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J Bacteriol. Dec 2010; 192(24): 6428–6438.
Published online Oct 15, 2010. doi:  10.1128/JB.00916-10
PMCID: PMC3008532

Expression of the Streptomyces coelicolor SoxR Regulon Is Intimately Linked with Actinorhodin Production[down-pointing small open triangle]

Abstract

The [2Fe-2S]-containing transcription factor SoxR is conserved in diverse bacteria. SoxR is traditionally known as the regulator of a global oxidative stress response in Escherichia coli, but recent studies suggest that this function may be restricted to enteric bacteria. In the vast majority of nonenterics, SoxR is predicted to mediate a response to endogenously produced redox-active metabolites. We have examined the regulation and function of the SoxR regulon in the model antibiotic-producing filamentous bacterium Streptomyces coelicolor. Unlike the E. coli soxR deletion mutant, the S. coelicolor equivalent is not hypersensitive to oxidants, indicating that SoxR does not potentiate antioxidant defense in the latter. SoxR regulates five genes in S. coelicolor, including those encoding a putative ABC transporter, two oxidoreductases, a monooxygenase, and a possible NAD-dependent epimerase/dehydratase. Expression of these genes depends on the production of the benzochromanequinone antibiotic actinorhodin and requires intact [2Fe-2S] clusters in SoxR. These data indicate that actinorhodin, or a redox-active precursor, modulates SoxR activity in S. coelicolor to stimulate the production of a membrane transporter and proteins with homology to actinorhodin-tailoring enzymes. While the role of SoxR in S. coelicolor remains under investigation, these studies support the notion that SoxR has been adapted to perform distinct physiological functions to serve the needs of organisms that occupy different ecological niches and face different environmental challenges.

The [2Fe-2S]-containing transcription factor SoxR belongs to the MerR family of transcriptional regulators. Members of this family mediate protection against various environmental insults, such as toxic metals and antibiotics and, in the case of SoxR, oxidative stress imposed by redox-cycling drugs and nitric oxide (reviewed in reference 4). Grouped together based on structural similarity, these homodimeric proteins also regulate gene expression via a common mechanism. Each subunit contains an N-terminal helix-turn-helix DNA binding domain, a coiled-coiled dimerization domain, and a C-terminal regulatory domain that senses the appropriate stimulus. MerR-type proteins activate transcription from promoters with a suboptimal spacing of 19 ± 1 bp between the −35 and −10 elements, which normally precludes open complex formation by RNA polymerase. MerR family members bind to an operator with dyad symmetry located between the −35 and −10 elements of their target promoters and, when activated, unwind the promoter, thereby facilitating initiation by RNA polymerase (25, 26, 27).

Best studied in Escherichia coli (and related enteric bacteria), SoxR senses redox stress via [2Fe-2S] clusters that are anchored by four cysteine residues located in the C-terminal regulatory domain of each monomer (3) (Fig. (Fig.1).1). The redox signal is transduced into a global cellular response via a second transcription factor, SoxS (reviewed in reference 45). SoxR is constitutively expressed and remains attached to the soxS promoter in the absence or presence of oxidative stress. However, in the absence of oxidants, SoxR exists in its resting state with reduced [2Fe-2S] clusters and soxS is not transcribed (28, 29). Exposure to redox-cycling drugs causes one-electron oxidation of SoxR's [2Fe-2S] centers, and the oxidized protein facilitates soxS transcription by mediating a structural change in the promoter DNA that stimulates initiation by RNA polymerase (14, 15, 21, 22, 28). SoxS in turn recruits RNA polymerase to transcribe the remaining members of the regulon (>100 genes), whose products facilitate the removal of reactive oxygen species and repair any damage that may have occurred during stress (46). Seemingly complex, this system is designed to orchestrate an efficient and rapid activation of the large repertoire of functions required to reestablish redox balance.

FIG. 1.
SoxR homologs from different bacteria are highly conserved. Alignment of SoxR proteins from E. coli (ECO), P. aeruginosa (PAU), S. griseus (GRI), S. scabies (SCA), S. clavuligerus (CLA), S. avermitilis (SAV), and S. coelicolor (SCO). The boxes below the ...

The soxR gene is conserved in a diverse population of bacteria, both Gram positive and Gram negative. Sequence comparison of SoxR proteins reveals a highly conserved DNA binding domain, suggesting that SoxR homologs will bind to similar DNA target sequences (Fig. (Fig.1).1). Furthermore, the four cysteine residues that anchor the [2Fe-2S] clusters in E. coli SoxR are also perfectly conserved, arguing that all SoxR homologs will be redox regulated (Fig. (Fig.1).1). Despite their biochemical similarity, SoxR homologs have been shown by recent studies to play distinct roles in different organisms. Pseudomonas putida and Pseudomonas aeruginosa, for instance, do not rely on SoxR for an oxidative stress response and, notably, also lack a soxS homolog (43, 44). While the function of SoxR in P. putida is still unknown, in P. aeruginosa, SoxR is activated by endogenously produced redox-active phenazine antibiotics and directly stimulates transcription of two efflux pumps and a putative flavin-dependent monooxygenase, believed to aid in phenazine transport and modification, respectively (12, 13, 44). Although the biological significance of this is still under investigation, a soxR-deficient mutant manifests an altered colony morphology and deregulated phenazine transport, oversecreting blue pyocyanin and undersecreting yellow and red phenazines (12).

The story that begins to unfold suggests that SoxR-mediated signal transduction pathways are distinctly adapted in different organisms. While soxR is highly conserved in a range of bacteria, soxS is found exclusively in enterics, where it is the sole SoxR target and where it assists SoxR in launching a global oxidative defense program. A comprehensive in silico search for putative SoxR-regulated genes in bacterial genomes predicts that the P. aeruginosa paradigm will predominate over the traditional E. coli model in the vast majority of bacterial species, all of which lack soxS (12). More specifically, for antibiotic-producing bacteria it is hypothesized that SoxR has evolved to respond to endogenous redox-active metabolites and activate machinery to process/transport these metabolites. This has been confirmed for P. aeruginosa but not for other model systems.

In this work, we explore if this prediction can be extended to the phylogenetically distant Gram-positive soil bacterium Streptomyces coelicolor. Members of this genus are notable for producing two-thirds of the biologically active secondary metabolites used in clinical medicine. S. coelicolor, the genetically best characterized member of this genus, produces at least four structurally diverse antibiotics, including the red-pigmented tripyrrole undecylprodigiosin (Red) and the blue-pigmented benzochromanequinone polyketide actinorhodin (Act). Production of these antibiotics is growth phase dependent and coincides with the onset of morphological differentiation on agar-grown cultures. Circumstantial evidence implicates SoxR in modulating gene expression in response to the pigmented antibiotics Red and/or Act in this bacterium. The expression levels of two genes, SCO2478 and SCO4266, identified bioinformatically as potential SoxR targets, were significantly reduced in an S. coelicolor mutant that does not produce either Red or Act (12). SCO2478 and SCO4266 are predicted to encode oxidoreductases with homology to enzymes that modify small molecules (including antibiotics) in other bacterial species (17, 55). These observations suggest that SoxR may be functionally conserved and similarly regulated in the evolutionarily distant antibiotic producers P. aeruginosa and S. coelicolor. To determine if this is true we sought to (i) identify additional SoxR-regulated genes in S. coelicolor and (ii) examine the dependence of SoxR activity on the specific antibiotics produced by S. coelicolor. Herein, we have provided evidence for a five-gene SoxR regulon whose expression depends on a functioning Act biosynthetic pathway. We note that the SoxR regulon is conserved in other members of the Streptomyces genus and consider possible physiological roles for SoxR in Streptomyces.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.

The S. coelicolor strains used in this study (Table (Table1)1) were cultured at 30°C on R2YE, SMMS, mannitol soya flour, or nutrient agar plates (36). E. coli ET12567 (38), BW25113 (11), and BT340 (9) were used for PCR-targeted disruptions (24), BL21 (λDE3) (Novagen) was used to overproduce recombinant SoxR, and DH5α (Gibco BRL) was used as the host for routine cloning. Media, culture conditions, and DNA manipulations for E. coli were carried out as described previously (49). The plasmids used were pIJ773 (24), pSET152 (2), pSE380 (Stratagene), and pET16b (Novagen).

TABLE 1.
Streptomyces coelicolor strains used in this study

Construction and complementation of soxR deletion strains.

The soxR open reading frame (from codons 1 to 141) was replaced with an apramycin resistance cassette in the chromosomes of M145 and M511 by use of the REDIRECT PCR-targeting methodology (24). A gene replacement cassette containing the RK2 origin of transfer (oriT) and an apramycin resistance gene (apr) was PCR amplified from pIJ773 by use of primers FOR-KO and REV-KO, which contain 39-nucleotide (nt) 5′ homology extensions corresponding to the soxR gene (see Table S1 in the supplemental material). Purified PCR product was recombined into the soxR-containing cosmid StI30A, using E. coli BW25113/pIJ790 as a host. The resulting recombinant cosmid, StI30M, was passaged through the methylation-deficient host, E. coli ET12567 carrying the nontransmissible oriT-mobilizing plasmid pUZ8002 (36), and then transferred into S. coelicolor strains M145 and M511 by conjugation. apramycin-resistant, kanamycin-sensitive double-crossover exconjugants (M145-1 and M511ΔsoxR, respectively) were identified and purified (Table (Table1).1). A markerless deletion allele of soxR was also constructed in the M145 background. Cosmid StI30M was introduced into E. coli BT340, and the apr-oriT cassette (which is flanked by FLP recognition target sites) was deleted by inducing FLP recombinase (24). The mutant cosmid StI30Mscar was introduced into M145-1 by protoplast transformation, and Kanr transformants were selected. After one round of nonselective growth, a colony that had lost both apramycin and kanamycin resistance was selected and purified (M145-1A) (Table (Table1).1). All soxR disruptions were confirmed by PCR and Southern analyses, and reverse transcription-PCR (RT-PCR) further confirmed the absence of soxR message (data not shown).

To complement M145-1A, SCO1697 (soxR), along with 300 bp of upstream sequence (which includes the soxR promoter), was PCR amplified (using primers 152F-Bam and 152R-Bam) (see Table S1 in the supplemental material) from M145 genomic DNA and cloned into the BamHI site of the integrating vector pSET152 to yield pSET152::soxR. Plasmids pSET152 and pSET152::soxR were introduced into M145-1A by conjugation from E. coli ET12567/pUZ8002.

Cloning, expression, and purification of recombinant SoxR protein.

The soxR coding region was PCR amplified from M145 chromosomal DNA by use of primers SC-Eco and SC-Hind (see Table S1 in the supplemental material) and cloned into the EcoRI/HindIII sites of pSE380 to yield pSE-SCSoxR. For overexpression in E. coli, the soxR coding region was PCR amplified from pSE-SCSoxR by use of primers SC-C and SC-D (Table S1) and cloned into the NdeI/BamHI sites of pET16b, downstream of a 10-histidine tag. The recombinant N-terminal His-tagged S. coelicolor SoxR protein was overexpressed in E. coli BL21(λDE3) and affinity purified on a 1-ml nickel-nitrilotriacetic acid agarose column (Qiagen) as described for the E. coli SoxR protein (7), except the S. coelicolor SoxR protein was eluted in buffer containing 350 mM imidazole. Protein concentrations were determined with a Bradford protein assay kit (Bio-Rad), using bovine serum albumin as a standard. Purified SoxR typically was >95% homogeneous, as estimated from visual inspection of Coomassie blue-stained SDS-polyacrylamide gels.

Construction of the C118A mutant.

The [2Fe-2S] clusters in SoxR were eliminated by changing the cysteine at position 118 to alanine, using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Plasmid pSET152::soxR was used as a template for mutagenesis along with primers C118A-F and C118A-R (see Table S1 in the supplemental material). The resulting mutant clone, pSET152::C118A, was introduced into M145-1A by conjugation from E. coli ET12567/pUZ8002 for in vivo studies. For overexpression and purification from E. coli, the C118A mutation was introduced as described above into soxR cloned in the pET16b vector. All mutations were confirmed by DNA sequence analysis, and the mutant protein was overexpressed and affinity purified from E. coli as described above.

Electrophoretic mobility shift assays (EMSAs).

Binding of SoxR to promoter DNA was assessed by gel mobility shift assays. DNA probes were generated by PCR amplification using M145 genomic DNA and the primers listed in Table S1 in the supplemental material. Purified probe DNAs were end labeled with digoxigenin (DIG; Roche) according to the manufacturer's instructions, and 5 fmol was incubated individually with histidine-tagged wild-type (WT) or C118A mutant SoxR (0 to 20 nM) for 15 min at 25°C in binding buffer [10 mM Tris-HCl, pH 7.5, 75 mM KCl, 2 mM dithiothreitol (DTT), 10% glycerol, 2 mM MgCl2, 0.1 μg poly(dI)-poly(dC)] in a total volume of 30 μl. For competition assays, 2,500 fmol of unlabeled probe (specific competitor) or an unlabeled, nonspecific target (160-bp fragment containing the SCO7009 promoter region) was also added to the reaction mixtures. Protein-bound and uncomplexed DNA products were separated on a 5% polyacrylamide gel (3.3 mM sodium acetate, pH 7.9, Tris-HCl, pH 8, 1 mM EDTA, 2% glycerol) that was run at 10°C and 180 V for 90 min. The DNA was transferred to nylon membranes (Roche) and detected chemiluminescently using a DIG gel shift 2nd-generation kit (Roche).

qRT-PCR.

Total RNA was extracted from cells cultured on cellophane-overlaid R2YE plates. For harvest, cells were incubated with RNAprotect bacterial reagent (Qiagen) for 5 min at room temperature, scraped off the cellophane membranes, centrifuged for 10 min at 5,000 × g, and frozen at −80°C. Cells were lysed by incubating them at 30°C for 30 min in TE buffer containing 3.3 mg/ml lysosome, followed by sonication. Total RNA was extracted from lysed cells by use of an RNeasy plant minikit (Qiagen) according to the manufacturer's instructions. Genomic DNA was removed by treatment with 5 units of RNase-free DNase I (Qiagen) for 1 h at 37°C. The RNA preparation was extracted once with acidic phenol-chloroform and ethanol precipitated. The absence of contaminating genomic DNA was confirmed by PCR using HrdB primers (see Table S1 in the supplemental material). cDNA was generated with iScript (Bio-Rad) and served as a template for quantitative real-time RT-PCR (qRT-PCR) (StepOne PCR machine; Applied Biosystems). The primers for qRT-PCR (Integrated DNA Technologies) were designed using Primer3 software (48), using a melting temperature of 60°C, an ~20-nt length, and an amplicon length of ~100 bp (Table S1). Each 20-μl qRT-PCR mixture contained 25 ng cDNA, 250 μM each forward and reverse primer, and 10 μl Power Sybr green PCR master mix (Applied Biosystems). The reaction parameters were as follows: 95°C for 10 min, followed by 40 two-step amplification cycles consisting of 15 s of denaturation at 95°C and 1 min of annealing and extension at 60°C. A final dissociation stage was run to generate a melting curve and verify the specificity of the amplification products. Samples were assayed in triplicate, and the target signal was standardized to the level for hrdB (housekeeping sigma factor).

Oxidant susceptibility tests.

The effects of various oxidants on the growth of M145 and M145-1A were determined using a disk diffusion assay. S. coelicolor spores (~108) were added to 4 ml of melted soft nutrient agar (Difco) and plated on nutrient agar plates (Difco). Six-millimeter Whatman paper disks impregnated with the various oxidants were then placed onto the agar. The plates were incubated at 30°C for 48 h, and the zone of growth inhibition around each disk was recorded. The drugs tested were paraquat, 4-nitroquinoline, phenazine methosulfate, plumbagin, menadione, diamide, H2O2, and tert-butyl hydroperoxide at the concentrations indicated (all reagents were purchased from Sigma). Pyocyanin was a generous gift from Lars Dietrich.

Solid-culture antibiotic quantification.

Wild-type and ΔsoxR mutant S. coelicolor spores (~108) were cultured on cellophane-overlaid R2YE plates for 3 and 5 days to assay for Red and Act, respectively. Cells were scraped off the cellophane, washed with water, retrieved by vacuum filtration, and dried at 75°C prior to weighing. Red was extracted with acidified methanol and Act with 1 N KOH as described previously (5, 30). Both pigments were quantified spectroscopically using published extinction coefficients (5, 30).

RESULTS

The S. coelicolor soxR mutant is not hypersensitive to oxidizing agents and lacks an obvious morphological phenotype.

With the notable exception of the pseudomonads, SoxR mediates protection against oxidative stress in all bacterial groups in which it has been studied (including E. coli, Salmonella enterica serovar Typhimurium, Agrobacterium tumefaciens, and Vibrio vulnificus) (16, 23, 35, 37). To determine if SoxR performs in a similar capacity in S. coelicolor, we tested the effects of various superoxide-generating compounds on the growth of wild-type and ΔsoxR S. coelicolor strains. An E. coli ΔsoxR mutant is hypersensitive to the drugs 4-nitroquinoline, phenazine methosulfate, and paraquat (23). In contrast, disk diffusion assays revealed that the wild-type and ΔsoxR S. coelicolor strains were equally sensitive to killing by these agents (Fig. (Fig.2)2) and other superoxide-generating drugs tested (data not shown for menadione, plumbagin, and pyocyanin). Treatment with other standard oxidizing agents, including H2O2, organic peroxides, and diamide, also did not reveal significant differences between the strains (Fig. (Fig.22 and data not shown). Thus, similar to the pseudomonads, S. coelicolor does not rely on SoxR for protection against oxidative stress.

FIG. 2.
The ΔsoxR S. coelicolor mutant is not hypersensitive to peroxide or superoxide-generating agents. Paper disks soaked with solutions of 4-nitroquinoline, phenazine methosulfate, paraquat, or hydrogen peroxide were placed on bacterial lawns of wild-type ...

As described earlier, it has been suggested that SoxR may be functionally conserved in S. coelicolor and P. aeruginosa, both soil-dwelling, antibiotic-producing bacterial species (12). SoxR modulates colony development and antibiotic secretion in the latter; as such, a P. aeruginosa soxR-null mutant exhibits increased secretion of the blue antibiotic pyocyanin and forms colonies with a smoother texture than the wild type (12). To determine if soxR deficiency results in a similar phenotype in S. coelicolor, we examined by light microscopy the morphology of wild-type and ΔsoxR S. coelicolor cells growing on different media (rich R2YE, minimal SMMS, and sporulation mannitol soya flour) over a 2-week period. Since it is well established that morphological and biochemical differentiation in Streptomyces is influenced by nutritional conditions, different media were used to determine whether any observed phenotype was medium specific. Our analyses revealed that there was no discernible phenotypic difference between the wild-type and soxR deletion strains (data not shown). Quantification of Red and Act isolated from surface-grown cultures also did not show a marked difference between the two strains (data not shown).

The putative SoxR regulon consists of five genes whose expression is developmentally regulated.

The phenotypic analysis of the soxR mutant described above failed to offer any clues as to the physiological role of SoxR in S. coelicolor. As such, we decided to adopt a bioinformatic approach and identify SoxR-regulated genes in this bacterium. We reasoned that the nature of these genes would provide insight into SoxR function. A previously conducted in silico search for SoxR-regulated genes in S. coelicolor, based on similarity with the E. coli soxS promoter, identified SCO2478 and SCO4266 as potential candidates (12). These two genes encode putative oxidoreductases with homology to antibiotic-tailoring enzymes (17, 53). The S. coelicolor genome contains three additional potential SoxR target genes, not reported by Lars Dietrich and coworkers, as they fell below the threshold set by these investigators (personal communication). These genes include a putative ABC transporter (SCO7008), a putative monooxygenase (SCO1909), and a predicted NAD-dependent epimerase/dehydratase (SCO1178) (Fig. (Fig.33 and Table Table2).2). Notably, SCO1909 is similar to PA2274, a confirmed SoxR-regulated gene in P. aeruginosa (12, 43). Like the E. coli soxS promoter and other promoters regulated by MerR family proteins, the promoters of the putative SoxR regulon in S. coelicolor have an elongated 19-bp spacer between their predicted −10 and −35 sequences (Fig. (Fig.33).

FIG. 3.
The promoters of putative SoxR-regulated genes in S. coelicolor contain conserved binding sites for SoxR. The E. coli soxS promoter is aligned with the promoters of predicted S. coelicolor SoxR-regulated genes, SCO2478, SCO4266 (ecaD), SCO7008 (ecaA), ...
TABLE 2.
The S. coelicolor SoxR regulon is conserved in other Streptomyces species

Interestingly, four of the putative SoxR target genes (SCO7008, SCO1909, SCO1178, and SCO4266) had previously been identified as genes whose expression was temporally coordinated with Act (eca) and whose levels were significantly reduced in a mutant that lacks the Act biosynthetic pathway-specific activator actII-ORF4 (32). We independently verified the developmental expression of SCO7008 (ecaA), SCO1909 (ecaB), SCO1178 (ecaC), and SCO4266 (ecaD) by qRT-PCR. In addition to these genes, we followed the expression of the fifth putative SoxR target, SCO2478, as well as SCO7009, an eca gene (ecaE) that is not predicted to be under SoxR control (because its promoter lacks an obvious SoxR binding sequence). Wild-type cells were grown on cellophane-overlaid R2YE agar medium, and samples were harvested after 1, 2, 3, or 5 days of incubation to obtain RNA for qRT-PCR. The five putative SoxR target genes exhibited growth phase-dependent expression and were maximally expressed between 3 and 5 days postinoculation (Fig. (Fig.4).4). This timing coincided with the appearance of Act under our experimental conditions (Red was apparent 24 h earlier) and is in agreement with the findings of Huang and coworkers for ecaA-ecaD (32). In contrast to reports by Huang et al. (31, 32), we did not observe significant changes in ecaE expression over the 5-day period (Fig. (Fig.4).4). The experiments described below also indicated that ecaE expression was not decreased in an actII-ORF4 mutant (see Fig. 7). The inconsistent findings for ecaE may be explained by the different methodologies employed (microarray versus qRT-PCR). Regardless, since ecaE serves only as a negative control in our experiments, we did not pursue this issue further.

FIG. 4.
The putative S. coelicolor SoxR regulon is upregulated late in development. RNA was extracted from plate-grown wild-type S. coelicolor after 1 (D1), 2 (D2), 3 (D3), or 5 (D5) days of incubation at 30°C and used to generate cDNA for qRT-PCR. Signals ...

SoxR binds specifically to the promoters of its putative target genes.

The ability of SoxR to bind to the promoter regions of its five putative target genes was assessed by EMSAs using purified N-terminally histidine-tagged SoxR and DIG-end-labeled DNA fragments spanning the various promoters. SoxR bound with high affinity to all five promoters; the amounts of protein needed to bind 50% of the DNA ranged from 1 to 5 nM (Fig. (Fig.5).5). This affinity compares with that of E. coli SoxR for the soxS promoter (7). In contrast, SoxR did not bind to a control DNA fragment spanning the promoter of ecaE (which is not predicted to be under SoxR control) (Fig. (Fig.5).5). Specificity of binding was examined through competition assays. The addition of a 500-fold excess of unlabeled probe abolished binding of SoxR to labeled probe DNA (Fig. (Fig.5).5). Addition of a 500-fold excess of nonspecific ecaE promoter probe had little effect on binding of SoxR to the promoters of SCO2478, ecaD, and ecaA but did compete slightly with binding to the promoters of ecaB and ecaC (Fig. (Fig.5).5). This probably reflects a reduced affinity of SoxR for the latter promoters, which harbor an imperfect inverted repeat sequence (Fig. (Fig.33).

FIG. 5.
SoxR binds to the promoters of its five predicted target genes. DIG-end-labeled DNA fragments spanning the promoter regions of SCO2478, ecaD, ecaA, ecaB, ecaC, and negative-control ecaE were incubated with various amounts of purified histidine-tagged ...

Quantitative real-time PCR confirms SoxR-dependent expression of the SoxR regulon.

The gel shift assays described above demonstrated that SoxR binds specifically to the promoters of its five putative target gene promoters in vitro. To examine in vivo regulation by SoxR, the expression levels of the five putative SoxR targets in the wild type and the ΔsoxR mutant were measured by qRT-PCR. Since the putative SoxR regulon was maximally expressed between days 3 and 5 postinoculation, total RNA was isolated from 4-day-old cultures (while Act was actively produced) and subjected to qRT-PCR. The analysis revealed that the mRNA levels of the five putative SoxR target genes were significantly reduced in the ΔsoxR mutant compared with the wild type (6- to 150-fold), confirming that these genes are indeed under SoxR control (Fig. (Fig.6A).6A). Conversely, ecaE expression levels were not significantly different in the two strains, as expected.

FIG. 6.
Quantitative RT-PCR confirms SoxR-dependent expression of the SoxR regulon in S. coelicolor. (A) The expression level of the five-gene SoxR regulon is significantly lower in a ΔsoxR mutant than in the wild type. Wild-type and ΔsoxR mutant ...

To confirm that the regulatory defect of the ΔsoxR mutant was due to the absence of soxR, and not to any polar effects resulting from the deletion, we conducted a complementation experiment. To this end, we utilized qRT-PCR to analyze the expression changes of SCO2478 and ecaD in the complemented ΔsoxR strain (which expresses soxR from a chromosomally integrated plasmid, pSET152). Figure Figure6B6B shows that episomal expression of soxR restored the soxR mutant's ability to induce SoxR-dependent genes. Together with the EMSA results, these results demonstrate that SoxR directly activates the expression of five genes in S. coelicolor.

Expression of the SoxR regulon is significantly reduced in an actinorhodin-deficient mutant.

SoxR is constitutively expressed in E. coli and P. aeruginosa, yet it activates transcription of its target genes only upon receiving the appropriate signal. This scenario is apparently preserved in S. coelicolor, where soxR mRNA was detected throughout development, but the SoxR regulon was significantly expressed only late in development, following the production of the pigmented antibiotics Red and Act (Fig. (Fig.4).4). This pattern suggested that Red and/or Act (or biosynthetic precursors of either antibiotic) might trigger SoxR activity. To test this hypothesis, expression of the SoxR regulon in M511 (a strain with an in-frame deletion of actII-ORF4, the pathway-specific regulator of Act biosynthesis), M510 (a ΔredD mutant lacking the pathway-specific activator of Red synthesis), and the pigment-deficient strain M512 (ΔredD ΔactII-ORF4) was analyzed (20). qRT-PCR analysis showed that while soxR was expressed at comparable levels in the wild type and the Act-null mutant M511 (data not shown), expression of the five SoxR target genes was significantly reduced in the Act-null mutant relative to the wild type (20- to 70-fold), demonstrating that expression of the Act pathway is necessary for SoxR activity (Fig. (Fig.7A).7A). In contrast, the SoxR regulon was expressed at similar levels in the wild type and the Red-null mutant M510, thereby eliminating Red as an effector of SoxR activity (Fig. (Fig.7A).7A). SoxR-regulated gene expression in M512 was not significantly different from that in M511, indicating that Red and Act do not act synergistically to regulate SoxR (Fig. (Fig.7A).7A). As mentioned earlier, expression of ecaE, which was identified by microarray analysis as a gene whose expression was coordinated with Act, was not reduced in the Act mutant strain M511 compared to the wild type in our experiments (Fig. (Fig.7A7A).

FIG. 7.
Expression of the SoxR regulon depends on production of actinorhodin. (A) Expression of the five-gene SoxR regulon is significantly decreased in an Act-deficient mutant. qRT-PCR was performed on RNA extracted from 4-day-old plate-grown wild-type, Red-deficient ...

The data presented so far demonstrate that the S. coelicolor SoxR regulon consists of five genes and that SoxR activity (and thus regulon expression) is dependent on a functional Act biosynthetic pathway. Huang and coworkers had independently observed the Act-dependent expression of four of these genes, ecaA-ecaD, and they postulated that these genes may be directly activated by the Act pathway-specific activator ActII-ORF4 (32). This suggestion was based on the observation that the promoters of ecaA, -B, and -C contain the sequence 5′-TCGAG, which is a potential ActII-ORF4 docking site (1) (Fig. (Fig.3).3). We entertained the possibility that these genes are under dual regulation by SoxR and ActII-ORF4. If true, then one would expect their expression to be more severely defective in a ΔactII-ORF4 ΔsoxR double mutant than in either of the single mutants. To test this dual-regulation mechanism, we compared the expression levels of ecaA, -B, and -C in M145-1A (ΔsoxR), M511 (ΔactII-ORF4), and M511ΔsoxRactII-ORF4 ΔsoxR) and found that these genes were expressed at comparable levels in ΔsoxR and the ΔactII-ORF4 ΔsoxR double mutant (Fig. (Fig.7B).7B). From this, we conclude that ecaA, -B, and -C are under the direct control of SoxR, while ActII-ORF4 acts indirectly by turning on the Act biosynthetic pathway, a product of which modulates SoxR activity.

The [2Fe-2S] clusters in SoxR are essential for transcriptional activity.

[2Fe-2S] clusters are essential for E. coli SoxR activity (3) and, although this has not been formally demonstrated (by mutagenic analysis), presumably also for the activity of the pseudomonad SoxR proteins. In these organisms, redox-active agents oxidize SoxR's [2Fe-2S] clusters to activate the protein. To confirm if SoxR is similarly redox regulated in S. coelicolor, we created a [2Fe-2S]-deficient mutant by replacing the cysteine at position 118 with alanine (Fig. (Fig.1).1). The equivalent mutation in E. coli SoxR results in elimination of [2Fe-2S] clusters and an inability to activate soxS transcription (3). We confirmed the absence of [2Fe-2S] clusters by monitoring the optical spectrum of the purified C118A mutant protein. While wild-type SoxR produced an absorption spectrum characteristic of [2Fe-2S]-containing proteins, the C118A variant did not (Fig. (Fig.8A).8A). Gel shift assays conducted with purified histidine-tagged proteins showed that while the C118A mutant protein retains the ability to bind to the promoters of ecaD and SCO2478, it binds with a lower level of affinity than wild-type SoxR (Fig. (Fig.8B8B and data not shown). This has also been observed with cluster-deficient E. coli SoxR, which exhibits a promoter-binding defect both in vivo and in vitro (7, 8). We introduced the C118A soxR mutant gene into the ΔsoxR strain via the pSET152 vector to allow chromosomal integration of the gene via the att site and monitored the transcription of soxR itself, and two SoxR target genes, SCO2478 and ecaD, by qRT-PCR. The results indicated in Fig. Fig.8C8C show that while complementation with wild-type soxR restored upregulation of SCO2478 and ecaD, the C118A mutant was unable to rescue the ΔsoxR defect. Therefore, [2Fe-2S] clusters are critical cofactors required for transcriptional activity of S. coelicolor SoxR.

FIG. 8.
[2Fe-2S] cofactors are critical for SoxR activity. (A) Absorbance spectra of wild-type and C118A SoxR proteins. The spectrum of wild-type SoxR shows four peaks, at 332, 414, 462, and 548 nm, which is characteristic of [2Fe-2S] proteins (42). The C118A ...

DISCUSSION

In the past decade, several groups have explored the function of SoxR homologs in different bacteria. This activity has been motivated by the observation that while soxR is highly conserved, its well-characterized partner in E. coli, soxS, is absent from the genomes of nonenteric species, suggesting that the physiological role and signal transduction pathways mediated by SoxR are distinctly adapted in different bacteria. The few species examined to date have revealed this to be true. In E. coli and related enterics, SoxR defends against superoxide stress generated by exogenous redox-cycling drugs. In these bacteria, SoxR's effect is mediated via SoxS. SoxR appears to function in a similar capacity in the Gram-negative pathogens V. vulnificus and A. tumefaciens, where soxR deficiency results in hypersensitivity to superoxide stress (16, 35, 37). Here, SoxR activates manganese-containing superoxide dismutases in a SoxS-independent manner (16, 35, 37). The two other organisms studied, P. aeruginosa and P. putida, do not employ SoxR for protection against oxidative stress (43, 44). Rather, SoxR regulates the expression of phenazine efflux and modification machinery in response to endogenously produced phenazine antibiotics in P. aeruginosa (12, 13). The P. putida SoxR regulon has not been elucidated empirically, but in silico analysis of the genome shows SoxR binding sites upstream of an RND transporter (encoded by PP2063-PP2065), an oxidoreductase (encoded by PP2507), and a GGDEF-containing protein (encoded by PP2505) (data not shown). Recently, it was demonstrated that the genes encoding these proteins (among other genes) are induced upon exposure to 2,4,6-trinitrotoluene, a nitroaromatic compound that causes toxicity by increasing cellular levels of reactive oxygen species and hydroxylamine compounds (18).

In this work, we have described a five-gene SoxR regulon in S. coelicolor whose products are predicted to encode an ABC transport protein (SCO7008 [ecaA]), two oxidoreductases (SCO2478 and SCO4266 [ecaD]), a monooxygenase (SCO1909 [ecaB]), and an NAD-dependent epimerase/dehydratase (SCO1178 [ecaC]) (Table (Table2).2). It is noteworthy that ecaD is similar to oxidoreductases (ActVI-ORF2 and ActVI-ORF4), and ecaB to a monooxygenase (ActVA-ORF6), that catalyze later steps in the Act biosynthetic pathway. These tailoring reactions lead to the production of a series of biologically active metabolites and finally Act (33, 41, 50). Also of note is that PA2274, a SoxR-regulated monooxygenase in P. aeruginosa, is predicted (based on sequence homology) to belong to the same class of monooxygenases as ecaB and ActVA-ORF6 (50). Thus, the S. coelicolor SoxR regulon resembles the P. aeruginosa equivalent in encoding a membrane transport protein and enzymes that modify small molecules. In these antibiotic producers, the SoxR regulons are expressed when specific redox-active antibiotics are made, Act in S. coelicolor and pyocyanin in P. aeruginosa, supporting the view that SoxR is activated by endogenously produced antibiotics and mediates the efflux and turnover of the same in the producer bacteria. It is interesting that the S. coelicolor SoxR regulon appears to be conserved in other members of this genus of prolific antibiotic producers, suggesting that SoxR plays an analogous role in all antibiotic producers where it is present. The completely sequenced genomes of Streptomyces avermitilis, S. clavuligerus, S. griseus, and S. scabies harbor all (or a subset) of the S. coelicolor SoxR-regulated genes (Table (Table2).2). The genes listed in Table Table22 all contain a putative SoxR binding site in their promoters (data not shown; sequences available from http://strepdb.streptomyces.org.uk/). ecaB and ecaC homologs are absent in S. avermitilis; however, this bacterium contains three other genes that are potentially under SoxR control, including SAV_1623 (putative transketolase), SAV_4018 (putative dehydrogenase), and SAV_4017 (putative TetR family transcriptional regulator). The last is intriguing and implies that SoxR could potentially control gene expression at a more global level via a second transcription factor, analogous to the situation in E. coli.

What is the biological significance of SoxR activity? On the basis of the identities of the SoxR regulon genes, the most obvious inference is protection of the producer organism from the toxic effects of endogenous antibiotics. Consistent with this theory, deletion of mexGHI-ompD (a SoxR-regulated transporter of phenazines in P. aeruginosa) results in reduced fitness (12). Given the apparent similarities between the S. coelicolor and P. aeruginosa systems, we were surprised to not observe any reduced viability (as assessed by colony size) or indeed any visible phenotype of the S. coelicolor soxR mutant. The simplest explanation to account for this is that S. coelicolor harbors multiple modes of self-resistance toward Act; in the event that one mechanism malfunctions, compensatory mechanisms would ensure that the producer organism remains safe. Indeed, genetic evidence implicates the products of actII-ORF2/ORF3 and actVA-ORF1 as significant players in the export of Act and derivative blue pigments (5, 19). An actII-ORF2/ORF3 actVA-ORF1 triple mutant strain showed a significant reduction in export of blue pigments compared to the wild type, but notably, efflux was not completely abolished (5). We speculate that in the absence of the major Act exporters, the SoxR-regulated ABC transporter ecaA compensates. It is interesting that ecaA is the least upregulated of the SoxR regulon genes. Thus, if involved in Act export, it may be a minor player. We were unable to detect any difference in the amount of blue pigments produced or secreted by wild-type or soxR mutant strains (data not shown).

The four other members of the SoxR regulon could also potentially mediate Act detoxification. As mentioned before, two of the predicted gene products are similar to enzymes that modify biosynthetic precursors of Act. It is feasible that the SoxR-regulated redox enzymes chemically modify Act precursors, converting them into less toxic species. This additional protective mechanism would function in conjunction with drug efflux. The use of multiple resistance mechanisms against a single toxic product is not uncommon (10). Streptomyces lavendulae, for example, protects itself against the deleterious effects of mitomycin via three distinct modes: drug inactivation by a flarin adenine dinucleotide-dependent oxidoreductase (34), drug export (51), and drug sequestration (52). That S. coelicolor employs multiple mechanisms to prevent self-destruction by Act would account for the fact that the actII-ORF2/ORF3 actVA-ORF1 triple mutant (which is defective in exporting Act) is viable (5), as is the soxR mutant.

One cannot discount an alternative function to Act-detoxification for the S. coelicolor SoxR regulon. The regulon products undoubtedly affect some process associated with Act, since their expression is intimately linked with the production of Act. But to what end? In recent years, there has been much debate about the ecological role of secondary metabolites. There is increasing appreciation that these metabolites, typically viewed as waste products or mediators of biowarfare, may in fact play important physiological roles in the producer organisms. The phenazine antibiotics produced by P. aeruginosa, for example, help in iron acquisition and energy generation under conditions of high cell density when oxygen availability becomes limited (47). Phenazines also act as intercellular signaling molecules and participate in a quorum-sensing network to coordinate biofilm formation in P. aeruginosa and Pseudomonas aureofaciens (12, 13, 39). SoxR may mediate some of these effects in the pseudomonads (12, 13). The physiological role of secondary metabolites in S. coelicolor is unknown, although it is postulated that they may act as differentiation effectors (i.e., promote sporulation) (6, 40, 54). This hypothesis is supported by the fact that secondary metabolite production is temporally associated with morphological differentiation, and many developmentally impaired mutants are also antibiotic deficient (and vice versa). Considering the complex genetic determinants encoding secondary metabolites and the metabolic resources devoted to their production, it is easy to envision that these metabolites play a beneficial role that confers a survival advantage in nature. SoxR may mediate part of the effects of Act in S. coelicolor, similar to its suggested role in P. aeruginosa. The reason we do not observe an associated phenotype with the soxR mutant is that laboratory conditions do not accurately mimic the natural ecological setting of S. coelicolor. We are designing screens to further investigate this.

What is the physiological signal that regulates SoxR activity in S. coelicolor? We can assume with reasonable certainty that the signal must be a redox-active molecule that regulates SoxR via its [2Fe-2S] clusters since the cluster-deficient SoxR mutant protein was defective in transcriptional activity (Fig. (Fig.8C).8C). We have provided evidence that this signal must be related to Act since SoxR does not induce its target genes in the absence of Act synthesis (Fig. (Fig.7A).7A). The trigger could be Act itself or a redox-active precursor or derivative of Act. It is interesting that the only documented Act resistance mechanism (the Act exporter described above) is activated by (S)-DNPA, a precursor of Act, and kalafungin, a molecule related to another precursor, dihydrokalafungin (53). It remains to be determined if these same molecules elicit SoxR activity.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by a start-up grant from Bryn Mawr College and an AREA grant from the National Institute of General Medical Sciences (R15GM093366) to M.C.

We are grateful to Lars Dietrich and Tracy Teal for sharing unpublished bioinformatic data and engaging in stimulating discussions, to Mervyn Bibb for providing strains M510, M511, and M512, to Amy Gehring for pSET152, to the John Innes Center for Redirect reagents, to William Malachowski for advice on redox-active molecules, and to Peter Setlow, Joanne Willey, and Lilian Hsu for helpful comments on the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 15 October 2010.

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

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