APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/97/$04.0010

July 1997, p. 2679–2685 Vol. 63, No. 7

Copyright © 1997, American Society for Microbiology

Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of theAcid Tolerance Response in Listeria monocytogenes LO28

BRID O’DRISCOLL, CORMAC G. M. GAHAN, AND COLIN HILL*

Microbiology Department & National Food Biotechnology Centre,University College Cork, Cork, Ireland

Received 31 January 1997/Accepted 12 April 1997

Listeria monocytogenes is capable of withstanding low pH after initial exposure to sublethal acidic conditions,a phenomenon termed the acid tolerance response (B. O’Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ.Microbiol. 62:1693–1698, 1996). Treatment of L. monocytogenes LO28 with chloramphenicol during acid adap-tation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tol-erance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresisrevealed changes in the levels of 53 proteins. Significant protein differences were also evident between non-adapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis re-vealed differences in protein expression between cells induced with a weak acid (lactic acid) and those inducedwith a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capableof maintaining their internal pH (pHi) at higher levels than nonadapted control cells during severe acid stress.Collectively, the data demonstrate the profound alterations in protein synthesis which take place during acidadaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions.

Listeria monocytogenes, a causative agent of both sporadicand epidemic food-borne illness, emerged as an importanthuman pathogen in the 1980s. Its ability to survive and grow inmany foods, in addition to its formidable capacity to overcomehost defense mechanisms and cause infection, has been closelymonitored in the last decade. L. monocytogenes is a neutralo-phile and will grow optimally only within a narrow pH range(optimum pH 6 to 7). The term “pH homeostasis” is used todescribe the ability of an organism to maintain its cytoplasmicpH (pHi) at a value close to neutrality despite fluctuations inthe external pH (pHo) (12). It has been proposed that whenthe pHi falls below a certain threshold value, cells will cease tofunction (16). The mechanism of action by which pH ho-meostasis is achieved is poorly understood, but both passiveand active mechanisms are thought to be involved.

Log-phase cells of L. monocytogenes are very sensitive to lowpH. The ability of these cells to withstand lethal pH conditionscan be dramatically improved following adaptation to a suble-thal pH (3, 15, 18). This adaptation is termed the acid toler-ance response (ATR). Spontaneous acid-tolerant mutants ofL. monocytogenes can also be recovered following exposure tosevere acid stress. Mouse infection studies involving one suchmutant, ATM56, revealed increased virulence relative to theparent strain, LO28 (18). Another trait attributable to the acid-tolerant mutant was an enhanced ability to survive in low-pHfoods (8). This study also demonstrated the significance of acidadaptation in the survival of L. monocytogenes in acid foodsand during milk fermentation.

Considerable data is available regarding the ATR phenom-enon in other food-borne pathogens, namely, Salmonella typhi-murium, Escherichia coli, and Aeromonas hydrophila (9, 13, 22).These studies have revealed that the ATR is a dauntinglycomplex biological phenomenon. Induction of the ATR in S.typhimurium involves the alteration of expression of as many as

52 separate proteins (4). It has previously been shown that theaddition of chloramphenicol impedes the development of theATR in L. monocytogenes, indicating the importance of denovo protein synthesis (3, 18). Davis et al. (3) have reportedthat the ATR induced in L. monocytogenes Scott A by a strongacid, HCl, involved the altered expression of at least 23 pro-teins. In this report, we describe the alterations in proteinlevels in L. monocytogenes during acid habituation with bothstrong and weak acids and also in the acid-tolerant mutantATM56, and we demonstrate the involvement of 60 proteins inthis complex phenomenon. We also describe the effect of pHo(adjusted with different acids) on the pHi of L. monocytogenesLO28 and comment on the role of pHi in protecting the or-ganisms from severe acid stress.

MATERIALS AND METHODS

Bacterial strains and media. L. monocytogenes LO28 (serotype 1/2c) is aclinical isolate obtained from P. Cossart, Pasteur Institute, Paris, France. Theisolation of the acid-tolerant mutant, designated ATM56, has been describedpreviously (18). This mutant is constitutively resistant to low pH at all stages ofgrowth. Bacteria were cultured in tryptone soy broth (Sigma Chemical Co., St.Louis, Mo.), supplemented with 0.6% yeast extract (TSB-YE). For solid media,agar was added to 1.5% (TSA-YE). Cell growth at 37°C in TSB-YE was moni-tored at 600 nm. All reagents used were obtained from Sigma unless otherwisespecified.

Adaptation of L. monocytogenes to two different acids and measurement of theATR. An overnight culture of L. monocytogenes was inoculated into freshTSB-YE (2.0% inoculum). Cells were grown statically to an absorbance at 600nm (A600) of 0.15 (early log phase). Triplicate samples were centrifuged andresuspended in TSB-YE adjusted with either 1 M lactic acid or 3 M HCl to pH5.5 and 5.0, respectively (acid adapted). Control cells were resuspended in pH 7.0broth (nonadapted). Following incubation at 37°C for 1 h, the cells were har-vested by centrifugation and resuspended in TSB-YE acidified to pH 3.5 (3 Mlactic acid) or pH 3.0 (5 M HCl). These acidified cultures were incubated for upto 120 min at 37°C, and viable plate counts were performed at intervals by serialdilution of samples in one-quarter-strength Ringer’s solution and enumerationon TSA-YE (8).

Preparation of protein samples. Overnight cultures of L. monocytogenes LO28and ATM56 were grown statically at 37°C in TSB-YE to early log phase (A600 50.15). The cells were adapted with either 1 M lactic acid or 3 M HCl ornonadapted as described above. They were incubated for 30 min at 20°C inprotoplast buffer (20 mM Tris-HCl [pH 7.5], 5 mM EDTA, 0.75 M sucrose, 10mg of lysozyme per ml, 50 U of mutanolysin per ml). The resulting protoplastswere then harvested by centrifugation at low speed for 10 min and subsequently

* Corresponding author. Mailing address: Department of Microbi-ology, University College Cork, Cork, Ireland. Phone: 353-21-902397.Fax: 353-21-903101. E-mail: c.hill@ucc.ie.

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FIG. 1. O’Farrell two-dimensional electrophoretic analysis of protein expression during the ATR in L. monocytogenes. (A) Cells grown at pH 7.0 (nonadapted); (B)cells adapted with lactic acid at pH 5.5; (C) cells adapted with HCl at pH 5.0; (D) nonadapted acid-tolerant mutant ATM56. Numbered circles refer to proteins whichare present in diminished amounts (relative to nonadapted cells), and numbered squares refer to proteins which are present in elevated amounts (relative to nonadaptedcells). Numbered arrows (A) refer to all protein alterations. Gels were run in the first dimension on a linear pH gradient; pH 5 (top left) to pH 7 (top right). Thenumbers on the left side of the gels indicate the molecular mass of the standards in kilodaltons.

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FIG. 1—Continued.

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lysed in 200 ml of Laemmli sample buffer (0.125 M Tris-HCl [pH 6.8], 10%glycerol, 5% b-mercaptoethanol, 2% [wt/vol] sodium dodecyl sulfate (SDS),0.01% bromophenol blue) at 90°C for 5 min. Proteins were precipitated withice-cold acetone and resolubilized in isoelectric focusing sample buffer (9.5 Murea, 0.2% [wt/vol] dithiothreitol, 1.6% 5-7 ampholytes, 0.4% 3-10 pharmalytes,0.5% [wt/vol] Triton X-100, 0.01% bromophenol blue). The samples were storedat 220°C.

Two-dimensional gel electrophoresis. Proteins were resolved on two-dimen-sional gels by the method of O’Farrell (19) with modifications as recommendedby the manufacturer (Pharmacia Biotech, Uppsala, Sweden). The proteins wereresolved by isoelectric focusing in the first dimension and discontinuous SDS-polyacrylamide gel electrophoresis in the second dimension with a Multiphor 11electrophoresis unit (Pharmacia Biotech). The first dimension involved a precastImmobiline DryStrip (Pharmacia Biotech) with a linear pH gradient (pH 5 to 7),while the second dimension was a precast ExcelGel SDS (Pharmacia Biotech)with a 12 to 14% (wt/vol) polyacrylamide gradient. Gels were stained with aSigma silver stain kit. Images of the gels were captured with a monochromecharge-coupled device camera linked to an image analysis software package(GlobalLab Data Translation).

Measurement of cytoplasmic volume. The cytoplasmic volume was determinedby the differential penetration of 3H2O (New England Nuclear, Boston, Mass.)and D-[U-14C]sorbitol (New England Nuclear) by the method of Patchett et al.(21). Cells were grown to an A600 of 0.15 and harvested by centrifugation. Thepellet was then resuspended in TSB-YE (adjusted to the appropriate pH) to givea final A600 of between 10 and 16. The cell suspensions were incubated at 37°C,and samples were taken at intervals. Aliquots (1 ml) were centrifuged (15,800 3g for 2 min), and the supernatant was removed. Aliquots (100 ml) of cell sus-pension and supernatant were counted in Ultima Gold scintillation fluid (Pack-ard Instrument Co., Rockville, Md.) in a Beckman liquid scintillation counterwith dual-label counting by full-spectrum analysis. The amount of protein perpellet (in milligrams) was also estimated by a Bio-Rad protein assay.

Measurement of pH. The pHi of cell suspensions was measured by the methodof Kroll and Booth (14), incorporating the internal volume previously calculated.The pHi was determined by measurement of the distribution of a radiolabelledweak acid, namely, [14C]benzoic acid. Cell suspensions were incubated at 37°Cwith [14C]benzoic acid (New England Nuclear) and [3H]sorbitol (New EnglandNuclear) as an extracellular water marker. At intervals, 1-ml samples were

centrifuged as previously described. A portion of the supernatant was removedand transferred to a centrifuge tube containing a similarly treated cell pellet thathad not been incubated with isotopes. This procedure ensured that the degreesof quenching in the supernatant and pellet samples were equivalent. Sampleswere counted as described above for measurement of intracellular volume. Datapoints indicate the mean of triplicate values, and the standard deviation is shownin each instance. Data sets at appropriate time points were subjected to a Studentt test.

RESULTS

Protein expression during acid induction and in the acid-tolerant mutant ATM56. Two-dimensional SDS-polyacryl-amide gel electrophoresis was used to examine protein alter-ations during acid adaptation and in the constitutively acid-tolerant mutant ATM56. Figure 1A indicates the positions ofall proteins shown to undergo alterations in levels under any ofthe conditions examined in this study. For convenience, theyhave been labelled p1, p2, p3, etc., in order of decreasingmolecular mass. The proteins are also listed in Table 1, to-gether with their response to induction with either lactic acidor hydrochloric acid. The fate of each protein in the acid-tolerant mutant is also presented in Table 1. Adaptation withlactic acid at pH 5.5 for 60 min (the optimum conditions forinduction with lactic acid) resulted in changes in the levels of34 proteins, 17 of which were induced by lactic acid and 17 ofwhich were repressed relative to the nonadapted cells (Fig. 1B;Table 1). Interestingly, adaptation with HCl at pH 5.0 for 60min (optimum conditions for induction with HCl) resulted in adifferent set of protein alterations in comparison to lactic acid,with 30 proteins present in increased amounts and 8 dimin-ished in concentration (Fig. 1C; Table 1). Furthermore, sixproteins (p1, p2, p25, p54, p57, and p59) appeared to be newlysynthesized in comparison to the nonadapted cells. Significantoverlap between the response to the two acids was evident, inthat 12 proteins showed increased levels in response to bothacids and 7 proteins were diminished in concentration underboth induction conditions (Table 1). One of the proteins syn-

FIG. 2. Acid tolerance response in L. monocytogenes LO28 with combina-tions of HCl and lactic acid as inducing and challenging acids. Cells were adaptedwith lactic acid and challenged with lactic acid (F) and HCl (å), adapted withHCl and challenged with HCl (■) and lactic acid (Ç); or nonadapted andchallenged with lactic acid (E) and HCl (h). The data shown are representativeof triplicate experiments. The variation was within 35% of the value given.

TABLE 1. Proteins which undergo changes in response to acidadaptation or are present in altered amounts in

the acid-tolerant mutant ATM56

Protein(molecular mass [kDa]a)

Responseb for:

Lacticacidc HClc ATM56

p3(86.8), p5(67.3), p21(37.0), p37(27.8),p38(28.0), p46(26.7)

1 1 1

p4(78.0), p11(53.0), p33(28.0), p34(28.7),p35(27.8), p52(23.3)

1 1 n

p6(65.3), p12(50.9), p18(44.2), p29(30.4),p56(,20)

1 n n

p1(102.0), p2(102.0), p10(53.2),p13(48.7), p25(33.7), p26(32.0),p30(30.0), p45(25.5), p54(20.8)

n 1 1

p7(56.0), p8(55.2), p23(34.1), p40(28.0),p42(28.0), p55(,20), p57(,20),p58(,20), p59(,20)

n 1 n

p15(47.8), p28(32.0), p44(25.6),p47(25.3), p50(24.0), p51(23.6)

n n 1

p36(28.0), p39(28.0), p41(27.8),p43(26.7), p60(,20)

2 2 2

p22(37.0), p53(21.3) 2 2 np9(54.2), p14(48.7), p16(46.9), p17(45.1),

p19(40.6), p20(37.0), p24(33.7),p48(25.0)

2 n n

p27(32.0), p49(25.0) 2 n 2p32(27.5) n 2 2p31(28.0) n n 2

a It was not possible to ascertain the molecular mass of proteins less than ca.20 kDa because the lower cutoff point of molecular mass markers used was 20.5kDa.

b 1, proteins present in increased amount or newly synthesized; 2, proteinspresent in diminished amount or absent; n, no significant change.

c L. monocytogenes adapted with lactic acid and HCl as described in Materialsand Methods.

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thesized exclusively after adaptation with HCl, p54, was alsoevident in a two-dimensional polyacrylamide gel profile of sta-tionary-phase cells (data not shown). The acid-tolerant mutantATM56 displayed an increased synthesis of 21 proteins, 15 ofwhich were also present in increased amounts in cells followinginduction with HCl (all 15) or lactic acid (6 of the 15 [Fig. 1D;Table 1]). Moreover, nine proteins were present in reducedamounts relative to those in uninduced LO28. It is also notablethat protein p54 (newly synthesized in HCl-induced cells and instationary-phase cells) was present in increased amounts inATM56.

Induction of the ATR by strong and weak acids. The proteinprofiles following induction with either lactic acid or HCl werenot identical, raising the possibility that the response inducedby the two acids also differs. To test this possibility, cells wereinduced separately with each acid and subsequently challengedwith both acid types. Previous experiments with a range of pHvalues have established that the optimal challenge and induc-tion conditions vary depending on the acidulent (data notshown). With lactic acid, optimal induction of acid toleranceoccurs at pH 5.5, while pH 3.5 is the optimal challenge pH. ForHCl, the optimal induction and challenge conditions are pH5.0 and 3.0, respectively. The extent of the ATR, as measuredby the percent survival after 90 min at the challenge pH, wasfound to be independent of the inducing acid in that a cultureoptimally induced with a strong acid is protected against chal-lenge with a weak acid and vice versa (Fig. 2). Therefore, nodifference in the ability of a culture to survive an acid challengecould be associated with the difference in protein patterns.This analysis was not extended to determine the extent ofprotection against other stresses.

Measurement of the pHi of LO28 and ATM56 during theATR. During severe acid stress, acid-adapted cells of LO28were capable of maintaining their pHi at slightly elevated levelscompared with nonadapted control cells (Table 2). Lactic acidwas more efficient than HCl in causing a decrease in the pHi(Fig. 3). This difference was predictable, given the differencesin the degree of dissociation between strong and weak acids atany given pH (2). While the degree of difference betweeninduced and noninduced cells was small (of the order of 0.12pH unit for lactic acid and 0.24 pH unit for HCl), it wasstatistically significant (P , 0.05). Nonetheless, it is difficult toascribe the differences in the percent survival to such smallvariations in pHi. Significantly, whereas nonadapted cells ex-posed to pH 3.0 (HCl) maintained a pHi of 5.74 after 90 min,they were unable to form colonies after this time. Cells initiallyadapted with lactic acid and subsequently challenged with lac-tic acid (pH 3.5) maintained their pHi at pH 5.70 and displayeda much higher percent survival. Thus, it is obvious that the pHialone does not determine whether a cell is viable (where via-bility is defined as the ability to give rise to a colony). Theacid-tolerant mutant ATM56 was capable of maintaining its

pHi at levels similar to those of induced cells in the presence ofboth HCl and lactic acid.

When chloramphenicol (100 mg/ml) is added immediatelyprior to the shift to the induction pH, the protective effectconferred by the ATR is almost eliminated (3, 18). Cellstreated with chloramphenicol during acid adaptation main-tained their pHi at values similar to those of nonadapted cells,suggesting that the inhibition of protein synthesis also inhibitsthe ability of L. monocytogenes to maintain intracellular ho-meostasis at lethal pH values. Similar results were also ob-tained whether HCl or lactic acid was used as the inducing andchallenging acid (Table 3).

DISCUSSION

Resistance to environmental stress in a diverse range ofbacteria has been linked to the induction of a unique set ofproteins (10, 11, 25, 26). Proteins which are induced by a rangeof stresses are considered to be members of global regulatorynetworks. It is conjectured that these global control systemscomprise multiple unlinked genes and operons coordinatelycontrolled by a common regulatory signal or regulatory gene.The term “stimulon” was coined to refer to a set of genes andultimately proteins which respond to a given environmentalstress (17). Surprisingly, the imposition of a single stress due toacid on L. monocytogenes appears to induce two differentstimulons depending on the acid type. This premise is based on

FIG. 3. The pHi of L. monocytogenes at different pHo values. The externalpH was adjusted with lactic acid (F) or HCl (■). Error bars represent thestandard deviations for triplicate experiments.

TABLE 2. pHi of LO28 and ATM56 during ATR

Listeria strain Induction conditions atsublethal pH for 60 mina Challenge pH pHi of cells exposed to

lethal pHo for 90 min % Survivalb

LO28 pH 3.5 (lactic acid) 5.58 6 0.031 0.01 6 0.002LO28 pH 5.5 (lactic acid) pH 3.5 (lactic acid) 5.70 6 0.028 26.6 6 5.44ATM56 pH 3.5 (lactic acid) 5.68 6 0.047 2.62 6 0.960LO28 pH 3.0 (HCl) 5.74 6 0.050 0.02 6 0.002LO28 pH 5.0 (HCl) pH 3.0 (HCl) 5.98 6 0.047 24.0 6 3.50ATM56 pH 3.0 (HCl) 5.84 6 0.045 2.10 6 0.360

a The initial pHo of cells was 7.0 prior to adjustment to induction conditions and subsequent exposure to challenging conditions as shown.b The percent survival of cells after 90 min is shown.

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two-dimensional protein analysis, which produced two differ-ent although overlapping protein profiles on induction withlactic acid and HCl at their optimal induction pH values forinitiating an ATR. It is noteworthy that in spite of these ap-parent differences in protein patterns, the degree of acid tol-erance conferred by both induction conditions appears identi-cal. Indeed, it is likely that an essential, overlapping group ofproteins is important for maintaining homeostasis followingeither HCl or lactic acid shock. Proteins that are induced byeither HCl or lactic acid alone may play peripheral roles inensuring cell survival.

Several proteins showing altered expression during the ATRin L. monocytogenes LO28 have comparable molecular weightsto proteins altered during the ATR in L. monocytogenes ScottA (3). However, the different natures of the gels used to sep-arate proteins in the first dimension and the second dimensionpreclude an accurate comparison of the two strains.

We have previously presented results on the efficacy of lacticacid as both an inducing and challenging acid in provoking asignificant ATR (18). In this study we demonstrate that HCl isequally efficient at demonstrating the presence of an ATR,albeit with different inducing and challenging pH values, aslactic acid. In spite of the evidence derived from the two-dimensional protein analysis suggesting that these acids mayinduce slightly different stimulons, both weak and strong acidsappear to be interchangeable in that HCl-induced cells areresistant to subsequent lactic acid challenge and vice versa.This observation may be particularly relevant when the infec-tious nature of L. monocytogenes is considered, in that the mostlikely acids that the organism will encounter in foods are weak(e.g., lactic and acetic acids) (23) whereas the initial barrier toa successful infection is presented by HCl in the stomach.Interestingly, recent work on S. typhimurium shows that induc-tion of the ATR with HCl protects against weak acids such asbenzoic acid and acetic acid (1).

Distinct protein differences were evident between the acid-tolerant mutant ATM56 and the parent strain, L. monocyto-genes LO28. Significant overlap was observed between the pro-teins altered in ATM56 and during induction of the ATR. Sixof the proteins which were present at elevated concentrationsand seven of the proteins which were present at diminishedconcentrations following induction with lactic acid were alsopresent in ATM56 in the absence of induction. In addition, 15of the proteins which were increased in concentration and 6 ofthe proteins which were reduced in concentration after induc-

tion with HCl were evident in ATM56. In all, changes in 30proteins were observed, 21 of which were present in increasedamounts (seven of which were exclusively synthesized) and 9 ofwhich were present in decreased amounts.

The ability to maintain pH homeostasis during severe acidstress is essential for the survival of neutralophilic bacteria. Ithas been shown for other genera that organisms adapted toacid maintain a higher pHi than do nonadapted cells whenchallenged with lethal acidic conditions. Foster and Hall dem-onstrated a superior pH homeostasis capacity in adapted S.typhimurium cells at low pHo in comparison to nonadaptedcells (6). At a pHo of 3.3, the pHi of adapted cells was main-tained at pH 5.0 in contrast to a pHi of 4.4 in nonadapted cells.In Lactococcus lactis, a less dramatic difference in pHi betweenadapted and nonadapted cells was evident. At a challenge pHoof 4.0, adapted cells maintained a pHi of 5.37, in contrast to apHi of 5.18 in nonadapted cells (20). This observation may alsobe extended to L. monocytogenes, since at a given lethal pHo,acid-adapted LO28 maintained a pHi at slightly higher levelsthan did nonadapted control cells. The extent of the differencein pHi was similar to that observed for L. lactis. pHi measure-ments of ATM56 at lethal pH values revealed the ability of thismutant to maintain a higher pHi in comparison to the parentstrain, LO28. This may be a contributory factor to its enhancedability to survive adverse acidic conditions. The relatively smalldegree of difference between the pHi of adapted and non-adapted cells and between nonadapted and mutant cells sug-gests that the pHi alone is not responsible for the difference inpercent survival displayed by these two cell types but that someother factor must also be involved.

As expected, the inhibition of protein synthesis with chlor-amphenicol prevented the development of pH homeostasis inacid-adapted cells, confirming the key role of de novo proteinsynthesis in regulation of pH homeostasis. Rapid shifts in pHolower the cytoplasmic pH transiently, and this change may besufficient to alter the rate of synthesis of acid-inducible geneproducts. Regulation of gene expression by cytoplasmic pH inS. typhimurium is well documented (7). Stationary-phase cellsare more tolerant to the adverse effects of acidic pH than arelog-phase cells for longer periods, and this growth phase-asso-ciated resistance is dependent on the alternative sigma factorRpoS (5). Acidification of the cytoplasm is one of the factorsknown to elicit increased synthesis of RpoS (24).

The present study demonstrates the complex physiology ofacid adaptation. Further studies on the molecular mechanismsgoverning the ATR and the enhanced acid resistance ofATM56 are under way and may provide an insight into theability of L. monocytogenes to withstand hostile environmentalconditions in foods and during infection.

ACKNOWLEDGMENTS

This work was supported by the Food Sub-Programme of the Op-erational Programme for Industrial Development which is adminis-tered by the Department of Agriculture, Food and Forestry and sup-ported by national and EU funds.

We thank Conor O’Byrne at Unilever Research in Colworth for hisassistance in two-dimensional polyacrylamide gel electrophoresis andhelpful discussions. We are also grateful to Eilis O’Sullivan and JohnO’Callaghan for helpful advice during pHi measurements.

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TABLE 3. Chloramphenicol inhibits the ability of adaptedcells to maintain intracellular pH homeostasis

Induction conditionsat sublethal pH

for 60 mina

Cma

present

pHi of cellsexposed to lethalpH for 90 minb

% Survivalc

2 5.58 6 0.031 0.01 6 0.0021 5.51 6 0.030 0.01 6 0.002

pH 5.5 (lactic acid) 2 5.70 6 0.040 26.6 6 5.44pH 5.5 (lactic acid) 1 5.60 6 0.031 0.05 6 0.007

2 5.74 6 0.050 0.02 6 0.0021 5.65 6 0.031 0.02 6 0.002

pH 5.0 (HCl) 2 5.98 6 0.007 24.0 6 3.50pH 5.0 (HCl) 1 5.88 6 0.030 0.06 6 0.003

a The initial pHo of cells was 7.0 prior to induction in the absence or presenceof chloramphenicol (Cm).

b Cells induced with lactic acid were challenged with lactic acid at pH 3.5. Cellsinduced with HCl were challenged with HCl at pH 3.0.

c The percent survival of cells after 90 min is shown.

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