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J Bacteriol. Apr 2001; 183(7): 2178–2186.
PMCID: PMC95122

Global Analysis of Escherichia coli Gene Expression during the Acetate-Induced Acid Tolerance Response

Abstract

The ability of Escherichia coli to survive at low pH is strongly affected by environmental factors, such as composition of the growth medium and growth phase. Exposure to short-chain fatty acids, such as acetate, proprionate, and butyrate, at neutral or nearly neutral pH has also been shown to increase acid survival of E. coli and Salmonella enterica serovar Typhimurium. To investigate the basis for acetate-induced acid tolerance in E. coli O157:H7, genes whose expression was altered by exposure to acetate were identified using gene arrays. The expression of 60 genes was reduced by at least twofold; of these, 48 encode components of the transcription-translation machinery. Expression of 26 genes increased twofold or greater following treatment with acetate. This included six genes whose products are known to be important for survival at low pH. Five of these genes, as well as six other acetate-induced genes, are members of the E. coli RpoS regulon. RpoS, the stress sigma factor, is known to be required for acid tolerance induced by growth at nonlethal low pH or by entry into stationary phase. Disruption of the rpoS gene by a transposon insertion mutation also prevented acetate-induced acid tolerance. However, induction of RpoS expression did not appear to be sufficient to activate the acid tolerance response. Treatment with either NaCl or sodium acetate (pH 7.0) increased expression of an rpoS::lacZ fusion protein, but only treatment with acetate increased acid survival.

In order to colonize their mammalian hosts, both commensal and pathogenic enteric bacteria must survive passage through the low-pH environment of the stomach. Enteric bacteria must also cope with acid stress in the intestine. Although the pH of the intestine is near neutral, there are high concentrations of short-chain fatty acids (SCFA), produced as fermentation products by the intestinal microflora. The concentration of organic acids in the human intestinal tract is estimated to be 12 mmol/kg in the ileum and ranges from 70 to 120 mmol/kg in the large intestine (14). Because the protonated form of SCFA equilibrates across the cytoplasmic membrane, SCFA can lower internal pH even when the external pH is neutral (12, 46, 48). In vitro, SCFA can have bacteriostatic and/or bactericidal effects depending upon their concentration, the pH, and other conditions (12, 47).

There is likely to be overlap in the mechanisms that confer resistance to acid pH and the mechanisms that confer resistance to SCFA. The ability to survive acid conditions is greatly enhanced by the addition of SCFA, such as acetate, proprionate, and butyrate, at neutral or near neutral pH to exponential-phase cultures of Escherichia coli (21) and Salmonella enterica serovar Typhimurium (27). In addition, induction of the acid tolerance response by growth at moderately acidic pH (pH 5.0 to 5.8), which dramatically increases acid survival in both E. coli and serovar Typhimurium (17, 19), has been shown to protect Salmonella against SCFA (5).

Expression profiling is a powerful tool for analyzing gene expression at a genomic scale. It can be used to compare global changes in gene expression that occur in response to an environmental stimulus or to compare the effects of genetic changes on gene expression. This analysis can provide important information about cell physiology and has the potential to identify connections between regulatory or metabolic pathways that were not previously known. The use of gene arrays to analyze gene expression has been used extensively for eukaryotic systems (16, 26). Recently its usefulness for analyzing gene expression in prokaryotes has also been demonstrated (3, 42, 53, 57).

To identify functions involved in the SCFA-induced acid tolerance response, gene arrays were used to characterize the changes in gene expression induced by acetate treatment of E. coli O157:H7. We also examined the ability of acetate at neutral pH to enhance resistance of E. coli O157:H7 and E. coli K-12 strains to oxidative stress and heat killing as well as to confer resistance to acid pH.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The bacterial strains used in this study were RZ4500 (E. coli K-12 MG1655 lacZΔ145) from Tricia Kiley, University of Wisconsin-Madison; DS352 (MG1655 lacZΔ145 rpoS::Tn10) (this laboratory); ATCC 43888 (E. coli O157:H7); JOE1 (ATCC 43888 ΔrpoS::kan); and RO91 (MC4100 [λRZ5:rpoS742::lacZ(Hyb)]), from Regine Hengge-Aronis, Free University of Berlin (28). The ΔrpoS::kan allele (7) was introduced into ATCC 43888 by conjugation with Hfr KL16 zed-3069::Tn10 ΔrpoS::kan (this laboratory). The origin of transfer for Hfr KL16 is near 64 min, and the rpoS locus is at 61.7 min (6, 36). Mating was interrupted after 5 min, so the O157:H7 ΔrpoS::kan transconjugants are expected to contain 5% or less E. coli K-12 DNA. None of the transconjugants received the zed-3069::Tn10 allele from the donor strain.

Cultures were grown aerobically at 37°C in M63 minimal glucose (0.2%) medium (40) supplemented with uridine (10 μg ml−1), 40 μg (each) of alanine, arginine, glutamate, glycine, histidine, isoleucine, leucine, lysine, proline, serine, threonine, and valine ml−1; and a vitamin mix containing 100 μg of biotin, nicotinamide, and thiamine ml−1 and 10 μg of riboflavin ml−1. Vitamin mix was added to the minimal medium because ATCC 43888 was found to have a vitamin auxotrophy. We did not determine what vitamin this strain requires. Growth was monitored by measuring culture turbidity with a Klett-Summerson colorimeter (Manostat, New York, N.Y.) equipped with a no. 54 filter (500 to 570 nm).

Survival assays.

Survival assays were performed to determine the extent to which SCFA adaptation affected the resistance of E. coli K-12 and E. coli O157:H7 to acid shock, oxidative stress, and heat shock. For all of the survival assays, single colonies were inoculated into 125-ml Erlenmeyer flasks containing 10 ml of supplemented M63 glucose medium and grown overnight at 37°C with aeration. Cells from the overnight cultures were diluted into 10 ml of fresh medium to a Klett value of 1. The cultures were grown until they reached a Klett value of 30 (ca. 2 × 108 cells ml−1). At that time, one-ninth volume of either deionized water, 1 M NaCl, or filter-sterilized SCFA stock solution (1.0 M [pH 7.0], adjusted with NaOH) was added, and the cultures were incubated for another hour at 37°C with aeration. The final concentration of NaCl or SCFA was 100 mM. NaCl was used as a control to reproduce the same osmolarity as in the experimental cultures. Because of day-to-day variability, untreated and NaCl controls were always run in each experiment. All experiments were performed at least three times on independent cultures except when noted otherwise.

Survival at pH 3.0 was determined as described previously (27) with the following modifications. After treatment for 1 h with either deionized water, NaCl, or SCFA, a 100-μl sample of each culture was diluted into 4 ml of M63 salts or 4 ml of 10 mM citric acid at pH 3.0 and incubated for 1 h in a 37°C waterbath without aeration. Each sample was serially diluted in M63 salts, and aliquots were plated onto Luria-Bertani (LB) plates to determine the number of viable cells. The percent survival was defined as the CFU per milliliter after acid shock divided by the CFU per milliliter in the buffer control.

Oxidative stress resistance was determined as described previously (29). After treatment for 1 h with either deionized water, NaCl, or sodium acetate, 2 ml of each culture was transferred to a test tube, H2O2 was added to a final concentration of 15 mM, and the cells were incubated for 1 h in a 37°C water bath without aeration. Samples were removed before and at 15-min intervals after the addition of H2O2 and serially diluted in M63 salts, and aliquots were plated onto LB plates to determine the number of viable cells at each time point. Percent survival was defined as the CFU per milliliter after the addition of H2O2 divided by the CFU per milliliter present before the addition of H2O2.

Thermotolerance was determined at 50°C as described previously (31). After treatment for 1 h with either deionized water, NaCl, or sodium acetate, a 100-μl sample of each culture was diluted into either 4 ml of M63 salts warmed at 50°C or 4 ml of M63 salts at room temperature. Samples were removed at 10-min intervals and serially diluted in M63 salts, and aliquots were plated onto LB plates to determine the number of viable cells. Percent survival was defined as the CFU per milliliter after incubation at 50°C divided by the CFU per milliliter in the room temperature control.

To determine whether acetate-inducible acid tolerance in E. coli K-12 and E. coli O157:H7 was dependent on new protein synthesis, the acid survival assay was performed as described above but with the addition of chloramphenicol (Sigma Chemical Co., St. Louis, Mo.) to a final concentration of 50 μg ml−1 to the cultures 5 min before or 30 or 55 min after the addition of sodium acetate (pH 7.0) to a final concentration of 100 mM.

β-Galactosidase assays.

β-Galactosidase activity was assayed as described by Miller (40) using cells permeabilized with sodium dodecyl sulfate (SDS) and CHCl3. β-Galactosidase activity is expressed as Miller units (optical density at 420 nm [OD420] per OD600 per min). Cultures were grown at 37°C in supplemented M63 glucose medium to ca. 2 × 108 CFU ml−1 when either NaCl or sodium acetate (pH 7.0) was added to a final concentration of 100 mM. Samples were removed immediately before and 30 and 60 min after the addition of NaCl or sodium acetate.

Analysis of gene expression using E. coli gene arrays.

Cultures were grown at 37°C in 100 ml of supplemented M63 glucose medium in 1-liter Erlenmeyer flasks to ca. 2 × 108 CFU ml−1. Cultures were harvested either immediately before or 30 min after the addition of sodium acetate (pH 7.0) to a final concentration of 100 mM. The 100-ml culture was poured over 100 g of ice in a 250-ml centrifuge bottle, and the cells were harvested by centrifugation. Cell pellets were resuspended in 5 ml of ice-cold 10 mM Tris-HCl (pH 7.5), 10 mM KCl, and 5 mM MgCl2. Total RNA was isolated as described previously (49). Briefly, after the addition of SDS (1%) and lysozyme (300 μg ml−1), the cell suspension was frozen in a dry ice-ethanol bath, thawed at 64°C, extracted three times with an equal volume of water-saturated phenol at 350 rpm at 64°C in a shaking water bath, ethanol precipitated three times, and resuspended in diethyl pyrocarbonate-treated deionized water, and RNA was quantitated by absorbance at 260 nm. 33P-labeled cDNA probes were prepared using E. coli gene-specific primers (Sigma-Genosys, The Woodlands, Tex.) following the protocol provided by Sigma-Genosys, except that the reaction buffer used consisted of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, and 10 mM dithiothreitol and the reaction mixture contained 40 μCi of [α-33P]dCTP (1,000 to 3,000 Ci mmol−1; Amersham Pharmacia), and 400 U of SuperScript II reverse transcriptase (Life Technologies, Rockville, Md.) in a 30-μl volume.

Panorama E. coli gene arrays were obtained from Sigma-Genosys, and hybridizations were done as recommended by the manufacturer. Briefly, the nylon membranes were prehybridized in 5 ml of hybridization buffer (5× SSPE [1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA, pH 7.7], 2% SDS, 1× Denhardt's reagent, and 100 μg of denatured, sonicated salmon sperm DNA per ml). After incubation for ≥1 h at 65°C, the prehybridization solution was replaced with the denatured radioactively labeled cDNA probe. The probes were denatured by adding equal counts per minute of each probe to 3 ml of hybridization buffer and incubating at 90 to 95°C for 10 min immediately before use. After hybridization for 16 h at 65°C in a hybridization incubator (Robbins Scientific Corp.), the filters were washed briefly three times at room temperature with 50 ml of wash solution (0.5× SSPE, 0.2% SDS) and then washed three times at 65°C in 100 ml of warmed wash solution. The washed filters were blotted between two pieces of Whatman 3MM paper for 5 min and then wrapped in Saran Wrap. For quantitation, filters were exposed to a phosphorimager screen, which was scanned at 100-μm resolution using a Fujix BAS2000 phosphorimager. For each filter, three exposure times were analyzed to determine that the majority of signals were within the linear detection range.

The Fujix BAS image files were analyzed using Visage HDG Analyzer software (R. M. Lupton, Inc., Jackson, Mich.) running on a Sun Microsystems ULTRA10 workstation. The signal intensity for each spot was determined using the integrated intensity function, which calculates the volume of each spot by summing the value of each pixel within the boundaries of the spot minus the local background. The integrated intensity (I.I.) values were exported to Microsoft Excel for further analysis. Each open reading frame (ORF) on the array is represented by duplicate spots, and the average I.I. for each ORF was calculated after determining that the values for the duplicate spots were within 50% of one another. (About 1% of the ORFs showed more than 50% variation between duplicates. These spots were examined individually to check that the boundaries had been placed correctly.) To compare the signal intensities between filters, a relative I.I. for each ORF was calculated by dividing the average I.I. for a given ORF by the total signal intensity on the filter and multiplying by 1,000. The total signal intensity on the filter was calculated by summing the integrated intensities of all ORFs on the array.

A background expression level was defined, which was the expression level seen for the uninduced lac operon. Control experiments showed that the relative I.I. for the genes in the lac operon were similar when cDNA probes were prepared from exponential-phase RNA isolated either from a Δlac strain or from an uninduced lac+ strain (data not shown). Signals below this threshold were not considered meaningful, and ORFs with a relative I.I. below this threshold in both growth conditions were filtered out of the data set. For the two experiments presented here, there were 2,867 and 3,419 ORFs, respectively, whose relative I.I. was above the background expression threshold in at least one growth condition.

Two criteria were used to define ORFs whose expression was altered by the addition of acetate. First, the relative I.I. had to be at least two times greater than the background expression threshold in one of the growth conditions. For genes whose expression was induced by acetate, the relative I.I. after exposure to acetate had to be at least twofold higher than the background expression level. For genes whose expression decreased after the addition of acetate, the relative I.I. before acetate addition had to be at least twofold higher than the background expression level. The second criterion used was that the relative expression level had to change at least twofold in two independent experiments. The following results were found for the two independent experiments. In one experiment, of 2,867 ORFs analyzed, the relative expression level of 46 (1.6%) ORFs increased ≥2-fold and the relative expression level of 144 (5.0%) decreased by at least 50%. In the second experiment, of 3,419 ORFs analyzed, the relative expression level of 53 (1.6%) increased ≥2-fold and the relative expression level of 74 (2.2%) decreased by 50% or more. In common to both experiments were 26 ORFs whose relative expression level increased ≥2-fold and 60 ORFs whose relative expression level decreased by 50% or more.

RESULTS

Acid tolerance after SCFA adaptation.

Exponentially growing cultures of E. coli O157:H7 or E. coli K-12 were treated with 100 mM acetate, butyrate, or proprionate at pH 7.0 for 1 h as described in Materials and Methods. SCFA treatment increased the doubling time from 30 to 35 min to about 110 min. In control cultures treated with 100 mM NaCl to cause the same change in osmolarity as the SCFA treatment, the doubling time increased to about 60 min.

Pretreatment with SCFAs increased the acid tolerance of both E. coli O157:H7 and E. coli K-12 (Table (Table1),1), while pretreatment with NaCl had no effect on survival compared to cultures pretreated with an equal volume of deionized water (data not shown). Only 0.03% ± 0.02% of the NaCl-treated E. coli O157:H7 cells survived acid shock. Exposure to acetate for 1 h increased the acid tolerance of E. coli O157:H7 400-fold to 11% ± 4.0% survival, while treatment with butyrate or proprionate increased percent survival 50- and 40-fold, respectively. Similar results were obtained for E. coli K-12. Survival in NaCl-treated controls was 0.1% ± 0.1%, while pretreatment with acetate, butyrate, and proprionate increased survival 300-, 100-, and 140-fold, respectively. The somewhat higher level of acid tolerance seen for E. coli K-12 compared to E. coli O157:H7 was unexpected but was consistently observed in three independent experiments.

TABLE 1
Exposure to SCFA increases survival at pH 3.0

To determine whether the increased acid tolerance was a specific effect of acetate or an indirect effect due to the large effect of 100 mM acetate on the growth rate, we examined lower concentrations of acetate, which had smaller effects on the growth rate (Fig. (Fig.1).1). In two independent experiments, exposure to 25 mM acetate at pH 7 for 1 h increased acid survival 100-fold (from 0.1% ± 0.05% to 14% ± 4%), while exposure to 50 mM acetate at pH 7 for 1 h increased acid survival 350-fold (from 0.1% ± 0.04% to 35% ± 15%) (Table (Table1).1).

FIG. 1
Effects of NaCl and sodium acetate on growth of E. coli O157:H7. Cultures of E. coli O157:H7 were grown at 37°C in supplemented M63 glucose medium as described in Materials and Methods. Culture growth was monitored by measuring turbidity with ...

Oxidative stress resistance after acetate adaptation.

In S. enterica serovar Typhimurium, induction of the acid tolerance response by growth at pH 5.8 confers resistance to other environmental stresses (31). Therefore, we tested whether adaptation of E. coli O157:H7 to SCFA provided cross-protection against oxidative or heat stress. Because acetate adaptation provided the greatest protection against acid shock, only acetate was tested for cross-protection. Resistance to H2O2 was determined as described in Materials and Methods. The results of a representative experiment are shown in Fig. Fig.2.2. Compared to the control culture, pretreatment with either 100 mM acetate (pH 7.0) or 100 mM NaCl increased survival almost 10-fold after 15 min of incubation with 15 mM H2O2. At longer incubation times, only adaptation to acetate increased survival.

FIG. 2
Pretreatment with sodium acetate provides protection against oxidative stress. Cultures of E. coli O157:H7 were grown at 37°C in supplemented M63 glucose medium as described in Materials and Methods to ca. 2 × 108 cells ml−1 prior ...

Thermotolerance after acetate adaptation.

We also tested whether exposure to acetate increased the resistance of E. coli O157:H7 to heat killing. Thermotolerance was determined as described in Materials and Methods. The results are shown in Fig. Fig.3.3. Pretreatment with 100 mM sodium acetate (pH 7) or 100 mM NaCl significantly increased the survival of exponential-phase cells at 50°C. In control cultures only 0.5% ± 0.2% of cells survived after 30 min at 50°C, while in the NaCl- and sodium acetate-treated cultures, 6% ± 4.0% and 5.0% ± 4.0%, respectively, of cells remained viable.

FIG. 3
Pretreatment with NaCl or sodium acetate provides protection against heat killing at 50°C. Cultures were grown at 37°C in supplemented M63 glucose medium as described in Materials and Methods to ca. 2 × 108 cells ml−1 prior ...

Role of protein synthesis in acetate-induced acid tolerance.

To determine whether the acetate-induced acid tolerance of E. coli O157:H7 and E. coli K-12 required new protein synthesis, we repeated the acid shock assay with the addition of chloramphenicol either 5 min before or 30 or 55 min after the addition of 100 mM acetate (pH 7.0) to exponential-phase cultures (Table (Table2).2). In the absence of chloramphenicol, 6.0% of acetate-treated exponential-phase E. coli O157:H7 survived acid shock. Treating the cells with chloramphenicol 5 min before the addition of acetate reduced survival to <0.05%, which was comparable to the survival of nonadapted cultures (Table (Table1).1). A low level of survival was also seen when chloramphenicol was added 30 min after the addition of acetate. In contrast, addition of chloramphenicol 55 min after the addition of acetate did not inhibit induction of acid tolerance (7% survival). Similar results were seen for E. coli K-12 cultures (Table (Table2).2).

TABLE 2
Effect of chloramphenicol on the development of acetate-induced acid tolerance

Effect of acetate treatment on gene expression.

To characterize the effect of acetate treatment on gene expression in E. coli O157:H7, we used E. coli gene arrays to compare the pattern of transcripts present before and after treatment with acetate. Transcriptional profiling was done as described in Materials and Methods.

The relative expression level for the majority of ORFs was not changed significantly by the addition of acetate. In one experiment, 87% of the ORFs showed less than a 50% change in the relative expression level before and after the addition of acetate. In the second experiment, 91% of the ORFs showed less than a 50% change after the addition of acetate. Genes whose relative expression level was induced ≥2.0-fold in two independent experiments are shown in Table Table3.3. Genes whose relative expression level decreased to 50% or less of the level seen prior to the addition of acetate in two independent experiments are listed in Tables Tables44 and and5.5.

TABLE 3
E. coli genes whose relative expression level increases after treatment with acetate
TABLE 4
Components of the transcription-translation machinery that showed decreased expression after acetate treatment
TABLE 5
Other E. coli genes whose relative expression level decreased after treatment with acetate

Role of rpoS in acetate-induced acid tolerance.

In E. coli, the rpoS gene, which codes for the alternate sigma factor ςS, is required for both stationary-phase-induced acid resistance and the exponential-phase acid tolerance response induced by growth at moderately low pH (13, 33, 51). Therefore, we expected that rpoS would also be required for acetate-induced acid tolerance. To test this hypothesis, we constructed rpoS::Tn10 derivatives of both E. coli O157:H7 and E. coli K-12 and measured survival at pH 3.0 before and after exposure to acetate. As expected, inactivation of rpoS greatly decreased the acetate-induced acid tolerance of both E. coli O157:H7 and E. coli K-12 (Table (Table6).6). However, exposure to acetate did increase survival compared to the control cultures that were treated with NaCl, suggesting that there is an rpoS-independent component of this response.

TABLE 6
RpoS is required for acetate-induced acid tolerance at pH 3.0

It was shown previously that acetate induces expression of RpoS (50). To determine the effects of acetate treatment on RpoS expression in our experiments, we assayed expression of the rpoS742::lacZ fusion protein. This fusion was chosen because it is expressed similarly to native RpoS upon entry to stationary phase in both minimal glucose and LB media (28) and also after osmotic shock (41). The results are shown in Fig. Fig.4.4. By 60 min after the addition of acetate (pH 7.0), β-galactosidase levels had increased fourfold. β-Galactosidase levels also increased after treatment with NaCl. There was an eightfold increase in β-galactosidase activity 60 min after the addition of NaCl.

FIG. 4
Effects of NaCl and sodium acetate treatment on expression of the rpoS742::lacZ fusion protein. β-Galactosidase activity is expressed in Miller units (ΔOD420 per OD600 per min). The results shown are the means of two independent experiments. ...

DISCUSSION

The ability of E. coli to survive at low pH is strongly affected by environmental factors. Exposure to SCFA at neutral or near neutral pH increases the acid survival of E. coli and S. enterica serovar Typhimurium (21, 27). Following the terminology proposed by Hall and colleagues to distinguish between constitutive and inducible acid protection systems (22), we refer to this response as SCFA-inducible acid tolerance. Induction of the acid tolerance response by growth of serovar Typhimurium at a low but nonlethal pH (pH 5.8) also provides cross-protection to a variety of stresses, including heat, high osmolarity, oxidative stress, and the cationic peptide polymyxin B (31). Here, we examined the molecular basis of SCFA-induced acid tolerance using gene arrays to characterize the changes in gene expression brought about by exposure to SCFA. First, however, we compared the ability of different SCFA to provide protection against acid shock. Exposure to acetate, butyrate, or proprionate at neutral pH increased the resistance of E. coli K-12 and E. coli O157:H7 at pH 3, with acetate providing the greatest protection. Treatment with acetate also enhanced the resistance of E. coli O157:H7 to oxidative stress and heat killing.

Genes whose expression was altered by exposure to acetate were identified using E. coli gene arrays. We identified 26 genes in E. coli O157:H7 whose transcript levels increased at least twofold after exposure to acetate at pH 7 (Table (Table3).3). This included 21 previously identified genes. Because the gene arrays used were based on the E. coli K-12 genomic sequence, our studies identified only those genes common to both E. coli K-12 and E. coli O157:H7. If there are O157:H7-specific genes whose expression is induced by SCFA, we would not have detected them.

Our analysis of the transcriptional response to acetate has identified induced genes, such as members of the RpoS regulon, whose expression was expected to increase based on previous work by other laboratories. We have also identified other induced genes whose products were not previously associated with acid survival. Future studies will determine whether any of these genes code for proteins involved in protecting E. coli against low pH or other environmental stresses.

Six of the genes whose expression was induced by addition of acetate at pH 7 code for proteins that are known to provide protection against acid stress in E. coli. These are the gadA, gadBC, cfa, and hdeAB genes. gadA and gadB encode isozymes of glutamate decarboxylase (Gad), enzymes that catalyze the conversion of glutamate to γ-aminobutyrate. gadC (xasA), which is located downstream of gadB, is predicted to code for a γ-aminobutyrate antiporter (25, 56). The GadA and GadB decarboxylases and the GadC antiporter are proposed to function together to help maintain a near neutral intracellular pH when cells are exposed to extreme acidic conditions (25, 52). Either GadA or GadB is sufficient for E. coli survival at pH 2.5, but both are needed for survival at pH 2.0 (10). Inactivation of gadC causes an acid-sensitive phenotype in E. coli (25) and Shigella flexneri (56). Stationary-phase induction of gadA and gadBC is dependent on ςS in both of these organisms (10, 15, 56). However, rpoS is not required for acid induction of gadA or gadB expression in either exponential- or stationary-phase E. coli (10). Although we see dramatic increases in expression of the gad genes, the Gad system is not likely to be important for acid survival under the conditions we tested, because the function of the Gad system is dependent upon the presence of external glutamate (32) and only a very low level of glutamate (6.8 μM) was present in our experiments.

The cfa gene codes for a cyclopropane fatty acid synthase, which adds a methylene group across the carbon-carbon double bond of unsaturated fatty acids in the inner membrane (20). In E. coli, cfa is strongly expressed during early stationary phase, and its induction in stationary phase is dependent upon ςS (55). The ability of E. coli to survive acid shock at pH 3 is correlated with the level of cyclopropane fatty acids in the inner membrane (9). Recently, it has been shown that mutants lacking cfa are sensitive to acid killing in early stationary phase (11). The wild-type level of acid resistance could be restored either by introduction of a functional cfa gene or by providing cyclopropane fatty acids in the growth medium, indicating that it is cyclopropane fatty acids themselves that are important for acid resistance. It has been suggested that cyclopropane fatty acids may help provide acid resistance by decreasing the permeability of the membrane to protons (11).

The hdeA, hdeB, and hdeD genes were first identified because their expression is strongly induced in E. coli cells lacking the nucleoid protein H-NS (59). The hdeAB operon is located immediately downstream of hdeD and is transcribed in the opposite direction (58). Expression of all three genes increases in stationary phase, and induction of the hdeAB operon is ςS dependent (4, 56, 59). HdeA and HdeB are predicted to be periplasmic proteins, and HdeD is a predicted membrane protein. The hdeA gene is required for stationary-phase acid resistance in both S. flexneri (56) and E. coli (18). Recently, HdeA has been shown to function as a chaperone in vitro at pH 2 but not at neutral pH (18). Thus, HdeA's role in acid resistance is likely to be to prevent the aggregation of periplasmic proteins denatured at low pH. Based on threading analysis, Gajiwala and Burley (18) predict that HdeB is a structural homolog of and forms heterodimers with HdeA. No function has been proposed for HdeD, but the chromosomal location of the gene and its expression pattern suggest that it may have a role in the same cellular process as HdeA and HdeB.

Two other genes whose expression is induced by exposure to acetate at neutral pH code for proteins that protect cells against oxidative damage. These are dps and katE. The dps gene codes for a nonspecific DNA-binding protein whose expression is induced in stationary phase as part of the RpoS regulon and also in response to oxidative stress, when its expression is controlled by OxyR (1, 2, 35). Dps has been shown to protect DNA against oxidative damage both in vivo and in vitro (38). The katE gene codes for the catalase hydroperoxidase (HPII), whose expression is regulated by RpoS (34).

The product of another acetate-induced gene, grxB, may also have a role in protecting cells against oxidative damage. The grxB gene codes for glutaredoxin 2, which, like other glutaredoxins, is a glutathione-dependent oxidoreductase (54). In the yeast Saccharomyces cerevisiae, glutaredoxins have been shown to protect cells against oxidative damage caused by superoxides and hydrogen peroxide (37, 45). Glutaredoxins may have a similar protective role in E. coli. Increased expression of the dps, katE, and grxB genes suggests that exposure to acetate leads to the production of reactive oxygen species. Alternatively, their induction may be part of a general cellular stress response.

Among the six unknown ORFs whose expression was strongly induced by acetate are yhiW and yhiX, which code for predicted AraC-type regulatory proteins. These putative transcription factors may be involved in controlling some of the changes in gene expression induced by SCFA. YhiX has been shown to be involved in upregulating expression of hdeAB, gadA, and gadB when E. coli is grown in minimal glucose medium at pH 5.5 (T. Conway, personal communication). It is likely that YhiX also activates expression of these genes in response to acetate treatment.

Among the genes whose transcription was induced by exposure to acetate at neutral pH were eight genes or operons known to be regulated by ςS, the stress sigma factor encoded by the rpoS gene (24, 39). These were adhE, cfa, dps, gadBC, hdeAB, katE, osmC, and osmY. However, we did not see induction of all known rpoS-dependent genes. Expression of some genes in the RpoS regulon is regulated by other factors in addition to ςS (23), so it is possible that expression of only a subset of RpoS-regulated genes is induced by acetate. Alternatively, we may have missed induction of some genes in the rpoS regulon because their transcripts are present at low levels even after induction or because of the timing of their expression. Cultures were harvested at only one time point after the addition of acetate, so it is possible that the maximal change in gene expression was missed in some cases. For example, we detected a twofold increase in the relative expression level of otsA and otsB in only one of two experiments. It is likely that expression of this operon was induced in both experiments but that in one experiment the peak expression level was missed.

RpoS has previously been shown to be crucial for acid survival in both E. coli and Salmonella (13, 30, 51). Therefore, it was not surprising that rpoS was also found to be required for acetate-induced acid tolerance. However, our results showed that induction of RpoS expression is not sufficient to confer acid tolerance. Addition of both NaCl and sodium acetate increased expression of an rpoS-lacZ fusion, but NaCl-treated cells did not show increased survival at pH 3.0 compared to untreated control cells. These results suggest that the acetate-induced acid tolerance response may involve gene products whose expression is independent of RpoS or that addition of NaCl or sodium acetate leads to induction of different members of the RpoS regulon.

We found 60 genes whose relative expression level decreased by at least 50% after the addition of acetate (Tables (Tables44 and and5).5). The majority of these genes (47 of 60) encode components of the transcription-translation machinery (Table (Table4).4). These included 12 genes or operons encoding a total of 41 ribosomal proteins, as well as the translation elongation factors EF-G, EF-Ts, and EF-Tu. The synthesis rate of many components of the transcription-translation machinery is growth rate regulated (8), so it is likely that the observed decrease in relative expression levels is due to the decreased growth rate caused by addition of acetate. Of the remaining 13 genes whose expression was repressed by acetate, 9 are previously identified genes. The repressed genes code for a variety of known or predicted cellular functions (Table (Table55).

Overall, the changes in gene expression seen for repressed genes were not as dramatic as those for genes whose expression was induced by the addition of acetate. For many genes, this probably accurately reflects the biology of the response to acetate. However, for genes or ORFs whose signal on the arrays is only a few fold above the background level of expression as defined in Materials and Methods, the change in expression will be underestimated.

ACKNOWLEDGMENTS

We thank Young Min Kwon and Steve Ricke for helpful discussions, T. S. Rama Subramanian for advice on cDNA synthesis and hybridizations, Genevieve Ledwell for help with the β-galactosidase assays, Terry Thomas for use of the Sun workstation and Visage HDG software, and Jim Hu for helpful discussions and critical advice on the manuscript.

This work was supported by grant RO1 GM55154 from the National Institutes of Health to D.A.S.

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