pH-Dependent Modulation of Cyclic AMP Levels and GadW ...

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Jun 6, 2003 - CRP and cAMP appear to influence GadX-GadW cross-repression from outside the loop ... suitable pH (Richard and Foster, unpublished data).
JOURNAL OF BACTERIOLOGY, Dec. 2003, p. 6852–6859 0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.23.6852–6859.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 185, No. 23

pH-Dependent Modulation of Cyclic AMP Levels and GadW-Dependent Repression of RpoS Affect Synthesis of the GadX Regulator and Escherichia coli Acid Resistance Zhuo Ma, Hope Richard, and John W. Foster* Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688 Received 6 June 2003/Accepted 29 August 2003

Extreme acid resistance is a remarkable property of virulent and avirulent Escherichia coli. The ability to resist environments in which the pH is 2.5 and below is predicted to contribute significantly to the survival of E. coli during passage through the gastric acid barrier. One acid resistance system imports glutamate from acidic environments and uses it as a proton sink during an intracellular decarboxylation reaction. Transcription of the genes encoding the glutamate decarboxylases and the substrate-product antiporter required for this system is induced under a variety of conditions, including the stationary phase and a low pH. Acid induction during log-phase growth in minimal medium appears to occur through multiple pathways. We recently demonstrated that GadE, the essential activator of the genes, was itself acid induced. In this report we present evidence that there is a regulatory loop involving cross-repression of two AraC-like regulators, GadX and GadW, that can either assist or interfere with GadE activation of the gad decarboxylase and antiporter genes, depending on the culture conditions. Balancing cross-repression appears to be dependent on cAMP and the cAMP regulator protein (CRP). The control loop involves the GadX protein repressing the expression of gadW and the GadW protein repressing or inhibiting RpoS, which is the alternative sigma factor that drives transcription of gadX. CRP and cAMP appear to influence GadX-GadW cross-repression from outside the loop by inhibiting production of RpoS. We found that GadW represses the decarboxylase genes in minimal medium and that growth under acidic conditions lowers the intracellular cAMP levels. These results indicate that CRP and cAMP can mediate pH control over gadX expression and, indirectly, expression of the decarboxylase genes. Mutational or physiological lowering of cAMP levels increases the level of RpoS and thereby increases the production of GadX. Higher GadX levels, in turn, repress gadW and contribute to induction of the gad decarboxylase genes. The presence of multiple pH control pathways governing expression of this acid resistance system is thought to reflect different environmental routes to a low pH. a complex regulatory network involving at least nine regulators. These regulators include two sigma factors (␴70 and ␴S), a two-component system (EvgAS), a critical LuxR-family regulator, GadE (previously designated YhiE), and two AraC-like regulators (GadX and GadW), as well as H-NS and cAMP regulator protein (CRP), which act as repressors (4–6, 12, 17, 19, 22). In addition, YdeO and TorR may be involved (3, 13). There are two basic regulatory circuits that control expression of the gadA and gadBC operons. One of these circuits involves the GadE activator, which has been shown to bind a 20-bp gad box sequence upstream of both gad decarboxylase operons (11). The expression of gadE is affected in two ways by the EvgAS two-component regulatory system—through direct binding to gadE (Ma and Foster, unpublished data) and indirectly via another AraC-like regulator, YdeO (13). A second, more complex circuit influences whether GadE successfully activates gadA and gadBC. The AraC-like regulator GadX, produced from a gene immediately downstream of the gadA decarboxylase gene, has been shown to activate gadA and gadBC and to bind in vitro to the region around the gad box (22). Expression of gadX proved to be dependent on the alternative sigma factor RpoS, making this circuit RpoS dependent (12, 22). CRP also inhibits gadA and gadBC expression via negative control of RpoS production (12).

Acid resistance is an important virulence property for commensal and pathogenic strains of Escherichia coli. The stomach, which serves as the usual portal of entry for these microbes, presents an acidic barrier consisting of a pH of 2 or below, a level of acid stress that is disastrous for most pathogens. E. coli, however, is armed with three acid resistance strategies designed to breach this barrier. The most effective of these systems involves a set of inducible genes encoding two nearly identical isoforms of glutamate decarboxylase (gadA and gadB) and an antiporter (gadC) that exchanges external glutamate for the intracellular decarboxylation product gamma-aminobutyric acid (2, 5, 7, 10). The process of decarboxylation consumes an intracellular proton and helps maintain a suitable ⌬pH (Richard and Foster, unpublished data). The importance of this system to survival in the gastrointestinal tract was recently demonstrated in calves (S. Price, J. C. Wright, F. J. Groves, M.-P. Castanie-Cornet, and J. W. Foster, unpublished data). The glutamate decarboxylase-antiporter system is subject to * Corresponding author. Mailing address: Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688. Phone: (251) 460-6323. Fax: (251) 460-7931. E-mail: [email protected]. 6852

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TABLE 1. Bacterial strains and oligonucleotides used in this study Strain or oligonucleotide

Strains EK227 EK344 EF362 EK395 EF528 EF530 EF828 EF861 EF863 EF864 EF928 EF1123 Oligonucleotides 377 378 379 465 166 507 508 511 512 540 541

Genotype or sequence

Source or reference

K-12 wild type ␭⫺ F⫺ ⌬cya::Cm K-12 (EK227) rpoS::Tn10 ⌬cyaA1400::Kan K-12 (EK227) ⌬crp::Cm K-12(EK227) ⌬cya::Km K-12 (EK227) ⌬gadX K-12 (EK227) ⌬gadW::Km K-12 (EK227) ⌬gadXW::Km K-12 (EK227) ⌬gadW::Km ⌬crp::Cm K-12 (EK227) ⌬cyaA1400::Km K-12 (EK227) ⌬crp::Cm ⌬gadW::Km rpoS::Tn10

A. C. Matin 5 5 J. Kaper EK227 ⫻ EK344 5 12 12 12 12 EK227 ⫻ EK395 EF864 ⫻ EF362

5⬘-GGAGTTCGAAATGGACCAGAAG-3⬘ 5⬘-AGTTTCGGGTGATCGCTGAG-3⬘ 5⬘-GGTGTCGACTATGAACCTGCTTCCCATCGACTAC-3⬘ 5⬘-CACCATGACTCATGTCTGCTCG-3⬘ 5⬘-GGAAAAGGTACCTGGCGAATG-3⬘ 5⬘-CAATACGCAAACCGCCTCTCC-3⬘ 5⬘-AGTGAATCCGTAATCATGGTCATA-3⬘ 5⬘-TCTGCGGAAGGAATAAGATTATAGAG-3⬘ 5⬘-GGATCACCGAGCAGACATGAGTCA-3⬘ 5⬘-CAAGTTATGATTTTTCTCATGACGAA-3⬘ 5⬘-CTAAAAATAAGATGTGATACCCAG-3⬘

GadW, another AraC-like regulator encoded by a gene downstream of gadX, was shown to bind to the gadA and gadBC promoter regions. GadW activates these genes under certain conditions and represses them under other conditions (12). The previous study also indicated that GadW can repress expression of gadX. In this intricate regulatory network overlapping systems are utilized to mediate pH and growth phase control of gadA and gadBC induction. A major question is how pH regulates expression of this system. Answering this question has been challenging because of the many regulators involved and because the regulatory needs that dictate expression change with changes in medium composition and growth phase. For example, gadA and gadBC are acid induced during exponential growth in minimal salts glucose medium, but growth in a rich complex medium (LuriaBertani [LB] medium) restricts expression to the stationary phase (5). Despite these complexities, some progress has been made in discerning targets of acid regulation. Previous results have demonstrated that there are at least two focal points of pH control. We recently obtained evidence that GadE is an activator protein essential for gadA and gadBC expression under any conditions (minimal or rich medium, log or stationary phase) and that gadE is itself acid inducible in minimal salts glucose medium (11). A second point of pH control appears to involve one of the AraC-like regulators, GadW. Unlike the situation in minimal glucose medium, in which GadE alone is needed for expression, GadX and/or GadW is a necessary coregulator in rich medium (12). The roles of these regulators are interwoven and sometimes at odds with each other. For instance, GadW and GadX have been shown to reciprocally repress expression of each other (12, 23). In the absence of GadX, GadW was shown to activate gadA and gadBC in rich medium, but only under alkaline conditions

(12). This result suggests that GadW senses or is regulated by some consequence of the growth pH. Here we report that gadX and gadW can themselves be acid induced in minimal medium if reciprocal repression is removed. Our results indicate that pH control of gadX occurs indirectly via pH-dependent effects on cAMP levels. When CRP is complexed with cAMP, it inhibits the production of RpoS, the alternative sigma factor needed for gadX transcription (8, 12). We also show here that an acid pH reduces intracellular cAMP levels, providing a mechanism by which pH can affect the regulatory balance of the RpoS-GadX-GadW control circuit. MATERIALS AND METHODS Bacterial strains and growth media. The bacterial strains and plasmids used in this study are listed in Table 1. The media used included minimal E medium containing 0.4% glucose (EG) (24) and the complex medium LB broth buffered with either 100 mM morpholinepropanesulfonic acid (MOPS) (pH 8.0) or 100 mM morpholinethanesulfonic acid (MES) (pH 5.5). Antibiotics were used as required at the following concentrations: ampicillin, 100 mg/ml; kanamycin, 25 mg/ml; tetracycline, 20 mg/ml; and chloramphenicol, 30 mg/ml. All strains were grown at 37°C with aeration. Molecular genetic techniques. Phage P1 transduction, transformation with CaCl2, and electroporation were performed by using standard methods (14). General DNA manipulations were also carried out as described previously (18). PCRs were performed by using whole cells with puReTaq Ready-To-Go PCR beads (Amersham Biosciences). PCR products were passed through Quantum Prep PCR Kleen spin columns (Bio-Rad) prior to subsequent treatments. Western blot analysis. Western blot analysis of glutamate decarboxylase, GadX, and RpoS was performed essentially as described previously (12). Strains were grown at 37°C in media containing the required antibiotics as indicated below. At an optical density at 600 nm (OD600) of 0.4 (log phase), cells were collected by centrifugation. Protein concentrations were determined by using the Bio-Rad protein assay reagent. Samples (5 mg of protein) were separated by Tris-HCl–10% polyacrylamide gel electrophoresis (PAGE). After semidry electrophoretic transfer of proteins onto polyvinylidene difluoride membranes (Mil-

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FIG. 1. Effect of Crp and GadW on gadA and gadBC expression. (A) Western blot analysis of GadA and GadB production in exponential-phase cells grown in EG. Sodium dodecyl sulfate-PAGE was performed with 5-␮g protein samples, and the gel was probed with anti-Gad antibody as described in Materials and Methods. The growth pHs for different cultures are indicated. (B) Northern blot analysis of gadA, gadAX, and gadBC. RNA extracted from exponential-phase cells grown in minimal glucose medium was probed for gadA and gadBC messages as described in Materials and Methods. Five micrograms of total RNA was loaded in each well. (C) Northern blot analysis of gadW message. Cells were grown as described above for panel B but were probed for the gadW message. WT, wild type.

lipore Co., Bedford, Mass.), the membranes were probed with either rat antiGad (2), mouse anti-RpoS, or rabbit anti-GadX (kindly provided by S. Shin and J. Kaper). Antibody-tagged protein bands on the probed membranes were detected by using an ECL Western blot detection kit (Amersham) (5). Northern blot analyses. Northern blot analyses of gadA, gadAX, gadBC, and gadW transcripts were performed as described previously (12). Total RNA was extracted from log-phase cultures (OD600, 0.4; 2 ⫻ 108 cells per ml) grown under both alkaline and acidic conditions in minimal medium by using an RNeasy kit (Qiagen). Aliquots (5 mg) of RNA were denatured at 65°C for 15 min and separated by electrophoresis through a 1.2% denaturing formaldehyde–agarose gel (18). The RNA was then transferred onto nylon membranes (AmershamPharmacia) and baked at 80°C for 2 h. The membranes were probed with a 1.4-kb gadA-gadB probe generated by PCR performed with oligonucleotides 377 and 378 or with a 0.733-kb gadW probe made by using oligonucleotides 465 and 466. Probes were labeled with [␣-32P]dCTP (ICN) by using a DECA Prime II random DNA priming kit (Ambion). The gadA-gadB probe corresponded to the entire open reading frame of gadA or gadB and hybridized to both gadA and gadB. As a control, the membranes were also hybridized with a 23S rRNA probe (oligonucleotide 379) that was end labeled with [␥-32P]ATP. EMSA. The ability of the MalE-GadX protein to bind to the gadW promoter region was tested by using an electrophoretic mobility shift assay (EMSA). The promoter target fragments were amplified with oligonucleotides 511 and 512. An unrelated lacZ promoter was also prepared as a control (oligonucleotides 507 and 508). The PCR-generated gadW promoter fragment extended from bp ⫺391 to 25 relative to the start codon of the GadW open reading frame. The fragment was end labeled with [␥-32P]ATP by using T4 polynucletide kinase. Radiolabeled DNA probes (5 ng; 5,000 cpm) were incubated with MalE-GadX fusion protein (provided by J. Kaper) at room temperature for 30 min in 20 ml of binding buffer (20 mM HEPES [pH 8.0], 5 mM MgCl2, 50 mM potassium glutamate, 0.01 mM EDTA, 1 mM NaH2PO4, 20 mM NaCl, 1 mM dithiothreitol, 30 mg of salmon sperm DNA per ml). Samples were loaded onto 5% Tris-borateEDTA nondenaturing Ready Gel (Bio-Rad) and electrophoresed at room temperature in 0.5⫻ Tris-borate-EDTA buffer with 1.2% glycerol. The gels were dried and exposed to X-OMAT Kodak film at ⫺70°C for 3 h or were scanned with a phosphorimager (FLA-5000; Fujifilm).

Intracellular cAMP measurements. E. coli wild-type cells and a cya-deficient mutant (as a control) were grown to the exponential phase (OD600, 0.4) and the stationary phase (22 h) in complex medium and minimal medium under both alkaline and acidic conditions, as indicated above. Cells were collected by centrifugation and resuspended in the lysis buffer provided with the cAMP enzyme immunoassay system (Amersham Pharmacia Biotech). After boiling at 100°C for 15 min in lysis buffer and centrifugation at 2,000 ⫻ g and 4°C for 5 min, the cAMP concentrations in supernatants were determined by using the kit according to the manufacturer’s instructions. The average intracellular cAMP concentration (determined in triplicate) was expressed in nanomoles per milligram of total protein.

RESULTS GadW and CRP cooperate to repress gadA and gadBC transcription in minimal salts glucose medium. Previous results demonstrated that GadW acts as a positive regulator of gadA and gadBC expression at a high pH in the absence of GadX or as a negative regulator at either a high pH or a low pH in the presence of GadX (12). Thus, positive control by GadW is GadX independent, while negative control occurs through GadW repression of gadX. Part of this regulation appears to be direct since GadW was shown to bind in vitro to the gadA and gadBC promoter regions (12). These studies were all performed by using log-phase cells grown in LB medium. As shown in Fig. 1, GadW also repressed gadA and gadBC expression during exponential growth in defined EG (Fig. 1A, compare lanes 5 and 6 with lanes 1 and 2), but in contrast to what took place in LB medium, this repression was not due to inhibition of GadX (Fig. 1A, compare lanes 3 and 4 with lanes

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7 and 8). Figure 1B shows that this control occurred at the mRNA level (Fig. 1B, compare the EF227 lanes with the EK861 lanes). Thus, GadW appears to be capable of directly and indirectly repressing the gadA and gadBC promoters depending on the growth medium. The previous study in which LB medium was used also showed that CRP is a negative regulator of gadA and gadBC. This was also true in minimal glucose medium (Fig. 1A and B, EF528 lanes). One difference from the previous LB medium results was that minimal medium expression of gadA and gadBC in crp or gadW mutants remained acid induced (Fig. 1A, compare lanes 3 and 4 with lanes 5 and 6 or with lanes 9 and 10). This was not the case in LB medium, in which both mutants expressed gad genes equally at pH 8 and 5.5 (12). Eliminating both gadW and crp, however, resulted in constitutively high levels of gadA and gadBC expression during growth in minimal glucose medium (Fig. 1A, lanes 11 and 12). These results indicate that CRP and GadW may both participate in alkaline repression of the gadA and gadBC genes during logphase growth in defined minimal medium. GadX control of gadA and gadBC expression in minimal medium is mainly indirect through effects on gadW. Previous work indicated that GadX is a direct activator of gadA and gadBC in rich LB medium (12, 22). However, the results shown in Fig. 1A suggest that in minimal medium GadX has the opposite effect, repressing gadA and gadBC at an alkaline pH. The evidence leading to this conclusion is that, compared to the production in wild-type cells (EK227), the gadX mutant displayed increased glutamate decarboxylase production at pH 7.7 (Fig. 1A, compare lanes 1 and 3). The gadX mutant exhibited only a slight decrease in glutamate decarboxylase production at pH 5.5, indicating that in this medium GadX is not much of an activator (Fig. 1A, compare lanes 2 and 4). This was confirmed (Fig. 1A) when we compared the glutamate decarboxylase expression in a gadX⫹ gadW strain (EF861) to that in a gadX gadW strain (EF863). When GadW repression was removed by mutation, deleting gadX had no effect on glutamate decarboxylase production (Fig. 1A, compare lanes 5 and 6 with lanes 7 and 8). This suggests that a major role of GadX in exponential-phase minimal medium-grown cells is to repress gadW at pH 5.5. GadX repression of gadW at pH 5.5 should allow elevated expression of gadA and gadBC. As shown in Fig. 1C, Northern blotting revealed that GadX does indeed repress gadW expression. If this repression was absent, gadW expression was acid induced. The mechanism of the acid induction is not known. Furthermore, the higher GadW levels produced in the gadX mutant at pH 5.5 corresponded to lower levels of GadA and GadB production, as shown in Fig. 1A, lanes 2 and 4, which is consistent with a direct repressing effect of GadW on gadA and gadBC transcription. The results shown in Fig. 1A (compare lane 1 with lanes 3 and 5) also suggested that a combination of GadX and GadW in wild-type cells is needed to represses gadA and gadB under alkaline conditions in exponential-phase cells growing in minimal medium. Again, this was not the case in LB medium, in which GadW was shown to activate gadA and gadBC expression under alkaline conditions (12). GadX binds to the GadW promoter region. Evidence described above indicated that GadX represses gadW expression (12). Data were obtained by using electrophoretic mobility

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FIG. 2. GadX binds to the gadW promoter. A promoter-containing fragment of gadW (426 bp; from position ⫺391 to position 25 relative to the translational start) was used in gel shift experiments with purified MalE-GadX protein (see Materials and Methods). The concentrations of hybrid MalE-GadX (also known as MBP-GadX) used and whether the reaction mixture contained cold specific competitor DNA (50 ng) are indicated above the lanes. A superscript P indicates promoter. Maltose binding protein is encoded by malE.

shifts that showed that this regulation was most likely direct. A 416-bp fragment of DNA containing the putative gadW promoter was used in DNA gel shift experiments with purified MalE-GadX protein. The results indicated that GadX does bind to this region of DNA (Fig. 2, lanes 5 through 9). An unlabeled gadW fragment also successfully competed with radiolabeled gadW DNA for binding of GadX. Expression of gadX is pH regulated via CRP. The data indicate that in minimal medium GadX represses gadW but has little direct effect on gadA and gadBC. GadW, however, clearly represses gadA and gadBC. If gadX were acid induced, this would lower GadW levels at pH 5.5. Lowering the GadW levels through GadX-dependent repression would translate into increased activation of gadA and gadBC by the GadE inducer (11). To examine the feasibility of this model, a Western blot analysis of GadX production was conducted (Fig. 3). Because GadX and GadW reciprocally repress each other, it was difficult to visualize the GadX protein in wild-type cells (Fig. 3, lanes 1 and 2). However, deleting gadW released gadX expression (12) and revealed that GadX production was, as pre-

FIG. 3. Crp and GadW repress gadX expression: Western blot analysis of GadX. Cultures of various strains were grown to the log phase in EG at the pHs indicated above the lanes. Sodium dodecyl sulfate-PAGE was performed by using 5-␮g protein samples, and the gel was probed with anti-GadX antibody. Wt, wild type.

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J. BACTERIOL. TABLE 2. Effects of pH, growth phase, and medium on intracellular cAMP levels Medium

EG

Phase

Log Stationary

LB

Log Stationary

FIG. 4. Effect of cya on gadA and gadBC expression. Cells were grown to the log phase in LB medium (A) or EG (B). Sodium dodecyl sulfate-PAGE was performed by using 5-␮g protein samples, and the gels were probed with anti-Gad antibody.

dicted, acid induced (Fig. 3, lanes 3 and 4). Previous work demonstrated that CRP also represses gadX, although this effect occurs indirectly through CRP-mediated repression of RpoS (12). The RpoS sigma factor then drives transcription of gadX. Interestingly, when the GadX level was measured in a crp mutant, the level was elevated, but the protein was not acid regulated (Fig. 3, compare lanes 1 and 2 with lanes 5 and 6). This suggested that the pH control of GadX production in minimal medium is mediated through CRP and that GadW can temper this control in some way (see below). Acid induction of GadX correlates with pH-dependent changes in cAMP levels. Previously published evidence suggested that cAMP levels might be elevated under alkaline conditions (1, 20), a situation that could explain the CRPdependent alkaline repression of gadA and gadBC (Fig. 1A, compare lanes 5 and 6 with lanes 11 and 12). Although CRP was shown previously to repress gadX, whether cAMP was also involved was never examined. Figure 4 shows that a cya mutant defective in cAMP synthesis did overexpress glutamate decarboxylase in a manner similar to a crp mutant. This was true in rich LB medium (Fig. 4A) and in minimal salts glucose medium (Fig. 4B). The data also demonstrated that elevated expression of gadA and gadBC in the absence of cya or crp was constitutive when the organism was growing in LB medium (Fig. 4A) but was acid induced in EG (Fig. 4B). Acid induction of glutamate decarboxylase in this situation (cya mutant in minimal glucose medium) is due to acid induction of the GadE activator (11). We then investigated whether pH could influence cAMP levels in different media and in different growth phases. Table 2 shows the results obtained. Overall, the levels of cAMP were three- to fivefold higher in log-phase, LB medium-grown cells than in EG-grown cells. It was clear that growth in either medium under acidic conditions lowered the intracellular cAMP concentration compared to the concentration measured in cells grown under alkaline conditions. A fourfold decrease was evident in EG, while growth in acidic LB medium lowered the cAMP concentration twofold. It has been reported previously that a gadW strain grown in LB medium had an elevated GadX level but that the level was not pH regulated (12). The high concentration of cAMP measured in LB medium-grown

pH

cAMP level (nmol/mg of protein)

7.7 5.5 7.7 5.5 8 5.5 8 5.5

119 ⫾ 4 30 ⫾ 1 13 ⫾ 1 1 ⫾ 0.2 320 ⫾ 1 164 ⫾ 2 296 ⫾ 5 133 ⫾ 7

Difference (fold)

4 13 2 2

cells is consistent with a lack of CRP-dependent pH control in this medium. However, the observed acid-repressed cAMP levels in minimal medium are consistent with the increases observed in the RpoS and GadX levels, as well as the concomitant decrease in the GadW repressor level, all of which translated into higher expression of the decarboxylase genes. Crp and GadW do not affect production of GadE. A previous study demonstrated that GadE is an essential activator of gadA and gadBC expression (11). Consequently, we asked whether CRP or GadW might represses gadA and gadBC expression at pH 7.7 by directly inhibiting gadE transcription. A Northern blot analysis in which the gadE mRNA levels in wild-type and crp-deficient strains were compared did not reveal any significant differences (data not shown). We also examined the possibility that GadW might mediate pH-dependent control of gadE. Again, a Northern blot analysis failed to reveal any effect of GadW on GadE transcript levels (data not shown). The only targets for GadW in this system appear to be gadX, gadA, and gadBC. Crp and GadW repression of GadX is RpoS dependent. We then asked if the CRP-dependent pH control of gadA and gadBC was RpoS and/or GadX dependent. As shown in Fig. 5, removing Crp and GadW resulted in constitutive expression of the GadA and GadB proteins and the GadX protein. However, deleting rpoS restored pH control of gadA and gadBC (this control is GadE dependent [data not shown]) and prevented expression of gadX. Thus, the Crp-dependent pH control of gadA and gadBC shown in Fig. 1A (compare lanes 11 and 12 with lanes 5 and 6) occurs through the RpoS-to-GadX pathway.

FIG. 5. Effects of GadW and CRP on GadX and GadAB production in minimal medium are RpoS dependent. Cells were grown to log phase in EG at the pHs indicated above the lanes. Sodium dodecyl sulfate-PAGE was performed by using 5-␮g protein samples, and the gels were probed with anti-Gad and anti-GadX antibodies.

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FIG. 6. GadW represses rpoS expression: Western blot analysis of RpoS. Cells were grown in EG to log phase at the pHs indicated above the lanes. Sodium dodecyl sulfate-PAGE was performed by using 5-␮g protein samples, and the gel was probed with anti-RpoS antibody.

GadW is a negative regulator of RpoS. GadW failed to bind to the gadX promoter region in EMSA experiments, indicating that repression was not direct (data not shown). We then examined whether GadW might indirectly control gadX through RpoS. Figure 6 shows the results of RpoS Western blot studies that indicated that GadW is a negative regulator of RpoS production. The increased production of RpoS in the gadW mutant was also acid induced, consistent with the studies described above which indicated that there is acid-regulated control of RpoS production by CRP-cAMP. It is unclear at what level GadW affects sigma S production.

FIG. 7. Model for pH control of glutamate-dependent acid resistance. There are at least three levels of pH control. (i) The expression of gadE is acid induced in minimal medium. (ii) Under alkaline conditions, cAMP levels are high and RpoS levels are low (log phase). GadX and GadW repress each other’s expression to establish an equilibrium. There is little expression of the gadA and gadBC genes because of direct GadW repression and because the GadE level is also low. Under acid conditions, cAMP levels fall and GadX levels rise, which further represses GadW. (iii). In LB medium, GadX is required for induction of gadA and gadBC. GadW, on the other hand, induces gadA and gadBC under alkaline conditions in the absence of GadX.

DISCUSSION There appear to be at least two regulatory arms that control production of the glutamate decarboxylase-dependent acid resistance system. The GadE activator arm is essential. Without GadE, the gadA and gadBC genes cannot be activated. The second arm, involving the AraC-like regulators GadX and GadW, either helps or hinders GadE depending on the environmental situation. We propose that GadX, GadW, and CRP sense different chemical signals in the cell and that the intracellular ratio of these signals alters the balance within the regulatory loop. Under some conditions GadX dominates, and under other conditions GadW dominates. The results presented above describe several new regulatory aspects of glutamate-dependent acid resistance. (i) An acid pH lowers cAMP levels in exponential-phase cells growing in minimal glucose medium. This could lead to elevated sigma S production that would drive increased production of GadX. (ii) However, GadW represses sigma S synthesis at low pH and, in turn, GadX synthesis. (iii) GadX, when not repressed by GadW, is clearly acid induced due to changes in cAMP. (iv) GadW is also acid induced by an unknown mechanism when it is not repressed by GadX. (v) GadX directly binds to the gadW promoter region. (vi) GadX and GadW collaborate to repress gadA and gadBC expression under alkaline conditions. In the developing model for the control of glutamate-dependent acid resistance in minimal glucose medium gadA and gadBC expression is potentially controlled by pH at several levels (Fig. 7). First, an acidic pH stimulates the production of GadE, the essential activator of these genes (11). How gadE is induced by low pH is not known. Second, the AraC-like regulatory protein GadW alters the function with respect to repression or induction of gadA and gadBC coincident with changes in the growth pH (12). A third level, demonstrated here, is that the growth pH can alter the synthesis of GadX, an

AraC-family regulator that can coactivate gadA and gadBC expression and represses GadW. This level of control is mediated by the effect of growth pH on the cAMP level and is only evident in minimal glucose medium in which the cAMP level is already low. Our model for regulation posits that artificially or naturally elevated levels of GadE (or of GadE activity) not only eliminate the need for the GadX coactivator but also overwhelm GadW-dependent or GadXW-dependent repression. However, when GadE activity is low, the balance between GadX and GadW and the signals that these proteins potentially sense become deciding factors in gadA and gadBC expression. GadX and GadW reciprocally repress each other’s expression in rapidly growing cells. But gadX expression is driven by sigma S, which itself is under considerable control. Conditions under which sigma S levels become elevated tend to increase GadX production relative to GadW production. The increase in GadX production can help activate the gadA and gadBC genes in the presence of low levels of GadE. Sigma S levels are controlled at many levels. One of these involves cAMP-CRP repression (9). Exponential-phase cells growing in rich medium possess high levels of cAMP, which ensure poor expression of rpoS. However, entry into stationary phase triggers increased translation of rpoS transcripts and decreased degradation of RpoS protein (9, 15, 16). Both of these processes occur independent of cAMP concentration. The stationary-phase increase in RpoS concentration causes GadX levels to rise and coactivate gadA and gadBC. Exponential-phase cells growing in minimal glucose medium have a much lower intracellular cAMP concentration than LB medium-grown cells have, but at pH 7.7 the level must be high enough to minimize GadX synthesis. Under acidic conditions, however, the cAMP level declines and the RpoS level can

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increase, even in log-phase cells. However, a concomitant increase in the GadX level may not be obvious if RpoS synthesis is repressed by other means (by GadW, for example) Many of the individual regulatory circuits described above were exposed by using mutants missing one of the network components. In the wild-type situation, however, many of the scenarios are not easily observed. This is because under the conditions tested (i.e., minimal medium) the network is set to counterbalance. For example, acid induction of gadX and acid induction of gadW were observed in mutants lacking one of the regulators. In wild-type situations when both regulators are present, the acid induction of one represses the acid induction of the other, and thus, in effect, the regulators cancel each other out. The balance might be influenced by acid effects on the cAMP-RpoS-gadX pathway, but GadW negative control of RpoS limits this. Thus, the system in minimal medium seems to be designed to rely primarily on GadE. Real world conditions under which acid can seriously tilt the balance of GadX and GadW in wild-type cells have not been clearly identified. Nevertheless, these control pathways are present and can help fine-tune gadA and gadBC expression. We previously demonstrated, for example, that gadA and gadBC expression retained residual pH control in situations where GadE was produced constitutively. This residual acid control required GadX and GadW (11). GadX and GadW are AraC-family regulators. Regulators in this family typically possess DNA-binding domains, dimerization domains, and sensing domains. Both GadX and GadW have been shown to bind DNA and dimerize in vivo and in vitro, but what chemical(s) they may sense remains a mystery (12, 21, 22). This raises the possibility of yet another layer of control, where the activity of GadX or GadW can be altered by the presence of cognate ligand molecules. Undefined growth conditions that change the concentrations of these molecules would likely alter the balance of power between GadX and GadW. This model predicts that there is a flexible regulatory network which is capable of engaging or thwarting glutamatedependent acid resistance under a variety of environmental conditions. Beyond acid resistance, the finding that cAMP levels and, thus, CRP activity can change with pH also has broad significance for understanding pH-regulated gene expression since CRP-cAMP regulates many genes. How acidic environments decrease cAMP levels or how alkaline environments increase cAMP levels is not yet understood. The obvious possibilities include effects on synthesis through adenylate cyclase, degradation through cAMP phosphodiesterase, or expulsion from the cell. The pH optima of adenylate cyclase and phosphodiesterase are both greater than 8.5. Thus, as the internal pH declines, synthesis of cAMP decreases. If the deleterious effect of acid pH on adenylate cyclase is greater than the effect on cAMP phosphodiesterase, cAMP levels decrease. A particularly interesting finding is that GadW appears to regulate RpoS production in some manner. The production of RpoS certainly does not suffer from a lack of regulatory oversight. Numerous mechanisms control the synthesis of this sigma factor at the transcription, translation, and posttranslation levels. Where GadW might fit into the picture is not known. EMSA studies have shown that purified GadW binds

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to the gadA and gadBC promoters, but we have not detected significant sequence homology between the rpoS promoters and the gadA and gadBC promoters. Thus, it seems unlikely that GadW directly affects RpoS expression. This protein more likely controls production of a component in the RpoS regulatory scheme. Part of the physiological rationale governing regulation of glutamate-dependent acid resistance is that rapidly growing E. coli does not need pH 2 acid resistance and, as a result, actively represses the gadA and gadBC genes. Encounters with low pH and entry into stationary phase harbinger future encounters with potentially lethal acidic conditions and dictate a need for gadA and gadBC derepression. The complexity and global reach of the gad regulatory web indicate that the cell requires redundant pH control pathways to anticipate and handle various environmental routes to low pH. ACKNOWLEDGMENT This work was supported by National Institutes of Health award R01-GM61147. REFERENCES 1. Alonzo, S., M. Heyde, P. Laloi, and R. Portalier. 1998. Analysis of the effect exerted by extracellular pH on the maltose regulon in Escherichia coli K-12. Microbiology 144:3317–3325. 2. Audia, J. P., C. C. Webb, and J. W. Foster. 2001. Breaking through the acid barrier: an orchestrated response to proton stress by enteric bacteria. Int. J. Med. Microbiol. 291:97–106. 3. Bordi, C., L. Theraulaz, V. Mejean, and C. Jourlin-Castelli. 2003. Anticipating an alkaline stress through the Tor phosphorelay system in Escherichia coli. Mol. Microbiol. 48:211–223. 4. Castanie-Cornet, M. P., and J. W. Foster. 2001. Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes. Microbiology 147:709–715. 5. Castanie-Cornet, M. P., T. A. Penfound, D. Smith, J. F. Elliott, and J. W. Foster. 1999. Control of acid resistance in Escherichia coli. J. Bacteriol. 181:3525–3535. 6. De Biase, D., A. Tramonti, F. Bossa, and P. Visca. 1999. The response to stationary-phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system. Mol. Microbiol. 32:1198–1211. 7. Hersh, B. M., F. T. Farooq, D. N. Barstad, D. L. Blankenshorn, and J. L. Slonczewski. 1996. A glutamate-dependent acid resistance gene in Escherichia coli. J. Bacteriol. 178:3978–3981. 8. Lange, R., M. Barth, and R. Hengge-Aronis. 1993. Complex transcriptional control of the ␴s-dependent stationary-phase-induced and osmotically regulated osmY (csi-5) gene suggests novel roles for Lrp, cyclic AMP (cAMP) receptor protein-cAMP complex, and integration host factor in the stationary-phase response of Escherichia coli. J. Bacteriol. 175:7910–7917. 9. Lange, R., and R. Hengge-Aronis. 1994. The cellular concentration of the ␴s subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability. Genes Dev. 8:1600–1612. 10. Lin, J., I. S. Lee, J. Frey, J. L. Slonczewski, and J. W. Foster. 1995. Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J. Bacteriol. 177:4097–4104. 11. Ma, Z., S. Gong, H. Richard, D. L. Tucker, T. Conway, and J. W. Foster. 2003. GadE (YhiE) activates glutamate-decarboxylase-dependent acid resistance in Escherichia coli K12. Mol. Microbiol. 49:1309–1320. 12. Ma, Z., H. Richard, D. L. Tucker, T. Conway, and J. W. Foster. 2002. Collaborative regulation of Escherichia coli glutamate-dependent acid resistance by two AraC-like regulators, GadX and GadW (YhiW). J. Bacteriol. 184:7001–7012. 13. Masuda, N., and G. M. Church. 2003. Regulatory network of acid resistance genes in Escherichia coli. Mol. Microbiol. 48:699–712. 14. Miller, J. H. 1992. A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 15. Muffler, A., D. Fischer, S. Altuvia, G. Storz, and R. Hengge-Aronis. 1996. The response regulator RssB controls stability of the sigma S subunit of RNA polymerase in Escherichia coli. EMBO J. 15:1333–1339. 16. Muffler, A., D. D. Traulsen, R. Lange, and R. Hengge-Aronis. 1996. Posttranscriptional osmotic regulation of the ␴s subunit of RNA polymerase in Escherichia coli. J. Bacteriol. 178:1607–1613. 17. Nishino, K., Y. Inazumi, and A. Yamaguchi. 2003. Global analysis of genes

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