus extracellular H20. An almost identical procedure was used to determine the distribution of weak acids (['4C]benzoic acid or ['4C]salicylic acid) or base (['4C]methylamine). The difference with this method is that the reaction mix (200 pll) contained 3,000 dpm each of 3H20 and ['4C]benzoic acid or methylamine per,u. The formula used to calculate ph, was total acid concentration in K ph o opk ph, log [total = acid concentration out (l)p Radiolabeled methylamine, benzoic acid, and salicylic acid were used to measure ph, at ph. levels of 7.7, 4.0 to 7.0, and 3.3 to 4.5, respectively. ph, measurements were made within 20 min of a pho shift. Measurement of acid-induced damage to f8-galactosidase. Cells constitutively synthesizing P-galactosidase were adjusted to the ph indicated in Fig. 3A and solubilized by the SDS-chloroform procedure of Miller (20). At the times indicated, the solution was neutralized and,-galactosidase activity was measured. RESULTS The ATR augments ph homeostasis. The fundamental question we must address is how adaptation to mild acidification protects the cell from subsequent extreme acid exposure at ph, 3.3. One possibility is that ph homeostasis mechanisms are enhanced during adaptation, thereby allowing the cell to maintain the ph, in the viable range for a longer period during severe acid stress. If this model is correct, one would expect to observe measurable ph, differ- 0t 01.5_\ CL~~~~~~~~~~~~~~~~a Dl~~~~~~~~~~~~~~~~~~~~~~~~( a Exp ralp J. BACTERIOL. 0~~~~~~~~~~~ External ph FIG. 1. Effects of phi on the phw of unadapted cells. Cells were grown to 2 x 8 cells per ml in E medium plus glucose (ph 7.7), and the medium ph was adjusted to the values indicated. After 20 mn of equilibration, thephl values were measured as described in Materials and Methods. The accuracy of each measurement, done in triplicate, was ±0o.1 unit. ences when comparing adapted with unadapted cells in acidic environments. To test this theory, we first monitored phi and ApH of unadapted cells at various ph. values by using radiolabeled weak acids (benzoic acid [pk = 4.2] and salicylic acid [pk = 3.0]) and a weak base (methylamine [pk =.16]) as outlined in Materials and Methods. ph1 values obtained for acid-shifted cells are somewhat lower than previously reported by Hickey and Hirshfield (14) because the cultures were grown under the semiaerobic conditions best suited to ATR development. As shown in Fig. 1, the ph, decreased from 7.8 to 5.8 when the ph. was shifted from 7.5 to 4.0. Concomitantly, the difference between pho and ph1 (ApH) increased from 0.3 to 1.8. We have recently shown that unadapted cells that are shifted to ph. 4.0 retain viability at or near % for several hours (8). However, once pho falls below 4.0, cells progressively lose viability as determined by viable plate count (8) and by vital staining with 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 this shift the ph, fell dramatically to 4.4 and ApH dropped from 1.8 to approximately 1.0 (Fig. 1). Cell viability remained above 80% during this short time frame, indicating that the decrease in ph, was not due to cell death. If the process of adaptation affects ph homeostasis mechanisms or buffering capacity, an increased (more alkaline) ph, should be evident when comparing adapted with unadapted cells, at least under low-ph stress. Initial comparisons between adapted and unadapted cells made at various pho values ranging from 7.5 to 4.0 revealed no differences in ph, (Table 2). However, a dramatic difference in ph, was observed when comparing adapted and unadapted cells at pho 3.3. Within minutes, the ph, of unadapted cell cultures fell 0.5 to 0.9 ph unit lower than that of adapted cells (Table 2). The data support the hypothesis that adaptation enables the cells to better maintain ph,. The ability of adapted cells to maintain a less acidic ph, could explain the tolerance to external acid conferred by the ATR. This adaptive ph, maintenance most probably reflects an increase in ph homeostasis capability. Although there are alternative possi- \ 6

3 VOL. 173, 1991 ph. TABLE 2. phi of S. typhimuriuma Unadapted cells phi" of: Adapted cells ± ± ± ± ± ± ± ± ± 0.1 a Radiolabeled benzoic acid assay or salicylic acid assay. Assayed at 15 min after shift. b Values are mean t range of variation for three experiments. bilities, they are unlikely and will be addressed below. In our model, the ATR system enhances ph homeostasis capacity only below (not above) ph Protein synthesis is required for ATR-enhanced homeostasis. The ph homeostasis mechanisms engaged above pho 4 function normally even in the absence of protein synthesis, as shown in Table 3. Unadapted cells shifted to pho 4.2 maintained a phi of 5.9 in either the presence or absence of chloramphenicol. We will refer to this form of ph homeostasis as constitutive. However, although ph homeostasis above pho 4.0 did not require de novo protein synthesis, ATR survival below pho 4 was shown previously to require de novo protein synthesis (). Therefore, to establish a link between inducible ph homeostasis capability and ATR survival, it was necessary to demonstrate that, contrary to what was observed with constitutive ph homeostasis, de novo protein synthesis was required for inducible homeostasis. The data in Table 3 provide that evidence. At pho 3.3, adapted cells maintained a 0.5 ph unit increase in phi when compared with unadapted cells. However, the addition of chloramphenicol 15 min prior to adaptation eliminated phi enhancement as well as survival. This observation must reflect either an additional ph homeostasis system induced during adaptation or a new protein(s) that protects preexisting homeostasis mechanisms. Previous studies with twodimensional PAGE analyses have shown that the production of 12 proteins is enhanced during adaptation. One or more of these proteins could be involved in the process described here. Lethal phi. The next logical question was to determine the approximate phi at which protein damage could be demonstrated and at which cells begin to lose viability. We examined the question of lethal ph1 by using the uncoupling agents DNP and CCCP. DNP and CCCP are protonophores that will tend to equilibrate ph1 and pho. Therefore, one can estimate 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 (unadapted) 4.6 ± NDd ND 3.3 (adapted) 5.1 ± <0.001 a Chloramphenicol (60 i.g/ml) was added 15 min prior to adaptation or ph shift 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 C b S 0.1 g pho = 5.0 INDUCIBLE ph HOMEOSTASIS 5131 F Dinitrophenol (um) B DNP pho ph FIG. 2. Effect of DNP on ph, and viability. (A) Cells were grown in minimal E medium plus glucose (ph 7.7) to an optical density at 600 nm of 0.4. Subsequently, the ph was adjusted to 5.0 and different 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 was added to cultures suspended in medium buffered to the indicated pho. Viability was measured 120 min after addition of DNP. cells suspended in medium at ph 5.0, a ph. that is ordinarily innocuous to the cell. Correlations made between measured ph, and viability will indicate the phi at which viability declines. As shown in Fig. 2A, the addition of increasing amounts of DNP to cells suspended in ph 5.0 medium resulted in a concomitant decrease in ph,. Allowing DNP to act for longer periods did not cause greater changes in phi beyond what is shown in the figure. Subsequent measurements of viability after removing DNP revealed that cell death occurred at ph, 5.4 and below (Fig. 2B). As a control, DNP at 400,uM did not cause a loss of viability when added at ph. 6 or above (Fig. 2B). Similar results were also obtained 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 damage proteins. Using permeabilized cells, we first determined that 3-galactosidase did not undergo significant irreversible acid denaturation until ph 5.5, at which point 30% of its activity was destroyed over 60 min (Fig. 3A). At ph 5.0, 90% of the activity was destroyed within the same time frame (Fig. 3A). We took advantage of this fact to correlate protein damage and loss of viability in an ATR experiment with strain JF1638 (Fig. 3B and C). JF1638 constitutively synthesizes P-galactosidase. Figure 3B reflects survival based upon viable counts, whereas Fig. 3C indicates the amount of P-galactosidase activity remaining in the same culture. The percentages of 3-galactosidase and surviving viable cells were almost identical over the 90-min exposure period to ph Consequently, P-galactosidase activity can be used to monitor phi-induced protein damage. In addition, the data confirm that the phi at which protein damage becomes evident most probably is between 5.0 and 5.5 since, in vitro, a-galactosidase begins to denature between ph 5.5 and 5.0. In vivo, as the phi decreased below 5.5, P-galactosidase activity began to be irreversibly destroyed. Unadapted cells which were unable to maintain a phi between 5 and 5.5 A

4 5132 FOSTER AND HALL J. BACTERIOL. (D120 Cl) CS o Co <n 40 o-0 20 I *0 1 (a! A LT2 unc::tnlo unc::tnolophf72 80 = 60 Co > 40 ol 20 0 a) 120 C') Cul1 00 Cf) 80 0 t 60 Co 40 m 20 o00 C Unadapted Adapted FIG. 3. Correlation between viability and the measurement of acid-damaged protein. (A) Cells (JF1638) constitutively producing,-galactosidase were suspended (37 C) at the ph indicated and then permeabilized to equilibrate phi and ph. with a chloroform-sds mixture described by Miller (19).,-Galactosidase activity was monitored at 0, 30, and 60 min as indicated by readjusting the ph to 7 prior to assay. (B and C) JF1638 was adapted at ph 5.8 or remained unadapted at ph 7.7. Following acid challenge (ph 3.3), viability (panel B) and active P-galactosidase (panel C) were measured at 0, 45, and 90 min after challenge. succumbed more quickly to the deleterious effects of acid phi, as evidenced by a more rapid denaturation of,-galactosidase. These data support the protonophore data indicating that phi levels below 5.5 are lethal and will damage internal proteins. ph 5.8 adaptation will not prevent acid-induced death caused by artificial acidification of the cytoplasm by protonophores. The evidence above indicated that adaptation enhanced the ability to maintain phi. However, it was considered possible that the ATR system also provided mechanisms for repair or prevention of acid damage. This was suggested by Goodson and Rowbury (13) in their work on acid habituation of E. coli. To test this hypothesis in Salmonella spp., adapted and unadapted cells were adjusted to pho 5.1 and then DNP or CCCP was added to 400 or 33,uM, respectively. These concentrations will almost equilibrate 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. However, no significant difference in viability could be demonstrated by this method (data not shown). Neither was there any difference in ph1, which was 5.3. Therefore, during ph 5.8 adaptation no internal system capable of repairing acid damage appears to be induced. The primary purpose of the S: JF1 638 iod icd/pfw75 atr-1 atr-12 FIG. 4. ATR mutants. (A) Complementation of the Atr- (acidsensitive) phenotype of atp::tnjo (second pair of bars) by cloned Salmonella atp+ operon (phf72). Survival data at ph 3.3 are given at 90 min. Symbols: L, unadapted cells; El, adapted cells. (B) Complementation of the Atr(Con) (constitutive acid-tolerant) phenotype of an icd mutant (second pair of bars) (JF1955) by cloned icd (third pair of bars) from S. typhimurium (pfw75). The fourth and fifth pairs of bars illustrate the constitutive acid tolerant phenotypes of JF1819 (atr-1) and JF1930 (atr-12 [ace]). preshock ATR system, then, is to maintain ph1 and, in so doing, to retain viability. Screening for spontaneous acid-tolerant mutants. To begin to understand acid tolerance, we screened survivors of long-term acid treatment to identify constitutively acidresistant mutants. Previous use of this strategy resulted in the discovery of one mutation, designated atr-j (strain JF1819), that resulted in the acid-resistant phenotype. A more detailed analysis of acid survivors was undertaken for this current search. Unadapted cells (JF1638 or LT2) grown to 2 x 8 cells per ml were exposed to ph 3.3 until only to 1,000 cells per ml survived. These were plated onto LB medium for further analysis. Surprisingly, 18% of the survivors screened proved to be auxotrophs. Of these, 44% required glutamate, 31% required uracil, and 25% required something else (methionine, proline, arginine, purines, or undetermined amino acids). All of the glutamate-requiring mutations tested mapped at 25 min on the S. typhimurium linkage map (cotransducible with phop [22]) and were found to be deficient in isocitrate dehydrogenase (data not shown). Another acid-tolerant mutant, atr-12, was discovered and found to require acetate for maximum growth. Cotransduction with zad: :TnJO placed the mutation at 3 min, suggesting the involvement of the aceef lpd operon and hence pyruvate dehydrogenase. Repair of this locus also restored a normal ATR. It is unclear why a deficiency in pyruvate dehydrogenase would lead to acid tolerance. icd mutations increase internal buffer capacity. The data in Fig. 4B illustrate that the icd mutants isolated by this technique were constitutively acid tolerant when compared with the icd' parental strain. Furthermore, a cloned icd' locus from Salmonella spp. not only complemented the glutamate auxotrophy, but also reestablished a normal ATR response. Why are icd mutants acid tolerant? Lakshmi and

5 VOL. 173, 1991 TABLE 4. phi of ATR mutants" phi at pho of: Strain Genotype (unadapted) (adapted) LT SF342 atp JF1819 atr-j NDb JF1930 atr-12 (ace) ND JF1791 icd ND a phi was measured as indicated in Materials and Methods by using radiolabeled weak acid or base distributions. The range of variation was 0.1 ph unit. b ND, not determined. Helling (17) reported that icd mutants accumulate enormous amounts of intracellular citrate and isocitrate (40 to 50 mm). Since the pk1 for these acids is 6.4, they could conceivably buffer the phi toward this value, preventing it from dropping below the critical 5.5 point vital for survival. Measurements of phi confirmed this (Table 4). At a ph. of 4.4, the phi of an icd mutant was 0.5 unit higher (phi 6.6) than that of its icd' parent (ph1 6.1), whereas little difference was noted at pho 7.5 (phi 7.8). A more dramatic difference was noted at pho 3.3, at which the phi of unadapted icd cells was 1.1 units more basic than that of unadapted LT2 cells. In accord with the accumulation of citrate as the means of acid resistance, secondary mutations in gita (citrate synthase) prevented the accumulation of citrate in an icd mutant and simultaneously eliminated the acid tolerance phenotype. Even though it would seem to be an attractive ATR mechanism, isocitrate dehydrogenase does not appear to be a major component of the adaptive ATR. This was suggested by the fact that the activity of this enzyme did not change during adaptation (data not shown). The effect of icd mutations on acid tolerance has also allowed us to address a fundamental question of acidinduced death. We have presumed that death results from internal acid damage caused by a lowered phi and that the ATR system prevents the phi from reaching these lethal levels. An alternative scenario would be that external acid could damage a cell surface component(s) essential for cell viability. As a subsequent, indirect consequence of cell death, the phi of the nonviable cell would decrease. In this case, lowering of the ph; would occur secondarily to death. The isolation of acid-tolerant icd mutants that possess an elevated internal buffer capacity provides elegant proof that acid-induced death is directly related to lowered ph,. Increased internal buffer capacity would not prevent external acid damage to the cell. atr-i and atr-12 (ace) mutations affect ATR ph homeostasis capability. Both the atr-j and atr-12 (ace) mutations result in a constitutive acid tolerance phenotype (Fig. 4B). To determine whether this may be due to increased buffer capacity, as was the case for icd mutants, or to an effect upon ATR ph homeostasis, we measured the phi values of both mutants at several different medium phs. The results (Table 4) indicate that even when unadapted, both atr(con) mutants have dramatically improved ph homeostasis capability at ph 3.3 when compared with LT2. Equally dramatic was the fact that, as opposed to the icd mutants, no significant differences were observed above ph 4 for either the atr-j or atr-12 (ace) mutant. This indicates that these mutations specifically enhance the ability of the cell to handle severe acid stress in the same range affected by the ATR and do not represent a INDUCIBLE ph HOMEOSTASIS 5133 K/ LT2 JF2280 Phenotype WT Ura- JF2276 lcd- JF1 930 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 either processed for adaptation as indicated in Materials and Methods or starved for a given nutrient as indicated in the figure. Carbon and uracil starvation was conducted at 0.02% and 5,ug/ml, respectively. Where indicated, acetate was added to 0.04%. Survival values are those following 2 h of ph 3.3 acid treatment. general increase in buffer capacity as was the case for icd mutants. 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 buffer capacity, why were so many other auxotrophic mutations associated with acid survival? An obvious possibility is that starvation for a given nutrient might elicit some degree of acid tolerance and thus provide a selective advantage. We have tested this theory by using several of the spontaneous mutants selected in this study. For example, carbon starvation of LT2 and uracil starvation of JF2278 did provide some tolerance to severe acid that was not evident when glucose or uracil was plentiful (Fig. 5). However, the degree of acid protection for carbon or uracil starvation was only a fraction of that afforded by ph 5.8 adaptation. It is reasonable to assume that survival of many of the auxotrophs was due to starvation-induced cross-protection. However, it is particularly interesting that the atr-12 (ace) mutant was acid tolerant whether or not it was starved for acetate. The indication is that the acid tolerance of this mutant must be due to an effect upon the ATR and not simply to starvation-induced cross-protection. The Mg2+-dependent proton-translocating ATPase is required for adaptive acid tolerance. The atp operon codes for the FoF1 proton-translocating ATPase required for oxidative synthesis of ATP and for the generation of proton motive force under anaerobic conditions. The Fo subunits are intrinsic membrane proteins that form a pore through which protons can pass. The F1 sector (ATPase) binds to Fo and protrudes into the cytoplasm. Protons passing into the cell through Fo will generate ATP via F1, whereas exiting protons will expend an ATP. Thus, FoF1 can pump protons out of the cell and conceivably raise the phi. It was shown previously that a TnJO insertion into the atp (formerly unc) operon conferred upon the cell an extreme acid-sensitive phenotype and an inability to undergo adaptive acid tolerance. The obvious question of whether the atp::tnlo insertion affects ph homeostasis is addressed in Table 4. Although the loss of ATPase did not influence ph homeostasis at pho 7.5 or 4.4, the atp::tnjo insertion clearly prevented

6 5134 FOSTER AND HALL J. BACTERIOL. o-o I Iv/ X77 I VI-17A LT2 LT2 LT2 atp::tn ph DgCD FIG. 6. Effect of DCCD on the development of acid tolerance. Cells were processed for adaptation as indicated in Materials and Methods. The challenge ph used for each set of experiments is shown below the figure. Where indicated, 5 mm DCCD was added 15 min prior to adjusting the culture to the challenge ph. SF342 was the atp::tnjo strain used. Symbols: C, unadapted cells; 1, adapted cells. ATR-enhanced homeostasis below ph. 4 (e.g., ph 3.3). Whether this implies a requirement for ATP or a direct involvement of the ATPase as a proton pump is unresolved. The atp operon from Salmonella species was cloned and subsequently shown to complement the succinate-negative phenotype that is characteristic of atp mutants, as well as to reestablish 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 into the role of the ATPase. DCCD is known to inhibit ATPase activity by covalently modifying subunit c of the intrinsic membrane sector Fo (24). DCCD prevents proton translocation through the Fo pore, thereby inhibiting the synthesis of ATP by the F1 sector (for a review, see reference 23). We reasoned that if the H+-translocating ATPase was important to acid tolerance, DCCD should prevent the development of acid tolerance, presumably by interfering with H+ translocation out of the cell. The results of these experiments are shown in Fig. 6. The second pair of bars shows that DCCD clearly prevented acid tolerance when added 45 min after adaptation of cells to ph 5.8 (15 min prior to the shift to ph 3.3). However, no loss in viability occurred when DCCD was added to cells shifted to ph 4.2 (third pair of bars). This suggests that constitutive ph homeostasis does not require the ATPase but that this pump is required for inducible homeostasis. 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, it seems unlikely that the atp mutant is acid sensitive as a result of a leaky F0. If some Fo pore is made, the addition of DCCD to the atp::tnjo strain should prevent proton movement through the pore into the cell and thus afford some protection against acid. This was not the case. The atp::tnjo mutant remained exquisitely sensitive to acid in the presence of DCCD (Fig. 6). DISCUSSION The data presented in this communication add to our knowledge of low-ph stress and the adaptive ATR. First, we have demonstrated that acid-induced death is the direct result of lowered phi. Loss of viability is not due to external damage to the cell. This conclusion is based on the fact that one can prevent acid-induced death by increasing the internal buffer capacity (e.g., accumulation of citrate by an icd mutant). Increased internal buffering will not prevent external acid damage. Our second finding is that acid damage causing inviability occurs when the ph1 decreases below 5.5 and rapidly accumulates when the ph1 drops below 5.0. This was demonstrated by artifically lowering the ph1 via protonophores as well as by measuring damage to internal 3-galactosidase. Third, the reason why adapted cells survive strong-acid conditions better than unadapted cells is due to an enhanced ability to maintain the ph1 above 5.0. The ph1 of adapted cells was maintained 0.5 to 0.9 unit more alkaline than that of unadapted cells. Evidence that the ATR system provides the mechanism(s) for augmenting ph, homeostasis was found in that adaptive enhancement of phi required de novo protein synthesis during adaptation at ph 5.8. Normal constitutive ph homeostasis that occurred down to ph. 4 did not depend upon the synthesis of new proteins. Several lines of evidence implicate the proton-translocating 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 of ph1 also indicate that atp::tnjo mutants cannot adaptively enhance ph1 as do wild-type cells. The fact that DCCD imparts an ATR- phenotype on wild-type cells suggests that the ATPase might serve as a proton pump to extrude protons during the ATR. Alternatively, DCCD might prevent the synthesis of ATP needed to power alternate homeostasis events. Proton-translocating ATPase activity from other organisms has been shown to increase in response to a decrease in cytoplasmic ph (16). It is not yet clear whether a similar phenomenon may occur here. One possibility is that a protein is induced during adaptation that interacts with preexisting H+-translocating ATPase complexes, rendering them more active or more stable under extremely acidic conditions. During this study, additional acid-resistant mutants have been found. Some of them (auxotrophs) can be explained by a partial cross-protection to acid as a result of starvation for a specific nutrient. There is evidence that starvation for carbon source can lead to general protection against other stress conditions such as heat shock (19). A possible reason for this is that starvation has been shown to induce the chaparonin class of stress proteins (e.g., DnaK and GroE), which are thought to renature improperly folded, denatured proteins. Thus, starvation could contribute to increased survival at low ph if these same chaparonins can refold acid-denatured proteins. In contrast to the auxotrophs noted above, other acidresistant mutants such as atr-j and atr-12 may more directly influence the ATR system. Although we cannot yet unambiguously attribute the acid tolerance of the atr-j and atr-12 (ace) mutants to an increased ATR, it is noteworthy that both mutants exhibited elevated ph1 at ph. 3.3 but not at ph This was unlike the obvious buffering effect caused by the icd mutations, when ph1 was higher at both ph. values. This evidence supports a link between these mutations and the ATR system. Previously we reported that the atr-j mutation affected the synthesis of several ATR poly-

7 VOL. 173, 1991 peptides (). These new mutants will be examined in a similar manner. In addition to what has been reported here, we have recently implicated the ferric uptake regulator (fur) as a major contributor to the regulation of the ATR (11). Mutations in the fur locus impart an Atr-, acid-sensitive phenotype, deregulate the production of eight ATR proteins, and eliminate the expression of several acid-regulated genes. fur mutations were also shown to prevent the development of the inducible phi homeostasis mechanism described here. The role of Fur in this system appears to be independent of iron, since the ATR is unaffected by iron availability. We have suggested that Fur may sense changes in phi and regulate a set of low-ph-inducible genes, including members of the ATR system. From the evidence obtained so far, a two-phase working model can be envisoned to explain how a cell copes with the eventuality 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 homeostasis system. This system will operate at pho values below 4.0. During severe acid stress, this mechanism will maintain the phi near 5.5 and minimize acid denaturation of internal proteins. The second phase of this protective model involves the acid shock proteins induced once the pho drops to between ph 5 and ph 3. Acid shock is a response distinct from the ATR. A completely different set of proteins are induced during acid shock. We and others have found that this shift also induces several of the heat shock proteins classified as chaparonins (8, 15). We predict that these proteins are important both in preventing acid denaturation and in refolding denatured proteins once the cells are relieved of severe acid stress. The,B-galactosidase experiments do provide evidence that protein denaturation will occur at the low phi values measured during severe acid stress (Fig. 3). Thus, the accumulated data indicate that the adaptive ATR is a complex system designed to shield the cell from excessive H+ ion concentrations through a form of inducible ph homeostasis. The potential contribution of this system, as well as other aspects of ph-regulated gene expression, to the pathogenesis of Salmonella species is formidable (1, 8-11, 14, 15, 25). During its life cycle, this neutrophilic organism encounters a variety of acidic environments, including pond water, stomach acid, and colon contents. Furthermore, as intracellular parasites, Salmonella species are exposed to low-ph conditions in the phagosomes and phagolysosomes of macrophages (7). Considerable work will be required to reveal the functional details of this response and its role in virulence. ACKNOWLEDGMENTS We thank Z. Aliabadi, H. Winkler, D. 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