The Rob protein, isolated on the basis of its ability to bind to the right arm of the Escherichia coli origin of chromosomal replication, is about 50% identical in amino acid sequence to SoxS and MarA, the direct regulators of the superoxide (soxRS) and multiple antibiotic resistance (mar) regulons, respectively. Having previously demonstrated that SoxS (as a MalE-SoxS fusion protein) and MarA are essentially identical in their abilities to activate in vitro transcription of genes of the sox-mar regulons, we investigated the properties of Rob as a transcriptional activator. We found that Rob (i) activates the transcription of zwf,fpr,fumC, micF, nfo, and sodA, (ii) requires a 21-bp soxbox-marbox-robbox sequence to activate zwf transcription, (iii) protects the soxbox/marbox/robbox from attack by DNase 1, (iv) is ambidextrous, i.e., requires the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF, (v) bends zwf and fumC DNA, and (vi) binds zwf and fumC DNA as a monomer. Since these transcription activation properties of Rob are virtually identical to those of MalE-SoxS and MarA, it appears as if the E. coli genome encodes three genes with the same functional capacity. However, in contrast to SoxS and MarA, whose syntheses are induced by specific environmental stimuli and elicit a clear defense response, Rob is expressed constitutively and its normal function is unknown.
Expression of the marA or soxS genes is induced by exposure of Escherichia coli to salicylate or superoxides, respectively. This, in turn, enhances the expression of a common set of promoters (the mar/soxRS regulons), resulting in both multiple antibiotic and superoxide resistance. Since MarA protein is highly homologous to SoxS, and since a MalE-SoxS fusion protein has recently been shown to activate soxRS regulon transcription, the ability of MarA to activate transcription of these genes was tested. MarA was overexpressed as a histidine-tagged fusion protein, purified, cleaved with thrombin (leaving one N-terminal histidine residue), and renatured. Like MalE-SoxS, MarA (i) activated the transcription of zwf, fpr, fumC, micF, nfo, and sodA; (ii) required a 21-bp "soxbox" sequence to activate zwf transcription; and (iii) was "ambidextrous," i.e., required the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF. Thus, the mar and soxRS systems use activators with very similar specificities and mechanisms of action to respond to different environmental signals.
Bacteria possess multiple mechanisms to survive exposure to various chemical stresses and antimicrobial compounds. In the enteric bacterium Escherichia coli, three homologous transcription factors—MarA, SoxS, and Rob—play a central role in coordinating this response. Three separate systems are known to regulate the expression and activities of MarA, SoxS, and Rob. However, a number of studies have shown that the three do not function in isolation but rather are coregulated through transcriptional cross talk. In this work, we systematically investigated the extent of transcriptional cross talk in the mar-sox-rob regulon. While the three transcription factors were found to have the potential to regulate each other's expression when ectopically expressed, the only significant interactions observed under physiological conditions were between mar and rob systems. MarA, SoxS, and Rob all activate the marRAB promoter, more so when they are induced by their respective inducers: salicylate, paraquat, and decanoate. None of the three proteins affects the soxS promoter, though unexpectedly, it was mildly repressed by decanoate by an unknown mechanism. SoxS is the only one of the three proteins to repress the rob promoter. Surprisingly, salicylate somewhat activates transcription of rob, while decanoate represses it a bit. Rob, in turn, activates not only its downstream promoters in response to salicylate but also the marRAB promoter. These results demonstrate that the mar and rob systems function together in response to salicylate.
Transcription of the multiple antibiotic resistance marRAB operon increases when one of the sequence-related activators, MarA, SoxS, or Rob, binds to the "marbox" centered at -61.5 relative to the transcriptional start site. Previous deletion analyses showed that an adjacent upstream "accessory region" was needed to augment the marbox-dependent activation. To analyze the roles of the marbox and accessory regions on mar transcription, thirteen promoters, each with a different 5-bp transversion of the -96 to -32 sequence, were synthesized, fused to lacZ, and assayed for beta-galactosidase production in single-copy lysogens with appropriate genotypes. The accessory region is shown here to be a binding site for Fis centered at -81 and to bind Fis, a small DNA-binding and -bending protein, with a Kd of approximately 5 nM. The binding of MarA to the marbox and that of Fis to its site were independent of each other. MarA, SoxS, and Rob each activated the mar promoter 1.5-to 2-fold when it had a wild-type marbox but Fis was absent. In the presence of MarA, SoxS, or Rob, Fis further enhanced the activity of the promoter twofold provided the promoter was also capable of binding Fis. However, in the absence of MarA, SoxS, or Rob or in the absence of a wild-type marbox, Fis nonspecifically lowered the activity of the mar promoter about 25% whether or not a wild-type Fis site was present. Thus, Fis acts as an accessory transcriptional activator at the mar promoter.
Transcriptional activation of the promoters of the mar/soxRS regulons by the sequence-related but independently inducible MarA and SoxS proteins renders Escherichia coli resistant to a broad spectrum of antibiotics and superoxide generators. Here, the effects of MarA and SoxS on transcription of the marRAB promoter itself were assayed in vitro by using a minimal transcription system and in vivo by assaying beta-galactosidase synthesized from marR::lacZ fusions. Purified MarA and MalE-SoxS proteins stimulated mar transcription about 6- and 15-fold, respectively, when the RNA polymerase/DNA ratio was 1. Purified MarA bound as a monomer to a 16-bp "marbox" located 69 to 54 nucleotides upstream of a putative RNA initiation site. Deletion of the marbox reduced MarA-mar binding 100-fold, abolished the stimulatory effects of MarA and SoxS on transcription in vitro, and reduced marR::lacZ synthesis about 4-fold in vivo. Deletion of upstream DNA adjoining the marbox reduced MarA binding efficiency 30-fold and transcriptional activation 2- to 3-fold, providing evidence for an accessory marbox. Although MarA and the mar operon repressor, MarR, bound to independent sites, they competed for promoter DNA in band shift experiments. Assays of marR::lacZ transcriptional fusions in marRAB deletion or soxRS deletion strains showed that the superoxide generator paraquat stimulates mar transcription via soxRS and that salicylate stimulates mar transcription both by antagonizing MarR and by a MarR-independent mechanism. Thus, transcription of the marRAB operon is autorepressed by MarR and autoactivated by MarA at a site that also can be activated by SoxS.
Efflux pumps function to rid bacteria of xenobiotics, including antibiotics, bile salts, and organic solvents. TolC, which forms an outer membrane channel, is an essential component of several efflux pumps in Escherichia coli. We asked whether TolC has a role during growth in the absence of xenobiotics. Because tolC transcription is activated by three paralogous activators, MarA, SoxS, and Rob, we examined the regulation of these activators in tolC mutants. Using transcriptional fusions, we detected significant upregulation of marRAB and soxS transcription and Rob protein activity in tolC mutants. Three mechanisms could be distinguished: (i) activation of marRAB transcription was independent of marRAB, soxR, and rob functions; (ii) activation of soxS transcription required SoxR, a sensor of oxidants; and (iii) Rob protein was activated posttranscriptionally. This mechanism is similar to the mechanisms of upregulation of marRAB, soxS, and Rob by treatment with certain phenolics, superoxides, and bile salts, respectively. The transcription of other marA/soxS/rob regulon promoters, including tolC itself, was also elevated in tolC mutants. We propose that TolC is involved in the efflux of certain cellular metabolites, not only xenobiotics. As these metabolites accumulate during growth, they trigger the upregulation of MarA, SoxS, and Rob, which in turn upregulate tolC and help rid the bacteria of these metabolites, thereby restoring homeostasis.
The AraC family transcription factor MarA activates ∼40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.
When environmental conditions change, cell survival can depend on sudden production of proteins that are normally in low demand. Protein production is controlled by transcription factors which bind to DNA near genes and either increase or decrease RNA production. Many puzzles remain concerning the ways transcription factors do this. Recently we collected data relating the intracellular level of a single transcription factor, MarA, to the increase in expression of several genes related to antibiotic and superoxide resistance in Escherichia coli. These data indicated that target genes are turned on in a well-defined order with respect to the level of MarA, enabling cells to mount a response that is commensurate to the level of threat detected in the environment. Here we develop a computational model to yield insight into how MarA turns on its target genes. The modeling suggests that MarA can increase the frequency with which a transcript is made while decreasing the overall presence of the transcription machinery at the start of a gene. This mechanism is opposite to the textbook model of transcriptional activation; nevertheless it enables cells to respond quickly to environmental challenges and is likely of general importance for gene regulation in E. coli and beyond.
A chromosomal gene of Enterobacter cloacae affecting the synthesis of major outer membrane proteins in E. cloacae and Escherichia coli was cloned by using selection for resistance to cefoxitin in E. coli. The presence of the gene, when plasmid-borne, led to a decrease in the amount of porin F in E. cloacae and the amount of OmpF in E. coli and caused 2- to 32-fold increases in the MICs of chloramphenicol, tetracycline, quinolones, and beta-lactam antibiotics. The gene encoded a 33-kDa protein, similar (83% identity) to the protein Rob involved in the initiation of DNA replication in E. coli, which was called RobA(EC1) by analogy. RobA from E. cloacae was found to inhibit ompF expression at the posttranscriptional level via activation of micF, a gene also apparently present in E. cloacae, as detected by PCR. As with its homolog from E. coli, RobA(EC1) is related to the XylS-AraC class of positive transcriptional regulators, along with MarA and SoxS, which also cause a micF-mediated decrease in the level of ampF expression.
The paralogous transcriptional activators, MarA, SoxS and Rob, activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between MarA concentration needed for half-maximal promoter activity in vivo with marbox binding affinity in vitro was poor and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS implying that the two activators interact with RNAP in different ways at the different promoters. Thus, the concentration and nature of activator determines which regulon promoters are activated and the extent of their activation.
gene regulation; AraC protein family; stress response
Escherichia coli K-12 strains are normally tolerant to n-hexane and susceptible to cyclohexane. Constitutive expression of marA of the multiple antibiotic resistance (mar) locus or of the soxS or robA gene product produced tolerance to cyclohexane. Inactivation of the mar locus or the robA locus, but not the soxRS locus, increased organic solvent susceptibility in the wild type and Mar mutants (to both n-hexane and cyclohexane). The organic solvent hypersusceptibility is a newly described phenotype for a robA-inactivated strain. Multicopy expression of mar, soxS, or robA induced cyclohexane tolerance in strains with a deleted or inactivated chromosomal mar, soxRS, or robA locus; thus, each transcriptional activator acts independently of the others. However, in a strain with 39 kb of chromosomal DNA, including the mar locus, deleted, only the multicopy complete mar locus, consisting of its two operons, produced cyclohexane tolerance. Deletion of acrAB from either wild-type E. coli K-12 or a Mar mutant resulted in loss of tolerance to both n-hexane and cyclohexane. Organic solvent tolerance mediated by mar, soxS, or robA was not restored in strains with acrAB deleted. These findings strongly suggest that active efflux specified by the acrAB locus is linked to intrinsic organic solvent tolerance and to tolerance mediated by the marA, soxS, or robA gene product in E. coli.
In Escherichia coli, Rob activates transcription of the SoxRS/MarA/Rob regulon. Previous work revealed that Rob resides in 3–4 immunostainable foci, that dipyridyl and bile salts are inducers of its activity, and that inducers bind to Rob’s C-terminal domain (CTD). We propose that sequestration inactivates Rob by blocking its access to the transcriptional machinery and that inducers activate Rob by mediating its dispersal, allowing interaction with RNA polymerase. To test “sequestration-dispersal” as a new mechanism for regulating the activity of transcriptional activators, we fused Rob’s CTD to SoxS and used indirect immunofluorescence microscopy to determine the effect of inducers on SoxS-Rob’s cellular localization. Unlike native SoxS, which is uniformly distributed throughout the cell, SoxS-Rob is sequestered without inducer, but is rapidly dispersed when cells are treated with inducer. In this manner, Rob’s CTD serves as an anti-sigma factor in regulating the co-sigma factor-like activity of SoxS when fused to it. Rob’s CTD also protects its N-terminus from Lon protease, since Lon’s normally rapid degradation of SoxS is blocked in the chimera. Accordingly, Rob’s CTD has novel regulatory properties that can be bestowed on another E. coli protein.
gene regulation; intracellular localization; immunofluorescence microscopy; anti-sigma factor; proteolysis
Transcriptional regulators of the AraC/XylS family have been associated with multidrug resistance, organic solvent tolerance, oxidative stress, and virulence in clinically relevant enterobacteria. In the present study, we identified four homologous AraC/XylS regulators, Rob, SoxS, PliA, and OpiA, from the fire blight pathogen Erwinia amylovora Ea1189. Previous studies have shown that the regulators MarA, Rob, and SoxS from Escherichia coli mediate multiple-antibiotic resistance, primarily by upregulating the AcrAB-TolC efflux system. However, none of the four AraC/XylS regulators from E. amylovora was able to induce a multidrug resistance phenotype in the plant pathogen. Overexpression of rob led to a 2-fold increased expression of the acrA gene. However, the rob-overexpressing strain showed increased resistance to only a limited number of antibiotics. Furthermore, Rob was able to induce tolerance to organic solvents in E. amylovora by mechanisms other than efflux. We demonstrated that SoxS from E. amylovora is involved in superoxide resistance. A soxS-deficient mutant of Ea1189 was not able to grow on agar plates supplemented with the superoxide-generating agent paraquat. Furthermore, expression of soxS was induced by redox cycling agents. We identified two novel members of the AraC/XylS family in E. amylovora. PliA was highly upregulated during the early infection phase in apple rootstock and immature pear fruits. Multiple compounds were able to induce the expression of pliA, including apple leaf extracts, phenolic compounds, redox cycling agents, heavy metals, and decanoate. OpiA was shown to play a role in the regulation of osmotic and alkaline pH stress responses.
OmpW is a minor porin whose biological function has not been clearly defined. Evidence obtained in our laboratory indicates that in Salmonella enterica serovar Typhimurium the expression of OmpW is activated by SoxS upon exposure to paraquat and it is required for resistance. SoxS belongs to the AraC family of transcriptional regulators, like MarA and Rob. Due to their high structural similarity, the genes under their control have been grouped in the mar/sox/rob regulon, which presents a DNA-binding consensus sequence denominated the marsox box. In this work, we evaluated the role of the transcription factors MarA, SoxS and Rob of S. enterica serovar Typhimurium in regulating ompW expression in response to menadione. We determined the transcript and protein levels of OmpW in different genetic backgrounds; in the wild-type and Δrob strains ompW was upregulated in response to menadione, while in the ΔmarA and ΔsoxS strains the induction was abolished. In a double marA soxS mutant, ompW transcript levels were lowered after exposure to menadione, and only complementation in trans with both genes restored the positive regulation. Using transcriptional fusions and electrophoretic mobility shift assays with mutant versions of the promoter region we demonstrated that two of the predicted sites were functional. Additionally, we demonstrated that MarA increases the affinity of SoxS for the ompW promoter region. In conclusion, our study shows that ompW is upregulated in response to menadione in a cooperative manner by MarA and SoxS through a direct interaction with the promoter region.
Bacterial transcription activators regulate transcription by making essential protein–protein interactions with RNA polymerase, for example, with region 4 of the σ70 subunit (σ70 R4). Rob, SoxS, and MarA comprise a closely related subset of members of the AraC/XylS family of transcription factors that activate transcription of both class I and class II promoters. Recently, we showed that interactions between SoxS and σ70 R4 occlude the binding of σ70 R4 to the −35 promoter element of class II promoters. Although Rob shares many similarities with SoxS, it contains a C-terminal domain (CTD) that the other paralogs do not. Thus, a goal of this study was to determine whether Rob makes protein–protein interactions with σ70 R4 at class II promoters and, if so, whether the interactions occlude the binding of σ70 R4 to the −35 hexamer despite the presence of the CTD. We found that although Rob makes fewer interactions with σ70 R4 than SoxS, the two proteins make the same, unusual, position-dependent interactions. Importantly, we found that Rob occludes σ70 R4 from binding the −35 hexamer, just as does SoxS. Thus, the CTD does not substantially alter the way Rob interacts with σ70 R4 at class II promoters. Moreover, in contrast to inferences drawn from the co-crystal structure of Rob bound to robbox DNA, which showed that only one of Rob’s dual helix–turn–helix (HTH) DNA binding motifs binds a recognition element of the promoter’s robbox, we determined that the two HTH motifs each bind a recognition element in vivo.
SoxS; genetic epistasis; σ70 R4; prerecruitment; Rob−micF crystal structure
The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.
The multiple antibiotic resistance gene pqrA was cloned from the chromosomal DNA of a clinical isolate of Proteus vulgaris 881051 into Escherichia coli KY2563. The MICs of quinolones tetracycline, cephalosporin, and chloramphenicol for transformant strain DNS7020 were from 8 to 32 times higher than those for the parent strain, KY2563. The level of expression of outer membrane protein F (OmpF) by DNS7020 was lower than that of KY2563 but not as low as that of an OmpF-deficient control strain. The 1.4-kb fragment containing the pqrA gene had an open reading frame encoding a polypeptide of 122 amino acid residues with a molecular weight of about 14,000, which was consistent with the experimental value identified by the Maxicell method. The putative PqrA polypeptide showed significant amino acid sequence similarity to the E. coli proteins SoxS and MarA. These polypeptides are strongly conserved in predicted helix-turn-helix DNA binding domains. The MarA protein, which is responsible for multiple antibiotic resistance in E. coli, also decreases OmpF expression. Moreover, the SoxS protein, which is characterized as a superoxide response regulon of E. coli, has also been shown to increase resistance to many structurally unrelated antibiotics. The soxS gene increases superoxide dismutase levels in addition to decreasing OmpF expression. The expression level of superoxide dismutase with DNS7020 was about 1.5 times higher than that with KY2563. These findings suggest that the pqrA gene in P. vulgaris confers multidrug resistance in a way similar to that of the soxS and marA genes in E. coli.
Multiple factors control the expression of the outer membrane porins OmpF and OmpC in Escherichia coli. In this work, we investigated the role of the mar-sox-rob regulon in regulating outer membrane porin expression in response to salicylate. We provide both genetic and physiological evidence that MarA and Rob can independently activate micF transcription in response to salicylate, leading to reduced OmpF expression. MarA was also found to repress OmpF expression through a MicF-independent pathway. In the case of OmpC, we found that its transcription was moderately increased in response to salicylate. However, this increase was independent of MarA and Rob. Finally, we found that the reduction in OmpF expression in a tolC mutant is due primarily to Rob. Collectively, this work further clarifies the coordinated role of MarA and Rob in regulating the expression of the outer membrane porins.
Rob is a member of the Sox/Mar subfamily of AraC/XylS-type transcriptional regulators implicated in bacterial multidrug, heavy metal, superoxide, and organic solvent resistance phenotypes. We demonstrate that, in Salmonella enterica, Rob overexpression upregulates the transcription of mgtA, which codes for the MgtA Mg2+ transporter. mgtA was previously characterized as a member of the Mg2+-modulated PhoPQ regulon. Here we demonstrate that Rob (but not its paralog protein SoxS or MarA) is able to induce mgtA transcription in a PhoP-independent fashion by binding to a conserved Mar/Sox/Rob motif localized downstream of the PhoP-box and overlapping the PhoP-dependent transcriptional start site. We found that Rob-induced mgtA expression confers low-level cyclohexane resistance on Salmonella. Because mgtA intactness is required for Rob-induced cyclohexane resistance, provided the AcrAB multidrug efflux pump can be expressed, we postulate that MgtA is involved in the AcrAB-mediated cyclohexane detoxification mechanism promoted by Rob in Salmonella.
The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase [encoded by aac(2')-Ia] in Providencia stuartii. Introduction of aarP into P. stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of beta-galactosidase from an aac(2')-lacZ fusion. Northern (RNA) blot analysis demonstrated that this increased aac(2')-Ia expression occurred at the level of mRNA accumulation. The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD,Rob, MarA, and SoxS. The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system. Introduction of aarP on a multicopy plasmid into either P. stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin. Multiple copies of aarP in E. coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E. coli. The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2')-Ia mRNA. Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2')-Ia gene. (P.N. Rather, E. Oroz, K.J. Shaw, R. Hare, and G. Miller, J. Bacteriol. 175:6492-6498).
Elevated levels of fluoroquinolone resistance are frequently found among Escherichia coli clinical isolates. This study investigated the antibiotic resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli clinical isolate, PS5, to two selection steps with increasing concentrations of norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by Western blotting that NorE5 has a multidrug resistance phenotype resulting from the overexpression of the antibiotic resistance efflux pump AcrAB-TolC. Microarray and gene fusion analyses revealed significantly increased expression in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS activity is attributable to a frameshift mutation that truncates SoxR, rendering it a constitutive transcriptional activator of soxS. Furthermore, microarray and reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter, showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox sequence is in the backward or class I orientation within the promoter, and its disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus, chromosomal mutations in parC and soxR are responsible for the increased antibiotic resistance of NorE5.
The Escherichia coli tolC encodes a major outer membrane protein with multiple functions in export (e. g., diverse xenobiotics, hemolysin) and as an attachment site for phage and colicins. tolC is regulated in part by MarA, SoxS and Rob, three paralogous transcriptional activators which bind a sequence called the marbox and which activate multiple antibiotic and superoxide resistance functions. Two previously identified tolC promoters, p1 and p2, are not regulated by MarA, SoxS or Rob but p2 is activated by EvgAS and PhoPQ which also regulate other functions. Using transcriptional fusions and primer extension assays, we show here that tolC has two additional strong overlapping promoters, p3 and p4, which are downstream of p1, p2 and the marbox and are activated by MarA, SoxS and Rob. p3 and p4 are configured so that a single marbox suffices to activate transcription from both promoters. At the p3 promoter, the marbox is separated by 20 bp from the −10 hexamer for RNA polymerase but at the p4 promoter, the same marbox is separated by 30 bp from the −10 hexamer. The multiple tolC promoters may allow the cell to respond to diverse environments by coordinating tolC transcription with other appropriate functions.
gene regulation; outer membrane protein; transcriptional start sites; efflux pumps; antibiotic resistance
Three paralogous transcriptional activators MarA, SoxS, and Rob, activate >40 Escherichia coli promoters. To understand why MarA does not activate certain promoters as strongly as SoxS, we compared MarA, MarA mutants, and SoxS for their abilities to activate 16 promoters and to bind their cognate marbox binding sites. Replacement of the MarA glutamic acid residue 89 with alanine greatly increased the marbox binding and activation of many class I promoters. Like cells constitutive for SoxS, cells expressing the MarA with the E89A mutation were more resistant to superoxides than those harboring WT MarA. The activities of several other E89 substitutions ranked as follows: E89A > E89G > E89V > WT > E89D. Increased binding and activation occurred only at class I promoters when the 12th base of the promoter's marbox (a position at which there is no known interaction between the marbox and MarA) was not a T residue. Furthermore, WT MarA binding to a synthetic marbox in vitro was enhanced when the phosphate group between positions 12 and 13 was eliminated on one strand. The results demonstrate that relatively minor changes in a single amino acid side chain (e.g., alanine to valine or glutamic acid to aspartic acid) can strongly influence activity despite any evidence that the side chain is involved in positive interactions with either DNA or RNA polymerase. We present a model which attributes the differences in binding and activation to the interference between the β- and γ-carbons of the amino acid at position 89 and the phosphate group between positions 12 and 13.
We previously reported that overexpression of the soxS or robA gene causes in several Escherichia coli strains the acquisition of higher organic solvent tolerance and also increased resistance to a number of antibiotics (H. Nakajima, K. Kobayashi, M. Kobayashi, H. Asako, and R. Aono, Appl. Environ. Microbiol. 61:2302-2307, 1995). Most E. coli strains cannot grow in the presence of cyclohexane. We isolated the marRAB genes from a Kohara lambda phage clone and cyclohexane-tolerant mutant strain OST3408. We found a substitution of serine for arginine at position 73 in the coding region of marR of OST3408 and designated the gene marR08. Our genetic analysis revealed that marR08 is responsible for the cyclohexane-tolerant phenotype. We observed that the marA gene on high-copy-number plasmids increased the organic solvent tolerance of E. coli strains. Furthermore, exposure of E. coli cells to salicylate, which activates the mar regulon genes, also raised organic solvent tolerance. Overexpression of the marA, soxS, or robA gene increased resistance to numerous antibiotics but not to hydrophilic aminoglycosides.
Cationic antimicrobial peptides (CAMPs), a component of the mammalian immune system, protect the host from bacterial infections. The roles of the Escherichia coli transcriptional regulators MarA, SoxS and Rob in susceptibility to these peptides were examined. Overexpression of marA, either in an antibiotic-resistant marR mutant or from a plasmid, decreased bacterial susceptibility to CAMPs. Overexpression of the soxS gene from a plasmid, which decreased susceptibility to antibiotics, unexpectedly caused no decrease in CAMP susceptibility; instead it produced increased susceptibility to different CAMPs. Deletion or overexpression of rob had little effect on CAMP susceptibility. The marRAB operon was upregulated when E. coli was incubated in sublethal amounts of CAMPs polymyxin B, LL-37 or human β-defensin-1; however, this upregulation required Rob. Deletion of acrAB increased bacterial susceptibility to polymyxin B, LL-37 and human β-defensin-1 peptides. Deletion of tolC yielded an even greater increase in susceptibility to these peptides and also led to increased susceptibility to human α-defensin-2. Inhibition of cellular proton-motive force increased peptide susceptibility for wild-type and acrAB deletion strains; however, it decreased susceptibility of tolC mutants. These findings demonstrate that CAMPs are both inducers of marA-mediated drug resistance through interaction with Rob and also substrates for efflux in E. coli. The three related transcriptional regulators show different effects on bacterial cell susceptibility to CAMPs.
Bacteria have a great capacity for adjusting their metabolism in response to environmental changes by linking extracellular stimuli to the regulation of genes by transcription factors. By working in a co-operative manner, transcription factors provide a rapid response to external threats, allowing the bacteria to survive. This review will focus on transcription factors MarA, SoxS and Rob in Escherichia coli, three members of the AraC family of proteins. These homologous proteins exemplify the ability to respond to multiple threats such as oxidative stress, drugs and toxic compounds, acidic pH, and host antimicrobial peptides. MarA, SoxS and Rob recognize similar DNA sequences in the promoter region of more than 40 regulatory target genes. As their regulons overlap, a finely tuned adaptive response allows E. coli to survive in the presence of different assaults in a co-ordinated manner. These regulators are well conserved amongst Enterobacteriaceae and due to their broad involvement in bacterial adaptation in the host, have recently been explored as targets to develop new anti-virulence agents. The regulators are also being examined for their roles in novel technologies such as biofuel production.
antibiotic resistance; AraC family regulators; Escherichia coli; Enterobacteriaceae; global regulators; drug development