WblA_ch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpA_ch

Abstract

Detailed mechanisms of WhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblA_ch, a Streptomyces chattanoogensis L10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression of wblA_ch was shown to be positively regulated by AdpA_ch. Gel retardation assays and DNase I footprinting experiments showed that AdpA_ch has specific DNA-binding activity for the promoter region of wblA_ch. Gene disruption and genetic complementation revealed that WblA_ch acts in a positive manner to regulate natamycin production. When wblA_ch was overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels of whi genes (including whiA, whiB, whiH, and whiI) were severely depressed in the ΔwblA_ch mutant, suggesting that WblA_ch plays a part in morphological differentiation by influencing the expression of the whi genes.

INTRODUCTION

The Gram-positive filamentous soil bacterial genus Streptomyces is characterized by its complex life cycle, which involves the formation of a substrate mycelium that goes on to develop aerial hyphae, the tips of which ultimately coil and septate into spores. These bacteria are well known for the ability to produce a variety of commercially valuable antibiotics and other secondary metabolites (1, 2). Previous investigations suggested that the triggering of antibiotic biosynthesis is closely coordinated with the initiation of morphological differentiation in Streptomyces species (1, 3, 4), and both processes have been shown to be stringently controlled via a hierarchical regulatory network involving the integration of various physiological and environmental signals (5, 6). Although a significant number of regulatory genes and mechanisms involved in the regulatory networks governing morphological development and antibiotic production in Streptomyces have been elucidated, relatively little is known about the correlation of these two physiological processes.

The whiB-like (wbl) genes, which encode homologues of WhiB, have received attention because of their important roles in diverse aspects of actinobacterial biology, such as morphological differentiation and antibiotic production (7, 8). The gene products are small cytoplasmic proteins that contain four conserved cysteine residues able to coordinate an Fe-S cluster and are found largely in actinomycetes, including some mycobacteria (9). It has been reported that wbl genes are induced by various stimuli, especially oxidative stress, suggesting a unique role in maintaining redox homeostasis (10–12). In Mycobacterium tuberculosis, WhiB3 acts as a metabolic regulator that binds to the promoters of polyketide biosynthetic genes (13), while WhiB6 acts as an initial phagosomal signal receptor (14). It has also been shown that WhiB7 is a critical protein for generating resistance to antibiotics in M. tuberculosis (15). Recently, whiB3 (16), whiB4 (17), whiB5 (18), and whiB2 (19) were shown to play essential roles in the virulence of M. tuberculosis during progressive infection.

In Streptomyces coelicolor M145, there are 11 WhiB-like proteins, among which WblA was reported to act as an important transcriptional regulator involved in antibiotic production and morphological differentiation (7, 20, 21). WblA and its orthologues have been described as crucial antibiotic downregulators for the biosynthesis of various antibiotics such as actinorhodin (21), doxorubicin (22), tautomycetin (23), and moenomycin (24). Moreover, it was discovered that wblA is negatively regulated by the pleiotropic regulator AdpA in S. coelicolor (25). However, in our study, we revealed differing functions of wblA_ch in antibiotic biosynthesis and in the AdpA-WblA regulatory relationship in Streptomyces chattanoogensis L10, an industrial strain used for natamycin production. We demonstrate that wblA_ch is an AdpA_ch regulon that is under general direct positive control of AdpA_ch. We also find that WblA_ch acts as a pivotal activator for natamycin biosynthesis and morphological differentiation in S. chattanoogensis L10.

MATERIALS AND METHODS

Media, plasmids, strains, and growth conditions.

The strains used in the present study are listed in Table 1. General techniques for bacterial growth and isolation and manipulation of nucleic acids were carried out according to standard protocols for Escherichia coli and S. coelicolor, respectively (26). S. chattanoogensis L10 strains were grown at 28°C on YMG agar (1% malt extract, 0.4% yeast extract, 0.4% glucose, 0.2% CaCO₃, and 2% agar, pH 7.2) for sporulation and at 30°C in YEME medium (0.3% yeast extract, 0.3% malt extract, 0.5% tryptone, 1% glucose) for natamycin production.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Description	Reference or source
Strains
S. chattanoogensis
WT	S. chattanoogensis L10 wild type	30
ΔwblAch	wblAch-disrupted mutant	This study
ΔadpAch	adpAch-disrupted mutant	28
YC1	wblAch-disrupted mutant complemented with wblAch and its own promoter	This study
YC2	Wild-type strain carrying wblAch overexpression	This study
YC3	Wild-type strain carrying an empty vector	This study
Saccharomyces cerevisiae BY4741	Indicator organism for natamycin bioassays	Laboratory stock
E. coli
TG1	General cloning host	Novagen
ET12567/pUZ8002	Methylation-deficient E. coli strain for conjugation with the helper plasmid	41
BL21(DE3)	Host for protein expression	Novagen
BW25113/pIJ790	Strain used for PCR-targeted mutagenesis	27
Plasmids
pMD19-T vector	General cloning vector	TaKaRa
pT-wblAch	pUC18 containing the promoter of wblAch	This study
pSET152	Integrative shuttle vector	26
pIJ8630	Integrative shuttle vector	42
pIJ773	Source of apramycin resistance cassette; aac(3)IV oriT	27
pMRD312	Cosmid containing wblAch	This study
pMRD313	wblAch replaced by aac(3)IV cassette in pMRD312	This study
pYP1	wblAch replaced by an 81-bp scar in pMRD312	This study
pYP2	pSET152 carrying wblAch and its promoter	This study
pYP3	pIJ8630 carrying wblAch and ermE* promoter	This study

In-frame deletion and complementation of wblA_ch.

Disruption of wblA_ch was performed by gene replacement according to a modified PCR targeting system (27). First, the cosmid pMRD312, containing the wblA_ch open reading frame (ORF) fragment, was introduced into E. coli BW25113/pIJ790. Second, the wblA_ch::FRT (FLP recombination target)-oriT-aac(3)IV-FRT disruption cassette was amplified from pIJ773 with primer pair wblA_ch-del-F/wblA_ch-del-R and electrotransformed into E. coli BW25113/pIJ790/cosmid pMRD312 to generate the disruption cosmid pMRD313, which replaced most of the wblA_ch coding region (amino acids 50 to 112). Third, the targeted cosmid pMRD313 was transformed into E. coli DH5α/BT340 in order to excise the aac(3)IV cassette. The resulting cosmid, pYP1, was conjugated by E. coli ET12567/pUZ8002 into S. chattanoogensis L10. The wblA_ch disruption mutant was selected by replica plating for thiostrepton-sensitive colonies and confirmed by PCR amplification with primer pair wblA_ch-F/wblA_ch-R.

For complementation, the integrative vector pSET152 was used. Primer pair wblA_ch-BamHI-F1/wblA_ch-EcoRV-R1 was used to amplify a 750-bp DNA fragment containing the wblA_ch ORF and its own promoter. The amplified PCR products were then inserted into pSET152, which was digested with BamHI and EcoRV, and the resultant plasmid, pYP2, was integrated into the chromosome of the wblA_ch deletion mutant at the phage ΦC31 attB site for complementation.

Overexpression of wblA_ch.

For wblA_ch overexpression, a 342-bp DNA fragment containing the complete wblA_ch gene was amplified by using primers wblA_ch-NdeI-F and wblA_ch-NotI-F. Afterwards, this PCR product was subcloned into the pMD19 vector (TaKaRa) after dA addition and then digested with NdeI and NotI to give a NdeI-NotI DNA fragment containing wblA_ch. This fragment was ligated into the same sites of pIJ8630, which contained the ermE* promoter inserted into the BamHI site. The resulting plasmid was then introduced into E. coli ET12567/pUZ8002 for E. coli-Streptomyces conjugation.

Microarray analysis.

Mycelia of S. chattanoogensis L10 wild-type (WT) and mutant strains grown in YEME medium were collected at 16 h, 24 h, and 36 h. Total RNA was isolated by using the RNA Extract kit (Qiagen, Germany) according to the manufacturer's instructions and checked for an RNA integrity number (RIN) to inspect RNA integration by using an Agilent Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA). Microarray assays, including primer design, labeling, hybridization, and washing, and microarray data normalization were performed by Shanghai Biotechnology Corporation (China) according to standard protocols.

Electrophoretic mobility shift assays.

Expression of the recombinant AdpA_ch protein with a His tag at its C terminus was performed with E. coli BL21, and the protein was purified by using Ni-nitrilotriacetic acid (NTA) His Bind resin (Novagen), as previously described (28). For probe preparation, probe A, probe B, and probe C, covering each binding site of the wblA_ch promoter, were amplified with the primers listed in Table 2. The promoter region (384 bp) of wblA_ch was amplified with primer pair wblA_ch-EMSA-F/wblA_ch-EMSA-R. Afterwards, the PCR products were cloned into the pMD19 vector (TaKaRa). The biotin-labeled probes were then prepared by PCR amplification with 5′-biotin-labeled M13 universal primers. About 1 ng of probe was incubated with purified His₆-AdpA_ch at 25°C for 30 min in buffer (20 mM Tris [pH 7.5], 0.01% bovine serum albumin [BSA], 5% glycerol, 50 μg ml⁻¹ sheared salmon sperm DNA); the protein concentrations used are indicated in Fig. 2. For the competition assay, 100-fold molar excesses of unlabeled probe and nonspecific DNA were each added to 0.2 μg purified His₆-AdpA_ch. Reactions were displayed on 5% acrylamide gels for separation in 0.5× Tris-borate-EDTA (TBE) buffer. Electrophoretic mobility shift assay (EMSA) gels were then electroblotted onto a nylon membrane and UV fixed by using a UV cross-linker. Labeled DNA was detected with streptavidin-horseradish peroxidase (HRP) and BeyoECL Plus (Beyotime, China) according to the manufacturer's instructions.

TABLE 2.

Oligonucleotides used in this study

^aSequences representing restriction sites and mutagenesis are underlined.

FIG 2.

Gel mobility shift assays using a labeled DNA fragment containing the wblA_ch promoter region and the His₆-AdpA_ch protein. Lane 1, labeled fragment; lanes 2 to 5, labeled fragment with 0.05 μg, 0.1 μg, 0.2 μg, and 0.4 μg of purified His₆-AdpA_ch protein, respectively; lane 6, labeled fragment with a 100-fold excess of nonspecific DNA and 0.2 μg purified His₆-AdpA_ch protein; lane 7, labeled fragment with a 100-fold excess of unlabeled wblA_ch promoter DNA and 0.2 μg purified His₆-AdpA_ch protein.

DNase I footprinting assay.

A DNase I footprinting assay was performed as previously described (29). First, the 6-carboxyfluorescein (FAM)-labeled wblA_ch probe was amplified by PCR using 5′-FAM-labeled M13 universal primers from plasmid pT-wblA_ch, followed by gel recovery. About 50 ng of fluorescently labeled probe was then added to the reaction mixture to a final volume of 50 μl together with the AdpA_ch protein, which was ultrafiltered with a centrifugal ultrafiltration device (YM-10; Millipore) for a 10-kDa cutoff and eluted in 20 mM Tris buffer (pH 7.5). After binding of the AdpA_ch protein to the wblA_ch probe (30°C for 30 min), 0.01 U of DNase I (Promega) was added for 1 min at 30°C. The reactions were then stopped with an equal volume of 100 mM EDTA, and samples were extracted by using phenol-chloroform. After precipitation with a combination of 10 μl 7.5 M ammonium acetate (NH₄Ac), 40 μg glycogen, and 100 μl 100% ethanol, the digested DNA mixture was loaded into an ABI 3130 DNA sequencer with the Liz-500 DNA marker (MCLAB). The DNA sequencing ladder was prepared according to instructions provided with the Thermo Sequenase dye primer manual cycle sequencing kit (USB).

Real-time quantitative PCR analysis.

Mycelium was harvested from strains grown in YEME medium or on YMG agar medium overlaid with cellophane discs for different times. Total RNAs were then isolated by using TRIzol reagent (Bio Basic Inc.) according to the manufacturer's guidelines. After treatment with DNase I (TaKaRa), PCR was carried out to ensure that there was no genomic DNA. The quality and quantity of RNA samples were then determined by UV spectroscopy and checked by agarose gel electrophoresis. A primeScript First Strand cDNA synthesis kit (TaKaRa) was used to generate cDNA from 2 μg of total RNAs according to the manufacturer's protocol. Quantitative real-time PCR of selected genes was performed on a Roche LightCycler 480 instrument (Roche), and ∼1 ng of synthesized cDNA was used as a DNA template in a 20-μl final reaction mixture volume for real-time quantitative PCR (qRT-PCR) with 10 μl Power SYBR green PCR master mix. hrdB was used as an internal control, and all experiments were done in triplicate.

Natamycin bioassay and HPLC analysis.

Natamycin produced by S. chattanoogensis on YMG agar was measured by bioassays using Saccharomyces cerevisiae as an indicator organism. Agar plugs were cut from the lawns of S. chattanoogensis that had been grown on YMG medium for 10 days by using a core borer (about 0.8 cm in diameter) and were placed onto a lawn of freshly plated Saccharomyces cerevisiae cells. After incubation at 30°C for 18 h, the inhibition zones of the bioassay plates were recorded. Natamycin production was further confirmed by high-performance liquid chromatography (HPLC) analysis using the Agilent 1100 HPLC system. An Agilent HC-C₁₈ column (5 μm, 4.6 by 250 mm) was used, and the UV detector was set at 303 nm. The mobile phase and gradient elution processes were previously described (30).

Scanning electron microscopy.

For scanning electron microscopy, agar plugs ∼1 cm in diameter were cut from the lawns of S. chattanoogensis strains grown on YMG medium for 10 days and then plunged into liquid nitrogen. The cut agar blocks were observed by scanning electron microscopy (S-3000N; Hitachi) after sputter coating with gold. The detailed process was performed as previously described (28).

Accession numbers.

The GenBank accession numbers for the sequences of wblA_ch, whi genes, and other genes in the microarray analysis are KM264326 to KM264342. Experimental details and data from the microarrays have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE59806.

RESULTS

Identification of wblA_ch as a member of the AdpA_ch regulon by transcriptional analysis.

AdpA is an important pleiotropic regulator in the A-factor regulatory cascade in Streptomyces griseus (31). Induction of adpA requires the dissociation of ArpA from the adpA promoter, and the members of the AdpA regulon are then activated to participate in several important cellular functions (32, 33). In our previous study, we showed that AdpA_ch plays an important role in natamycin biosynthesis and morphological differentiation in S. chattanoogensis L10 (28). For a more detailed analysis of AdpA_ch, we compared the pattern of gene expression of the wild-type (WT) strain to that of the ΔadpA_ch mutant strain. Mycelium was harvested after 16 h, 24 h, and 36 h of growth, and RNA was isolated from three independent cultures of each strain.

In the DNA microarray analysis, we used the criterion of a fold change of ≥2 and ≤0.5 (P < 0.05) to discriminate AdpA_ch-responsive genes. According to this criterion, approximately 809 and 431 genes were transcriptionally downregulated and upregulated, respectively, in the adpA_ch mutant strain compared to the WT strain at all tested time points. Among these genes, we noticed that L10.orf0502, which encodes a WhiB-type transcription regulator, was downregulated 79-fold at 16 h, 23-fold at 24 h, and 6.4-fold at 36 h in the ΔadpA_ch mutant strain (Fig. 1A). These results suggested that L10.orf0502 is positively regulated by AdpA_ch. A sequence similarity search showed that L10.orf0502 is an orthologous protein of the WblA protein (GenBank accession no. NP_627776.1) (90% identity) of Streptomyces coelicolor A3(2). Thus, it was designated wblA_ch. To further verify the microarray data and clarify the relationship between the wblA_ch and AdpA_ch in S. chattanoogensis L10, we carried out real-time quantitative PCR (qRT-PCR) to test the expression of the wblA_ch gene in the ΔadpA_ch mutant and in the WT strain. As expected, the transcript levels of wblA_ch in the ΔadpA_ch disruption mutant had decreased significantly in contrast to that of the WT strain at all tested time points (Fig. 1B). These results further confirmed that AdpA_ch acts as an activator of wblA_ch transcription in S. chattanoogensis L10.

FIG 1.

Expression profiles of wblA_ch differentially expressed during growth of the WT strain and the ΔadpA_ch mutant. (A) Microarray analysis of the transcription profiles of selected genes. RNA samples were isolated at 16 h, 24 h, and 36 h during growth on YEME medium. (B) RT-PCR analysis of wblA_ch transcript levels in the WT strain and the ΔadpA_ch mutant. The expression level of wblA_ch is presented relative to the wild-type sample at 16 h, which was arbitrarily assigned a value of 1. The transcription level of hrdB was assayed as an internal control. Error bars were calculated by measuring the standard deviations among data from three replicates of each sample.

Interaction of AdpA_ch with the promoter region of wblA_ch.

To determine whether AdpA_ch plays a direct role in the regulation of the wblA_ch gene, electrophoretic mobility shift assays (EMSAs) were performed. For this purpose, AdpA_ch was overexpressed in the E. coli BL21 strain as a recombinant His₆-tagged protein and purified by Ni-NTA agarose chromatography. A biotin-labeled wblA_ch probe covering the wblA_ch promoter region from positions −321 to +63 was prepared. Results from EMSAs showed that purified His₆-AdpA_ch can bind to the upstream regions of wblA_ch to form a complex (Fig. 2). There was only one shifted band for the wblA_ch probe at a low His₆-AdpA_ch protein concentration. However, when the protein concentration was increased, two other shifted bands were observed, accompanied by the disappearance of the lower band (Fig. 2, lanes 2 to 5). It is therefore likely that there is more than one His₆-AdpA_ch-binding site located on the wblA_ch promoter region. In order to examine binding specificity, EMSAs with an excess of unlabeled specific and nonspecific competitor DNA were performed. As shown in Fig. 2, the retarded bands (lanes 6 and 7) disappeared in the presence of excess unlabeled wblA_ch probe but not in the presence of nonspecific DNA. These results suggest that purified AdpA_ch regulates the transcription of wblA_ch by specifically binding to the upstream regions of wblA_ch.

To further define the accurate binding sites of AdpA_ch in the upstream region of wblA_ch, DNase I footprinting assays were carried out. The experiments revealed that His₆-AdpA_ch protected three regions, from nucleotides −83 to −44, −182 to −160, and −226 to −192 relative to the wblA_ch transcription start point (Fig. 3A and C). After further analysis of the protected DNA regions, we found that there was a highly conserved AdpA_ch-binding sequence in each binding site, which is identical to that found in S. griseus (Fig. 3B) (34). Therefore, series of mutations were constructed to determine the actual AdpA_ch-binding sites. As shown in Fig. S4 in the supplemental material, no binding shift was detected for mutated sites A to C compared with their corresponding wild-type targets. Thus, these consensus sequences are essential for the binding activity of AdpA_ch.

FIG 3.

DNase I footprinting assay for determination of the AdpA_ch-binding site. (A) A 5′-FAM-labeled wblA_ch probe was used in the DNase I footprinting assay without or with purified AdpA_ch (10 μg). The protected regions are underlined. (B) Sequences of the determined AdpA_ch-binding sites and the consensus AdpA_ch-binding sequence (in boldface type). (C) Nucleotide sequences of the wblA_ch promoter region and the predicted AdpA_ch-binding sites. The transcription start point (tsp) is marked by a bent arrow, the AdpA_ch-binding sites are overlined, and the ribosome-binding site (RBS) is boxed.

wblA_ch is a pivotal activator of natamycin biosynthesis.

Previous reports have shown that WblA and its orthologues act as negative regulators for antibiotic biosynthesis in various Streptomyces species and that wblA deletion mutants lead to overproduction of the corresponding antibiotics (21–24). In order to identify the function of wblA_ch in natamycin biosynthesis in S. chattanoogensis L10, a wblA_ch deletion mutant was constructed via homologous recombination, as described Materials and Methods. The disruption mutant was confirmed by PCR analysis and Southern blot analysis (see Fig. S1 in the supplemental material). Meanwhile, the complemented strain was constructed by reintroducing a 750-bp DNA fragment containing wblA_ch and its own promoter into the mutant strain. To assess natamycin production, Saccharomyces cerevisiae was used as an indicator organism for the bioassay. As shown in Fig. 4A, no inhibition was observed with the agar plug from the wblA_ch deletion mutant, whereas the growth-inhibiting activity against S. cerevisiae was restored when plasmid pYP2 was reintroduced into the ΔwblA_ch mutant. To further confirm this phenotype, high-performance liquid chromatography (HPLC) analysis was carried out. No peak of natamycin was present in the culture filtrates of the wblA_ch deletion mutant, in contrast to the culture filtrates of the WT strain, and the complemented strain had restored the production of natamycin to a level similar to that of the WT strain (Fig. 4B). Nevertheless, these three strains had comparable growth rates and similar biomasses (Fig. 4C). These results indicate that WblA_ch may act as a positive regulator for natamycin biosynthesis.

FIG 4.

Effect of wblA_ch disruption on growth and production of natamycin. (A and B) Natamycin bioassay (A) and HPLC analysis of fermentation filtrates (B) from the WT strain, the wblA_ch deletion mutant, and the wblA_ch-complemented mutant. The peak of natamycin is marked by an arrow. mAU, milli-absorbance units. (C) Growth of the wild-type strain and mutant strains.

To test this hypothesis, we carried out qRT-PCR to evaluate the effects of WblA_ch on the transcription of scnRI and scnRII, pathway-specific regulators for natamycin biosynthesis (28). As expected, the transcript levels of scnRI and scnRII were lower in the wblA_ch mutant strain than in the WT strain at all tested time points from 16 h to 48 h (Fig. 5). Moreover, WblA_ch was expressed as an N-terminally His₆-tagged protein in order to test whether WblA_ch directly regulates natamycin biosynthesis by its binding activity. Unfortunately, the EMSA result showed that WblA_ch cannot directly bind to the promoters of scnRI, scnRII, or any of the other structural genes in the cluster (data not shown).

FIG 5.

Quantitative RT-PCR analysis of scnRI (A) and scnRII (B) in the WT strain and the wblA_ch deletion mutant. The RNA samples were obtained from cultures grown in YEME medium for 16 h, 24 h, 36 h, and 48 h. The expression levels of scnRI and scnRII are presented relative to the levels in the wild-type sample at 16 h, which was arbitrarily assigned a value of 1. The transcription level of hrdB was assayed as an internal control, and error bars were calculated by measuring the standard deviations among data from three replicates of each sample.

Overexpression of wblA_ch results in increased natamycin production.

Previous investigations indicated that overexpression of wblA resulted in decreased production of the corresponding antibiotic (21–24). To evaluate the function of wblA_ch in S. chattanoogensis L10, pIJ8630::wblA_ch was constructed, in which wblA_ch was under the control of the strong constitutive ermE* promoter, and this recombinant plasmid was then introduced into the WT strain. As shown in Fig. 6, the level of natamycin production of the resulting mutant (YC2) was increased ∼1.3-fold compared to that of the WT strain after 96 h of incubation, although the biomasses of these strains were similar (Fig. 4C). This result reinforced the evidence that wblA_ch is a pivotal activator for natamycin production.

FIG 6.

Effect of wblA_ch overexpression on production of natamycin. Natamycin production of fermentation filtrates in the wild-type strain (WT), the wild-type strain with pIJ8630 (pIJ8630/WT), and the wblA_ch overexpression strain (pYP3/WT) at different incubation times is shown.

Pleiotropic effects of wblA_ch on morphology differentiation.

To investigate the effect of a deletion of wblA_ch on development, the mutant was compared to the wild-type strain on YMG medium. As shown in Fig. S2A in the supplemental material, disruption of wblA_ch caused a whi phenotype, being able to erect aerial hyphae but defective in sporulation. Scanning electron microscopy of the wblA_ch mutant grown on YMG medium revealed thin, sparse aerial hyphae, with characteristic aberrant sporulation septation (see Fig. S2B in the supplemental material). The morphological deficiency in the mutant strain was fully complemented by the pSET152 derivative carrying a 750-bp DNA fragment containing wblA_ch and its own promoter. In addition, we further evaluated the transcription of whi genes, which are related to spore formation in Streptomyces. Our data showed that the transcription levels of the whi genes were severely decreased in the ΔwblA_ch mutant, with the exception of whiD, which exhibited a slight increase (Fig. 7). Other whi genes, such as whiA and whiE, showed low transcriptional activity in the ΔwblA_ch mutant, ∼20% of the wild-type levels, while transcriptional activities of whiB, whiH, and whiI were almost absent in the mutant. These results implied that WblA_ch may play a part in morphological differentiation through interacting with other whi genes.

FIG 7.

Effect of wblA_ch disruption on the transcription of whi genes. The RNA samples were harvested from strains grown on YMG agar medium overlaid with cellophane discs for 4 days. The expression level of whiA is presented relative to the wild-type sample, which was arbitrarily assigned a value of 1. The transcription level of hrdB was assayed as an internal control, and error bars were calculated by measuring the standard deviations among data from three replicates of each sample.

DISCUSSION

The biochemical and genetic mechanisms of WblA with regard to secondary metabolite and morphological differentiation have not been determined previously. In this study, we found that AdpA_ch, a pleiotropic regulator in S. chattanoogensis L10, acts as a positive regulator of the expression of wblA_ch. However, wblA was previously reported to be negatively regulated by AdpA in S. coelicolor, a model organism for studying bacterial differentiation (25). This finding is directly contradictory to our own findings. AdpA_ch shares 92% sequence identity with AdpA_sc (28), and WblA_ch shares 90% sequence identity with WblA_sc. Although these two types of protein share good amino acid sequence homology, different regulatory patterns are observed in the AdpA-WblA regulatory relationship in these two strains. The host-specific characteristics of the WblA orthologues in different strain backgrounds seem inconceivable because of the important roles of these proteins in controlling antibiotic production and morphological differentiation in Streptomycetes. Notably, results of microarray and chromatin immunoprecipitation (ChIP)/chromatin affinity precipitation (ChAP)-seq analysis of Streptomyces griseus coincide with our results (35, 36). Moreover, wblA_ch was shown to encode a positive regulator participating in natamycin biosynthesis in S. chattanoogensis L10, whereas wblA and its orthologues were previously determined to be novel antibiotic downregulators in various Streptomyces species. Based on these cumulative evidences, WblA_ch in S. chattanoogensis L10, an industrial strain for natamycin production, is suggested to be involved in a number of functions that differ from those observed in other Streptomyces species in previous reports.

The Wbl proteins, which harbor a predicted helix-turn-helix motif, are supposed to be able to bind DNA as transcription factors. A series of Wbl proteins, including WhiB1 (37), WhiB2 (38), WhiB3 (13), and WhiB4 (17), have been experimentally demonstrated to bind with DNA. However, the EMSA results did not support the hypothesis that WblA_ch has the binding ability to regulate the expression of genes involved in natamycin biosynthesis directly. Similar to our results, previous genetic studies on WblA orthologues did not reveal the mechanisms by which they regulate antibiotic production in Streptomyces. Previous reports showed that Wbl proteins in general may change their regulatory properties in response to dormancy signals, including O₂ and nitric oxide (NO), via their iron-sulfur cluster (39). It was also shown that the Wbl proteins can bind specific target proteins as ligands to modify their activity (40). Thus, it is likely that the binding activity of WblA orthologues may require a redox state or interaction with other cellular proteins.

Natamycin is the major secondary metabolite of S. chattanoogensis L10 in YEME medium; simultaneously, a yellow pigment is generated in the same medium. Several lines of evidence imply that these two pathways compete for precursors originating from primary metabolism. In our study, overexpression of wblA_ch resulted in increased production of natamycin, whereas the production of yellow pigment had decreased severely (see Fig. S3 and Table S1 in the supplemental material). Notably, the available literature reported that Wbl proteins are involved in the metabolic switchover, such as WhiB3, which was shown to act as an intracellular redox sensor and regulates fatty acid metabolism by binding directly to its promoter sequence in M. tuberculosis (13), and WhiB5, which was shown to be involved in metabolic regulation during starvation in M. tuberculosis (18). WblA was reported to be able to sense and respond to the intracellular redox environment in a manner considered to be coupled to central metabolism (7, 39). Based on this characteristic of the Wbl protein, we speculated that WblA_ch may act as a metabolic regulator where overexpression of wblA_ch results in a redirection of the flux toward the biosynthesis of natamycin. Thus, it could provide a strategy for engineering new overproducer strains of natural products where regulation of the enzymes involved in metabolic flux becomes a realistic way to generate improved strains. In sum, our findings in this study should lead to an increased understanding of the biological function of the WhiB-like protein WblA, and further experiments will provide more accurate information on the biological functions of these proteins.