Role of the Multidrug Resistance Regulator MarA in Global Regulation of the hdeAB Acid Resistance Operon in Escherichia coli

MarA, a transcriptional regulator in Escherichia coli, affectsfunctions such as multiple-antibiotic resistance (Mar) and virulence.Usually an activator, MarA is a repressor of hdeAB and otheracid resistance genes. We found that, in wild-type cells grownin LB medium at pH 7.0 or pH 5.5, repression of hdeAB by MarAoccurred only in stationary phase and was reduced in the absenceof H-NS and GadE, the main regulators of hdeAB. Moreover, repressionof hdeAB by MarA was greater in the absence of GadX or Lrp inexponential phase at pH 7.0 and in the absence of GadW or RpoSin stationary phase at pH 5.5. In turn, MarA enhanced repressionof hdeAB by H-NS and hindered activation by GadE in stationaryphase and also reduced the activity of GadX, GadW, RpoS, andLrp on hdeAB under some conditions. As a result of its directand indirect effects, overexpression of MarA prevented mostof the induction of hdeAB expression as cells entered stationaryphase and made the cells sevenfold more sensitive to acid challengeat pH 2.5. These findings show that repression of hdeAB by MarAdepends on pH, growth phase, and other regulators of hdeAB andis associated with reduced resistance to acid conditions.

INTRODUCTION

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INTRODUCTION

MarA, an AraC/XylS transcriptional regulator of Escherichiacoli, directly activates or represses multiple chromosomal genes.As a result, it affects many functions including multiple-antibioticresistance (Mar), virulence, and survival (1, 5). MarA is specifiedby the marRAB operon, whose expression is induced by sodiumsalicylate, menadione, and other chemicals which inactivatethe autorepressor MarR (1, 10, 33, 38). Using gene arrays, Pomposielloet al. (33) found that overexpression of MarA from an isopropyl-β-D-thiogalactopyranoside(IPTG)-inducible plasmid repressed hdeAB and other genes involvedin acid resistance (gadA, gadB, gadC, hdeD, and slp), whereassodium salicylate activated these same genes. Direct repressionof hdeAB by MarA has been demonstrated previously (37). However,whether MarA affects cell resistance to acid conditions remainedunknown.

The hdeAB operon of E. coli specifies two periplasmic chaperonesthat are essential for cell survival in rich media at extremelylow pH, such as that in the stomach (pH 1.5 to 3). Both chaperonesare only active below pH 3 and prevent irreversible aggregationof acid-denatured periplasmic proteins (14, 21, 23, 28). HdeAseems to play a major role at pH 2, whereas HdeB is more activeat pH 3 (23). The determined HdeA crystal structure and themodel HdeB structure are similar (14, 45), in agreement withtheir genetic and functional similarities. HdeA is one of themost abundant periplasmic proteins in stationary phase (25).

Expression of hdeAB is affected by different environmental conditions.The hdeAB operon is induced in stationary phase (39, 44) andat acid pH (19, 43). Transcription of hdeAB depends on complexcircuits of regulation involving RpoD ( ${sigma}$ ⁷⁰), RpoS ( ${sigma}$ ³⁸), H-NS,cyclic AMP receptor protein, GadE, GadX, GadW, GadY, EvgAS,YdeO, Lrp, MarA, SoxRS, TorRS, and TrmE, among other regulators(see Fig. 1 and references in the legend; see Fig. 2 for theDNA binding sites of some of these regulators in the hdeAB promoter).

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FIG. 1. Regulation of the hdeAB operon. The figure has been produced using data from the literature (8, 13, 16, 20, 22, 24, 26, 27, 30, 33, 37, 39, 42, 43, 44, 46) as well as the results obtained in this study. Thick arrows represent genes. Direct regulation is shown as a continuous thin arrow. Indirect regulation, or regulation in which a direct effect has not been confirmed, is shown as a dotted thin arrow. See the supplemental material for a more detailed explanation of this figure. cAMP, cyclic AMP; CRP, cAMP receptor protein.

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FIG. 2. Binding sites in the hdeAB promoter for the selected regulators. The hdeAB promoter was analyzed from positions –168 to +54 as described in Materials and Methods. The binding sites of the selected transcriptional regulators are shown below the corresponding sequence as continuous arrows (known) or dotted arrows (putative). In parentheses are the number of nucleotides that match the consensus sequence/number of nucleotides of the consensus sequence. Even though hdeAB is transcribed from RNA polymerase containing ${sigma}$ ⁷⁰ or ${sigma}$ ³⁸ (39), its promoter does not have the –10 sequence (CTATACT) found in many promoters regulated by ${sigma}$ ³⁸ that replaces the –10 sequence (XTATAAT) found in ${sigma}$ ⁷⁰ promoters (12). TSS, transcription start site.

We have studied how MarA fits into the complex transcriptionalcontrol of hdeAB. We report the effect of pH, growth phase,and known regulators of hdeAB on hdeAB expression and repressionby MarA, as well as the effect of MarA on the activities ofthese other regulators. We also describe the effect of MarAon acid resistance.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strains, plasmids, and growth media. The bacterial strains and plasmids used in this study are listedin Table 1. Because HdeA and HdeB seem to be mainly importantin rich medium (28), we used LB medium (per liter: 10 g of tryptone,5 g of yeast extract, 10 g of NaCl) at pH 7.0 or buffered topH 5.5 with 100 mM MES (morpholinoethanesulfonic acid). Allcultures were grown at 37°C with agitation. Cells grownovernight (18 h) were subsequently diluted 1:100 in fresh mediato an optical density at 600 nm (OD₆₀₀) of 0.03. According tothe growth curves obtained, exponential-phase cells were definedas those obtained from cultures in fresh media grown for 2 to2.5 h (0.3 to 0.6 OD₆₀₀) and early-stationary-phase cells asthose obtained after 6 h (1.4 to 2.9 OD₆₀₀). The final pH ofthe cultures was 7.0 to 8.0 in LB at pH 7.0 and 5.6 to 6.0 inLB at pH 5.5. The antibiotics were used at 100 µg ml^–1(ampicillin) and 25 µg ml^–1 (chloramphenicol, kanamycin,and tetracycline). MarA expression from plasmid pMB102 was inducedby inoculation of cells from the overnight cultures into freshmedium containing 0.5 mM IPTG.

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TABLE 1. Bacterial strains and plasmids used in this study

Construction of the hdeABp-lacZ chromosomal transcriptional fusion and other DNA manipulations. A 372-bp fragment from the hdeAB promoter (from –349 to+23 relative to the transcriptional start site) was amplifiedby PCR using chromosomal DNA from strain AG100 as template,primers HdeALF (5'AGGgaattcAAAATATCGCCAGAGACGAAC, EcoRI sitein lowercase) and HdeALR (5'TATggatccAGCCGTCACGAATCAAT, BamHIsite in lowercase), and Turbo Pfu polymerase (Stratagene, LaJolla, CA), using a T_m of 55°C. The amplified DNA was clonedinto the vector pRS415 between the EcoRI and BamHI sites, yieldingthe plasmid pHdeA415, whose insert sequence was verified atthe Tufts University Core Facility. Recombination between thehdeABp-lacZ region of pHdeA415 and ${lambda}$ RZ5 (35) resulted in a lysatebearing ${lambda}$ RZ5 (hdeABp-lacZ). This was used to infect strain JHC1096,and Amp^r Lac⁺ lysogens were selected and purified on LB agarcontaining ampicillin (50 µg ml^–1) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside;40 µg ml^–1). Lysates from these lysogens were thenused to infect strain JHC1096, and Amp^r Lac⁺ lysogens were againisolated. The resulting strain, HdeA96, was confirmed by PCR,as previously described (34), to have a single copy of the transcriptionalfusion located in the ${lambda}$ att site on the chromosome. Strain HdeA96was subsequently transformed with plasmid pMB102 to obtain strainHdeA100. Plasmid CaCl₂ transformation and phage P1 transductionto transfer mutations in different hdeAB regulatory genes tothe strain HdeA100 were performed according to standard procedures(36).

Identification of the putative binding sites for the hdeAB regulators. The hdeAB promoter was analyzed from positions –168 (transcriptionalstart site of the divergent gene hdeD) to +54 (translation startcodon for HdeA) (Fig. 2). Putative binding sites were identifiedusing the "search patterns" utility of Colibri (genolist.pasteur.fr/Colibri/).MarA (37) and H-NS (39) binding sites in the hdeAB promoterregion have already been identified. The GadE binding site hasbeen located between –158 and +4 but not identified (20).The consensus binding sequences we used were the following:5'WYAGGMWWWWDYTWYWWW for binding of GadE, GadX, and GadW (gadbox[26, 43]); 5'YAGHAWATTWTDCTR for Lrp (11); and 5'CTGTTCATATfor TorR (2, 40); where D is A, T, or G; H is A, C, or T; Mis A or C; R is A or G; W is A or T; and Y is C or T. Only sequenceswith 70% or higher identity to the consensus sequences wereselected.

β-Galactosidase assays. Expression of the marO-lacZ and hdeABp-lacZ chromosomal reporterswas tested using exponential-phase and stationary-phase cellsprepared as described above. The assays were performed usingcells permeabilized with sodium dodecyl sulfate-chloroform aspreviously described (29). All assays were performed at leastin triplicate; each replicate was the result of an independentassay performed in duplicate. Statistically significant differenceswere determined by t test (two independent samples with equalvariance, two-tailed distribution) using Microsoft Excel 2003software. Unless otherwise stated, differences were consideredto be statistically significant only when P was <0.01.

Acid resistance assays at very low pH. The assays were performed using early-stationary-phase cells(OD₆₀₀ of 1.3 to 2.5) prepared as described above and obtainedfrom cultures in fresh media grown for 6 h (cultures in LB broth)or 7 to 8 h (cultures in LB broth containing 5 mM sodium salicylate).Control cells were directly plated on LB pH 7.0 agar after serialdilution in fresh LB broth at pH 7.0. To mimic the conditionsthat E. coli cells would encounter when passing through thestomach, sample cells were diluted to about 10⁶ CFU per ml inLB at pH 2.5 (acidified with HCl) and incubated for 2 h at 37°Cwithout shaking. Cells were then serially diluted in fresh LBbroth at pH 7.0 and plated on LB pH 7.0 agar plates to determineviability. The assays were performed at least in triplicate.

RESULTS

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RESULTS

Effect of stationary phase and pH on hdeAB expression and repression by MarA. We chose conditions that are known to have a major effect onthe expression of hdeAB (exponential versus stationary phaseand pH 7.0 versus pH 5.5 [19, 39, 43, 44]) and on the physiologyof the HdeA and HdeB proteins (LB rich medium [28]). In a ${Delta}$ marRABstrain bearing an hdeABp-lacZ chromosomal reporter, the expressionof hdeAB at pH 7.0 increased 1.7-fold from exponential phaseto early stationary phase (Fig. 3). The β-galactosidaseactivity obtained and the increase in hdeAB expression observedare in agreement with a previous report (39). When the assayswere performed using late-stationary-phase cells (overnightcultures), the activity found was about 10-fold higher thanthat in exponential-phase cells (data not shown). Such highexpression is consistent with the high amount of HdeA proteinreported under similar conditions (25). At pH 5.5, the expressionof hdeAB in exponential phase showed no significant increasecompared to that at pH 7.0, even though other authors have foundacid induction in exponential-phase cells grown under differentconditions (19, 43). In stationary phase, hdeAB expression atpH 5.5 was larger than that at pH 7.0 (a 2.5-fold increase comparedto expression in exponential phase at pH 5.5; Fig. 3).

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FIG. 3. Effect of MarA on the expression of hdeAB. The β-galactosidase activity in Miller units of the hdeABp-lacZ chromosomal reporter was determined using HdeA100 cells grown in LB at pH 7.0 (left) or in LB at pH 5.5 (right) to exponential or stationary phase. The assays were performed in the absence of MarA (without IPTG) or in the presence of MarA (with IPTG). The results are presented as the averages ± the standard errors of the means (n $≥$ 3). Statistically significant differences (P < 0.01) between related conditions (exponential versus stationary phase at the same pH; pH 7.0 versus pH 5.5 in the same growth phase; and presence versus absence of MarA under the same pH and growth phase) are indicated by asterisks.

When HdeA100 cells (bearing pMB102) were induced by IPTG tooverexpress MarA, we found that MarA prevented nearly all ofthe 1.7- to 2.5-fold increase in hdeAB expression as cells enteredstationary phase (Fig. 3). MarA did not repress hdeAB in exponentialphase but did repress about twofold in stationary phase regardlessof the pH (Fig. 3) to nearly the exponential-phase level. Withthe control plasmid pJPBH, IPTG led to no effect (data not shown).

Effect of known regulators on hdeAB expression: H-NS is the main repressor and GadE is the main activator of hdeAB. We asked whether the changes in the repression produced by MarAon hdeAB occurred via other known regulators of hdeAB. Sevenregulators were selected for the study: H-NS, RpoS ( ${sigma}$ ³⁸), andGadE, which are known direct regulators of the hdeAB operon(see Fig. 1 and 2); and GadX, GadW, Lrp, and TorR, which arethought to be both direct (not demonstrated, but all of themhave several putative binding sites in the hdeAB promoter; Fig.2) and indirect regulators of hdeAB (they regulate H-NS, RpoS,or GadE; Fig. 1).

The effects of these regulators on hdeAB expression obtainedearlier by others cannot be compared since these studies havebeen performed using dissimilar methods and conditions (in somecases only with microarrays of exponential-phase cells grownin minimal medium). Therefore, before studying their effectson MarA control of hdeAB expression, we evaluated the relativeimportance of each of these regulators individually on hdeABexpression under the same conditions. We chose the conditionsdescribed above that have a major effect on the expression ofhdeAB and on the physiology of HdeA and HdeB.

The results obtained and their statistical significance aredisplayed in Table 2 (data in the absence of MarA). Under allthe conditions studied, H-NS was the main repressor of hdeAB,although repression by H-NS was about half as great at pH 5.5as at pH 7.0. GadE was the main activator. GadX was a director indirect activator of hdeAB only in stationary-phase cellsat pH 5.5, but it was a repressor in exponential phase at pH7.0; GadW was a repressor of hdeAB mainly in stationary phaseat pH 5.5. RpoS had a small effect on hdeAB expression and onlyunder certain conditions. Lrp only repressed hdeAB in exponentialphase at pH 7.0. TorR did not significantly affect hdeAB expression.

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TABLE 2. Activation or repression of hdeAB expression by selected regulators in the absence and the presence of MarA^a

MarA also controls hdeAB expression by modifying the effect of other regulators of hdeAB. We studied whether MarA was able to alter the effect of theother regulators on hdeAB expression. Under each condition,we compared the regulator-inactivated strain/parental strainratio obtained in the presence of MarA with that obtained withoutMarA (Table 2).

The activity of two major regulators, H-NS and GadE, under theseconditions was strongly affected by MarA (Table 2). MarA enhancedhdeAB repression by H-NS in stationary phase at both pH 7.0and pH 5.5, which indicates that MarA assists or collaborateswith H-NS to repress hdeAB in stationary phase. On the contrary,MarA reduced the ability of GadE to activate hdeAB in stationaryphase at pH 5.5, which suggests that MarA hinders hdeAB activationby GadE.

MarA also reduced the repression produced by GadX and Lrp inexponential phase at pH 7.0 and the repression produced by GadWand RpoS in stationary phase at pH 5.5 (Table 2).

Effect of the other regulators of hdeAB on repression by MarA. After finding that MarA was able to alter the effect of theother regulators on hdeAB, we studied the converse possibility:whether the absence of the other regulators affected the abilityof MarA to repress hdeAB (Table 3). A dramatic decrease in therepression produced by MarA in stationary phase was found inthe hns and gadE mutants at both pH 7.0 and pH 5.5 (Table 3).Therefore, both H-NS and GadE were involved in the increasedhdeAB repression by MarA that occurred as cells enter stationaryphase.

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TABLE 3. hdeAB repression by MarA in strains inactivated for selected regulators of hdeAB

On the contrary, repression by MarA increased in the gadX andlrp mutants in exponential phase at pH 7.0 and in the gadW,rpoS, and lrp mutants in stationary phase at pH 5.5 (Table 3).These results might be due to competition of GadX, GadW, andLrp with MarA, while the decreased repression by MarA producedby RpoS may be a direct or indirect effect (see Fig. 1).

Effect of growth phase and pH on the expression of marA without and with the inducer sodium salicylate. To understand how the native promoter of marA responded to growthphase and pH, we used marO-lacZ, a chromosomal reporter of themarRAB operon (in SPC105). We found that expression of marAat pH 7.0 was slightly, but significantly, induced in stationaryphase compared to the level in exponential phase; greater inductionoccurred at pH 5.5 (Table 4). Induction of marA expression bysodium salicylate (5 mM) depended also on the growth phase andthe pH. At pH 7.0, salicylate produced a strong induction after1 h when added to cells growing in exponential phase (Table4), as previously reported (10, 38), whereas it had much lesseffect when added to stationary-phase cells (as measured relativeto expression in cells in stationary phase without salicylate)(Table 4). At pH 5.5, salicylate produced little or no effecton marA expression after 1 h when added to cells in exponentialor in stationary phase. When cells grown overnight without salicylatewere inoculated into medium at pH 7.0 with 5 mM salicylate,cell growth was slower than that without inducer. Under theseconditions, the induction of marA expression by salicylate wasvery strong when tested in exponential phase and even strongerin stationary phase (Table 4). Note that this set of cells wasexposed longer to salicylate (Table 4). Growth was arrestedwhen overnight cultures grown without salicylate were dilutedinto medium at pH 5.5 with 5 mM salicylate.

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TABLE 4. Effect of different growth conditions on the expression of marRAB^a with and without the inducer sodium salicylate

Overexpression of marA decreases acid resistance in E. coli. To know whether repression of hdeAB by MarA affects acid resistancein E. coli under conditions similar to those in the stomach,we compared the survivals at pH 2.5 for 2 h at 37°C of awild-type strain and a ${Delta}$ marRAB strain (Fig. 4). We used cellsgrown in LB with or without 5 mM sodium salicylate to earlystationary phase (where MarA produces the highest repressionof hdeAB). In the absence of salicylate, the two strains showedsimilar survivals (20 to 34%) when grown at both pH 7.0 (Fig.4) and pH 5.5 (not shown) prior to the acid challenge. However,growing the cells in LB at pH 7.0 in the presence of the marRABinducer salicylate produced a fivefold decrease in the acidresistance of the ${Delta}$ marRAB strain but a 35-fold decrease in theacid resistance of the wild-type strain (ratio between grayand black bars in Fig. 4). These results show that (i) althoughsalicylate induces the expression of hdeAB and other acid resistancegenes (33), salicylate itself was toxic, decreasing the acidresistance of the cells, and (ii) the strong overexpressionof marA produced by salicylate in wild-type cells made themsevenfold more sensitive than ${Delta}$ marRAB cells to the acid challenge.

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FIG. 4. Effect of MarA on the acid resistance of E. coli. The acid challenge at pH 2.5 was performed as described in Materials and Methods, using early-stationary-phase cells grown in LB broth at pH 7.0 without (gray) or with (black) 5 mM sodium salicylate to induce marRAB. Two different strains were compared: the ${Delta}$ marRAB strain JHC1096 ( ${Delta}$ marRAB) and the wild-type strain GC4468 (WT). The percent survival at pH 2.5 after incubation for 2 h was determined in comparison with untreated samples. The results are presented as the averages ± the standard errors of the means (n = 3). The n-fold decrease in survival produced by sodium salicylate in each strain (ratio between gray and black bars) is indicated above the bars.

DISCUSSION

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DISCUSSION

We studied the role of MarA in the complex regulation of hdeABand in acid resistance of E. coli growing in rich medium atboth pH 7.0 and pH 5.5. Overexpression of MarA largely preventedthe induction of hdeAB expression as cells enter stationaryphase, in part because MarA-mediated repression of hdeAB increasedin stationary phase. Growth phase or pH-dependent changes ontranscriptional regulation by MarA have not been reported beforefor any other member of the mar regulon. Thus, we asked if suchchanges occurred via other regulators of hdeAB.

Seven other regulators were selected to study how they affectedhdeAB repression by MarA. These regulators were chosen becausethey regulate hdeAB directly and/or indirectly (Fig. 1), andbecause all of them have known or putative DNA binding sitesin the hdeAB promoter (Fig. 2). MarA is known to bind a "marbox"partially overlapping the –35 hexamer of the promoter(37). Therefore, competition with RNA polymerase for DNA bindingand/or direct interactions between MarA and ${sigma}$ ⁷⁰, ${sigma}$ ³⁸, or othersubunits of RNA polymerase are possible. Moreover, several putativebinding sites for the other selected regulators, except H-NS,are adjacent to or partially overlap the marbox (Fig. 2). Thus,interaction and/or competition between MarA and these regulatorsis also possible.

Before studying how these regulators affected MarA repressionon hdeAB, we examined their own effect on hdeAB under the chosengrowth conditions. H-NS was the main repressor of hdeAB underall the conditions studied, in agreement with previous findingsat pH 7.0 (3, 39). However, hdeAB repression by H-NS was significantlylower at pH 5.5, which has not been described before.

GadE was the main activator of hdeAB in LB medium under allthe conditions studied, whereas GadX repressed or activatedhdeAB depending on the growth conditions, and GadW repressedhdeAB. The different effects that GadX and GadW had under diverseconditions, sometimes opposite to that of GadE, support theidea that they can regulate hdeAB independently of GadE. Theeffect of the Gad regulators on hdeAB has not been studied beforein stationary phase; thus we can only compare our results withprevious data obtained using exponential-phase cells grown inminimal medium (20, 43). In general, our results are in agreementwith the previous findings (20, 43), although some differenceswere found, probably because of the different culture mediaused. (i) In exponential phase, we found that GadX repressedhdeAB in LB medium at pH 7.0 (Table 2), while others observedno significant effect of GadX in minimal medium at pH 7.4 (43).(ii) In exponential phase, we found that GadX produced no effecton hdeAB expression in LB medium at pH 5.5 (Table 2), whileothers reported activation of hdeAB in minimal medium at pH5.5 (20, 43) to be even greater than that produced by GadE (20).

RpoS had little effect on hdeAB expression under the conditionsstudied, probably because of a balance between its direct andindirect effects (Fig. 1). Lrp had little effect on hdeAB expressionin LB medium, in contrast with the strong repression of hdeABby Lrp that occurs in minimal media (42). TorR had no effecton hdeAB expression, which suggests that TorR only regulateshdeAB after induction of the TorRS system by trimethylamineN-oxide and anaerobiosis (8).

We then found that some of the hdeAB regulators examined canaffect MarA-mediated repression. Moreover, in turn, MarA canmodify the effect of some of these other regulators. This isthe first report of functional interactions between MarA andother gene regulators. H-NS and GadE were associated with theincrease in hdeAB repression by MarA that occurred in stationaryphase. Conversely, MarA helped H-NS repression and interferedwith GadE activation of hdeAB in stationary phase. Given thatthe H-NS and MarA binding sites are not close (Fig. 2), andthat H-NS can produce complex DNA superstructures in the DNA(39), it is not yet clear how H-NS and MarA interact to represshdeAB.

It seems unlikely that GadE, the main activator of hdeAB, assistsMarA in repression. More likely, MarA interferes with GadE-mediatedactivation of hdeAB directly (the marbox partially overlapsone "gadbox" [Fig. 2]; thus, MarA and GadE might compete forDNA binding or for interacting with RNA polymerase) and/or indirectly(MarA represses the expression of gadE and the gadE activatorsgadX and evgA [33]).

MarA seems also to compete functionally with GadX, GadW, RpoS,and Lrp under some conditions, which might ensure that hdeABexpression is not repressed more than needed. Such interferencemight be direct, given the partial overlap between the MarAbinding site and some binding sites of these regulators (Fig.2), or indirect by means of an intermediary (Fig. 1).

To know whether repression of hdeAB by MarA was correlated withdecreased acid resistance, we studied the effect of pH and growthphase on the expression of marA and the effect of MarA on acidresistance. We found that, like hdeAB expression (Fig. 3), marAexpression was induced in stationary phase and at acid pH (Table4). However, this moderate induction was presumably not enoughto significantly change hdeAB expression, since the acid resistanceof the cells remained almost unaffected (Fig. 4); but it mightbe important in affecting the expression of other genes of themar regulon with a less complex regulation or with marboxeswith higher affinity for MarA.

Sodium salicylate induced the expression of marA at pH 7.0 butnot at pH 5.5 (Table 4), which might relate to a change in itsinteraction with MarR (5) or to the unknown mechanisms involvedin the induction of marRAB by acid pH. However, this lack ofinduction at pH 5.5 could be important to maintain hdeAB expressionand the resistance of the cells under acid conditions.

At pH 7.0, induction of marA expression by salicylate was verystrong, mainly when the cells were exposed to salicylate forlonger periods of time. This strong induction made stationary-phasecells sevenfold more sensitive to acid challenge at pH 2.5 inthe presence of marA than in its absence (Fig. 4), consistentwith the repression produced by MarA on hdeAB (and probablyother acid resistance genes) in stationary phase.

Further research is needed to understand the biological meaningof MarA repression of hdeAB at pH 7.0 in response to salicylate.Such repression might allow cell resources to be directed tothe (unknown, but perhaps more important) MarA-mediated responseto salicylate. Once salicylate was no longer present, the fastdegradation of MarA (18) would allow a rapid recovery of theexpression of hdeAB and other acid resistance genes, minimizingthe impact of MarA on acid resistance.

In conclusion, MarA plays an active and complex role in theregulation of hdeAB and acid resistance in E. coli that dependson the growth conditions, on other regulators of hdeAB, andon the degree of induction of marA expression.

ACKNOWLEDGMENTS

We thank J. Calvo, V. Mejean, J. Foster, A. Sayad, R. Kolter,and P. Bertin for kindly providing the strains mutated in thedifferent hdeAB regulators.

This work was supported by United States Public Health Servicegrant AI56021 from the National Institutes of Health.

FOOTNOTES

* Corresponding author. Mailing address: Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6764. Fax: (617) 636-0458. E-mail: stuart.levy{at}tufts.edu

Published ahead of print on 14 December 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

REFERENCES

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