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Appl Environ Microbiol. Dec 2010; 76(23): 7741–7753.
Published online Oct 8, 2010. doi:  10.1128/AEM.00819-10
PMCID: PMC2988594

Repression of Antibiotic Production and Sporulation in Streptomyces coelicolor by Overexpression of a TetR Family Transcriptional Regulator [down-pointing small open triangle]

Abstract

The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its −35 promoter sequence. The interruption of this gene in S. lividans or S. coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches.

The development of the Gram-positive genus Streptomyces is characterized by a complex morphological differentiation process thought to be triggered by conditions of nutritional limitation that often correlate with high cell density (41). This process includes the arising of an aerial mycelium from the vegetative mycelium and then the differentiation of the tips of the aerial hyphae into spores (10, 15). This process is coupled to a metabolic differentiation that correlates with the production of a wide range of pharmaceutically important secondary metabolites, including antibacterial, anticancer, and immunosuppressive drugs (8, 10). The genes responsible for the biosynthesis of these secondary metabolites are clustered in the genome and coordinately regulated by pathway-specific transcriptional regulators (1, 4, 18, 19, 62, 70). The expression of these specific regulators linked to the biosynthetic pathways is directly or indirectly controlled either by positive pleiotropic regulators, such as AfsR2 (69), AfsS (29), AtrA (65), PtpA (67), PkaD (68), and the two-component systems AfsK/AfsR (66) and EcrA1/EcrA2 (39), by negative regulators, including the two-component systems AbsA1/AbsA2 (44), PhoR/PhoP (55), and CutR/CutS (9), or by enzymatic systems, such as Ppk (16). The pleiotropic regulators, thought to sense a variety of extracellular or intracellular signals related to nutriment availability, cell crowding, or energy shortage, are necessary to trigger the necessary metabolic adjustments to adapt to these conditions (27, 43), whereas the ppk gene is thought to act as an ATP-regenerating enzyme (54).

Streptomyces coelicolor A3(2) is usually used as the reference strain to study morphological and metabolic differentiation in relation with antibiotic biosynthesis (10, 15). S. coelicolor has long been known to produce four major antibiotics, actinorhodin (ACT) (40), undecylprodigiosin (RED) (13), methylenomycin (MMY) (36), and calcium-dependent antibiotic (CDA) (21), and recently, two novel ones were characterized, CPK, a putative type I polyketide (17, 50), and albaflavenone, a sesquiterpene antibiotic (72). ACT is a secondary metabolite of the polyketide family that is strongly produced by S. coelicolor but only weakly produced by its close relative, Streptomyces lividans. However, S. lividans has the genetic capability to produce this compound since the weak production of ACT could be stimulated by various genetic manipulations that include the overexpression of the pathway-specific activator gene actII-ORF4 (7), the afsR and afsR2 (now afsS) genes (35), the rep gene (42), and the phosphotyrosine protein phosphatase-encoding gene ptpA (67). The inactivation of other genes, such as ppk (11), or mutations in the rpoB gene (25) or in the ribosomal protein S12 (23) are also leading to an enhancement of ACT production, indicating that ACT production was subjected to complex positive and negative controls. Furthermore, the extracellular addition of the signal molecule S-adenosylmethionine (34) or of ATP (38) or the replacement of glucose by glycerol in a minimal medium (35) was also shown to enhance ACT production in S. lividans. However, a systemic understanding of the regulation of antibiotic biosynthesis and in particular of the molecular basis of the very different abilities of S. lividans and S. coelicolor to produce ACT is still lacking. In order to tackle this question, we hypothesized that a major negative regulator of ACT production might be present in S. lividans but absent or nonfunctional in S. coelicolor. In an attempt to get this putative negative regulator, a genomic library of S. lividans DNA was constructed into the high-copy-number replicative plasmid pIJ702 (33) and introduced into S. coelicolor. An unique white transformant (ACT) was obtained. This phenotype was shown to result from the overexpression of a regulator of the TetR family (equivalent to SCO3201 of S. coelicolor). The consequences of the overexpression and of the deletion of SCO3201 on the metabolic and morphological differentiation processes of S. coelicolor and S. lividans (wild type [wt] and ppk mutant) were assessed. The ability of SCO3201 to negatively regulate its own transcription was demonstrated by both in vitro and in vivo approaches, and the sequence of its operator site located in its own promoter region was determined. Sequences related to the SCO3201 operator site were found in the promoter region of scbA, a gene encoding a butyrolactone-synthesizing enzyme (61). These sequences were shown to be part of the binding site of ScbR, a TetR family regulator, positively controlling scbA expression, as SCO3201 does when overexpressed.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. S. coelicolor M145 (SCP1 SCP2) (33), S. lividans TK24 (str-6 SLP2 SLP3) (33), and its derived ppk mutant, the Δppk::Ωhyg strain (11), were used in this study. Escherichia coli strains DH5α and D41 were used for routine subcloning work and as a host for protein expression, respectively. For the biological assay of calcium-dependent antibiotic (CDA), Oxoid nutrient agar (ONA) medium (33) was used for cultivation of Streptomyces strains, and Micrococcus luteus DSM1790 was used as an indicator strain. S. lividans TK24 chromosomal DNA was isolated from HT medium (33)-grown cultures. SFM medium (33) was used for the assessment of the completion of the sporulation process as well as for spore collection. Protoplast generation, transformation, and regeneration on R2YE medium (33) were performed according to a previously documented procedure (26). Spores of various Streptomyces strains were spread on the surfaces of cellophane discs (from Cannings Packaging, Limited, United Kingdom) laid on the surface of R2YE agar medium with no phosphate supplementation (condition of phosphate limitation). When necessary, ampicillin, apramycin, and thiostrepton were included in the solid R2YE medium at a final concentration of 50 μg/μl, whereas thiostrepton was added at a concentration of 5 μg/μl in a liquid R2YE culture.

TABLE 1.
Strains and plasmids used in this study

Construction of a genomic library of S. lividans TK24.

Fragmented mycelium originating from a 36-h preculture of S. lividans TK24 in 10 ml HT medium was used to inoculate 100 ml of HT medium in 500-ml flasks. The culture was incubated overnight at 30°C, and chromosomal DNA was extracted from the mycelium using the CTAB method described in Practical Streptomyces Genetics (33). Total DNA (5 μg) was partially digested with the addition of 30 units of Sau3AI (Fermentas), followed by incubation at 37°C for 5 min. The digestion products were separated on a 0.8% agarose gel in Tris-acetate-EDTA buffer. Approximately 3- to 5-kb-long DNA fragments were electroeluted from the gel, treated with phenol-chloroform-isoamyl alcohol, and precipitated. The recovered Sau3AI DNA fragments were ligated into the high-copy-number vector pIJ702 (32), digested with BglII, and dephosphorylated. Subsequently, the ligation mixture was used to transform S. coelicolor M145 protoplasts, and transformants were selected in the presence of thiostrepton (50-μg/ml final concentration) on R2YE medium.

Overexpression of SCO3201SL/SC in Streptomyces.

The S. lividans/S. coelicolor SCO3201 (SCO3201SL/SC) gene fused to a six-histidine tag at its 3′ end (corresponding to the carboxy-terminal end of the protein) was retrieved as an XbaI-PstI fragment from pET21a+-SCO3201SL and pET21a+-SCO3201SC, respectively (see below), and ligated into pWHM3-ermE cut by XbaI and PstI to yield pWHM3-SCO3201SL and pWHM3-SCO3201SC, respectively. pWHM3-ermE is a derivative of pWHM3 with a 279-bp KpnI-BamHI ermE fragment containing the ermE promoter (5) as well as a plausible ribosomal binding site (GenBank accession no. M11200). Protoplasts of S. coelicolor M145 as well as the wild type and the ppk mutant of S. lividans were transformed by pWHM3-SCO3201SL and pWHM3-SCO3201SC.

Determination of CDA, RED, and ACT production.

In order to evaluate the CDA production, 2- by 2-cm patches made with 106 spores of the various strains were made on Oxoid nutrient agar (ONA) (33) and incubated at 30°C for 48 h. ONA plates were overlaid with an overnight culture of Micrococcus luteus in LB medium diluted 1/100 in soft nutrient agar (SNA) (33) with or without Ca(NO3)2 at 12 mM (final concentration). Plates were incubated at 37°C overnight. The diameters of the inhibition zones, formed only in the presence of Ca(NO3)2, were measured to estimate CDA production.

In order to quantify the production of RED (mostly intracellular) and ACT (both intra and extracellular), 107 spores of the various strains were plated on cellophane discs laid on the surface of R2YE solid medium (no Pi supplementation) and incubated at 30°C for 84 h. In order to assay the intracellular concentrations of RED and ACT, the mycelium corresponding to one-quarter of an 8.5-cm-diameter plate (approximately 40 mg of dry cell weight) was extracted by the addition of 1 ml of methanol and of 1 ml of KOH (1 N), respectively, and continuous vortexing for 30 min at 4°C. The resulting RED extract was acidified to pH 2 to 3 with the addition of 1 ml HCl (1 N), and the optical density at 530 nm (OD530) was measured with a spectrophotometer (Beckman) against a blank constituted by methanol and 1 N HCl (1:1 ratio in volume). The OD640 of the resulting ACT extract was measured with the same spectrophotometer against a blank constituted by 1 N KOH (33). The mycelium corresponding to the remaining half of the plate was saved for dry-cell-weight (biomass) determination. In order to assay for extracellular ACT, a volume of R2YE agar medium corresponding to one-quarter of the individual plate was smashed and allowed to diffuse in 10 ml water for 1 h at 4°C. After centrifugation, supernatant was collected and 10 ml 1 N KOH was added and mixed by inversion. Subsequently, 10 ml HCl (3 N) was added. The resultant mixture was incubated on ice for 10 min. Precipitated ACT was collected by centrifugation at 13,000 rpm for 10 min. The resulting pellet was diluted in 1 ml of KOH (1 N), and the OD640 of the solution was determined. The assays for each antibiotic were completed in triplicate for each strain. The mean values ± standard deviations were calculated and are shown in Tables Tables22 to to55.

TABLE 2.
Quantitative analysis of calcium-dependent antibiotic
TABLE 3.
Quantitative analysis of undecylprodigiosin
TABLE 4.
Quantitative analysis of actinorhodin
TABLE 5.
Quantitative analysis of actinorhodin (intracellular and extracellular values)

Construction of disrupted mutants of SCO3201 in S. coelicolor M145 and in S. lividans TK24 (wild type and ppk mutant).

In order to inactivate SCO3201 in the wild-type and the ppk mutant strains of S. lividans TK24 as well as in S. coelicolor M145, two 1-kb DNA fragments flanking the SCO3201 coding sequence were amplified by PCR from the chromosome DNA of S. lividans TK24 and S. coelicolor M145, using the two pairs of primers, one pair comprising UtetR-BamHI and UtetR-XbaI and one pair comprising DtetR-HindIII and DtetR-XbaI (Table (Table6).6). The resulting PCR fragments were digested with the corresponding restriction enzymes and ligated in the presence of an apramycin resistance cassette (recovered as an XbaI fragment from pHp45Ωapra [6]) into the thermosensitive pGM160 vector (47) cut by BamHI and HindIII. The resulting plasmids suitable for gene interruption [pGM160/SCO3201SC/SL::acc(3)IV] were introduced into S. coelicolor M145 as well as into S. lividans TK24 (wild type and ppk mutant). After three rounds of sporulation at 37°C, the progeny was screened for the apramycin-resistant and thiostrepton-sensitive phenotype to yield the expected SCO3201 disruptive mutants. The primer pair comprising ItetR1 and ItetR2 (Table (Table6)6) was used to confirm the replacement of the SCO3201 gene by the aac(3)IV cassette in the three strains by PCR.

TABLE 6.
Synthetic oligonucleotides used in this study

Purification of His-tagged SCO3201SL/SC in E. coli.

In order to purify SCO3201SL/SC from E. coli, the SCO3201 coding genes of S. coelicolor M145 (SCO3201SC) and S. lividans TK24 (SCO3201SL) were amplified by PCR from the genomic DNA of each strain, using the primers ExpTetR1-NdeI and ExpTetR2-XhoI (Table (Table6).6). The PCR products were cloned into pET21a+ cut by NdeI and XhoI to yield pET21a+-SCO3201SC and pET21a+-SCO3201SL, in which the transcription of the cloned gene is under the control of the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible lac promoter (58). The two plasmids were transformed into E. coli D41, a convenient host for protein expression (46). The resulting transformants were cultivated in LB medium at 37°C to give an OD600 of 0.6. IPTG was added at a final concentration of 1 mM for induction, and incubation was pursued for 4 extra hours. The cells were collected by centrifugation, resuspended in lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole), and disrupted by sonication. The cell debris was removed by centrifugation at 12,000 rpm at 4°C for 15 min. The resulting cell extract was passed through HIS-Select nickel affinity Gel (P6611; Sigma), and six-histidine-tagged SCO3201 (His6-SCO3201) was purified to near-homogeneity in accordance with the protocol of the manufacturer.

Preparation of Streptomyces cell crude extracts.

Two hundred milligrams of mycelia of 24-h-grown cultures from each individual strain was collected from lawns grown on R2YE solid medium and washed with ice-cold water containing 5 mM EDTA. The mycelium was suspended in 2 ml sonication buffer (10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol [DTT], 10% glycerol, and 1 mM phenylmethanesulfonyl fluoride). The mixture was then subjected to sonication to obtain a homogeneous solution, and cell debris was removed by centrifugation. The protein concentration of each sonicate was determined using the Bradford reagent (catalogue no. 500-0006; Bio-Rad). The protein concentrations of all samples were adjusted to 10 μg/μl before utilization for the electrophoretic mobility shift assay (EMSA).

EMSA.

The 5′-biotin-labeled SCO3201 promoter region was obtained by PCR based on the chromosome DNA of S. coelicolor M145, using primers PtetR2 (5′ biotin labeled; Eurogentec) and PtetR2′ (Table (Table6).6). The SCO3201 promoter region (SCO3201p; 0.1 pmol) was incubated with purified His6-SCO3201 or cell crude extracts from individual strains for 15 min at room temperature in a buffer containing 10 mM Tris-HCl (pH 7.0), 50 mM KCl, 1 mM EDTA, 1 mM DTT and 1 μg poly(dI-dC) in a total volume of 20 μl. After incubation, complexes and free DNA were resolved on a native polyacrylamide (5%) gel prerun at 100 V for 30 min with a running buffer containing 45 mM Tris-HCl (pH 8.3), 45 mM boric acid, and 10 mM EDTA and then run for 40 min at 100 V. Subsequently, the complexes and free DNA were transferred to a positively charged membrane (catalogue no. 130291; Appligene) using an electrophoretic transfer cell. After cross-linking under UV light at 254 nm for 10 min, visualization of the DNA signal from membrane was carried out using a LightShift chemiluminescence EMSA kit (catalogue no. 20148; Pierce, Rockford, IL).

The promoter regions of scbA, scbR, and cpkO were amplified by PCR from S. coelicolor genomic DNA, using primer pairs PscbA1/PscbA2, PscbR1/PscbR2, and PcpkO1/PcpkO2 (Table (Table6).6). The resulted DNA fragments were purified by a NucleoSpin Extract II column (catalogue no. 740 609.50; Macherey-Nagel). Fragment labeling was performed by PCR as described by Tiffert et al. (64), using the 5′-AGCCAGTGGCGATAAG-3′ primer that was labeled at its 5′ end with Cy5. These labeled fragments were used for EMSA. The DNA bands were visualized by fluorescence imaging using a Typhoon Trio+ variable-mode imager.

DNase I footprinting.

DNase I footprinting assays with the SCO3201 promoter region were performed using the fluorescence labeling procedure described by Yindeeyoungyeon and Schell (71). DNA probes for both coding and complementary strands were amplified by PCR from the genomic DNA of S. coelicolor M145 using the primers ItetR1 and GSP3 (Table (Table6),6), which were fluorescein labeled at the 5′ end of the coding and complementary strands, respectively. The resultant PCR products were purified from agarose gel (catalogue no. 740 609.50; Macherey-Nagel). The DNA probe (0.3 pmol) was incubated with 0.8 pmol of purified SCO3201 under the same conditions as those described above for EMSA, except that the reaction scale was enlarged to 50 μl. After the binding reaction, 5 μl of 5 mM CaCl2 was added to each reaction mixture and DNase I (catalogue no. AM2222; Ambion) digestion was carried out with 2 μl of the 1:1,000 dilution (4 × 10−3 units) at 37°C for 1 min. The reaction was stopped with the addition of EDTA at a final concentration of 5 mM, and the reaction mixture was subsequently heated at 75°C for 10 min to totally inactivate DNase I. DNA was then purified with a NucleoSpin Extract II column (catalogue no. 740 609.50; Macherey-Nagel). After ethanol precipitation, the samples were resuspended in 30 μl of SLS buffer (Beckman Coulter) and subjected to capillary electrophoresis by loading into a CEQ 8000 sequencing apparatus (Beckman Coulter). The elucidation of the DNA sequence of the protected region was carried out by comigration of the footprint assay, the molecular standard (catalogue no. 608095; Beckman Coulter), and the corresponding sequencing reaction (catalogue no. 608120; Beckman Coulter).

5′ rapid-amplification-of-cDNA-ends (5′-RACE) PCR.

In order to determine the transcriptional start site of SCO3201, total RNA was extracted from a 24-h-old culture of S. coelicolor M145 grown on solid R2YE medium using the NucleoSpin RNAII isolation kit (catalogue no. 740 955.50; Macherey-Nagel). Five micrograms of total RNA was used for reverse transcription with 2 pmol of a gene-specific primer, GSP1 (Table (Table6),6), using Superscript III first-strand synthesis supermix (catalogue no. 18080-400; Invitrogen). The sample was subsequently purified with a NucleoSpin Extract II column (catalogue no. 740 609.50; Macherey-Nagel). A homopolymeric A tail was added to the 3′ end of cDNA using terminal deoxynucleotidyl transferase (catalogue no. EP0161; Fermentas) in accordance with the manufacturer's instructions, and the sample was again purified with a NucleoSpin column. The poly(dA)-tailed cDNA was then used as a template for the PCR amplification using a poly(dT) primer (70 homopolymeric T) and a second inner-gene-specific primer, GSP2 (Table (Table6).6). An additional round of PCR was subsequently performed with a 1,000-fold dilution of the original PCR product as a template, using the poly(dT) primer and a nested GSP3 primer (Table (Table6),6), to obtain a single specific band. The final PCR products were sequenced using the nested GSP3 primer and the reaction kit (catalogue no. 608120; Beckman Coulter) according to the manufacturer's protocol on a CEQ8000 (Beckman Coulter) apparatus. The transcriptional start site was identified as the nucleotide immediately preceding the stretch of T residues complementary to the A tail in the sequence of the PCR product.

SCO3201 promoter probing using the promoter-less xylE gene encoding a catechol 2,3-dioxygenase.

The SCO3201 promoter region was amplified by PCR from the S. coelicolor M145 chromosome using primers PtetR1-XbaI and PtetR1-HindIII (Table (Table6).6). The resulting PCR product was ligated into the promoter probe vector pIJ4083 (33) cut by XbaI and HindIII. The ligation mixture was used to transform S. lividans TK24, and the correct structure of the promoter probe construct isolated from these primary transformants was checked by restriction analysis. This plasmid was then transformed into the three strains of interest and their corresponding SCO3201 knockout mutants. These six strains were cultivated in R2YE liquid medium, and the xylE activity was assayed in the cell extract as previously described (28). Total proteins were measured using the Bradford reagent (catalogue no. 500-0006; Bio-Rad) to calculate the specific enzymatic activity.

Isolation of total RNA and nonquantitative RT-PCR.

Streptomyces mycelia were collected from R2YE agar plates, and total RNA was isolated using a NucleoSpin RNA II kit (catalogue no. 740955.50; Macherey-Nagel) in accordance with the protocol provided by the manufacturer. The RNA samples were subsequently treated with RNase-free DNase (catalogue no. AM2222; Ambion) and purified with a NucleoSpin RNA cleanup kit (catalogue no. 740948.50; Macherey-Nagel). Two micrograms of each RNA samples was used as a template for cDNA synthesis with random primers, using the ThermoScript reverse transcription-PCR (RT-PCR) system (catalogue no. 11146-016; Invitrogen). The program for cDNA synthesis was 25°C for 10 min, followed by 50 min at 50°C. Ten percent of the cDNA synthesis reaction mixture (2 μl) was used as a template for each subsequent PCR, using the following gene-specific primer pairs: RT-hrdB1/RT-hrdB2, RT-scbA1/RT-scbA2, RT-scbR1/RT-scbR2, and RT-cpkO1/RT-cpkO2 (Table (Table6).6). The PCR program used was as follows: (i) 95°C for 5 min, (ii) 95°C for 40 s, (iii) 62°C for 40 s, (iv) 72°C for 40 s, and (v) 72°C for 10 min, with the second to fourth steps repeated for 35 cycles. Negative controls were carried out without the addition of reverse transcriptase in order to demonstrate that amplified products were derived from RNA transcripts and not from chromosomal DNA.

RESULTS

The cloning of SCO3201SL on a multicopy plasmid strongly inhibits ACT biosynthesis in S. coelicolor.

S. coelicolor produces copious amount of the blue-pigmented antibiotic actinorhodin (ACT), whereas S. lividans hardly produces any detectable amount of this compound. Since it is now known from the comparison of the S. coelicolor (http://www.sanger.ac.uk/) and S. lividans (http://www.broadinstitute.org/) genome sequences that approximately 600 genes that are present in the S. lividans genome are absent in the S. coelicolor genome and vice versa, we hypothesized that the very different abilities of these two strains to produce antibiotics might be due to the presence in the S. lividans genome of a major negative regulator of ACT production that would be absent or not functional in S. coelicolor. In an attempt to clone this putative negative regulator, a genomic library of S. lividans DNA was constructed in the high-copy-number replicative plasmid pIJ702 (33). Transformation of S. coelicolor M145 with 10 μg of this library yielded approximately 4,000 transformants. The average size of genomic DNA inserts being approximately 4 kb, the library coverage was the equivalent of two genomes. An unique colorless transformant (ACT) was obtained. The ACT phenotype was confirmed upon retransformation of S. coelicolor with the plasmid extracted from this unique white transformant. Sequence analysis of the insert carried by the plasmid revealed that the plasmid contains two complete genes homologous to SCO3201 and SCO3200 of S. coelicolor (Fig. (Fig.1).1). These two converging genes encode regulators of the TetR and DeoR families, respectively. In order to determine which of these two genes was responsible for the ACT phenotype, each gene and its upstream region as well as the upstream regions alone were cloned separately on the multicopy plasmid pIJ702 (33) and introduced into S. coelicolor. These experiments clearly demonstrated that only the overexpression of SCO3201 of S. lividans (SCO3201SL), encoding a regulator of the TetR family, was responsible for the ACT phenotype.

FIG. 1.
Schematic representation of the genetic surroundings of SCO3201.

The overexpression of SCO3201SC also strongly inhibits ACT biosynthesis of S. coelicolor.

In order to determine whether the equivalent gene of S. coelicolor was or was not functional, SCO3201 of S. coelicolor (SCO3201SC) and SCO3201SL were both cloned in the high-copy-number plasmid pWHM3 under the control of the strong constitutive ermE promoter. These constructs were introduced into S. coelicolor M145, and indeed, both conferred the ACT phenotype (Fig. (Fig.2),2), indicating that SCO3201SC was functional despite the 7 nucleotide changes leading to 3 amino acid changes (besides the start codon) compared with SCO3201SL (http://www.broadinstitute.org/) (see Fig. S1 in the supplemental material). These results invalidated our hypothesis demonstrating that SCO3201 obviously does not encode the hypothetical negative regulator of ACT biosynthesis that we were looking for.

FIG. 2.
Phenotypes resulting from SCO3201 overexpression. Effects of the introduction of pWHM3, pWHM3-SCO3201SC, and pWHM3-SCO3201SL in S. coelicolor M145 (A) and S. lividans TK24/ppk::Ωhyg (B) on ACT production (lawns grown on R2YE medium for 48 and ...

The overexpression of SCO3201SL/SC inhibits both metabolic and morphological differentiation of S. coelicolor.

In order to assess the effect of the overexpression of SCO3201SL/SC on the global ability of S. coelicolor M145 as well as of the ppk mutant of S. lividans TK24 to overproduce antibiotics, these strains as well as S. lividans TK24 were transformed either by the plasmid pWHM3-SCO3201SC or pWHM3-SCO3201SL or by pWHM3-ermE (empty vector as a control). The presence of any of the pWHM3-based multicopy plasmids did not change the growth pattern of the studied strains that were grown on the R2YE solid medium for 84 h (data not shown). ACT, RED, and CDA were assayed with these cultures as described in Materials and Methods. The results shown in Table Table44 indicate that the introduction of pWHM3-SCO3201SL/SC led to 74% and 95% reductions of ACT production of S. coelicolor M145/pWHM3 and of S. lividans TK24/Δppk::Ωhyg/pWHM3, respectively. Similarly, the results shown in Table Table33 indicate that the introduction of pWHM3-SCO3201SL/SC led to 36% and 67% reductions of RED production of S. coelicolor M145/pWHM3 and of S. lividans TK24/Δppk::Ωhyg/pWHM3, respectively. Regarding CDA, the diameters of the growth inhibition zones of M. luteus attributed to CDA production around patches of S. coelicolor M145 were approximately 1.5-fold larger than those seen around patches of the two S. lividans strains (Table (Table2;2; see also Fig. S3 in the supplemental material), indicating that S. coelicolor was a stronger producer of CDA than S. lividans (wild type and ppk mutant). The introduction of pWHM3-SCO3201SL/SC in these strains led to a total absence of the growth inhibition zone of M. luteus, suggesting that the overexpression of SCO3201SC/SL led to a strong repression of CDA production.

Furthermore, since we noticed that aerial mycelium formation and sporulation were delayed in S. coelicolor M145/pWHM3-SCO3201SC/SL grown on R2YE, we tested the sporulation ability of this strain on SFM medium (20), a medium known to promote good sporulation. Indeed, we confirmed that the introduction of this plasmid into S. coelicolor M145 strongly inhibited the morphological differentiation process and sporulation in this strain (Fig. (Fig.2).2). Curiously, the introduction of this plasmid into S. lividans TK24, the wt (Fig. (Fig.2),2), and the ppk mutant (data not shown) did not lead to an obvious inhibition of the morphological differentiation process (sporulation) of these strains. These observations confirm that the regulatory circuits controlling morphological and metabolic differentiation in these two closely related species are somehow different.

The interruption of SCO3201 did not correlate with any obvious phenotype.

Since the overexpression of SCO3201 had a strong negative effect on the antibiotic production of S. coelicolor M145 as well as of the ppk mutant strain of S. lividans TK24, we thought that conversely, its interruption might correlate with an enhanced level of antibiotic production, at least in S. lividans TK24. In order to test this hypothesis, this gene was interrupted in S. lividans TK24 (wt and ppk mutant) as well as in S. coelicolor M145 as described in Materials and Methods. The interruption of SCO3201 did not lead to the expected phenotype of ACT overproduction in S. lividans TK24 or to any other obvious phenotype in that strain, in the ppk mutant, or in S. coelicolor M145 (Tables (Tables22 to to5).5). This lack of an obvious phenotype again confirmed that this gene was not encoding the master negative regulator of ACT biosynthesis that we sought. However, a slightly more precocious sporulation of the SCO3201 knockout mutant of S. coelicolor M145 could be noticed on SFM medium at 48 h (data not shown). These unexpected negative results suggest that under the condition of overexpression, this TetR regulator might interact with the promoter regions of the target genes of some of the 151 TetR regulators present in the S. coelicolor genome (51), those that are most likely to play a role in the regulation of the metabolic and morphological differentiation process of that bacteria (37, 56, 60, 61). In order to test this hypothesis, and considering that this “experimental artifact” could prove to be quite useful in elucidating the regulatory circuits governing metabolic and physiological differentiation in Streptomyces, the study of this regulatory gene was pursued.

In vitro, SCO3201 interacts with its own promoter region.

Since TetR regulators often negatively regulate their own expression by binding, as dimers, to operator sites located in their promoter region (3, 12, 31, 53), the ability of SCO3201SC to interact with its own promoter region was tested with the EMSA technique using a biotin-labeled SCO3201 promoter region (fragment SCO3201p) and His6-SCO3201SC purified from E. coli. The result shown in Fig. Fig.3A3A clearly demonstrated that purified His6-SCO3201SC was indeed able to specifically bind to and delay its own promoter region since two shifted bands were detected. The more abundant shifted band, band II, might correspond to the binding of a dimer of SCO3201SC, whereas the much less abundant shifted band, band I, whose amount increases with increasing amounts of SCO3201SC (Fig. (Fig.3A,3A, lanes 2 to 5), might correspond to the binding of more than one dimer to secondary operator sites present in the promoter region or to a tetramer to the core binding site (Fig. (Fig.4).4). The addition of an excess amount of an unlabeled SCO3201 promoter fragment to the reaction mixture indeed led to the disappearance of the shifted bands (Fig. (Fig.3A,3A, lanes 6 and 7), demonstrating the specificity of the SCO3201SC-SCO3201 promoter interaction. A biotinylated DNA fragment internal to the SCO3201SC coding sequence (positions +4 to +250) was also used as a negative control in the EMSA, and indeed, no significant shifting of this fragment was observed in the presence of SCO3201SC (data not shown). Similar results were obtained with purified His6-SCO3201SL (data not shown), indicating that both proteins possess the ability to interact with the SCO3201 promoter region that is identical in S. coelicolor and S. lividans.

FIG. 3.
Electrophoretic mobility shift assays of the SCO3201 promoter region with purified His6-SCO3201SC/SL (A) and with cell crude extracts (B) from S. lividans TK24 (WT), S. lividans TK24/pWHM3-SCO3201SL, and S. lividans TK24/SCO3201::aac(3)IV. In both cases, ...
FIG. 4.
Sequence of the SCO3201 promoter region. The transcription start point is indicated by a bent arrow. The translation start codon GTG is shown in boldface letters. The putative −10 and −35 boxes are boxed. The DNA region protected by SCO3201 ...

When similar EMSA experiments were carried out with crude protein extracts (Fig. (Fig.3B)3B) originating from the wild-type strain of S. lividans grown on solid R2YE, two clearly delayed discrete bands were seen besides a very abundant but less delayed band. When crude extracts of the SCO3201 mutant of that strain were used, the topmost delayed band disappeared, whereas it was reinforced when crude extracts were prepared from the strain overexpressing SCO3201SL. These results clearly demonstrated that SCO3201SL was responsible for the retardation of the most delayed band but that another transcriptional regulator might be responsible for the retardation of the second most delayed band. The less delayed but very intense band is similarly abundant with all crude extracts, suggesting that it might be due to the interaction of the SCO3201 promoter region with the RNA polymerase.

The core of the SCO3201 binding site overlaps with the −35 box of its promoter region.

In order to position the SCO3201 binding site relative to the promoter region, the transcriptional start site of SCO3201 was determined using 5′-RACE PCR as described in Materials and Methods. The results (Fig. (Fig.4;4; see also Fig. S2A in the supplemental material) indicated that the transcriptional start site coincides with the first G of the putative GTG translation initiation codon of SCO3201, which is frequently used in Streptomyces. SCO3201 is thus transcribed as a leaderless mRNA, like many other Streptomyces genes (24, 52). The determination of the transcription start point of SCO3201 led to the proposal of the putative −10 and −35 promoter sequences boxed in Fig. Fig.4.4. These promoter sequences show similarity to the vegetative promoters recognized by Eσ70 of E. coli and have a classical spacing of 17 nucleotides (nt) (57).

In order to determine the SCO3201SC binding site in the SCO3201 promoter region, DNase I footprinting assays of the fluorescently labeled SCO3201 promoter region were performed in the presence and absence of SCO3201SC and analyzed with a capillary DNA sequencer, as described in Materials and Methods. The results showed three closely located intermittently protected regions, extending from position −13 to position −56 relative to the transcription start point (a total of 44 bp) on both the coding and the complementary strands (see Fig. S2B in the supplemental material). Full protection was achieved at a very low His6-SCO3201SC concentration (0.02 μM) in each case. The central SCO3201 binding site (BSC) encompasses an 11-nt palindromic sequence (TGGCAGATTCTGCCA) that overlaps the −35 sequence of the promoter region (Fig. (Fig.4).4). A second protected site (BSR), located to the right of BSC, encompasses the left part of a 5-nt inverted repeat (CGGCA), located 12 nt upstream of the transcriptional start site. This short palindromic sequence has four nucleotides (GGCA) in common with BSC. The third protected site (BSL) is located on the left of BSC and encompasses the 10-nt very-GC-rich palindromic sequence (ACCCCGGGGT). The three nucleotides at each extremity of this short palindromic sequence are the same as those of BSC (but in the opposite orientation). The footprint analysis confirmed the direct interaction of SCO3201 with its own promoter region, indicating that SCO3201 was negatively regulating its own transcription by blocking the access of the RNA polymerase to its promoter region.

The negative autoregulation of SCO3201 was confirmed, in vivo.

In order to confirm, in vivo, the negative autoregulation of SCO3201 expression, a transcriptional fusion of the promoter region of SCO3201SC (which shows 100% identity in nucleotide sequence to that of SCO3201SL) with the xylE reporter gene was constructed using the high-copy-number promoter probe vector pIJ4083 (33). This plasmid was introduced into the wt and ppk mutant strains of S. lividans TK24, into S. coelicolor M145, and into their corresponding SCO3201 knockout mutants. The XylE activities were assayed for these strains grown in liquid R2YE medium as described in Materials and Methods, and the quantitative results are shown in Fig. Fig.5.5. The expression profiles of the SCO3201p-xylE fusion were quite different in the three strains. However, in all strains, the expression level of the xylE fusion was higher during active growth (<50 h) than in stationary phase (>60 h) and was higher in the antibiotic-producing strains (and higher in S. coelicolor than in the ppk mutant of S. lividans) than in the non-antibiotic-producing strain (S. lividans). In the three strains, the interruption of SCO3201 led to a clear increase of the xylE fusion expression, demonstrating the negative autoregulation of SCO3201. However, whereas the expression of the xylE fusion continued during stationary phase in the SCO3201 mutant of S. coelicolor, the expression of this gene in the SCO3201 mutants of the wt and the ppk mutant of S. lividans was rather transitory and centered on the 36-h point. These observations suggest that the regulatory circuits governing SCO3201 expression are quite different in these two species.

FIG. 5.
(A) Growth curves of S. coelicolor M145 (circle), S. lividans TK24 (triangle), S. lividans TK24/ppk::hyg (square), and their corresponding SCO3201 knockout mutants. (B) XylE activities resulting from the expression of the xylE reporter gene put under ...

SCO3201 directly interacts with the scbA promoter region in vitro and regulates its transcription in vivo.

Sequences similar to the SCO3201 operator site were searched in the promoter regions of other known Streptomyces regulators of the TetR family using the MEME algorithm (multiple expectation maximization [EM] for motif elicitation) (2). The best hit found was in the promoter region of scbA, a gene encoding a protein required for γ-butyrolactone SCB1 biosynthesis (61). The expression of scbA is known to be positively regulated by the divergently located TetR-like regulator ScbR through direct interaction (45, 61). ScbR was also shown to negatively regulate its own expression and that of cpkO, a gene encoding the putative pathway-specific transcriptional regulator of the cryptic type I polyketide gene cluster, also through direct interaction (63). The ability of SCO3201 to shift the promoter regions of scbA, scbR, and cpkO was then tested. Only the promoter region of scbA was shifted by the addition of histidine-tagged SCO3201 (Fig. (Fig.6A).6A). The shifting was abolished by the addition, in large excess, of the unlabeled probe (Fig. (Fig.6A,6A, lane 6) demonstrating the specificity of the protein-DNA interaction. With the MEME software program, the scbA promoter region was found to share the highest level of similarity with the SCO3201 binding site since a region of 26 nt, with 17 identical nucleotides, was overlapping with most of BSC and BSR (Fig. (Fig.6C),6C), whereas the ScbR and cpkO promoter regions shared a shorter region of similarities, encompassing mostly BSL and/or the left part of BSC (see Fig. S4 in the supplemental material). SCO3201 is thus predicted to have less affinity for the scbR and cpkO promoter regions than for the scbA promoter region.

FIG. 6.
(A) Electrophoretic mobility shift assay of the scbA promoter region with purified His6-SCO3201. Cy5-labeled DNA probe (0.1 pmol) was used for each reaction. The shifted bands are indicated by arrows. (B) Assessment of scbA, scbR, and cpkO transcript ...

In order to determine whether SCO3201 was or was not interfering with the ScbR/ScbA/CpkO regulatory circuit in vivo, the levels of transcription of scbA, scbR, and cpkO were analyzed by RT-PCR in S. coelicolor/pWHM3 and S. coelicolor/pWHM3-SCO3201 at 24 h and 48 h. As shown in Fig. Fig.6B,6B, the overexpression of SCO3201 correlates with a downregulation of scbR and cpkO expression at 24 h but not at 48 h and with the upregulation of scbA expression at both 24 h and 48 h. The relief of SCO3201-mediated repression of scbR and cpkO expression at 48 h might be related to the weak affinity of SCO3201 for its target sites in the scbR and cpkO promoter region. This weak affinity might be further destabilized by the presence of the SCO3201 ligand at late time points (48 h).

Overexpression of SCO3201 reduces the efficiency of ACT export, at least in S. lividans.

In Table Table5,5, the ratios between extracellular (exported) and intracellular ACT were calculated for the antibiotic-producing S. coelicolor M145 and S. lividans Δppk::Ωhyg strains containing either pWHM3 or pWHM3-SCO3201. This ratio was found to be 14-fold (11.91/0.84) lower in the S. lividans Δppk::Ωhyg strain containing pWHM3-SCO3201 than in that strain containing empty pWHM3 but only 1.4-fold (5.52/3.92) lower in the equivalent S. coelicolor strains. This observation suggests a possible involvement of SCO3201 in the regulation of the expression of the genes encoding the ACT export machinery, at least in S. lividans. Indeed, the ACT export machinery, encoded by the actAB operon, is under the control of the TetR regulator actII-ORF1 (actR) (14, 59, 60), and SCO3201 might be able to control, in vivo, the expression of this operon. The strikingly differential effects of SCO3201 overexpression in the two species on the export of ACT is not understood but might be related to differences in the regulatory circuits governing ACT export in the two species or to the greater abundance in S. coelicolor than in the ppk mutant of S. lividans of ligands able to dissociate SCO3201 from its binding sites.

DISCUSSION

The present paper describes the characterization of a novel regulator of the TetR-family, SCO3201, one among the 151 TetR regulators present in the S. coelicolor genome (51), whose overexpression was shown to have a strong negative effect on the metabolic (antibiotic production) and morphological (sporulation) differentiation process of S. coelicolor M145. The overexpression of SCO3201 led to an apparent total shutoff of CDA biosynthesis in the three strains tested, to a strong reduction of ACT biosynthesis, and to a more moderate reduction of RED biosynthesis. The reduction of ACT and RED biosynthesis was always more drastic in the ppk mutant strain of S. lividans TK24 than in S. coelicolor M145. These differences might indicate the existence of different regulatory circuits controlling antibiotic production in these two species or a greater abundance in S. coelicolor than in S. lividans of the ligand able to dissociate SCO3201 from its target sites. Furthermore, these observations confirm the existence of common regulatory circuits governing the biosynthesis of secondary metabolites that require different types of precursors for their biosynthesis (amino acids versus acetyl-coenzyme A [CoA] derivatives).

Interestingly, the overexpression of SCO3201 had also a negative effect on the morphological differentiation process of S. coelicolor M145 but not of S. lividans TK24 (wt and ppk mutant). These observations demonstrate that the regulatory circuits controlling the morphological differentiation process are somehow different in these two closely related species. Despite the overexpression of SCO3201, S. lividans sporulates, indicating either that some of the SCO3201 operator sites are missing in front of key genes involved in the positive regulation of sporulation of that strain or that some negative regulatory elements of the sporulation process are missing in S. lividans. Indeed, it was demonstrated that some genomic islands larger than 25 kb and comprising more than 600 genes were present in the S. coelicolor genome but absent in the S. lividans genome (30).

The SCO3201 regulator was shown to negatively autoregulate its own synthesis, and its operator site in its own promoter region was identified as the 15-bp palindromic sequence (TGGCAGATTCTGCCA) that is a typical binding site for homodimer transcriptional regulators of the TetR family (51). This binding site overlaps with the −35 promoter sequence, impairing RNA polymerase binding. Footprinting experiments revealed that the binding of SCO3201 wobbles between its core binding site and two lateral sequences with reduced sequence conservation with the core sequence and imperfect symmetry (Fig. (Fig.4).4). The binding of TetR regulators to their target sites is usually relieved by small inducer molecules that trigger drastic conformational changes (22). In the case of SCO3201, the “inducer molecule” is not known, but it is interesting to note that the C terminus of this regulator, which usually includes the ligand binding motif in other TetR regulators, bears some weak similarities with leucine/isoleucine/valine-binding protein (LIVBP), suggesting that some branched amino acids (Leu, Ile, and Val) might be able to dissociate SCO3201 from its target sites. However, in the multicopy situation under the condition of overexpression, the TetR ligands might not be abundant enough to relieve the SCO3201-mediated repression of gene expression.

Furthermore, our band-shifting experiments with the SCO3201promoter region carried out in the presence of crude extracts of the different strains (Fig. (Fig.3B)3B) indicated that the regulation of SCO3201 expression might involve another regulator besides SCO3201. Indeed, in the SCO3201 mutant of the S. lividans, the expression of the xylE reporter gene under the control of the SCO3201 promoter was shown to be transitory (peak at 36 h), suggesting the contribution of another positive or negative regulator besides SCO3201 mediating transitory activation or repression relief, respectively. The time point of 36 h usually corresponds to the transition between a first phase of active growth and a second phase of slower growth that precedes the entry into stationary phase. If a second negative regulator controls SCO3201 expression, it might be another TetR-like regulator recognizing the SCO3201 operator sequence. Alternatively, we cannot exclude that the second putative regulator is a positive one, since three rather well conserved direct repeats of the sequence GAACC were found 36 bp upstream of the −35 sequence, the usual position of activator sites (Fig. (Fig.4).4). In the two antibiotic-producing strains (the S. coelicolor strain and the ppk mutant of S. lividans), the expression of SCO3201 is higher than in the S. lividans strain, suggesting that either repression of SCO3201 expression by both SCO3201 and the other putative negative regulator is partially relieved or that activation by an unknown regulator takes place concomitantly to the relief of the SCO3201-dependent repression.

However, the most surprising outcome of this study is that whereas the overexpression of SCO3201 leads to a very strong phenotype, the interruption of this gene did not correlate with any obvious phenotype in any of the three studied strains. This indicated that the strong phenotypic consequences of the overexpression of SCO3201 were somehow an “experimental artifact” linked to SCO3201 abundance. Under conditions of great SCO3201 abundance, the latter might be able to interact with the operator sequences of other TetR regulators not closely related to its own operator site. Indeed, other TetR regulators, including ScbR, CprA, CprB, RrdA, SCO1712, and ActR, were shown to be involved in the regulation of antibiotic production or antibiotic export (37, 48, 49, 59, 61). We demonstrated that SCO3201 was able to mimic ScbR activity, enhancing scbA expression in vivo, indicating that this “experimental artifact” could prove quite useful in deciphering the regulatory circuits controlling the metabolic and morphological differentiation in S. coelicolor and S. lividans. Indeed, the deletion of scbA was previously shown to lead to an increase in ACT and RED biosynthesis (61), so its overexpression is likely to contribute to the observed reductions of ACT and RED biosyntheses in S. coelicolor overexpressing SCO3201. Other targets of SCO3201 remain to be characterized. This characterization is in process by in silico as well as by in vivo and in vitro capture of genomic DNA-SCO3201 cross-link approaches.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by the Université Paris Sud 11, the PRES UniverSud Paris, and the CNRS.

We are most grateful to Marc Galimand's group (Pasteur Institute), which kindly provided the sequencing kit, as well as to Gilles Vergnaud's group for the use of their CEQ8000 apparatus for the footprint experiment.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 October 2010.

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