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

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1997, p. 2679 2685 Vol. 63, No. 7 0099-2240/97/$04.00 0 Copyright 1997, American Society for Microbiology Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of the Acid 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 adaptation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tolerance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresis revealed changes in the levels of 53 proteins. Significant protein differences were also evident between nonadapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis revealed differences in protein expression between cells induced with a weak acid (lactic acid) and those induced with a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capable of maintaining their internal ph (ph i ) 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 acid adaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions. Listeria monocytogenes, a causative agent of both sporadic and epidemic food-borne illness, emerged as an important human pathogen in the 1980s. Its ability to survive and grow in many foods, in addition to its formidable capacity to overcome host defense mechanisms and cause infection, has been closely monitored in the last decade. L. monocytogenes is a neutralophile and will grow optimally only within a narrow ph range (optimum ph 6 to 7). The term ph homeostasis is used to describe the ability of an organism to maintain its cytoplasmic ph (ph i ) at a value close to neutrality despite fluctuations in the external ph (ph o ) (12). It has been proposed that when the ph i falls below a certain threshold value, cells will cease to function (16). The mechanism of action by which ph homeostasis is achieved is poorly understood, but both passive and active mechanisms are thought to be involved. Log-phase cells of L. monocytogenes are very sensitive to low ph. The ability of these cells to withstand lethal ph conditions can be dramatically improved following adaptation to a sublethal ph (3, 15, 18). This adaptation is termed the acid tolerance response (ATR). Spontaneous acid-tolerant mutants of L. monocytogenes can also be recovered following exposure to severe acid stress. Mouse infection studies involving one such mutant, ATM56, revealed increased virulence relative to the parent strain, LO28 (18). Another trait attributable to the acidtolerant mutant was an enhanced ability to survive in low-ph foods (8). This study also demonstrated the significance of acid adaptation in the survival of L. monocytogenes in acid foods and during milk fermentation. Considerable data is available regarding the ATR phenomenon in other food-borne pathogens, namely, Salmonella typhimurium, Escherichia coli, and Aeromonas hydrophila (9, 13, 22). These studies have revealed that the ATR is a dauntingly complex biological phenomenon. Induction of the ATR in S. typhimurium involves the alteration of expression of as many as * Corresponding author. Mailing address: Department of Microbiology, University College Cork, Cork, Ireland. Phone: 353-21-902397. Fax: 353-21-903101. E-mail: c.hill@ucc.ie. 52 separate proteins (4). It has previously been shown that the addition of chloramphenicol impedes the development of the ATR in L. monocytogenes, indicating the importance of de novo protein synthesis (3, 18). Davis et al. (3) have reported that the ATR induced in L. monocytogenes Scott A by a strong acid, HCl, involved the altered expression of at least 23 proteins. In this report, we describe the alterations in protein levels in L. monocytogenes during acid habituation with both strong and weak acids and also in the acid-tolerant mutant ATM56, and we demonstrate the involvement of 60 proteins in this complex phenomenon. We also describe the effect of ph o (adjusted with different acids) on the ph i of L. monocytogenes LO28 and comment on the role of ph i in protecting the organisms from severe acid stress. MATERIALS AND METHODS Bacterial strains and media. L. monocytogenes LO28 (serotype 1/2c) is a clinical isolate obtained from P. Cossart, Pasteur Institute, Paris, France. The isolation of the acid-tolerant mutant, designated ATM56, has been described previously (18). This mutant is constitutively resistant to low ph at all stages of growth. 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 monitored at 600 nm. All reagents used were obtained from Sigma unless otherwise specified. Adaptation of L. monocytogenes to two different acids and measurement of the ATR. An overnight culture of L. monocytogenes was inoculated into fresh TSB-YE (2.0% inoculum). Cells were grown statically to an absorbance at 600 nm (A 600 ) of 0.15 (early log phase). Triplicate samples were centrifuged and resuspended in TSB-YE adjusted with either 1 M lactic acid or 3 M HCl to ph 5.5 and 5.0, respectively (acid adapted). Control cells were resuspended in ph 7.0 broth (nonadapted). Following incubation at 37 C for 1 h, the cells were harvested by centrifugation and resuspended in TSB-YE acidified to ph 3.5 (3 M lactic acid) or ph 3.0 (5 M HCl). These acidified cultures were incubated for up to 120 min at 37 C, and viable plate counts were performed at intervals by serial dilution of samples in one-quarter-strength Ringer s solution and enumeration on TSA-YE (8). Preparation of protein samples. Overnight cultures of L. monocytogenes LO28 and ATM56 were grown statically at 37 C in TSB-YE to early log phase (A 600 0.15). The cells were adapted with either 1 M lactic acid or 3 M HCl or nonadapted as described above. They were incubated for 30 min at 20 C in protoplast buffer (20 mm Tris-HCl [ph 7.5], 5 mm EDTA, 0.75 M sucrose, 10 mg of lysozyme per ml, 50 U of mutanolysin per ml). The resulting protoplasts were then harvested by centrifugation at low speed for 10 min and subsequently 2679

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 which are present in diminished amounts (relative to nonadapted cells), and numbered squares refer to proteins which are present in elevated amounts (relative to nonadapted cells). 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). The numbers on the left side of the gels indicate the molecular mass of the standards in kilodaltons. 2680

VOL. 63, 1997 ACID-INDUCED PROTEINS IN L. MONOCYTOGENES 2681 FIG. 1 Continued.

2682 O DRISCOLL ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Proteins which undergo changes in response to acid adaptation or are present in altered amounts in the acid-tolerant mutant ATM56 Protein (molecular mass [kda] a ) Response b for: Lactic acid c HCl c ATM56 p3(86.8), p5(67.3), p21(37.0), p37(27.8), p38(28.0), p46(26.7) p4(78.0), p11(53.0), p33(28.0), p34(28.7), n p35(27.8), p52(23.3) p6(65.3), p12(50.9), p18(44.2), p29(30.4), n n p56( 20) p1(102.0), p2(102.0), p10(53.2), n p13(48.7), p25(33.7), p26(32.0), p30(30.0), p45(25.5), p54(20.8) p7(56.0), p8(55.2), p23(34.1), p40(28.0), n n p42(28.0), p55( 20), p57( 20), p58( 20), p59( 20) p15(47.8), p28(32.0), p44(25.6), n n p47(25.3), p50(24.0), p51(23.6) p36(28.0), p39(28.0), p41(27.8), p43(26.7), p60( 20) p22(37.0), p53(21.3) n p9(54.2), p14(48.7), p16(46.9), p17(45.1), n n p19(40.6), p20(37.0), p24(33.7), p48(25.0) p27(32.0), p49(25.0) n p32(27.5) n p31(28.0) n n 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 20.5 kda. b, proteins present in increased amount or newly synthesized;, proteins present in diminished amount or absent; n, no significant change. c L. monocytogenes adapted with lactic acid and HCl as described in Materials and Methods. lysed in 200 l of Laemmli sample buffer (0.125 M Tris-HCl [ph 6.8], 10% glycerol, 5% -mercaptoethanol, 2% [wt/vol] sodium dodecyl sulfate (SDS), 0.01% bromophenol blue) at 90 C for 5 min. Proteins were precipitated with ice-cold acetone and resolubilized in isoelectric focusing sample buffer (9.5 M urea, 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 stored at 20 C. Two-dimensional gel electrophoresis. Proteins were resolved on two-dimensional gels by the method of O Farrell (19) with modifications as recommended by the manufacturer (Pharmacia Biotech, Uppsala, Sweden). The proteins were resolved by isoelectric focusing in the first dimension and discontinuous SDSpolyacrylamide gel electrophoresis in the second dimension with a Multiphor 11 electrophoresis unit (Pharmacia Biotech). The first dimension involved a precast Immobiline 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 a Sigma silver stain kit. Images of the gels were captured with a monochrome charge-coupled device camera linked to an image analysis software package (GlobalLab Data Translation). Measurement of cytoplasmic volume. The cytoplasmic volume was determined by the differential penetration of 3 H 2 O (New England Nuclear, Boston, Mass.) and D-[U- 14 C]sorbitol (New England Nuclear) by the method of Patchett et al. (21). Cells were grown to an A 600 of 0.15 and harvested by centrifugation. The pellet was then resuspended in TSB-YE (adjusted to the appropriate ph) to give a final A 600 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 g for 2 min), and the supernatant was removed. Aliquots (100 l) of cell suspension and supernatant were counted in Ultima Gold scintillation fluid (Packard Instrument Co., Rockville, Md.) in a Beckman liquid scintillation counter with dual-label counting by full-spectrum analysis. The amount of protein per pellet (in milligrams) was also estimated by a Bio-Rad protein assay. Measurement of ph. The ph i of cell suspensions was measured by the method of Kroll and Booth (14), incorporating the internal volume previously calculated. The ph i was determined by measurement of the distribution of a radiolabelled weak acid, namely, [ 14 C]benzoic acid. Cell suspensions were incubated at 37 C with [ 14 C]benzoic acid (New England Nuclear) and [ 3 H]sorbitol (New England Nuclear) as an extracellular water marker. At intervals, 1-ml samples were centrifuged as previously described. A portion of the supernatant was removed and transferred to a centrifuge tube containing a similarly treated cell pellet that had not been incubated with isotopes. This procedure ensured that the degrees of quenching in the supernatant and pellet samples were equivalent. Samples were counted as described above for measurement of intracellular volume. Data points indicate the mean of triplicate values, and the standard deviation is shown in each instance. Data sets at appropriate time points were subjected to a Student t test. RESULTS Protein expression during acid induction and in the acidtolerant mutant ATM56. Two-dimensional SDS-polyacrylamide gel electrophoresis was used to examine protein alterations during acid adaptation and in the constitutively acidtolerant mutant ATM56. Figure 1A indicates the positions of all proteins shown to undergo alterations in levels under any of the conditions examined in this study. For convenience, they have been labelled p1, p2, p3, etc., in order of decreasing molecular mass. The proteins are also listed in Table 1, together with their response to induction with either lactic acid or hydrochloric acid. The fate of each protein in the acidtolerant mutant is also presented in Table 1. Adaptation with lactic acid at ph 5.5 for 60 min (the optimum conditions for induction with lactic acid) resulted in changes in the levels of 34 proteins, 17 of which were induced by lactic acid and 17 of which were repressed relative to the nonadapted cells (Fig. 1B; Table 1). Interestingly, adaptation with HCl at ph 5.0 for 60 min (optimum conditions for induction with HCl) resulted in a different set of protein alterations in comparison to lactic acid, with 30 proteins present in increased amounts and 8 diminished in concentration (Fig. 1C; Table 1). Furthermore, six proteins (p1, p2, p25, p54, p57, and p59) appeared to be newly synthesized in comparison to the nonadapted cells. Significant overlap between the response to the two acids was evident, in that 12 proteins showed increased levels in response to both acids and 7 proteins were diminished in concentration under both induction conditions (Table 1). One of the proteins syn- FIG. 2. Acid tolerance response in L. monocytogenes LO28 with combinations of HCl and lactic acid as inducing and challenging acids. Cells were adapted with lactic acid and challenged with lactic acid (F) and HCl (å), adapted with HCl and challenged with HCl ( ) and lactic acid (Ç); or nonadapted and challenged with lactic acid (E) and HCl ( ). The data shown are representative of triplicate experiments. The variation was within 35% of the value given.

VOL. 63, 1997 ACID-INDUCED PROTEINS IN L. MONOCYTOGENES 2683 Listeria strain Induction conditions at sublethal ph for 60 min a TABLE 2. ph i of LO28 and ATM56 during ATR Challenge ph ph i of cells exposed to lethal ph o for 90 min % Survival b LO28 ph 3.5 (lactic acid) 5.58 0.031 0.01 0.002 LO28 ph 5.5 (lactic acid) ph 3.5 (lactic acid) 5.70 0.028 26.6 5.44 ATM56 ph 3.5 (lactic acid) 5.68 0.047 2.62 0.960 LO28 ph 3.0 (HCl) 5.74 0.050 0.02 0.002 LO28 ph 5.0 (HCl) ph 3.0 (HCl) 5.98 0.047 24.0 3.50 ATM56 ph 3.0 (HCl) 5.84 0.045 2.10 0.360 a The initial ph o 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. thesized exclusively after adaptation with HCl, p54, was also evident in a two-dimensional polyacrylamide gel profile of stationary-phase cells (data not shown). The acid-tolerant mutant ATM56 displayed an increased synthesis of 21 proteins, 15 of which were also present in increased amounts in cells following induction with HCl (all 15) or lactic acid (6 of the 15 [Fig. 1D; Table 1]). Moreover, nine proteins were present in reduced amounts relative to those in uninduced LO28. It is also notable that protein p54 (newly synthesized in HCl-induced cells and in stationary-phase cells) was present in increased amounts in ATM56. Induction of the ATR by strong and weak acids. The protein profiles following induction with either lactic acid or HCl were not identical, raising the possibility that the response induced by the two acids also differs. To test this possibility, cells were induced separately with each acid and subsequently challenged with both acid types. Previous experiments with a range of ph values have established that the optimal challenge and induction conditions vary depending on the acidulent (data not shown). With lactic acid, optimal induction of acid tolerance occurs at ph 5.5, while ph 3.5 is the optimal challenge ph. For HCl, the optimal induction and challenge conditions are ph 5.0 and 3.0, respectively. The extent of the ATR, as measured by the percent survival after 90 min at the challenge ph, was found to be independent of the inducing acid in that a culture optimally induced with a strong acid is protected against challenge with a weak acid and vice versa (Fig. 2). Therefore, no difference in the ability of a culture to survive an acid challenge could be associated with the difference in protein patterns. This analysis was not extended to determine the extent of protection against other stresses. Measurement of the ph i of LO28 and ATM56 during the ATR. During severe acid stress, acid-adapted cells of LO28 were capable of maintaining their ph i at slightly elevated levels compared with nonadapted control cells (Table 2). Lactic acid was more efficient than HCl in causing a decrease in the ph i (Fig. 3). This difference was predictable, given the differences in the degree of dissociation between strong and weak acids at any given ph (2). While the degree of difference between induced and noninduced cells was small (of the order of 0.12 ph unit for lactic acid and 0.24 ph unit for HCl), it was statistically significant (P 0.05). Nonetheless, it is difficult to ascribe the differences in the percent survival to such small variations in ph i. Significantly, whereas nonadapted cells exposed to ph 3.0 (HCl) maintained a ph i of 5.74 after 90 min, they were unable to form colonies after this time. Cells initially adapted with lactic acid and subsequently challenged with lactic acid (ph 3.5) maintained their ph i at ph 5.70 and displayed a much higher percent survival. Thus, it is obvious that the ph i alone does not determine whether a cell is viable (where viability is defined as the ability to give rise to a colony). The acid-tolerant mutant ATM56 was capable of maintaining its ph i at levels similar to those of induced cells in the presence of both HCl and lactic acid. When chloramphenicol (100 g/ml) is added immediately prior to the shift to the induction ph, the protective effect conferred by the ATR is almost eliminated (3, 18). Cells treated with chloramphenicol during acid adaptation maintained their ph i at values similar to those of nonadapted cells, suggesting that the inhibition of protein synthesis also inhibits the ability of L. monocytogenes to maintain intracellular homeostasis at lethal ph values. Similar results were also obtained whether HCl or lactic acid was used as the inducing and challenging acid (Table 3). DISCUSSION Resistance to environmental stress in a diverse range of bacteria has been linked to the induction of a unique set of proteins (10, 11, 25, 26). Proteins which are induced by a range of stresses are considered to be members of global regulatory networks. It is conjectured that these global control systems comprise multiple unlinked genes and operons coordinately controlled by a common regulatory signal or regulatory gene. The term stimulon was coined to refer to a set of genes and ultimately proteins which respond to a given environmental stress (17). Surprisingly, the imposition of a single stress due to acid on L. monocytogenes appears to induce two different stimulons depending on the acid type. This premise is based on FIG. 3. The ph i of L. monocytogenes at different ph o values. The external ph was adjusted with lactic acid (F) or HCl ( ). Error bars represent the standard deviations for triplicate experiments.

2684 O DRISCOLL ET AL. APPL. ENVIRON. MICROBIOL. TABLE 3. Chloramphenicol inhibits the ability of adapted cells to maintain intracellular ph homeostasis Induction conditions at sublethal ph for 60 min a Cma present ph i of cells exposed to lethal ph for 90 min b % Survival c 5.58 0.031 0.01 0.002 5.51 0.030 0.01 0.002 ph 5.5 (lactic acid) 5.70 0.040 26.6 5.44 ph 5.5 (lactic acid) 5.60 0.031 0.05 0.007 5.74 0.050 0.02 0.002 5.65 0.031 0.02 0.002 ph 5.0 (HCl) 5.98 0.007 24.0 3.50 ph 5.0 (HCl) 5.88 0.030 0.06 0.003 a The initial ph o of cells was 7.0 prior to induction in the absence or presence of chloramphenicol (Cm). b Cells induced with lactic acid were challenged with lactic acid at ph 3.5. Cells induced with HCl were challenged with HCl at ph 3.0. c The percent survival of cells after 90 min is shown. two-dimensional protein analysis, which produced two different although overlapping protein profiles on induction with lactic acid and HCl at their optimal induction ph values for initiating an ATR. It is noteworthy that in spite of these apparent differences in protein patterns, the degree of acid tolerance conferred by both induction conditions appears identical. Indeed, it is likely that an essential, overlapping group of proteins is important for maintaining homeostasis following either HCl or lactic acid shock. Proteins that are induced by either HCl or lactic acid alone may play peripheral roles in ensuring cell survival. Several proteins showing altered expression during the ATR in L. monocytogenes LO28 have comparable molecular weights to proteins altered during the ATR in L. monocytogenes Scott A (3). However, the different natures of the gels used to separate proteins in the first dimension and the second dimension preclude an accurate comparison of the two strains. We have previously presented results on the efficacy of lactic acid as both an inducing and challenging acid in provoking a significant ATR (18). In this study we demonstrate that HCl is equally efficient at demonstrating the presence of an ATR, albeit with different inducing and challenging ph values, as lactic acid. In spite of the evidence derived from the twodimensional protein analysis suggesting that these acids may induce slightly different stimulons, both weak and strong acids appear to be interchangeable in that HCl-induced cells are resistant to subsequent lactic acid challenge and vice versa. This observation may be particularly relevant when the infectious nature of L. monocytogenes is considered, in that the most likely acids that the organism will encounter in foods are weak (e.g., lactic and acetic acids) (23) whereas the initial barrier to a successful infection is presented by HCl in the stomach. Interestingly, recent work on S. typhimurium shows that induction of the ATR with HCl protects against weak acids such as benzoic acid and acetic acid (1). Distinct protein differences were evident between the acidtolerant mutant ATM56 and the parent strain, L. monocytogenes LO28. Significant overlap was observed between the proteins altered in ATM56 and during induction of the ATR. Six of the proteins which were present at elevated concentrations and seven of the proteins which were present at diminished concentrations following induction with lactic acid were also present in ATM56 in the absence of induction. In addition, 15 of the proteins which were increased in concentration and 6 of the proteins which were reduced in concentration after induction with HCl were evident in ATM56. In all, changes in 30 proteins were observed, 21 of which were present in increased amounts (seven of which were exclusively synthesized) and 9 of which were present in decreased amounts. The ability to maintain ph homeostasis during severe acid stress is essential for the survival of neutralophilic bacteria. It has been shown for other genera that organisms adapted to acid maintain a higher ph i than do nonadapted cells when challenged with lethal acidic conditions. Foster and Hall demonstrated a superior ph homeostasis capacity in adapted S. typhimurium cells at low ph o in comparison to nonadapted cells (6). At a ph o of 3.3, the ph i of adapted cells was maintained at ph 5.0 in contrast to a ph i of 4.4 in nonadapted cells. In Lactococcus lactis, a less dramatic difference in ph i between adapted and nonadapted cells was evident. At a challenge ph o of 4.0, adapted cells maintained a ph i of 5.37, in contrast to a ph i of 5.18 in nonadapted cells (20). This observation may also be extended to L. monocytogenes, since at a given lethal ph o, acid-adapted LO28 maintained a ph i at slightly higher levels than did nonadapted control cells. The extent of the difference in ph i was similar to that observed for L. lactis. ph i measurements of ATM56 at lethal ph values revealed the ability of this mutant to maintain a higher ph i in comparison to the parent strain, LO28. This may be a contributory factor to its enhanced ability to survive adverse acidic conditions. The relatively small degree of difference between the ph i of adapted and nonadapted cells and between nonadapted and mutant cells suggests that the ph i alone is not responsible for the difference in percent survival displayed by these two cell types but that some other factor must also be involved. As expected, the inhibition of protein synthesis with chloramphenicol prevented the development of ph homeostasis in acid-adapted cells, confirming the key role of de novo protein synthesis in regulation of ph homeostasis. Rapid shifts in ph o lower the cytoplasmic ph transiently, and this change may be sufficient to alter the rate of synthesis of acid-inducible gene products. Regulation of gene expression by cytoplasmic ph in S. typhimurium is well documented (7). Stationary-phase cells are more tolerant to the adverse effects of acidic ph than are log-phase cells for longer periods, and this growth phase-associated resistance is dependent on the alternative sigma factor RpoS (5). Acidification of the cytoplasm is one of the factors known to elicit increased synthesis of RpoS (24). The present study demonstrates the complex physiology of acid adaptation. Further studies on the molecular mechanisms governing the ATR and the enhanced acid resistance of ATM56 are under way and may provide an insight into the ability of L. monocytogenes to withstand hostile environmental conditions in foods and during infection. ACKNOWLEDGMENTS This work was supported by the Food Sub-Programme of the Operational Programme for Industrial Development which is administered by the Department of Agriculture, Food and Forestry and supported by national and EU funds. We thank Conor O Byrne at Unilever Research in Colworth for his assistance in two-dimensional polyacrylamide gel electrophoresis and helpful discussions. We are also grateful to Eilis O Sullivan and John O Callaghan for helpful advice during ph i measurements. REFERENCES 1. Baik, H. S., S. Bearson, S. Dunbar, and J. W. Foster. 1996. The acid tolerance response of Salmonella typhimurium provides protection against organic acids. Microbiology 142:3195 3200. 2. Cherrington, C. A., M. Hinton, G. C. Mead, and I. Chopra. 1991. Organic acids: chemistry, antibacterial activity and practical applications. Adv. Microb. Physiol. 32:87 108.

VOL. 63, 1997 ACID-INDUCED PROTEINS IN L. MONOCYTOGENES 2685 3. Davis, M. J., P. J. Coote, and C. P. O Byrne. 1996. Acid tolerance in Listeria monocytogenes: the adaptive acid tolerance response (ATR) and growthphase-dependent acid resistance. Microbiology 142:2975 2982. 4. Foster, J. W. 1991. Salmonella acid shock proteins are required for the adaptive acid tolerance response. J. Bacteriol. 173:6896 6902. 5. Foster, J. W. 1995. Low ph adaptation and the acid tolerance response of Salmonella typhimurium. Crit. Rev. Microbiol. 21:215 237. 6. Foster, J. W., and H. K. Hall. 1992. Effect of Salmonella typhimurium ferric uptake regulator (fur) mutations on iron- and ph-regulated protein synthesis. J. Bacteriol. 174:4317 4323. 7. Foster, J. W., Y. K. Park, I. S. Bang, K. Karem, H. Betts, H. K. Hall, and E. Shaw. 1994. Regulatory circuits involved with ph-regulated gene expression in Salmonella typhimurium. Microbiology 140:341 352. 8. Gahan, C. G. M., B. O Driscoll, and C. Hill. 1996. Acid adaptation of Listeria monocytogenes can enhance survival in acid foods and during milk fermentation. Appl. Environ. Microbiol. 62:3128 3132. 9. Goodson, M., and R. J. Rowbury. 1989. Habituation to normal lethal acidity by prior growth of Escherichia coli at a sublethal ph value. Lett. Appl. Microbiol. 8:77 79. 10. Heyde, M., and R. Portalier. 1990. Acid shock proteins of Escherichia coli. FEMS Microbiol. Lett. 69:19 26. 11. Hickey, E. W., and I. N. Hirshfield. 1990. Low-pH-induced effects on patterns of protein synthesis and on internal ph in Escherichia coli and Salmonella typhimurium. Appl. Environ. Microbiol. 56:1038 1045. 12. Hill, C., B. O Driscoll, and I. R. Booth. 1995. Acid adaptation and food poisoning microorganisms. Int. J. Food. Microbiol. 28:245 254. 13. Karem, K. J., J. W. Foster, and A. K. Bej. 1994. Adaptive acid tolerance response (ATR) in Aeromonas hydrophila. Microbiology 140:1731 1736. 14. Kroll, R. G., and I. R. Booth. 1981. The role of potassium transport in the generation of a ph gradient in Escherichia coli. Biochem. J. 198:691 698. 15. Kroll, R. G., and R. A. Patchett. 1992. Induced acid tolerance response (ATR) in Listeria monocytogenes. Lett. Appl. Microbiol. 14:224 227. 16. McDonald, L. C., H. M. Hassan, H. P. Fleming, and M. A. Daeschel. 1991. Use of continuous culture for internal ph determination of lactic acid bacteria. Food Microbiol. 8:137 142. 17. Nystrom, T., and F. C. Neidhardt. 1993. Isolation and properties of a mutant of Escherichia coli with an insertional inactivation of the uspa gene, which encodes a universal stress protein. J. Bacteriol. 175:3949 3956. 18. O Driscoll, B., C. G. M. Gahan, and C. Hill. 1996. Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl. Environ. Microbiol. 62:1693 1698. 19. O Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007 4021. 20. O Sullivan, E. 1996. The response of Lactococcus lactis subsp. cremoris 712 to acid stress, p. 103. Ph.D. thesis. National University of Ireland, Dublin. 21. Patchett, R. A., A. F. Kelly, and R. G. Kroll. 1992. Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes. Appl. Environ. Microbiol. 58:3959 3963. 22. Rowbury, R. J. 1995. An assessment of environmental factors influencing acid tolerance and sensitivity in Escherichia coli, Salmonella spp. and other enterobacteria. Lett. Appl. Microbiol. 20:333 337. 23. Salmond, C. V., R. G. Kroll, and I. R. Booth. 1984. The effect of food preservatives on ph homeostasis in Escherichia coli. J. Gen. Microbiol. 130:2845 2850. 24. Schellhorn, H. E., and V. L. Stones. 1992. Regulation of KatF and KatE in Escherichia coli K-12 by weak acids. J. Bacteriol. 174:4769 4776. 25. Spector, M. P., Z. Aliabadi, T. Gonzalez, and J. W. Foster. 1986. Global control in Salmonella typhimurium: two-dimensional electrophoretic analysis of starvation-, anaerobiosis-, and heat-shock-inducible proteins. J. Bacteriol. 168:420 424. 26. Volker, U., S. Engelmann, B. Maul, S. Riethdorf, A. Volker, R. Schmid, H. Mach, and M. Hecker. 1994. Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology 140:741 752. Downloaded from http://aem.asm.org/ on May 8, 2018 by guest