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Journal of Bacteriology, July 2008, p. 4951-4958, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00195-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

mgtA Expression Is Induced by Rob Overexpression and Mediates a Salmonella enterica Resistance Phenotype

Julieta Barchiesi, María E. Castelli, Fernando C. Soncini, and Eleonora García Véscovi^*

Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas (IBR-CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, S2002LRK Rosario, Argentina

Received 7 February 2008/ Accepted 7 May 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Rob protein is a member of a subfamily of AraC/XylS transcriptional regulators which also includes MarA, SoxS, and TetD, a more recently described protein (14, 27). Although all of the proteins in this subfamily recognize the same asymmetric degenerate binding motif known as the Mar/Sox/Rob box, they are able to differentially induce each individual promoter, depending on the activator concentration (24) and the relative affinity that each regulator has for each operator region (26, 27). It has been demonstrated that MarA, SoxS, and Rob are able to promote low-level resistance to multiple drugs, heavy metals, and superoxide and also to induce organic solvent tolerance in Escherichia coli. In contrast to what has been described for SoxS or MarA, the intracellular levels of Rob do not appear to be subject to extensive variations under most of the conditions tested, although a moderate increase has been observed in stationary phase or in phosphate- or glucose-restricted medium (17). In E. coli, it was demonstrated that Rob inducers such as dipyridyl, bile salts, or fatty acids trigger its activity by provoking a conformational switch in the regulator (37, 38). This activation can also be achieved in vitro or in vivo by overexpressing either the N-terminal portion (which harbors the DNA-binding domain) or the full-length protein (3, 16, 32).

While searching for factors that would modulate the expression of Salmonella mgtA independently of the PhoP/PhoQ system, we identified Rob. We found that Rob was able to induce mgtA transcription by a direct interaction with its promoter. mgtA codes for a P-type ATPase which is known as one of the three Mg²⁺ transporters in Salmonella enterica, together with MgtB and CorA (see reference 19 for a comprehensive review). It has been previously demonstrated that mgtA expression is regulated at the transcriptional level by the extracellular Mg²⁺-responsive PhoP/PhoQ two-component system (13, 39) and posttranscriptionally by an intracellular Mg²⁺-sensitive riboswitch that governs the extent of transcript elongation and stability (9, 40). Several lines of evidence have converged to suggest that MgtA plays an essential role in maintaining Salmonella Mg²⁺ homeostasis. mgtA is required for normal cell growth in Mg²⁺-restricted minimal liquid medium (39). Additionally, mgtA transcription is differentially and more sensitively regulated in response to environmental Mg²⁺ availability compared to other PhoP-regulated genes, with its promoter displaying a higher affinity for the PhoP regulator (31, 40). The fine and multilevel regulation of mgtA expression is indicative of MgtA relevance in the physiology of the cell.

In this work, we show that the mgtA regulatory region also harbors a Mar/Sox/Rob box downstream the PhoP recognition motif and that its expression is selectively induced by Rob and not modulated by MarA or SoxS. We demonstrate that basal Rob levels contribute to MgtA expression in late exponential phase and show that Rob overexpression confers enhanced resistance to cyclohexane on Salmonella. We also provide evidence that mgtA expression is involved in this solvent resistance phenotype, disclosing a novel role for MgtA in Salmonella.

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Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were grown in Miller's Luria-Bertani (LB) medium or on LB agar plates overnight at 37°C. The antibiotics used were ampicillin (100 µg ml^–1), kanamycin (50 µg ml^–1), and chloramphenicol (20 µg ml^–1). All of the reagents, chemicals, and oligonucleotides used were from Sigma. For the β-galactosidase activity assay, bacteria were grown overnight with shaking at 37°C in LB medium with the addition of different concentrations of isopropyl-β-D-thiogalactopyranoside (IPTG), with and without 50 mM MgCl₂, as indicated. β-Galactosidase levels were determined as described previously (30).

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TABLE 1. Bacterial strains, vectors, and plasmids used in this study

Genetic and molecular biology techniques. Gene disruptions or lacZ reporter fusions were carried out as described previously (10) in strain LB5010 (4). All constructions were transferred to wild-type (WT) strain 14028s by P22 transduction (11). When necessary, the antibiotic resistance cassette inserted at the deletion point was removed by using temperature-sensitive plasmid pCP20 carrying the FLP recombinase (8).

The marA soxS double mutant was generated by transducing the soxS::Cm^r deletion into the marA mutant strain by phage P22-mediated transduction. pCE36 was used to introduce the transcriptional lacZ fusion as previously described (12). The same procedure was used to construct the marA soxS rob mutant strain. All mutated DNA fragments were sequenced to confirm the required mutation and to screen against undesired mutations.

To construct plasmids pRob, pMarA, and pSoxS (Table 1), the rob, marA, and soxS genes were amplified from the S. enterica serovar Typhimurium ATCC 14028s chromosome by PCR with primers rob BglII (F) and rob HindIII (R) for pRob, marA BglII (F) and marA HindIII (R) for pMarA, and soxS BglII (F) and soxS HindIII (R) for pSoxS (Table 2). Each fragment obtained was cloned into BamHI-HindIII-digested pUHE21-2laqI^q. Plasmid DNA was introduced into bacterial strains by electroporation with a Bio-Rad apparatus according to the manufacturer's recommendations.

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TABLE 2. Oligonucleotide primers used in this study

An adaptation of the method described by Horton et al. (15) was used to generate chromosomal deletions in the mgtA untranslated region (UTR). Briefly, a first round of PCR was carried out with the mgtA-tF oligonucleotide as the 5' primer with mgtA-1-2A, mgtA-1A, or mgtA-2A and the mgtA-tR oligonucleotide as a 3' primer with mgtA-1-2B, mgtA-1B, or mgtA-2B to generate the first 5' or 3' amplification product for UTR, UTR1, or UTR2, respectively. A second round of amplification was performed with mgtA-tF and mgtA-tR and the 5' or 3' amplification product as the template. The PCR products were purified, cloned into the pGEM-T (Promega Corp.) plasmid, and verified by DNA sequence analysis. To introduce these mutations into the chromosome, a second round of overlap extension PCR was performed with a treB-F/TreR-R-amplified fragment containing a Cm^r cassette from pKD3 and the mgtA-tF/mgtA-tR PCR products of the fragments cloned in pGEM-T. The treB-F/mgtA-tR PCR fragments containing the mutated mgtA promoter sequence were introduced into the mgtA::MudJ treBR phoP strain (40). All of the mutations were verified by chromosomal DNA sequence analysis. All of the primer sequences used are provided in Table 2.

RNA purification and primer extension. Total RNA was extracted from late-exponential-phase cultures (optical density at 630 nm = 0.9) grown in LB at 37°C with 100 µg ml^–1 ampicillin and 0.1 mM IPTG as previously described (5). A final RNA cleanup step with an RNeasy kit (Qiagen), used according to the manufacturer's protocol, was included. cDNA synthesis was performed with 1.0 pmol of ³²P-end-labeled primer MgtA Primext 2, with 50 µg of total RNA and 1 U of SuperScript II RNaseH2 reverse transcriptase (Life Technologies, Inc.). The extension products were analyzed by electrophoresis on a 6% polyacrylamide-8.0 M urea gel and compared with sequence ladders initiated with the same ³²P-labeled primer that was used for primer extension.

DNase I footprinting assay. DNase I protection assays were done for both DNA strands with labeled primers mgtA-tF and #384 mgtA2. Binding reaction mixtures with 0 or 132 nmol of purified Rob-HisX6 protein and 6.0 fmol of labeled DNA in a 20-µl volume were incubated at room temperature for 30 min. Rob-HisX6 protein was purified by our previously described protocol (22). The binding buffer used for protein-DNA incubations contained 25 mM Tris-HCl (pH 8), 50 mM NaCl, 5 mM MgCl₂, 5 mM dithiothreitol, 10% glycerol, 2.0 ng µl^–1 salmon sperm DNA, and 0.025 mg ml^–1 bovine serum albumin. DNase I (0.05 U; Life Technologies, Inc.) was added, and the mixture was incubated for 60 s at room temperature in a final volume of 100 µl. The reaction was stopped by adding 90 µl of 20 mM EDTA (pH 8.0)-200 mM NaCl-100 µg ml^–1 tRNA. DNA fragments were purified by phenol-chloroform extraction and resuspended in 7.0 µl of H₂O. Samples (3 µl) were analyzed by denaturing polyacrylamide (6%) gel electrophoresis by comparison with a DNA sequence ladder generated with the appropriate primer (1).

Solvent tolerance assay. A method termed the efficiency-of-plating assay was used to determine solvent tolerance (23). Briefly, stationary-phase LB broth cultures were diluted into the same medium to yield a suspension of approximately 10⁴ cells ml^–1. A 30-µl volume of the cell suspension was spread over the surface of a 20-ml LB-agar plate containing 0.1 mM IPTG and ampicillin which was subsequently overlaid with 1 ml of cyclohexane or 2 ml of n-hexane. The plates were sealed, and growth was assessed following incubation at 30°C for 24 h.

We plotted the percent survival of each strain (WT or otherwise isogenic mgtA, marA soxS, or acrAB mutant harboring the vector plasmid or the pRob plasmid) grown in 20 ml LB-agar-ampicillin-0.1 mM IPTG plates with a 1-ml cyclohexane or a 2-ml n-hexane overlay, relative to the growth in plates without solvent, which was taken as 100%.

Antibiotic and heavy metal susceptibility assays. For antibiotic sensitivity assays, 50 µl of a 2 x 10^–2 dilution of overnight cultures was mixed with 50 µl of antibiotic solution dissolved in the same medium at final concentrations that ranged between 0 and 15 µg ml^–1 for kanamycin, 0 and 3.0 µg ml^–1 for tetracycline, 0 and 0.6 µg ml^–1for ciprofloxacin, and 0 and 45 µg ml^–1 for nalidixic acid. The mixtures were incubated in 96-well microtiter plate at 37°C with shaking for 18 h. Optical density at 630 nm was determined with a Dynatech Laboratories microplate reader.

For metal sensitivity assays, a 5 x 10^–7 dilution of overnight culture of the strains was done in phosphate-buffered saline. A 30-µl aliquot was applied to LB plates containing increasing concentrations of CdCl₂, AgNO₃, or HgCl₂. Plates were incubated at 37°C for 24 h. Alter incubation, the number of CFU per milliliter was calculated and percent survival was estimated on the basis of the count of the corresponding strain grown in the absence of added metal (7).

Statistical analysis. Statistical analysis was performed by one-way analysis of variance and the Holm-Sidak test.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
mgtA expression is specifically upregulated by Rob in a PhoP-independent manner. We have previously observed that, when expressing PhoP from multicopy plasmid pBP1019, the induction of the PhoP-controlled gene expression turns out to be PhoQ and extracellular Mg²⁺ concentration independent (22), with the exception of mgtCB and mgtA (40). This observation coincided with other reported results (9, 42). With the aim of identifying regulatory factors other than the PhoP/PhoQ system or the posttranscriptional riboswitch controlling mgtA expression (9), we transformed an mgtA::MudJ phoP mutant Salmonella strain (39) with a partially Sau3AI-digested Salmonella phoPQ DNA chromosomal library generated in plasmid pBBR1-MCS2 (20). Transformants were selected in 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal)-LB-agar plates. Out of approximately 5,000 transformants, we isolated one positive blue clone for the induction of mgtA expression. DNA sequence analysis and a subsequent search of the Salmonella genome database (29) showed that the insert harbored the entire rob gene sequence and approximately 1,000-bp flanking sequences upstream and downstream of rob. The rob gene codes for Rob, a transcriptional regulator member of the AraC/XylS family of proteins that also includes MarA and SoxS.

To verify that Rob expression was responsible for mgtA induction, we PCR amplified and cloned rob into medium-copy plasmid pUHE21-2lacI^q (39) under the control of the IPTG-inducible promoter. We determined mgtA transcriptional activities in the Salmonella WT and phoP mutant backgrounds. As shown in Fig. 1A, rob overexpression upregulated mgtA transcription in a PhoP-independent manner. When a high extracellular Mg²⁺ concentration was present in the growth medium, Rob induction of mgtA was still evident. Nevertheless, an eightfold decrease in mgtA transcription, presumably due to the Mg²⁺ riboswitch mechanism, was dominant under the latter condition. On the other hand, only when cells were grown to late exponential phase was a 30% reduction in mgtA expression consistently observed in the rob mutant compared to the otherwise isogenic WT strain (Fig. 1C).

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FIG. 1. Rob induces the expression of mgtA in an Mg2+- and PhoP/PhoQ-independent manner. (A) β-Galactosidase activity from the mgtA::lacZ transcriptional fusion was determined in WT and phoP, rob, marA, and soxS mutant background strains harboring plasmid pUHE21-2lacIq (vector), pRob, pMarA, or pSoxS, as indicated. Cells were grown overnight in LB medium without (gray bars) or with the addition of 50 mM MgCl2 (black bars). β-Galactosidase activity values of strains grown under low-Mg2+ conditions differ significantly from those of their counterparts grown under high-Mg2+ conditions (*, P < 0.001; **, P = 0.017; ***, P = 0.015). (B) Transcriptional activity determined from the acrAB::lacZ fusion in a marA soxS strain harboring each indicated plasmid and grown overnight in LB medium. (C) β-Galactosidase activity from the mgtA::lacZ transcriptional fusion was determined in the WT and rob mutant strains. Cells were treated as described for panel A, except that they were grown to late exponential phase. β-Galactosidase activity is in Miller units. Data correspond to mean values of at least three independent experiments performed in triplicate. Error bars correspond to standard deviations. β-Galactosidase activity values from WT and rob strains were significantly different (*, P < 0.001).

Rob, SoxS, and MarA display overlapping functionality, and all three recognize a degenerate 20-bp binding site in the regulatory region of the target genes that is known as the Mar/Sox/Rob box (28). Therefore, to examine whether the three paralog proteins share the ability to upregulate mgtA, we determined mgtA expression when SoxS, MarA, or Rob was overexpressed in the soxS, marA, or rob mutant background. As shown in Fig. 1A, and in contrast to Rob, neither SoxS nor MarA overexpression upregulated mgtA transcription. To verify the functionality of the three expressed proteins, we measured their abilities to promote acrAB expression, which is known to be stimulated by Rob, MarA, or SoxS (37, 43). We used an acrAB-lacZ transcriptional fusion in a marA soxS double-mutant background strain. As shown in Fig. 1B, the three overexpressed regulators were functional in the ability to promote acrAB expression. To exclude the effects of mutual interference or cross-regulation among the three proteins, we also verified that Rob was able to induce mgtA expression when endogenous marA, soxS, and rob expression was simultaneously abolished (Fig. 2). A 0.1 mM IPTG concentration was selected to induce Rob expression in subsequent experiments. We also examined whether Rob could affect the transcriptional activity of other PhoP-regulated genes. By measuring the β-galactosidase activity from strains harboring lacZ transcriptional fusions to pcgF, virK, pcgM, pbgF, and mgtCB (39) transformed with either pRob or the empty vector, we observed that their expression remained unaltered when Rob expression was induced (data not shown).

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FIG. 2. Rob-controlled transcription of mgtA does not require marA or soxS integrity. Transcriptional activity from the mgtA::lacZ fusion was determined as described for Fig. 1 in a marA soxS rob mutant strain harboring pUHE21-2lacIq (vector) or the pRob plasmid and grown overnight in LB with the indicated concentrations of IPTG. Data correspond to mean values of three independent experiments performed in triplicate. Error bars correspond to standard deviations.

Together, these results demonstrate that overexpression of Rob, but not of SoxS or MarA, is able to specifically induce mgtA expression in a PhoP-independent fashion.

Rob-dependent transcriptional start site of mgtA. We mapped the Rob-dependent transcriptional start site of mgtA by primer extension analysis. We compared the WT versus the phoPQ mutant harboring either pRob or the empty plasmid. Two products were detected in the WT strain (Fig. 3A). For the largest one, the transcription start site corresponded to a T residue located 264 bp upstream of the mgtA start codon, which coincides with the PhoP-dependent transcription start site previously identified in both S. enterica serovar Typhimurium and E. coli (18, 21, 39). As expected, this product was not detected in the phoPQ mutant strain. The second transcript was initiated at a G residue located 221 bp upstream of the mgtA start codon. This shorter product was detected either in the WT strain or in the phoPQ strain expressing Rob from the pRob plasmid (Fig. 3A).

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FIG. 3. Mapping of the Rob-regulated region in the mgtA promoter. (A) Primer extension analysis of mgtA with RNA isolated from WT or from phoPQ cells harboring pUHE21-2lacIq (vector) or the pRob plasmid and grown to the mid-exponential phase in LB medium. The sequences spanning the two transcription start sites (bold) are shown on the left. (B) β-Galactosidase activities determined in the mgtA::MudJ treBR phoP mutant strain (–) and otherwise isogenic strains carrying deletions in the mgtA 5' UTR (UTR, UTR1, and UTR2) harboring pUHE21-2lacIq (vector) or the pRob plasmid and grown overnight in LB medium with 0.1 mM IPTG without (gray bars) or with the addition of 50 mM MgCl2 (black bars).

Identification of the Rob-binding site in the mgtA promoter region. To define the region that is required for Rob-dependent induction in the mgtA promoter, we initially took advantage of three deletion mutants previously generated in our laboratory that lack sequences from nucleotide (nt) –21 to nt –260 (UTR), from nt –121 to nt –260 (UTR1), and from nt –21 to nt –120 (UTR2) relative to the mgtA translational start ATG site (see reference 40 and Materials and Methods for details). As shown in Fig. 3B, Rob-dependent induction of mgtA transcription was detected in the UTR2 deletion-containing strain, irrespective of the Mg²⁺ concentration used, while it was abrogated in the strains harboring the UTR and UTR1 deletions. This result indicates that the region required for Rob-dependent regulation is at least partially encompassed in the region between nt –121 and nt –260 relative to the mgtA translational start codon (see Fig. 4C for a scheme including the UTR deletions used).

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FIG. 4. Identification of the Rob-regulated promoter of mgtA. (A) DNA footprinting analysis of the promoter region of mgtA performed on the end-labeled coding and noncoding strands. The DNA fragments were incubated with purified Rob at a final concentration of 0 or 132 nM. Solid lines and stars indicate the Rob-protected region and hypersensitive sites, respectively. (B) DNA sequence of the mgtA promoter region. The PhoP- and Rob-dependent transcription start sites (pphoP and prob) are indicated. Position numbering refers to the Rob-dependent transcription start site. Solid lines indicate the Rob-protected region. The region harboring the mgtA sequence that matches the Rob-binding consensus (gray shaded) and the hypersensitive sites (boldface italics) are also indicated. (C) Schematic representation of the 5' region of mgtA, including the PhoP- and Rob-controlled promoters with their transcription start sites (PhoP-BS in white and Rob-BS in black, respectively), the 5' UTR (UTR1, light gray bar; UTR2, dark gray bar; rbsw, riboswitch encompassed region), and the predicted mgtA ribosome-binding site ( SD). The promoters harboring the UTR, UTR1, and UTR2 deletions are also schematized.

To define whether Rob directly controls the expression of mgtA, a DNase I footprinting assay was performed with purified Rob as a His₆-tagged protein, as described in Materials and Methods. As shown in Fig. 4B, Rob protected an overlapping region from nt –28 to nt –53 relative to the Rob-dependent transcriptional start site. The C residue at position –34 in the direct strand and the corresponding G residue at position –34 in the reverse strand showed hypersensitivity to DNase I. The protected region (Fig. 4A) encompassed a sequence that displays a 15-of-20 match to the AHRGCACRWWNNRYYAAAHN Mar/Sox/Rob revised consensus binding motif (28). These results demonstrate the presence of a Mar/Sox/Rob binding motif in the –28 to –53 protected region of this promoter relative to the start site of the Rob-dependent transcript, providing evidence of a direct interaction of Rob within the mgtA promoter (see schemes in Fig. 4B and C depicting the locations of the regulatory regions determined in the mgtA promoter region).

Rob-induced mgtA expression confers resistance to cyclohexane on Salmonella. It has been previously demonstrated that overexpression of Rob confers multidrug, heavy metal, and organic solvent resistance on E. coli (3, 16, 32, 43). To gain an insight into the physiological role of the Rob-dependent induction of mgtA in Salmonella, we compared the resistances of the WT and mgtA mutant strains to diverse compounds in the absence or presence of overexpressed Rob. Antibiotic and metal resistances were determined by MIC assays, while resistance to organic solvents was assayed by the efficiency-of-plating method as previously described (23, 43). We found that Rob overexpression did not affect WT Salmonella resistance to kanamycin, while it conferred augmented resistance to tetracycline, ciprofloxacin, and nalidixic acid (Table 3). No differential effects were observed in heavy metal resistance (Ag, Cd, and Hg; data not shown), while increased tolerance was detected when the cells were challenged with n-hexane and cyclohexane (Table 3 and Fig. 5). The Rob-dependent induction of cyclohexane resistance was unaltered in a marA soxS mutant background. The Rob-mediated cyclohexane resistance was approximately 10-fold reduced in the mgtA strain compared to that of the WT strain (Fig. 5). In contrast, mgtA deletion did not affect the Rob-mediated resistance to the other compounds tested.

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TABLE 3. Rob-induced resistance phenotypesa

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FIG. 5. MgtA-dependent resistance to cyclohexane. Relative survival of Salmonella strains on cyclohexane-containing LB-agar plates was performed as described in Materials and Methods. The WT and mutant strains carrying the pUHE21-2lacIq (vector) or pRob plasmid are shown. Data correspond to mean values of five independent experiments performed in triplicate. Error bars correspond to standard deviations. The survival values of the mgtA mutant strain harboring the vector and pRob plasmids were significantly different (*, P < 0.001).

It has been previously demonstrated that deletion of the multicomponent multidrug efflux pump-encoding operon acrAB results in loss of organic solvent tolerance (2, 43). Therefore, we tested whether Rob-induced cyclohexane tolerance was detectable in an acrAB background. Under our conditions, the resistance of S. enterica serovar Typhimurium to cyclohexane was abrogated in the acrAB mutant strain compared to the WT strain (Fig. 5). This result suggests that mgtA expression contributes to acrAB-mediated cyclohexane resistance.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Previous studies have provided ample demonstration that mgtA expression is subject to two different regulatory mechanisms in response to the availability of Mg²⁺. One is mediated by the phosphorylation status of the PhoP regulator, which modulates mgtA transcription in response to the kinase/phosphatase activity balance of the environmental Mg²⁺-responsive sensor PhoQ (6, 22). The other posttranscriptionally regulates the levels of the mgtA transcript, depending on the intracellular Mg²⁺ content (9, 40).

In this work, we show that the mgtA promoter region harbors a Mar/Sox/Rob motif which is responsive to Rob overexpression, not being significantly affected by overexpression of the MarA or SoxS paralogs. Interestingly, the resultant Rob-dependent mgtA transcript is distinct and independent of that induced by the PhoP regulatory activity. However, it preserves unmodified the 5' UTR, which is the target of the Mg²⁺-dependent posttranslational regulatory mechanisms. This suggests that mgtA downregulation by high intracellular Mg²⁺ concentrations would dominate over a (currently unknown) Rob-inducing condition(s). In contrast, in a phoP background, when the region required for the riboswitch is disrupted while preserving the Rob-binding site (UTR2 strain), mgtA is induced by Rob in an Mg²⁺-independent manner. Neither Rob-modulated expression nor a Mar/Sox/Rob conserved box in the respective regulatory region was detected in other PhoP-dependent genes examined (including mgtCB), suggesting that this feature is unique to mgtA among the Salmonella PhoPQ regulon members. Both the absence of a sequence matching the Mar/Sox/Rob motif in the promoter region of corA and β-galactosidase activities measured from a strain harboring a corA::lacZ transcriptional reporter in which Rob was overexpressed indicated that corA is not under Rob regulation (our unpublished results). Therefore, of the three Mg²⁺ transporters present in Salmonella, MgtA is the only one whose expression is subject to Rob-dependent regulation.

Upregulation of mgtA was only observed during Rob overexpression. This was not unexpected, since most Rob-dependent phenotypes have been detected only when the regulator was overexpressed (3, 32, 43). Consistent with this result, when the rob mutant was compared to the otherwise WT strain in late exponential phase, downregulation of mgtA expression was observed, showing that, under this condition, physiological Rob levels contribute approximately 30% of mgtA expression.

No mgtA induction was observed when testing compounds previously described to posttranslationally activate Rob in E. coli (i.e., salicylate, deoxycholate, and dipyridyl) (37, 38; data not shown). In this regard, the E. coli and Salmonella Rob amino acid sequences display 92% identity. However, they show only one nonidentical amino acid along the N-terminal domain which harbors the 106-residue DNA-binding region, while the 183-amino-acid C-terminal domain that encompasses the putative regulatory domain which would bind the inducers shows 20 nonidentical residues. Therefore, a dissimilar physiological function of Rob in E. coli or Salmonella might also explain why the inducers tested were ineffective in activating Salmonella Rob.

Interestingly, when the sequence that matches the Mar/Sox/Rob motif in the S. enterica mgtA promoter is compared to that region in other members of the family Enterobacteriaceae, an identical box can be found upstream of Citrobacter koseri mgtA. E. coli, Shigella, Klebsiella, and Enterobacter strains show the Mar/Sox/Rob motif with a few scattered mismatches compared to the S. enterica Rob motif (Fig. 6). This observation suggests that regulation of mgtA by Rob is an ancestral trait of related enterobacteria.

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FIG. 6. Alignment of sequences that match the Mar/Sox/Rob motif in the promoter region of mgtA in S. enterica and other enterobacteria. DNA sequence alignment of the S. enterica serovar Typhimurium LT2, C. koseri ATCC BAA-895, E. coli W3110, Shigella flexneri 2a strain 2457T, Enterobacter sp. strain 638, and Klebsiella pneumoniae MCG 78578 mgtA promoter regions. Predicted Rob-binding sites are located 250, 398, 249, 249, 255, and 256 nt upstream of the mgtA translational start site, respectively. Nucleotides that match the Mar/Sox/Rob consensus motif (sequence at the top) are in boldface.

Because the overexpression of Rob was demonstrated to increase bacterial resistance to organic solvents, heavy metals, and antibiotics, we examined whether mgtA expression somehow contributes to the expression of these phenotypes. Deletion of mgtA greatly diminishes Rob-mediated induction of Salmonella resistance to cyclohexane. The induction of multidrug resistance and of organic solvent tolerance by the AraC/XylS-type regulators has been essentially associated with the abilities of these regulators to activate the expression of AcrAB, an RND family efflux pump which displays a broad drug specificity, and to simultaneously downregulate the expression of the major porin OmpF (36, 41). As shown in Fig. 1B, Rob overexpression is able to upregulate acrAB transcriptional activity even in a marA soxS background. On the other hand, the transcriptional activity of acrAB remained unchanged in an mgtA mutant strain compared to that in the otherwise isogenic WT strain (not shown).

Traditionally, testing for cyclohexane resistance has been considered a quick and easy procedure to detect intrinsic fluoroquinolone resistance phenotypes in both E. coli and Salmonella isolates. However, it seems that this association is valid only in those cases in which resistance is linked to active efflux mechanisms, mainly mediated by a high acrAB expression level. But a cyclohexane tolerance assay is not appropriate when mutations in the gyrase or topoisomerase gene are the essential components of the fluoroquinolone resistance phenotype (25, 33, 35, 36). Although we observed that, under our conditions, Rob-induced cyclohexane resistance was dependent on the intactness of acrAB, we did not find a concomitant Rob-enhanced mgtA-dependent resistance to fluoroquinolones (i.e., ciprofloxacin and nalidixic acid).

In sum, these last results reveal that mgtA contributes to the mechanism of Rob-mediated resistance to cyclohexane in Salmonella, most probably because of its role in preserving Mg²⁺ homeostasis when the cation is scarce. In light of our results, we favor the possibility that, under certain environmental conditions, mgtA might contribute to the acrAB-dependent solvent efflux mechanism. Lastly, the multilevel regulation mechanisms that converge to control mgtA expression, comprising posttranscriptional and transcriptional regulatory mechanisms (i.e., PhoPQ, attenuation, degradation by RNase E, and Rob regulation), are indicative of a requirement for MgtA expression in Salmonella under precise physiological conditions.

 
We are indebt to John S. Gunn for the generous gift of the S. enterica rob mutant strain. We thank the reviewers for their suggestions during the elaboration of the manuscript.

This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica and from the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET, Argentina). E.G.V., F.C.S., and M.E.C. are career investigators of the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET, Argentina). F.C.S. is a member of the Consejo de Investigaciones de la Universidad Nacional de Rosario (CIUNR, Argentina). J.B. has a fellowship from the Agencia Nacional de Promoción Científica y Tecnológica.

 
* Corresponding author. Mailing address: Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR) Suipacha 531, S2002LRK Rosario, Argentina. Phone: 54-341-4356369, ext. 131. Fax: 54-341-4804598. E-mail: garciavescovi{at}ibr.gov.ar

Published ahead of print on 16 May 2008.

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Journal of Bacteriology, July 2008, p. 4951-4958, Vol. 190, No. 14
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