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The MarR Repressor of the Multiple Antibiotic Resistance (mar) Operon in Escherichia coli: Prototypic Member of a Family of Bacterial Regulatory Proteins Involved in Sensing Phenolic Compounds
Molecular Medicine volume 1, pages436–446 (1995)
The marR gene of Escherichia coli encodes a repressor of the marRAB operon, a regulatory locus controlling multiple antibiotic resistance in this organism. Inactivation of marR results in increased expression of marA, which acts at several target genes in the cell leading to reduced antibiotic accumulation. Exposure of E. coli to sodium salicylate (SAL) induces marRAB operon transcription and antibiotic resistance. The mechanism by which SAL antagonizes MarR repressor activity is unclear.
Materials and Methods
Recombinant plasmid libraries were introduced into a reporter strain designed to identify cloned genes encoding MarR repressor activity. Computer analysis of sequence databases was also used to search for proteins related to MarR.
A second E. coli gene, MprA, that exhibits MarR repressor activity was identified. Subsequent database searching revealed a family of 10 proteins from a variety of bacteria that share significant amino acid sequence similarity to MarR and MprA. At least four of these proteins are transcriptional repressors whose activity is antagonized by SAL or by phenolic agents structurally related to SAL.
The MarR family is identified as a group of regulatory factors whose activity is modulated in response to environmental signals in the form of phenolic compounds. Many of these agents are plant derived. Some of the MarR homologs appear more likely to control systems expressed in animal hosts, suggesting that phenolic sensing by bacteria is important in a variety of environments and in the regulation of numerous processes.
In Escherichia coli, a simultaneous increase in resistance to a number of structurally unrelated antibiotics is associated with mutations at the multiple antibiotic resistance (mar) locus, located at 34′ on the E. coli chromosome (1). Susceptibility to a number of antibiotic classes is affected by this system, including tetracycline, chloramphenicol, β-lactams, and fluoroquinolones (2,3). Reduced drug accumulation, resulting from activation of efflux systems along with down-regulation of outer membrane porins, appears to be the primary resistance mechanism (2–5). A Mar phenotype has also been observed in a variety of gram-negative, quinolone resistant clinical isolates, suggesting that nonspecific multidrug resistance systems may play a role in the outcome of antibiotic therapy regimens with this class of antibiotics (6).
Recent efforts from several labs have helped clarify the identity and function of genes encoded at the mar locus that play a role in the resistance scheme. These studies have drawn attention to a small operon encoding the marRAB genes as the critical element in this system (Fig. 1). This locus appears to be regulatory in nature. marA encodes a positive regulator of antibiotic resistance whose level of expression seems to be the critical determinant of antibiotic resistance. A Tn5 insertion in marA reverses the resistant phenotype of mar mutants (1), and overexpression of this gene confers resistance even in a strain containing a large deletion of the mar region of the chromosome (7). MarA shares significant amino acid sequence similarity to members of the AraC family of transcriptional activators (7,8), most notably SoxS, a positive regulator of the superoxide stress response whose induction also leads to a Mar phenotype (9,10).
Control of marRAB operon transcription, and thus of marA expression, is the role of the marR gene product. Mutations that result in increased antibiotic resistance are frequently located in marR (8,11), and marR is necessary to maintain marRAB operon expression at a low level (11,12). These findings indicate that MarR is a negative regulator of the marRAB operon, and imply that marRAB operon expression is inducible.
Support for this notion has recently emerged. A conditional antibiotic resistant phenotype similar to that associated with mar mutants can be elicited by culturing E. coli in the presence of the aromatic weak acid sodium salicylate (SAL) (13). Moreover, exposure to SAL results in the rapid induction of marRAB operon transcription and of expression of a mar-lacZ fusion, providing a partial explanation for the effects of this agent (14). Strains deleted for the mar locus are defective in SAL-inducible antibiotic resistance (14), and the only genes encoded at the mar locus that are required for the salicylate effect are marR and marA (12). A recent report indicated that a hybrid protein consisting of a fusion between maltose binding protein and MarR can bind the marRAB promoter region in vitro, and that this binding is antagonized by a variety of chemical agents including SAL (15). These observations suggest a simple model in which MarR directly represses marRAB operon transcription, and SAL antagonizes MarR repressor activity, presumably by dissociating MarR from the operator region (Fig. 1). Since exposure to acetylsalicylic acid (aspirin) can induce the same antibiotic resistance phenotype (13), an awareness of the importance of the mar locus during infection and antibiotic therapy seems warranted.
Thus, an understanding of the physiological signals governing MarR repressor activity would be very informative with respect to the control of intrinsic antibiotic resistance in enteric organisms. We report here the finding that MarR is related, both in primary amino acid sequence and in function, to a family of bacterial regulatory proteins that are involved in specific responses to environmental phenolic compounds. This discovery significantly expands the context in which the mar system should be considered and suggests that the marRAB operon is part of a larger stimulon that may be important in virulence and resistance processes in bacteria.
Materials and Methods
Bacterial Strains and Plasmids
All strains described in this study are E. coli Kl2 derivatives of MC4100 (F− araD139 Δ(lac)U169 rpsL relA thi) (16). Strain B318, used extensively herein, contains the Δ1738 deletion (17) which removes ∼39 kbp of DNA from the mar region of the chromosome including the marRAB operon. It is also lysogenic for a recombinant lambda phage carrying a marR-lacZ protein fusion, in which lacZ is fused in frame after the 75th codon of marR, and expression of the fusion is under the control of the marRAB operon promoter (12). Expression of this fusion is constitutive in strain B318 due to the absence of a source of MarR repressor.
E. coli and Salmonella typhimurium plasmid libraries were constructed from size-selected fragments of Sau3A partial digests of chromosomal DNA from either strain W3110 (E. coli) or SR-11 (S. typhimurium ), cloned into the BamRl site of pBR322 (for E. coli ) or pBluescript SK− (for S. typhimurium; Stratagene). pmarR (12) consists of a 570 bp PCR fragment containing the marRAB operon operator/promoter region and all of marR, cloned into pBR322. The pS plasmids shown in Fig. 2 were constructed by subcloning the indicated restriction fragments of pmarD (see text) into pBluescript SK− (Stratagene). pmprA was constructed by PCR synthesis of the mprA gene using primers based on the published sequence (20). The vector was pBluescript SK−. The mprA::cat allele was constructed by first isolating the cat gene from λ1324 (21) by digestion of the phage DNA with BamHI, converting the BamHI sites into SphI sites using linkers, and cloning the fragment into the Sphl site in mprA in plasmid pS6 (Fig. 2). This results in disruption of mprA after the 34th codon. The mprA::cat plasmid was linearized by digestion with PstI and used to replace the normal mprA gene by linear transformation of a recD::Tn10 strain (22). Subsequent strains containing the mprA::cat allele were constructed by P1 transduction. pEc17kd was constructed by PCR synthesis of a DNA fragment corresponding to the coding region of the 17kd gene from the prs locus (23) using chromosomal DNA from strain ECOR 55 (24) as a template. This fragment was then cloned into the IPTG-inducible expression plasmid pTrc99A (Pharmacia, Piscataway, NJ).
Reagents and Growth Conditions
Bacteria were propagated in LB media (25), which, when needed, was solidified with 1.5% Bacto-Agar (DIFCO, Detroit, MI). All antibiotics were from Sigma Chemical Co. (St. Louis, MO, U.S.A.) other reagents were from commercial sources and were of the highest purity available.
Other Genetic and Biochemical Methods
DNA hybridizations were performed following standard protocols (26). Probes were labeled with digoxigenin using the Genius kit (Boehringer-Mannheim, Indianapolis, IN) following the manufacturers instructions. For Southern hybridizations, DNA samples were resolved on agarose gels and transferred to Nytran membranes (Schleicher and Schuell, Keene, NH) by capillary transfer following protocols from the membrane supplier. The miniset blot of the ordered recombinant phage collection of Kohara (27) was from TaKaRa. β-Galactosidase assays were performed as described by Miller (25) using the chloroform-SDS method of cell permeabilization. Briefly, cultures were grown in L broth to an A600 = 0.3, at which time inducers were added to 2.5 mM. Incubation was continued for 1 hr, and β-galactosidase activity, expressed as Miller Units, was determined.
Alignments of promoter regions shown in Fig. 3 were performed using MacVector version 4.5 software for the Macintosh (Eastman Kodak Co., Rochester, NY, U.S.A.). The computer searches that generated the sequences shown in Fig. 4 were performed using TFASTA (28) in the GCG package of sequence analysis programs (29). Initial searches used MarR or MprA as the query sequence. New matches found in these searches were then used as queries in additional searches. This iterative process was repeated until no new members were found. Alignment of the sequences was performed using the PILEUP program (29).
Identification of a Functional Homolog of marR
In the course of genetic studies on the regulation of mar operon transcription, we constructed an E. coli strain, B318, which proved useful for cloning mar homologs from related enteric organisms. B318 contains a 39-kbp deletion of the mar region of the chromosome including the marRAB operon, and also carries a λ phage containing a marR-lacZ translational fusion under the control of the mar operator/promoter region (12). Expression of this fusion is constitutively derepressed (Lac+) in this strain due to the absence of MarR repressor. Introduction of a plasmid containing marR restores both repression of fusion expression and inducibility by SAL (12). The presence of marA on the same plasmid restores normal levels of SAL-inducible antibiotic resistance (14).
Our initial objective was to use B318 to clone mar sequences from virulent enteric organisms, and then construct deletion strains for the purpose of testing the importance of the mar system in various pathogenic models. When B318 was transformed with a plasmid library containing random S. typhimurium chromosomal DNA, nine white (Lac−) transformants were obtained on MacConkey-lactose indicator plates. These were further tested for the presence of marRAB operon DNA by Southern blotting. Two classes of clone were identified. The first class (five transformants) contained sequences that hybridized with an E. coli mar probe, and included the S. typhimurium marR gene as judged by DNA sequence analysis. The remaining four transformants contained cloned DNA sequences that were not detected by the E. coli mar probe, but cross-hybridized with each other, indicating that they were derived from the same locus and possibly encoded a second mar repressor. This new marR-like activity was designated marD.
To determine if E. coli also had a marD gene the cloning strategy was repeated, this time using an E. coli plasmid library. White transformants were again identified and, as in the Salmonella experiment, the population was mixed. Fourteen of fifteen clones were shown by hybridization to carry mar sequences. The remaining isolate contained a cloned insert that cross-hybridized with the S. typhimurium marD fragment, indicating that this locus was also present in E. coli. The corresponding plasmid was designated pmarD (Fig. 2).
To find the location of marD on the E. coli chromosome, pmarD was used as a hybridization probe against the Kohara miniset blot (27). Positive hybridization signals localized the cloned sequence to the 52′ region of the chromosome. Comparison of the restriction map for this region of the chromosome with that of pmarD indicated that three known genes, mprA, emrA, and emrB, were present within the 6 kbp cloned segment (Fig. 2). To localize marD repressor activity within this interval, deletion and subcloning experiments were performed. As is shown in Figure 2, this activity was associated with the mprA gene, previously identified as a repressor of genes involved in the biosynthesis of the peptide antibiotic microcin B17 (20,30).
One feature of MarR repressor activity is its antagonism by SAL (14). To determine if MprA-mediated repression of marR-lacZ was similarly affected, B318 derivatives carrying either mprA or marR plasmids were treated with SAL and assayed for β-galactosidase activity. As is shown in Table 1, SAL relieved repression mediated by either marR or mprA. These results indicate that mprA, when present on a multicopy plasmid, can functionally replace marR and restore regulation to a marR-lacZ fusion.
It seemed unlikely that mprA played a significant role in regulating mar gene expression in wild type cells, since a deletion of marR alone results in high level derepression of a marR-lacZ fusion. To determine if mprA played a minor role in regulating mar gene expression, a chloramphenicol resistance determinant was inserted after the 34th codon of the cloned mprA gene, and the disrupted allele was recombined onto the chromosome in place of the normal gene (see Materials and Methods). In an otherwise wild-type strain (i.e., marR+), the mprA::cat allele had no affect on marR-lacZ fusion expression. In addition, expression of this fusion in a strain containing a marR::Tn10 allele was also unaffected by the introduction of mprA::cat (data not shown). Thus, marR and mprA are not redundant regulators in the E. coli strains used here. The complementation of a marR deficiency by mprA requires that the latter be present in multicopy.
In wild-type cells, MprA is known to repress expression from two specific promoters: that of the microcin B17 biosynthetic gene cluster (PmcbA)(30) and its own promoter (PmcbA)(20). A recent report indicated that mprA expression is significantly induced following salicylate treatment (31). To confirm that this is the case in the strain background used here, we measured expression of a mprA-lacZ transcriptional fusion present in single copy on a λ prophage under conditions identical to those used to follow marR-lacZ fusion expression; namely, 1-hr exposure to 2.5 mM SAL using cells grown to mid-log prior to SAL addition. A 5-fold induction of fusion enzyme expression was consistently observed under these conditions (data not shown). Thus, SAL stimulates the expression of a normal cellular target of MprA.
MarR and MprA may thus act as sequence-specific repressors. As such, promoters controlled by these regulators may contain conserved sequences at appropriate positions with respect to known or suspected recognition sites for RNA polymerase (32). A comparison of the promoter regions for mprA (20) and mcbA (33) revealed such a sequence that was identical at 19 of 24 positions (Fig. 3A). In PmprA′ this element is located 5 bp upstream from the predicted −35 sequence. In contrast, this same sequence is found 77 bp upstream from the −35 element of a promoter known to direct the post-exponential expression of mcbA (33). It seemed possible that the increase in mcbA expression observed in an mprA mutant during exponential growth results from activation of a different promoter element than that used for postexponential expression. Consistent with this notion, good −35 and −10 elements could be found in the vicinity of the putative MprA recognition sequence (−35′ and −10′ in Fig. 3B).The −35′ was buried within the putative MprA recognition sequence.
Comparison of the promoter regions of marR (8) and mprA indicated that the region of greatest similarity corresponded to the conserved sequence found in the PmprA/PmcbA alignment. Figure 3A shows that the PmarR element is identical at 11 of 24 positions with the other two sequences. Moreover, the putative −35 sequence of PmarR is nested within this conserved element (Fig. 3B).
Identification of Other Bacterial Homologs of MarR
Previous computer-assisted searches had failed to identify any significant similarities between MarR and translated sequences in the available databases (8). However, an alignment of MarR and MprA showed that the two proteins share 28% identity at the amino acid sequence level. Inclusion of conservative amino acid replacements increased the similarity to 47%. Since the complementation results (Table 1) indicated that this level of similarity is significant and related to function, we reexamined the databases for evidence of other homologs, using the program functional relationship between MarR and MprA, evidence for analogous properties of the other two E. coli proteins was sought.
Relevant information for the third entry, EcHpcR, was obtained from the literature. HpcR is found in E. coli C strains and is a repressor of adjacent genes involved in the catabolism of homoprotocatechuate (HPC) (34), a plant-derived phenolic that is structurally related to SAL. Importantly, HpcR repressor activity is antagonized by HPC (34) in a manner similar to the effect of SAL on MarR and MprA.
Ec17kd, the fourth entry shown, is the putative product of an open reading frame linked to the pap/prs cluster of genes associated with pilus formation in certain pyelonephritic E. coli strains. Although no function has been associated with this ORF as yet, its similarity to MprA has been noted previously (23). In order to determine if Ec17kd could restore MarR repressor activity to strain B318, the coding region for Ec17kd was synthesized by PCR and cloned into the IPTG-inducible expression plasmid pTrc99A However, we found that even in the absence of induction with IPTG, the Ec17kd construct repressed marR-lacZ expression in strain B318 (Table 2), suggesting that leaky expression from the trc promoter produced sufficient 17kd protein to complement the marR deficiency in this strain. Furthermore, this repressor activity was antagonized by SAL. Thus, Ec17kd is also functionally related to MarR.
The findings presented in this study identify a new family of bacterial regulatory proteins related not only by primary amino acid sequence similarity but also, in the cases tested here, by function. The prototypic member of this family, MarR, is a repressor of a regulatory operon involved in controlling the intrinsic level of antibiotic resistance in E. coli. Salicylate induction of marRAB operon transcription, and consequently of increased antibiotic resistance, must somehow involve antagonism of MarR. The results presented here demonstrate that two other E. coli gene products, MprA and Ec17kd, can also repress expression from the mar promoter. Moreover, this repressor activity is sensitive to antagonism by SAL. Since MprA-mediated repression of a normal target for this protein (namely, its own promoter) is also sensitive to SAL, the activities associated with MprA and Ec17kd at the mar promoter likely reflect the normal cellular functions of these proteins. The identification of conserved sequences located in the promoter regions of genes controlled by MprA (Fig. 3) is consistent with this protein acting as a sequence-specific repressor. Therefore, we conclude that the family of proteins identified in Fig. 4 likely share common functions as regulators of gene expression whose activity is modulated by phenolic agents. In addition, it is plausable to propose that a stimulon exists in E. coli that is responsive to SAL (Fig. 5). Thus, exposure of E. coli strains to this agent would derepress the expression of genes controlled by MarR and its related repressors, and thus affect the activity of several independently regulated genetic pathways. Such a treatment would result in an increase in antibiotic resistance (MarR controlled) and microcin B17 antibiotic production (MprA controlled), along with the target genes controlled by Ec17kd, should the particular strain contain the pap/prs cluster. The proximity of mprA to the downstream emrA and emrB genes, which encode an efflux pump for xenobiotic agents (42), is similar to the physical arrangement of marR and marA, and raises the possibility that expression of the EmrAB pump is also controlled by MprA. The cellular target(s) for Ec17kd regulation is not known; however, its association with a virulence locus suggests that it may participate in a pathogenic function. Since the mar locus is known to be present in a large number of enteric species (43) and the experiments reported here indicate that mprA is also found in S. typhimurium, it seems reasonable to propose that the SAL stimulon is conserved among the enterics. SAL has also been reported to induce increased antibiotic resistance in Pseudomonas cepacia, suggesting that drug resistance associated with sensing of this phenolic is widespread (44).
Although not specifically related to pathogenesis, the identification of HpcR as a member of this family may help clarify other issues regarding the mechanism of repressor antagonism. HpcR is known to regulate expression of a catabolic pathway that allows soil-dwelling E. coli C strains to metabolize homoprotocatechuate (HPC) (34,45). As HPC is thought to be the authentic inducer for the hpc system, the possibility that SAL is structurally related to the natural inducer of the mar system is raised. The observation that the mar and hpc systems have nothing in common other than their respective repressors and inducers is consistent with the concept of direct recognition of the inducer by the repressor, as has been suggested for MarR (15).
The remaining proteins listed in Fig. 4 are from bacteria other than E. coli. These include gram-positive and gram-negative species as well as mycobacteria, indicating that MarR family members are not restricted to enterics. The PecS protein from Erwinnia chrysanthemi (ErcPecS) is associated with the transcriptional control of pectate lyase and cellulase production, which are involved in invasion of plant tissues (35). petP from Rhodobacter capsulatus is part of a two-gene operon with a second putative transcriptional regulator petR, a genetic organization similar to that of marR and marA (36). The remaining proteins are inferred from open reading frames present in database entries, but for which no genetic information exists. However, since the non-E. coli members of this family exhibit similar levels of sequence conservation and include the same highly conserved residues as the E. coli proteins, a reasonable expectation is that they are also regulatory proteins and that their activity is affected by a phenolic compound. The target genes for this latter group remain to be identified, but the finding that they can be associated by sequence similarity with the MarR family should provide a means for their detection. It is worth noting that other as yet unidentified MarR family members may exist in E. coli and could have escaped detection in the cloning experiments described here, since the screen we employed demanded that the cloned gene product recognize and repress expression from the marRAB promoter. MarR homologs capable of binding SAL but that have different promoter specificities would not have been identified.
The organisms in which the gene products listed in Fig. 4 are found are largely free-living bacteria or plant pathogens. In addition, SAL is an important signaling molecule in plants (46). It thus seems likely that phenolic sensing is an important component of environmental surveillance by free-living bacteria, particularly when considering the variety of noxious aromatic agents produced by plant species (47). However, the association of Ec17kd with the MarR family suggests that phenolic sensing may play a role in animal infections as well, since the presence of the pap cluster with which the gene encoding Ec17kd is associated correlates strongly with the pyelonephritic capability of E. coli (23). Thus, MarR family members may also carry out important roles in phenolic sensing in animal hosts, and so might monitor either plant-derived compounds ingested by the host or host-synthesized phenolic metabolites. Such inducers might themselves be toxic compounds against which these organisms need to protect themselves; alternatively, they might serve as cues that the bacterium is in the intestinal environment, and could signal the induction of protective (MarR, MprA) and colonization (Ec17kd) factors. Experiments directed towards determining the importance of specific MarR family members in these processes should be extremely informative in this regard.
Preliminary experiments indicate that there is some specificity with respect to the inducing agent. Syringaldehyde, a substituted phenolic that has been identified as an inducer of virulence gene expression in Agrobacterium tumefaciens (48), is also a potent inducer of the mar system (PFM, unpublished observations). Dinitrophenol has also been shown to efficiently stimulate marRAB operon transcription (14). In contrast, HPC is not an effective inducer of mar expression (PFM and MCS, unpublished observations). Moreover, while SAL is a good inducer of mar expression, salicyl alcohol is not (14). In this light, it is interesting to speculate on the role of the conserved amino acids shared by MarR family members (Fig. 4). These residues could be involved in phenolic binding, operator/promoter recognition, or protein-protein interactions (e.g. dimerization of repressor molecules, interaction with an accessory factor, etc). Since the MarR homologs are likely to exhibit different specificities for phenolics and promoters, conserved amino acids should be involved in interactions that are carried out in very similar ways among these repressors.
The identity of the natural inducers for most of the proteins in the MarR family remains to be determined. However, the finding that the repressor activities of MarR, MprA, and Ec17kd can be antagonized by SAL, a plant-derived phenolic, raises the possibility that plant products are the natural ligands for many, if not all, of these systems. For HpcR this is clearly the case. Moreover, syringaldehyde and related compounds were first identified as stimulants of virulence gene expression in Agrobacterium tumefaciens (48); similar signals may be recognized by MarR homologs from other plant pathogens and symbionts (e.g., ErcPecS, RcPetP, CfExoO) and may play a role in regulating virulence gene expression in these organisms. MarR and its relatives found in enteric bacteria such as E. coli (EcMprA, Ec17kd) might modulate gene expression in response to similar phenolics encountered in the gut of mammalian hosts as products of plant diets. In any case, the results reported here suggest that environmental surveillance of phenolic compounds is highly conserved in bacteria and may be important in a variety of signal transduction pathways.
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The authors would like to thank Leonard Post, Steve Gracheck, and Eric Olson for critically reading the manuscript, two anonymous reviewers for their insightful comments, and to our colleagues in the Infectious Diseases Section and the Olson lab for their support throughout.