INTRODUCTION

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Aerobic organisms obtain energy by the oxidation of organic compounds, with oxygen as the final electron acceptor. As a by-product of this process, reactive oxygen species are generated, with potentially damaging consequences for the cell. Escherichia coli responds to the intracellular increase in reactive oxygen species by inducing sets of genes whose products either avert or repair the eventual oxidative damage (19). The response to increased levels of superoxide is regulated by the soxRS system, a pair of regulatory genes that, together with their downstream-regulated genes, define the soxRS regulon (3, 37).

The SoxR protein is expressed constitutively and is a homodimeric transcriptional regulator that contains redox-active iron-sulfur clusters (18, 38). The oxidation state of the iron-sulfur clusters in SoxR regulates the transcriptional activity of the protein (20): while reduced SoxR does not affect transcription, oxidized SoxR dramatically enhances the transcription rate of soxS, a gene that codes for a second transcriptional activator (22, 23). The SoxS protein is a member of the AraC/XylS family of transcriptional regulators, and enhanced expression of SoxS activates at least 15 genes (20, 24), including sodA (Mn-containing superoxide dismutase), zwf (glucose-6-phosphate dehydrogenase), micF (antisense RNA to the porin OmpF mRNA), nfo (DNA repair endonuclease IV), fpr (NADPH:ferredoxin oxidoreductase), acrAB (efflux pump), acn (aconitase), fumC (heat-resistant fumarase), and nfsA (nitroreductase A). The induction of SoxS also results in enhanced resistance to multiple antibiotics (3, 4), dependent on micF (11) and acrAB (25).

Recently, the soxRS genes from Salmonella enterica serovar Typhimurium were cloned (B. L. Martins, P. J. Pomposiello, Z. Li, and B. Demple, unpublished data). Their nucleotide sequence (GenBank accession no. U61147) showed 97% identity of the predicted polypeptides with SoxR and SoxS of E. coli. Moreover, the regulatory region between the two genes is nearly identical to that of E. coli. The cloned soxRS genes were used to generate soxRS deletion mutants by allelic exchange (Martins et al., unpublished data). These soxRS strains are hypersensitive to the superoxide-generating agent paraquat (PQ), fail to induce the expression of nfo and sodA, and do not show increased resistance to antibiotics in response to treatment with PQ. Additionally, an independent study (16) showed that the soxS gene from S. enterica serovar Typhimurium is required for activation of sodA expression and resistance to redoxcycling agents.

Despite the similarity of the soxRS genes between S. enterica serovar Typhimurium and E. coli, enzyme activity measurements suggested that the soxRS regulons from E. coli and S. enterica serovar Typhimurium are different: while incubation with PQ induced the SodA protein and endonuclease IV, it failed to induce glucose-6-phosphate dehydrogenase or fumarase C in wild-type S. enterica serovar Typhimurium (Martins et al., unpublished data). The evolution of E. coli and S. enterica serovar Typhimurium under different environmental pressures might have led to differential recruitment of genes into the soxRS regulon. Therefore, a detailed description of the S. enterica serovar Typhimurium soxRS regulon might yield insights into the mechanisms of resistance to antibiotics and antimicrobial compounds generated by the immune system.

In order to characterize the soxRS regulon of S. enterica serovar Typhimurium, we built an inducible soxS expression system that is uncoupled from oxidative stress. We used this system both to measure the effect of the expression of SoxS on genes known to be under soxRS control in E. coli and to identify novel targets of soxS regulation in S. enterica serovar Typhimurium.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Table 1 shows the bacterial strains and plasmids used in this work. Bacterial cultures were grown at 37°C in Luria-Bertani (LB) broth (27) with vigorous aeration by shaking at 250 rpm. Ampicillin (100 µg/ml) and kanamycin (50 µg/ml) were added when necessary. The lacZ inducer isopropyl--D-thiogalactopyranoside (IPTG) and PQ were added at the final concentrations described in the figure legends. The indicator 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) was added to plates at a final concentration of 25 µg/ml.

DNA manipulation and construction of pJP105. DNA purification, incubation with restriction enzymes, electrophoresis in agarose gels, ligation, and bacterial transformation were performed according to standard protocols (5). Phage P22 transductions were performed by standard methods (26). To construct plasmid pJP105, the S. enterica serovar Typhimurium soxS gene was subcloned from pBCKpn (Martins et al., unpublished data) as an HpaI-SalI fragment into pBR322 (9) digested with EcoRV and SalI, generating plasmid pJP103. The lacI gene from pSE380 (Novagen) was excised as a PshAI-SalI fragment and ligated to pJP103 previously digested with AvaI, followed by treatment with T4 DNA polymerase and SalI, to generate plasmid pJP104. The lacZ promoter from pRZ4004 (32) was liberated by digestion with EcoRI, filled in by Klenow fragment DNA polymerase, and digested with HindIII. Plasmid pJP104 was digested with ClaI, followed by incubation with T4 DNA polymerase and HindIII, and ligated to the lacZ promoter fragment from pRZ4004 to generate plasmid pJP105 (5.4 kbp). This construct was used to transform the intermediary S. enterica serovar Typhimurium strain SL4213 (restriction deficient), and plasmid DNA extracted from this transformant was used to transform strain EM1 (soxRS) to ampicillin resistance.

Northern blot analysis. Bacterial cultures were grown overnight in LB broth at 37°C with the appropriate antibiotics, diluted to 1/100 in 2 ml of LB broth, and grown at 250 rpm in 18-mm-diameter tubes to mid-log phase. At an optical density at 600 nm of ~0.5, growing cultures were exposed to different concentrations of PQ or IPTG for 30 min. Total RNA was extracted with an RNAeasy kit (Qiagen) and resuspended in RNAse-free water. The concentration of the total RNA preparations was determined by measuring the absorbance at 260 nm, and 2 µg of total RNA was run per lane in 1.25% agarose gels containing formaldehyde and transferred to Nytran membranes with a Turboblotter setup (Schleicher & Schuell). The RNA was cross-linked to the membrane by UV irradiation, and the membranes were then hybridized at 68°C with radioactively labeled DNA fragments in cylindrical tubes by using QuickHyb solution (Stratagene). The membranes were washed according to the instructions from the manufacturer. X-ray films were exposed to the membranes at 70°C and developed with a Fuji automatic developer. The radioactive signals were quantitated with an Applied Biosystems phosphorimager.

MudJ mutagenesis. The MudJ (promoterless lacZ, Kan^r) phage was delivered to the soxRS/pJP105 strain by P22 transduction from strain TT10288 (hisD::MudJ hisA::MudI) as described by Hughes and Roth (21). Kanamycin-resistant transductants were isolated on LB agar plates containing kanamycin and ampicillin. Each selection plate was replica plated onto LB agar plates containing kanamycin, ampicillin, and X-Gal. One of these plates also contained 1 mM IPTG to promote expression of SoxS from pJP105. Colonies displaying differences in blue coloration between the two plates were selected as candidates for harboring lacZ insertions in SoxS-regulated genes.

PQ sensitivity test on gradient plates. The sensitivities to PQ of different strains were determined by the extent of linear growth on LB agar plates containing a gradient of PQ (13). Briefly, overnight cultures of the different strains were diluted 1/100 and grown in LB broth with aeration for 8 h and stamped on gradient plates containing different maximal concentrations of PQ. The gradient plates were incubated at 37°C for 16 h, and the length of confluent growth along the gradient was measured.

-Galactosidase assay. -Galactosidase-specific activity in chloroform-treated cells was determined as described by Miller (27). Samples were taken from bacterial cultures grown overnight in LB broth, diluted 1/100, and grown with aeration for 2 h to an optical density at 600 nm of ~0.5. At that point, the cultures were either exposed to PQ or IPTG for 30 min or left untreated.

Nonspecific PCR amplification. Chromosomal DNA was extracted from each strain carrying a Mu insertion and used as a template in a PCR that included primer MuLEFT (5'-ATAATCCAATGTCCTCCCGG-3'), derived from the sequence of the left end of phage Mu, and a primer of nonspecific sequence (5'-ATTGGAGCGCAGGTTAGTCG-3'). Conditions for these first-round PCRs were as follows: 1.5 mM MgCl₂, 0.5 U of Taq DNA polymerase (Sigma), 2 mM deoxynucleoside triphosphates (dNTPs), ~0.2 µg of chromosomal DNA, and 50 pmol of each primer. Reaction mixtures were incubated for 10 cycles of 1 min at 40°C (annealing), 30 s at 72°C (extension), and 30 s at 95°C (denaturation), followed by 30 cycles of the same steps but with an annealing temperature of 55°C. A second PCR was conducted with 5 µl of the first reaction mixture as the template, the nested primer MuEND (5'-TACTTCAAGTGAATCAATACA-3'), derived from the sequence of the left end of phage Mu, and the same nonspecific primer used in the first reaction. Conditions for these second-round PCRs were as follows: 1.5 mM MgCl₂, 0.5 U of Taq DNA polymerase (Sigma), 2 mM dNTPs, ~0.2 µg of chromosomal DNA, and 50 pmol of each primer. Reaction mixtures were incubated for 30 cycles of 1 min at 55°C (annealing), 30 s at 72°C (extension), and 30 s at 95°C (denaturation). The extension products from these reactions were isolated from agarose gels and sequenced at the Molecular Biology Core Facilities of the Dana-Farber Cancer Institute.

	RESULTS

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An engineered soxS expression system: SoxS is sufficient for enhanced resistance to PQ. In order to analyze the direct contribution of SoxS to the activation of antioxidant defenses, we built a soxS expression system that uncoupled soxS transcription from oxidative stress. In plasmid pJP105, the lacZ promoter (33) drives the transcription of the soxS cistron. As this plasmid also bears the lacI gene, the lacZ promoter is expected to be repressed in the absence of the inducer IPTG. When pJP105 was used to transform S. enterica serovar Typhimurium EM1 (soxRS) and the expression of the plasmid-borne soxS gene was monitored by Northern analysis, soxS mRNA showed strong induction (up to 90-fold) by the gratuitous lac inducer IPTG (Fig. 1). As expected, the soxS message from pJP105 is longer than the wild-type message, due to the displaced transcriptional start site provided by the lacZ promoter and intervening cloning fragments, which add a total of 273 nucleotides. The induction level of the IPTG-regulated soxS transcript was comparable to that of the soxS transcript from wild-type strain ATCC 14028 treated with increasing concentrations of PQ (Fig. 1). In control experiments, untransformed strain EM1 showed no soxS message either in the presence or the absence of PQ (data not shown). We conclude from these results that the IPTG-induced expression of the soxS gene from plasmid pJP105 mimics the induction of wild-type soxS by PQ and that this heterologous expression can be regulated by the concentration of inducer in the growth medium.

View larger version (62K): [in this window] [in a new window]   FIG. 1.   Northern blotting analysis of soxS expression from strains ATCC 14028 and EM1/pJP105. (A) Northern blotting. A radioactively labeled 450-bp EcoRI-HpaI fragment of plasmid pBCKpn (Martins et al., unpublished data) containing the S. enterica serovar Typhimurium soxS coding region was hybridized with total RNA from strain ATCC 14028 (wild type) treated with increasing concentrations of PQ (0, 25, 100, or 250 µM; lanes 1 to 4, respectively) or strain EM1(soxRS)/pJP105 treated with increasing concentrations of IPTG (0, 0.125, 0.50, or 1.00 mM; lanes 6 to 9, respectively). The control strain, EM1/pBR322, was treated with 1.00 mM IPTG (lane 5). (B) Quantitation of the soxS radioactive signal. The radioactivity for the soxS band in each lane was quantitated by phosphorimaging. The values are the relative activations for the treatments, normalized to the signal in the absence of treatment in the wild-type samples and to the signal from lane 5 in panel A. The lane numbers correspond to those in panel A. (C) Total RNA loading. Shown is an ethidium bromide stain of a duplicate gel to that used for panel A.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Complementation of the sensitivity to PQ by pJP105. Cultures of strains ATCC 14028, EM1, and EM1/pJP105 were grown overnight in LB broth and stamped onto PQ gradient plates with increasing concentrations of IPTG (0 to 2 mM) as indicated. The plates were incubated at 37°C for 14 h, and the extent of growth of each strain was measured. The values are the averages of one representative experiment done in duplicate, and the error bars are the standard deviations.

Induction of soxS and transcriptional activation of sodA and micF. Enzyme assays have shown that SodA activity is induced in S. enterica serovar Typhimurium upon exposure to PQ in a soxRS-dependent manner (16; Martins et al., unpublished data). We probed the activation of the sodA gene in S. enterica serovar Typhimurium either by treatment of cells with PQ or by the regulated expression of SoxS in the absence of oxidative stress. Northern blotting shows a direct correlation between sodA induction (Fig. 3) and soxS expression (Fig. 1), either in response to PQ in the wild-type strain or by IPTG induction in EM1/pJP105. The maximal induction ratio for sodA expression in the wild-type strain after exposure to PQ was 13-fold, while the maximum in the soxRS/pJP105 strain treated with IPTG was 8-fold (Fig. 3). Thus, other pathways such as the marAB system (1) may contribute to the PQ-induced expression of sodA in S. enterica serovar Typhimurium. In fact, the induction ratio for sodA in a soxRS strain exposed to 0.25 mM PQ was threefold (data not shown). Nevertheless, transcriptional induction of sodA expression by SoxS is sufficient to account for most of the PQ-inducible SodA activity.

View larger version (76K): [in this window] [in a new window]   FIG. 3.   Northern blotting analysis of sodA transcription. (A) Northern blotting. The membrane used for Fig. 1 was stripped and rehybridized with a ~1-kb AvaI fragment from plasmid pDT1.5 (12) containing the coding region of the sodA gene from E. coli. Strain ATCC 14028 (wild type) was treated with increasing concentrations of PQ (0, 25, 100, or 250 µM; lanes 1 to 4, respectively), and strain EM1(soxRS)/pJP105 was treated with increasing concentrations of IPTG (0, 0.125, 0.50, or 1.00 mM; lanes 6 to 9, respectively). The control strain, EM1/pBR322, was treated with 1.00 mM IPTG (lane 5). (B) Quantitation of the radioactive signals. The radioactivity for the soxS band in each lane was quantitated by phosphorimaging. The values are the relative activations for the treatments as described in the legend for Fig. 1. The lane numbers correspond to those used for panel A.

View larger version (69K): [in this window] [in a new window]   FIG. 4.   Northern blotting analysis of micF transcription. (A) Northern blotting. A 51-mer synthetic oligonucleotide derived from the sequence of the micF gene from S. enterica serovar Typhimurium (15) was labeled with T4 polynucleotide kinase and [32P]dATP. The probe was hybridized with total RNA from strain ATCC 14028 (wild type) treated with increasing concentrations of PQ (0, 25, 100, or 250 µM; lanes 1 to 4, respectively) or strain PP120(soxRS)/pJP105 treated with increasing concentrations of IPTG (0, 0.125, 0.50, or 1.00 mM; lanes 6 to 9, respectively). The control strain, PP120/pBR322, was treated with 1.00 mM IPTG (lane 5). (B) Quantitation of the radioactive signals. The radioactivity for the micF band in each lane was quantitated by phosphorimaging. The values are the relative activations for the treatments as described in the legend for Fig. 1. The lane numbers correspond to those used for panel A. (C) Total RNA loading. Shown is an ethidium bromide stain of a duplicate gel of total RNA.

MudJ mutagenesis and screening for SoxS-regulated genes. In order to identify novel SoxS-regulated genes, we screened a library of random lacZ insertions for differential expression in the presence or absence of SoxS. A soxRS strain was transformed with pJP105 and then mutagenized with MudJ, a bacteriophage Mu derivative with a promoterless lacZ operon for the generation of transcriptional fusions (10). Primary candidates for SoxS-regulated insertions were selected as described in Materials and Methods. From approximately 56,000 colonies, 114 candidates were picked, purified by single-colony isolation, and tested for -galactosidase activity in liquid culture in either the absence or the presence of IPTG (1 mM). We disregarded any transductant showing less than twofold activation by IPTG. From the group of 114 candidates, 32 Kan^r transductants showed increased -galactosidase activity after treatment with IPTG. In order to confirm regulation by SoxS in a physiologically relevant setting, the MudJ (Kan^r) insertion from each of these 32 mutants was transduced into a wild-type S. enterica serovar Typhimurium strain and the -galactosidase activity of each transductant was measured from LB cultures in the presence or absence of 0.25 mM PQ. Only six transductants showed increased -galactosidase activity after treatment with PQ (Fig. 5A). The rest of the transduced insertions were confirmed as Mu transpositions onto plasmid pJP105, as evidenced by three observations: first, the cotransduction of the plasmid-encoded resistance to ampicillin together with the Mu-encoded resistance to kanamycin; second, the presence of plasmid DNA in the Kan^r transductants; third, the increased sizes of these plasmids with respect to pJP105, as revealed by restriction analysis, consistent with a MudJ insertion (data not shown).

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Induction of -galactosidase activity by PQ in six independent lacZ insertions. Liquid cultures of the mutant strains were assayed for -galactosidase activity as explained in Materials and Methods. (A) Sin fusions in a soxRS+ background. (B) Sin fusions in a soxRS background. The results are the averages of three independent experiments, and the error bars correspond to the standard deviations.

View larger version (82K): [in this window] [in a new window]   FIG. 6.   Sensitivity to PQ of six independent lacZ insertions. Linear growth of different strains of S. enterica serovar Typhimurium on PQ gradient plates is shown. Maximum concentrations are shown below the graph. The maximum possible growth was 8 cm.

Molecular characterization of SoxS-regulated genes. To establish the identity of the soxS-regulated loci, we obtained the nucleotide sequence of the regions adjacent to the left end of the MudJ insertions by a nonspecific PCR method (see Materials and Methods). We then compared these nucleotide sequences with DNA databases by using the BLAST program (2). The Sin1 and Sin2 insertions were located in the sodA gene, which encodes a polypeptide with 97% identity to E. coli SodA (36). Both insertions were located at the 3'-untranslated end of the reported sodA sequence.

	DISCUSSION

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The structural similarity between the soxRS genes from E. coli and those from S. enterica serovar Typhimurium (GenBank accession no., U61147) predicted that the soxRS regulon serves similar functions in both species. We constructed an IPTG-regulated expression system that uncouples soxS expression from oxidative stress in order to study the direct contribution of SoxS protein to the activation of antioxidant defenses. By using this expression system, the steady-state levels of soxS message could be titrated by increasing the concentration of IPTG, which mimicked the physiological range of soxS expression in response to oxidative stress. The PQ-induced expression of soxS from wild-type strain ATCC 14028 seemed to saturate at an induction ratio of ~70-fold, while IPTG-induced expression of soxS from pJP105 was linear up to ~100-fold. The somewhat higher level attained for the artificial construct could be due to the absence of soxS autorepression (30) in the IPTG-driven lacZ promoter.

The heterologous expression of SoxS in S. enterica serovar Typhimurium resulted in enhanced resistance to PQ (Fig. 2). This result demonstrates that, as in E. coli, an elevated level of SoxS protein is sufficient for activation of important antioxidant genes in S. enterica serovar Typhimurium. Expression of SoxS in the absence of oxidative stress was sufficient for transcriptional activation of sodA to a high level. Interestingly, exposure of a soxRS strain to PQ elicited a small but reproducible increase in sodA transcription (data not shown). A modest, PQ-dependent, soxRS-independent induction of sodA was also observed by Martins et al. (unpublished data) and might be due to MarA, a transcriptional activator closely related to SoxS (1). The MarA protein activates sodA expression in E. coli (1) and may also do so in S. enterica serovar Typhimurium. The synthesis of MarA in E. coli was reported to be induced slightly by PQ (34), an effect that could underlie SoxS-independent induction of sodA in both organisms. The S. enterica serovar Typhimurium marA gene (35) codes for a predicted polypeptide 86% identical to E. coli MarA. Additionally, sodA in E. coli is regulated by ArcA, Fnr, and Fur (12). Of these, Fur is a reasonable candidate for contributing to PQ-induced sodA expression under aerobic growth. Expression of SoxS in soxRS strains of S. enterica serovar Typhimurium in the absence of oxidative stress was sufficient for transcriptional activation of micF, which reached the same maximum level as that observed in wild-type cells exposed to PQ. This result demonstrates that SoxS is sufficient for full, PQ-induced expression of micF. The induction of micF has not been directly connected to oxidative stress resistance. However, induction of micF in E. coli is necessary for enhanced resistance to multiple antibiotics. This phenotype depends on repression of the synthesis of porin OmpF, with a consequent reduction in permeability to small hydrophilic molecules (29). The apparent existence of the same mechanism in S. enterica serovar Typhimurium could be relevant in the development of antibiotic resistance during bacterial infection (see below).

SoxS also activates antibiotic resistance mechanisms in other species. In preliminary experiments, Klebsiella aerogenes transformed with pJP105 displayed increased resistance to chloramphenicol, nalidixic acid, tetracycline, and kanamycin in the presence of IPTG (P. J. Pomposiello and B. Demple, unpublished data). This result also suggests the existence of a soxRS regulon in Klebsiella. There is also evidence for a soxRS regulon in clinical strains of Enterobacter agglomerans, Shigella flexneri, and Shigella dysenteriae (B. L. Martins, A. Koutsolioutsou, and B. Demple, unpublished data).

Our screening for Sox-regulated genes with MudJ yielded lacZ insertions in three different genes. Of these, sodA was the only gene already characterized in S. enterica serovar Typhimurium (36), but its role in antioxidant defense is not completely understood. However, we have shown that the insertion of MudJ in sodA and the other two genes rendered the corresponding strains hypersensitive to oxidative stress in comparison to the isogenic wild-type strain. These results suggest strongly that all three genes have roles in protecting the cell against PQ-elicited oxidative stress. The relative PQ resistances of the Sin1 and Sin2 insertions (Fig. 6) are likely due to their locations in the 3'-untranslated region of sodA, which are expected to preserve substantial sodA function.

The remaining Mu insertions map to two uncharacterized S. enterica serovar Typhimurium ORFs. For Sin3, Sin4, and Sin5, the strong homology of the disrupted gene with the E. coli fpr gene and the high sensitivity of the Sin3, Sin4, and Sin5 strains to PQ are consistent with the identification of this locus as the S. enterica serovar Typhimurium fpr gene. The E. coli fpr gene was originally isolated in a screening for PQ-sensitive mutants (28). The E. coli fpr is a NADPH:ferredoxin oxidoreductase required for anaerobic ribonucleotide reduction (7). Finally, Sin6 maps to a gene with a sequence similar to that of the E. coli ydbK ORF. The annotation of this ORF as a putative iron-sulfur-containing oxidoreductase, together with the hypersensitivity to PQ of the corresponding mutant, argues in favor of a role for this S. enterica serovar Typhimurium gene in protection against superoxide stress. Oxidoreductases contribute to resistance to oxidative damage by redirecting electron transfer, rereducing oxidized iron-sulfur centers, and replacing damaged components (20, 24).

There are 15 known genes in E. coli under soxS regulation (20, 24). This figure led us to expect a higher yield of SoxS-regulated genes for our mutagenesis and screening in S. enterica serovar Typhimurium. There are at least three possible reasons for this discrepancy. First, ~80% of the insertions identified as IPTG responsive were in plasmid pJP105 rather than in chromosomal sites. Thus, the MudJ mutagenesis was not saturating. Second, the strain used in the screening, EM1, has increased basal expression of micF for unknown reasons (unpublished data). If this effect extends to other genes in EM1, the difference between the basal and SoxS-activated levels of expression for various genes might be obscured. Third, it is possible that S. enterica serovar Typhimurium has fewer genes under soxRS control than does E. coli. As noted earlier, glucose-6-phosphate dehydrogenase and fumarase C are regulated by SoxS in E. coli but do not seem to be members of the soxRS regulon in S. enterica serovar Typhimurium (Martins et al., unpublished data).

SoxS does not seem to be a virulence factor in S. enterica serovar Typhimurium. Both soxRS (Martins et al., unpublished data) and soxS (16) strains were not attenuated in mouse infection experiments and had no disadvantage in survival inside activated macrophages. Of the S. enterica serovar Typhimurium genes under soxRS control, sodA confers some protection against early killing by macrophages, but a sodA strain of S. enterica serovar Typhimurium was not found to be attenuated in virulence assays (36). However, there is evidence that the soxRS regulon could be contributing to the development of infection in humans through conferring elevated levels of multiple antibiotic resistance. Preliminary results show that expression of SoxS in S. enterica serovar Typhimurium is sufficient to enhance resistance to nalidixic acid and chloramphenicol (Pomposiello and Demple, unpublished data). The enhancement of antibiotic resistance via activation of the soxRS regulon could be a stepping-stone in the acquisition of clinically relevant antibiotic resistance, as has been proposed for the marA regulon (1). This hypothesis is supported by the isolation of soxS-constitutive S. enterica serovar Typhimurium (Martins et al., unpublished data) and pathogenic E. coli strains (31) from human patients with systemic infections. In one case of S. enterica serovar Typhimurium that developed increased antibiotic resistance during an infection, a mutation that contributes to the phenotype and that was mapped to a single-nucleotide change in the soxR gene arose (Martins et al., unpublished data). Thus, activation of the soxRS regulon by redox-active compounds generated by the immune system (19) could paradoxically enhance the antibiotic resistance of enteropathogenic bacteria and complicate the treatment of bacterial infections.