Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis

ABSTRACT

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5′-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence.

IMPORTANCE BldC is a global transcriptional regulator that acts as a “brake” in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.

INTRODUCTION

Members of the genus Actinoplanes are Gram-positive filamentous actinomycetes that grow into a substrate mycelium. Typically, they produce terminal sporangia in a wide variety of shapes that arise from the substrate mycelium through a short sporangiophore. The spores released from sporangia can swim as zoospores using flagella and exhibit chemotactic behavior toward various types of compounds, such as aromatic compounds, sugars, and amino acids (1). Eventually, the zoospores germinate and grow into the substrate mycelium. Due to this complex life cycle, Actinoplanes spp. are of great interest for studying the molecular mechanisms of morphological development in prokaryotes. Actinoplanes missouriensis is a well characterized member of the genus Actinoplanes; its complete genome sequence has been determined (2). Following vegetative growth, A. missouriensis produces a few hundred dormant spores encased within a membranous substance (spore sheath) inside each terminal sporangium (3). Spores are released from sporangia upon contact with water via a process known as sporangium dehiscence (3). After release from sporangia, the spore sheath is removed to generate each zoospore, which starts swimming using flagella at a high speed (3). Zoospores display chemotactic properties for various types of substrates, including sugars, amino acids, aromatic compounds, and mineral ions, and stop swimming to germinate within a few hours (4). Because sporangium dehiscence is a process that can be considered a part of the transition from dormant spores to active cells, there is considerable interest in understanding the mechanisms that control this physiological transition.

Streptomyces bacteria are primarily soil-dwelling, ubiquitous actinomycetes that undergo morphological differentiation from vegetative mycelia to spore chains via aerial hyphae (5, 6). In several model species, the regulatory gene loci that control entry into the morphological development phase have been comprehensively identified. Genes located in such loci are called bld (bald) because mutations in these genes prevent the formation of aerial hyphae from substrate mycelium (7, 8). One of the Bld proteins, BldC, is a highly conserved small (68 amino acid residues in Streptomyces spp.) protein containing a winged helix-turn-helix (wHTH) motif, which shows a sequence similarity to the wHTH motifs of the MerR-family transcriptional regulators (9). Because BldC acts as a transcriptional regulator to sustain vegetative growth before the onset of morphological development, the deletion of bldC causes precocious hypersporulation without the formation of aerial hyphae (9, 10). MerR forms a homodimer, and two units connect via a long α helix between an N-terminal wHTH domain and a C-terminal effector-recognition domain. Canonical MerR-family regulators bind operator sequences in the spacer region between the −10 and −35 regions of promoters to untwist and shorten the unusually long spacers for transcriptional activation in response to the binding of cognate ligands to the effector-recognition domain (11–13). In contrast, BldC consists of the wHTH motif with no obvious effector-recognition or dimerization domain. Structural analysis of BldC from Streptomyces coelicolor A3(2) in the DNA-bound form revealed that the closest relative of BldC is Xis from bacteriophage lambda with respect to their DNA-binding mode. Xis is a DNA architectural protein required for the excision of the phage lambda. BldC and Xis asymmetrically oligomerize in a head-to-tail manner on direct repeats to produce a nucleoprotein filament, leading to pronounced distortion of DNA (14, 15). Although the number of direct repeat sequences in BldC-binding sites is significantly variable, this DNA-binding mode allows the cooperative binding of additional BldC monomers (15). Because BldC-like proteins are widely distributed among eubacteria, they have been proposed to be members of a new structural family of transcriptional regulators (15).

In a previous study, we reported functional analysis of a BldD ortholog: AmBldD, which is essential for normal developmental processes in A. missouriensis. AmBldD is a global transcriptional regulator that represses the transcription of the genes required for sporangium formation (16). In this study, we conducted a functional analysis of one of the AmBldD regulons, AMIS_1980, which encodes a 91-amino acid BldC ortholog in A. missouriensis.

RESULTS

Transcription of bldC is activated during sporangium formation.

We determined the transcriptional start point of bldC to be 71 nucleotides upstream of the translational start codon using high-resolution S1 nuclease mapping (Fig. 1A). In a previous study, we reported that AmBldD directly binds to the upstream region of bldC to repress its transcription (16). Consistent with this, a palindromic sequence similar to the sequence of the AmBldD box was found in the 5′-untranslated region of bldC (Fig. 1B). In S. coelicolor A3(2), bldC is a member of the BldD regulon (17). Because AmBldD is abundantly produced during vegetative growth, and its levels decrease during sporangium formation (16), the transcription of bldC was expected to be induced during sporangium formation. Thus, we quantified the transcript levels of bldC in the wild-type (wt) strain using reverse transcription-quantitative PCR (qRT-PCR). The transcripts of bldC were slightly detected in vegetative hyphae cultivated in nutrient-rich peptone-yeast extract-MgSO₄ (PYM) liquid broth at 30°C for 72 h and in substrate hyphae cultivated on sporangium-forming humic acid-trace element (HAT) agar at 30°C for 24 h (Fig. 1C, bars V and S₁). The transcripts were abundantly detected in the mixture of substrate hyphae and sporangia formed on HAT agar on day 3, and the transcript levels further increased on day 7 (Fig. 1C, bars S₃ and S₇). The most abundant transcripts were detected in the mixture of hyphae and sporangia just after the suspension into 25 mM histidine solution to induce sporangium dehiscence (Fig. 1C, bar D₀). Then, the transcript levels decreased during sporangium dehiscence and increased again after the completion of sporangium dehiscence (Fig. 1C, bars D₁₅ and D₆₀). The physiological significance of this reactivation is unknown, but several genes that are transcriptionally activated during sporangium formation show a similar transcriptional pattern.

FIG 1.

Transcriptional analysis of bldC. (A) Transcriptional start point of bldC determined by high-resolution S1 nuclease mapping. The S1 nuclease-digested fragment is flanked by a Maxam-Gilbert sequence ladder (G+A). The arrowhead indicates the position of the S1 nuclease-protected fragment. The 5′ terminus of the mRNA was assigned to the position indicated by the bent arrow because the fragment generated by the chemical sequencing reaction migrates 1.5 nucleotides ahead of the corresponding fragment generated by S1 nuclease digestion of the DNA-RNA hybrid. (B) Nucleotide sequence of the upstream region of bldC. The transcriptional start point is shown by the bent arrow. The putative AmBldD box (composed of an inverted repeat sequence) is indicated by a rectangle; a consensus AmBldD box sequence is shown below the sequence. The translational start codon is underlined. (C) Transcript levels of bldC. The levels of the transcript were examined using reverse transcription-quantitative PCR (qRT-PCR) analysis. The rpoB transcript was used as an internal standard. The data are the mean values from three biological replicates ± standard deviations. RNA samples were prepared from vegetative mycelia grown in PYM liquid broth for 72 h (V), substrate hyphae grown on HAT agar for 24 h (S₁), mixtures of substrate hyphae and sporangia grown on HAT agar for 3 and 7 days (S₃ and S₇), and sporangia (including some amount of substrate hyphae) incubated in 25 mM histidine solution for sporangium dehiscence for 0, 15, and 60 min (D₀, D₁₅, and D₆₀).

Null mutant of bldC is defective in sporangium dehiscence.

To examine the in vivo function of BldC, we generated a bldC null mutant (ΔbldC) strain. No difference was observed between the wt and ΔbldC strains by macroscopic observation of mycelia or sporangia grown on nutrient-rich yeast extract-beef extract-NZ amine-maltose monohydrate (YBNM) and HAT agar (data not shown). To examine sporangium formation in detail, we observed the mycelia of the wt and ΔbldC strains grown on HAT agar at 30°C for 7 days using scanning electron microscopy (SEM). Contrary to our hypothesis that bldC is involved in sporangium formation, both strains produced normal globose or subglobose sporangia with short sporangiophores (Fig. S1 in the supplemental material), indicating that BldC is not required for the formation of normally shaped sporangia under the examined conditions. Then, we observed ultrathin sections of the wt and ΔbldC sporangia, which were grown under the same conditions used for SEM analysis, by transmission electron microscopy (TEM) to examine spore maturation inside sporangia. Consequently, both strains produced normal round sporangiospores of similar sizes (Fig. 2), further supporting that BldC is not required for sporangium formation.

FIG 2.

Transmission electron microscopy (TEM) observation of the ultrathin sections of the wild-type (wt) (A) and bldC null mutant (ΔbldC) (B) sporangia produced on HAT agar through 7 days of cultivation.

Next, we examined sporangium dehiscence and motility of zoospores released from sporangia using optical microscopy. Under dehiscence-inducing conditions, few spores were released from the sporangia of the ΔbldC strain, whereas most of the sporangia from the wt strain opened up and released a large number of spores (Fig. 3A and B). In this experiment, the tested strains harbored the integration vector pTYM19-Apra on the chromosome because they were used as control strains in the complementation test, as described below. This observation suggests that sporangium dehiscence was severely inhibited in the ΔbldC strain. Therefore, we quantified the spores released from sporangia of the wt and ΔbldC strains, both of which contained pTYM19-Apra on the chromosome, by counting the colonies formed on YBNM agar after incubation at 30°C for 2 days. Sporangia of the ΔbldC strain formed on one HAT agar plate released approximately 10⁵ spores, whereas the sporangia of the wt strain released approximately 10⁷ spores under the same conditions (Fig. 3D). In the gene complementation test, sporangium dehiscence and the number of spores released from sporangia of the ΔbldC strain were restored by the introduction of bldC with its own promoter on pTYM19-Apra (Fig. 3C and D). These results support the hypothesis that BldC is required for sporangium dehiscence. Considering the possibility that the ΔbldC zoospores failed to germinate under a nutrient-rich condition, we cultivated spores released from sporangia of the wt and ΔbldC strains by suspending the spores into PYM liquid broth. After shaking at 30°C for 5 h, most spores of both strains germinated similarly when observed by optical microscopy (Fig. S2 in the supplemental material). This result indicated that BldC is not required for spore germination.

FIG 3.

Sporangium dehiscence and number of spores released from sporangia. (A to C) Sporangium dehiscence observed by optical microscopy for the wt (A) and ΔbldC (B) strains harboring the empty plasmid (pTYM19-Apra) and the ΔbldC strain harboring the complementation plasmid (C) on the chromosome. Each strain was cultivated on HAT agar at 30°C for 7 days. Sporangia scraped from HAT agar were suspended into 25 mM histidine solution and incubated for 1 h. The left and right panels show the microscopic images of sporangia or spores obtained just after the suspension and after incubation for 1 h, respectively. Sporangia and spores are shown by arrows and arrowheads, respectively. (D) Number of spores released from sporangia. Zoospores released from sporangia by pouring 25 mM NH₄HCO₃ solution were counted as CFU on YBNM agar. Data are mean values ± standard errors from three biological replicates.

Analysis of the BldC regulon.

Considering the significant phenotypic changes under the dehiscence-inducing condition in the ΔbldC strain, we postulated that BldC regulates the transcription of genes involved in sporangium dehiscence. We hypothesized that the ΔbldC strain produces physiologically immature sporangia (despite their normal shapes) that are deficient in sporangium dehiscence when placed under dehiscence-inducing conditions. Therefore, we compared the transcriptomes during sporangium formation between the wt and ΔbldC strains using RNA sequencing (RNA-Seq) analysis. Total RNA was extracted from a mixture of substrate hyphae and sporangia grown on HAT agar at 30°C for 3 days in triplicate for each strain. From the obtained data, we identified the genes that met the following criteria as up- and downregulated genes in the ΔbldC strain compared to the wt strain: (i) average number of reads per kilobase of coding sequence per million mapped reads (RPKM) in the ΔbldC strain higher than 2.0-fold (upregulation) or lower than 0.5-fold (downregulation) compared to that of the wt strain, and (ii) q value less than 0.05. Based on these criteria, we observed that the transcript levels of 116 genes were significantly changed, with 90 and 26 genes being downregulated and upregulated in the ΔbldC strain, respectively (Fig. 4; Tables S1 and S2 in the supplemental material).

FIG 4.

Volcano plot of differential gene expression analyzed by RNA sequencing (RNA-Seq). Each gene was plotted based on the fold change in expression of the ΔbldC strain versus the gene expression in the wt strain and the q value. Differentially expressed genes are highlighted by blue and red dots, which indicate the upregulated and downregulated genes in the ΔbldC strain, respectively. The dotted line indicates the threshold of the q value (0.05).

Among the 90 downregulated genes in the ΔbldC strain, we found five genes belonging to the flagellar gene cluster (fliC, flhA, fliO, flgC, and AMIS_76340), a gene belonging to the pilus gene cluster (pilO), six genes probably related to chemotaxis (cheA2, cheY2, cheY4, cheW4, mcp18, and mlp8), and six genes that are or are predicted to be involved in transcriptional regulation (tcrA, hhkA, fliA4, AMIS_6890, AMIS_37060, and AMIS_75240; Table S1). Notably, hhkA and tcrA were downregulated in the ΔbldC strain (2.5- and 3.6-fold downregulation, respectively). These genes, which most likely encode a pair of proteins constituting a two-component regulatory system, are globally involved in sporangium formation, spore dormancy, and sporangium dehiscence (18, 19). In addition, we identified a gene (AMIS_65300) encoding an ortholog of SsgB (4.5-fold downregulation in the ΔbldC strain), which is the key developmental protein required for sporulation in Streptomyces (20, 21). Eight genes that are predicted to be involved in transcriptional regulation (AMIS_10340, AMIS_22170, AMIS_23110, AMIS_37680, AMIS_47320, AMIS_55890, AMIS_72730, and AMIS_72740) were among the 26 genes upregulated in the ΔbldC strain (Table S2). Among these, the gene products of AMIS_72730 and AMIS_72740 are expected to constitute a two-component regulatory system (Table S2).

BldC directly activates transcription of at least 18 genes.

A previous structural analysis of the S. coelicolor A3(2) BldC-DNA complex revealed two major sequence elements for the specificity of the binding of BldC to DNA: a 4-bp AT-rich sequence, followed by a C or G 4 or 5 nucleotides downstream. Therefore, the S. coelicolor A3(2) BldC-binding consensus sequence is 5′-AATT-N_3-4-(C/G)-3′ (15). However, conservation of this consensus sequence is not critical to BldC binding because the narrow minor groove formed by the AT-rich sequence is important instead of the direct base recognition by BldC (15). Therefore, it is difficult to predict BldC-binding sites using in silico analyses. Consistent with this report, a computational search of upstream regions of the genes regulated by BldC using the MEME algorithm (http://meme-suite.org/tools/meme) failed to produce highly enriched sequences (data not shown). Therefore, to examine whether BldC directly regulates the transcription of genes upregulated or downregulated in the ΔbldC strain, we produced and purified a recombinant BldC protein with a polyhistidine tag at its C-terminal end (BldC-His) using Escherichia coli as the host (Fig. S3A in the supplemental material). We prepared ³²P-labeled DNA probes containing the upstream regions from each of the 29 genes that were downregulated (28 genes) or upregulated (1 gene) in the ΔbldC strain (Tables S1 and S2) and used them for electrophoretic mobility shift assay (EMSA). In this assay, we selected these 29 genes from the putative 116 BldC-dependent genes (Tables S1 and S2) because their transcriptional start points have been determined by our unpublished differential RNA-Seq (dRNA-Seq) analysis and a previous study (19). Retarded bands were detected for 18 of the 29 probes (Fig. 5). All the 18 probes were derived from genes downregulated in the ΔbldC strain (Table S1). In contrast, no retarded signal was detected using the remaining 11 probes (Fig. S3B). Because BldC-His did not bind to the upstream regions from hhkA and ssgB (Fig. S3B), we concluded that BldC indirectly activates the transcription of hhkA and ssgB.

FIG 5.

Binding of BldC-His to regions upstream of the BldC-regulated genes. Electrophoretic mobility shift assay (EMSA) was performed using the ³²P-labeled DNA probes. Corresponding gene names or identifiers and amount of BldC-His used are shown above the lanes. The positions of the wells and the BldC-His-bound probes are shown by open and closed triangles, respectively.

BldC in A. missouriensis shares 43% amino acid identity with the S. coelicolor A3(2) BldC, and the helix-turn-helix motifs of both proteins are highly conserved (Fig. S4 in the supplemental material). Structural analysis of the S. coelicolor A3(2) BldC-DNA complex demonstrated that the side chains of Thr27, Thr29, Trp31, and Lys33, and the amide nitrogen of Ala15 and Asp24 anchor wHTH on the DNA through interaction with the phosphate backbone of both DNA strands (15). The basic residues Arg47 and Arg48 aid docking between the S. coelicolor A3(2) BldC wing and the DNA minor groove, and the His46 side chain fits within the AT-rich narrowed minor groove. Furthermore, Arg30, located in the recognition helix, recognizes C or G 4 or 5 nucleotides downstream from the 4-bp AT-rich sequence (15). These residues, except for Ala15 and Lys33, are conserved in the A. missouriensis BldC (Fig. S4), suggesting that the A. missouriensis BldC binds to a direct repeat sequence similar to that of the S. coelicolor A3(2) BldC. To obtain insight into the BldC-binding sequence, we performed a DNase I footprinting assay on the upstream regions of AMIS_65940 and AMIS_6350; their transcript levels were downregulated in the ΔbldC strain (Table S1). The assay suggested that BldC-His protected large regions extending approximately from −22 to +20 and from −55 to −30 in AMIS_65940 and AMIS_6350, respectively, relative to the transcriptional start points (Fig. S5A and B in the supplemental material). In DNase I footprinting assay and EMSA using mutated DNA probes (described below), the nucleotide positions were described by referring to the transcriptional start points predicted from RNA-Seq and unpublished dRNA-Seq analyses. In AMIS_65940, four and two sites of enhanced DNase I cleavage were observed on the top and bottom strands, respectively (Fig. S5A and C). In AMIS_6350, four and one sites of enhanced cleavage were observed on the top and bottom strands, respectively (Fig. S5B and D). These results demonstrate that BldC-His binds to long sequences, leading to DNA distortion. The BldC-His-binding sites in the AMIS_65940 and AMIS_6350 promoter regions contained five and three imperfect direct repeat sequences, respectively, on the top strands (Fig. S5C and D), suggesting that BldC cooperatively binds to the direct repeat sequences in a head-to-tail fashion, similar to the S. coelicolor A3(2) BldC (15).

To confirm that BldC-His binds to the direct repeats in the other regions examined by EMSA, we prepared mutated DNA probes with all or most of the predicted BldC-binding motifs deleted. In this experiment, we selected five probes (upstream regions from AMIS_270, AMIS_55760, AMIS_48270, AMIS_76480, and AMIS_76820 in Fig. 5) for target regions, in which up to five imperfect repeats were identified visually (Fig. 6A). In EMSA using the mutated probes, no retarded signal was produced for the upstream regions from AMIS_270 and AMIS_76820, and the affinity of BldC-His was severely lowered for the upstream regions from AMIS_55760, AMIS_48270, and AMIS_76480 (Fig. 6B), indicating that BldC-His binds to the direct repeat sequences in these regions. Structural analysis of the S. coelicolor A3(2) BldC-DNA complex suggested that dimerization on one direct repeat is the minimum requirement for the DNA binding of BldC, and further multimerization would occur at the binding sites carrying additional repeat sequences (15). Thus, cooperative binding of BldC would lead to the detection of single retarded signals in EMSA, irrespective of the number of repeat sequences within the probes (Fig. 5). Therefore, BldC directly activates the transcription of at least 18 genes by binding to their promoter regions.

FIG 6.

Binding of BldC-His to direct repeat sequences. (A) Nucleotide sequences of regions upstream from AMIS_270, AMIS_55760, AMIS_48270, AMIS_76480, and AMIS_76820. Direct repeat sequences identified by visual inspection are shaded in light blue. Arrows indicate the direction of each repeat sequence. Bent arrows indicate the transcriptional start points. Translational start codons are underlined. (B) EMSA using mutated probes. DNA fragments were ³²P-labeled and used as probes. Schematics of the probes are shown above the panels. DNA regions amplified by PCR are shown by thick lines (probes 1, 2, 3, 4, and 5), and deleted sequences in the mutated probes are shown by thin lines (probes 1Δ, 2Δ, 3Δ, 4Δ, and 5Δ). Transcriptional start sites and direct repeat sequences are shown as in panel A. The amount of BldC-His used is shown above the lanes. The positions of the wells and the BldC-His-bound probes are shown by open and closed triangles, respectively.

Introduction of multiple copies of tcrA into the ΔbldC strain.

As described above, the transcript level of tcrA was significantly downregulated in the ΔbldC strain (3.6-fold; Table S1). Because we found an imperfect direct repeat in the upstream region of tcrA that overlapped with the sequence of the AmBldD box (Fig. S6 in the supplemental material), we postulated that BldC binds to this direct repeat to activate the transcription of tcrA (Fig. 5). Considering that sporangium dehiscence is severely inhibited in the ΔtcrA strain (18), we hypothesized that the decreased expression of tcrA may be a reason for the deficiency in sporangium dehiscence in the ΔbldC strain. To verify this, we introduced tcrA with its own promoter into the ΔbldC strain using pCAM2, which is an E. coli-A. missouriensis shuttle vector with the copy number of 4 per chromosome in A. missouriensis (22). Because tcrA transcripts were detected in the ΔbldC strain to a certain extent (Table S1), we assumed that the introduction of multiple copies of tcrA led to the increased expression of tcrA in the ΔbldC strain. Contrary to our hypothesis, however, the number of spores released from the ΔbldC sporangia was not restored to the wt level by the introduction of tcrA (Fig. S7 in the supplemental material), suggesting that the strictly controlled expression of tcrA is important for sporangium dehiscence (see Discussion).

DISCUSSION

Although the DNA-binding domain of BldC is related to that of MerR-family transcription factors, recent transcriptional, biochemical, and structural studies on BldC in S. coelicolor A3(2) and Streptomyces venezuelae revealed a wider relationship of BldC with nucleoid-associated proteins and DNA architectural proteins (10, 14, 15). In this study, however, we conducted detailed characterization of the BldC ortholog in A. missouriensis, focusing on the function of BldC as a transcription factor, and revealed the effect of BldC on gene expression and morphological development in A. missouriensis. Although BldC-like proteins are widely distributed throughout eubacteria, no BldC orthologs have been studied outside the streptomycetes. To the best of our knowledge, this is the first report on the detailed characterization of the BldC-family proteins in the actinomycetes other than Streptomyces. In Streptomyces, BldC functions as a developmental “brake” to produce a prolonged period of vegetative growth before entry into developmental stages (10). In contrast, BldC is essential for sporangium dehiscence, which is the activation process of dormant sporangia, but is not required for the formation of sporangia with normal shapes (Fig. 2 and 3; Fig. S1). Because precocious sporangium formation was not observed in the ΔbldC strain (Fig. S1; data not shown), BldC does not seem to function as a developmental “brake.” We conclude that BldC is a key factor in the production of mature sporangia that can release spores under dehiscence-inducing conditions instead of maintaining vegetative growth. Thus, in the evolutionary process, BldC seems to have evolved to play different roles in the morphological differentiation between Streptomyces and Actinoplanes.

The BldC regulon has been extensively studied in S. venezuelae using ChIP-Seq and RNA-Seq (10). BldC in S. venezuelae functions as both an activator and a repressor of many developmental genes. It represses the transcription of a subset of target genes, including whiI, smeA-sffA, whiD, sigF, dynAB, ssgB, and sigI, whereas it activates a subset of target genes, including whiH, bldM, wblA, and ssgR (10). Among these, an ortholog of ssgB (AMIS_65300), which encodes the only SsgA-like protein in A. missouriensis, was found among the genes downregulated in the ΔbldC strain (Table S1). Thus, ssgB is differently regulated by BldC between S. venezuelae and A. missouriensis. In addition, orthologs of whiD (AMIS_6880), sigF (AMIS_43830), dynAB (AMIS_810-820), and wblA (AMIS_78290) are conserved in A. missouriensis, but the transcription of all these genes was not significantly changed in the ΔbldC strain in this study (Tables S1 and S2). Next, we examined whether S. venezuelae has orthologs of the putative 116 BldC-dependent A. missouriensis genes (Tables S1 and S2); we defined orthologs in accordance with the following two criteria: (i) the reciprocal best hits in the BLASTP analysis with over 40% amino acid identity of the gene products and (ii) synteny (preserved order of gene on the chromosome). As a result, a total of seven genes (4 of the 90 genes and 3 of the 26 genes down- and upregulated, respectively, in the ΔbldC strain) were conserved in S. venezuelae (Tables S1 and S2). Within the four S. venezuelae orthologs, three genes were downregulated in the ΔbldC strain, as in A. missouriensis, whereas the remaining one gene was upregulated in the ΔbldC strain (10) (Table S1). Meanwhile, all of the three S. venezuelae orthologs were upregulated in the ΔbldC strain (10), as in A. missouriensis (Table S2). These gene products are predicted to function as components of ABC transporters, although their physiological roles in vegetative growth or morphological development remain elusive. The difference in the gene composition of the BldC regulon between Streptomyces and Actinoplanes also supports the different regulatory roles of BldC between these two genera. It should be noted that a large part of the BldD regulon also differs between Streptomyces and Actinoplanes, whereas BldD seems to have similar functions in these two genera (BldD represses the expression of genes required for the onset of morphological differentiation) (16). Thus, several global regulators of morphological differentiation are conserved among actinomycetes, but their functions and regulons are not necessarily similar.

In the ΔbldC strain, the transcript levels of tcrA, which encodes a global transcriptional activator controlling sporangium formation, sporangium dehiscence, and spore dormancy (18), were significantly downregulated (3.6-fold; Table S1). We postulate that the downregulation of tcrA led to the decreased transcript levels of several genes of the TcrA regulon in the ΔbldC strain; a total of 11 genes, including three flagellar genes (fliC, fliO, and flgC), three chemotaxis-related genes (cheA2, cheY4, and mcp18), and a pilus gene (pilO), all of which were downregulated in the ΔbldC strain in this study, were also downregulated significantly in a tcrA null (ΔtcrA) mutant analyzed in our previous study (Table S1) (19). We found an imperfect direct repeat in the upstream region of tcrA that overlapped with the sequence of the AmBldD box (Fig. S6 in the supplemental material). Because BldC-His was bound to the upstream region of tcrA in EMSA (Fig. 5), we postulated that BldC binds to this direct repeat to activate the transcription of tcrA. Although we hypothesized that the reduced expression of tcrA would result in a defect in sporangium dehiscence in the ΔbldC strain, this defect was not restored by the introduction of multiple copies of tcrA with its own promoter (Fig. S7). Therefore, we concluded that the lack of BldC affects the expression of a large number of genes directly or indirectly and proposed that the sum of these effects reduces the ability to form physiologically mature sporangia that can release spores under dehiscence-inducing conditions. Nevertheless, further analysis of the BldC regulon will provide clues to the molecular mechanisms of sporangium dehiscence.

MATERIALS AND METHODS

General methods.

The bacterial strains, plasmid vectors, and media used in this study were described previously (16, 23). The primers used in this study are listed in Table S3 in the supplemental material. The preparation of A. missouriensis cells and RNA extraction were described previously (18). qRT-PCR and high-resolution S1 nuclease mapping were performed as described previously (24). The rpoB gene was used as an internal standard in qRT-PCR. All reactions were performed in triplicate, and the data were normalized using the average for the internal standard. SEM was performed with an S-4800 scanning electron microscope (Hitachi, Tokyo, Japan) as described previously (25). TEM was performed with an H-7600 electron microscope (Hitachi) as described previously (18).

Construction of the ΔbldC mutant.

For the construction of ΔbldC mutant, the upstream and downstream regions of bldC were amplified by PCR. The amplified DNA fragments were cloned into pUC19 and sequenced to confirm that no PCR-derived error had been introduced. The cloned fragments were digested and cloned together into pK19mobsacB (26), the kanamycin resistance gene of which had been replaced with the apramycin resistance gene aac(3)IV (16). Using the plasmid, ΔbldC mutant was generated by the method described previously (23). The disruption of the gene was confirmed by PCR (data not shown).

Construction of the recombinant strain for complementation test.

A DNA fragment containing the promoter and coding sequences of bldC was amplified by PCR. The amplified fragment was cloned into pUC19 and sequenced to confirm that no PCR-derived error had been introduced. The cloned fragment was digested and cloned into pTYM19-Apra (18). The generated plasmid was introduced into the ΔbldC strain by conjugation as described previously (27). Plasmid pTYM19-Apra was also introduced into the wild-type and ΔbldC strains for the vector control strains. Then, apramycin-resistant colonies were obtained.

Microscopic observation.

The sporangia and mycelia formed on the HAT agar were scraped with a spatula and suspended into 25 mM histidine solution. The solution was incubated at room temperature for 1 h. Before and after the incubation, the sporangium-containing solution was observed with a BH-2 light microscope equipped with a DP22 digital camera (Olympus, Tokyo, Japan). To observe spore germination, zoospores of the wt and ΔbldC strains released from sporangia were suspended into PYM liquid broth and cultivated with shaking at 30°C for 5 h. Before and after the cultivation, the zoospore-containing solution was observed with the light microscope.

Counting of spores released from sporangia.

Strains were precultured in PYM broth with shaking at 30°C for 2 days. A fixed amount of the mycelium (1 mL of preculture) was inoculated onto a HAT plate, and the plate was incubated at 30°C for 7 days to produce sporangia. Zoospores were released from the sporangia by pouring 10 mL of 25 mM NH₄HCO₃ solution onto one HAT plate and incubating the plate at room temperature for 1 h. After being collected from the plate, the zoospore-containing solution was filtrated through a 5-μm Acrodisc membrane filter (Pall Corporation, NY, USA) to eliminate hyphae and sporangia. A portion of the filtrate (or its diluted solution) was inoculated onto YBNM agar, and the plate was incubated at 30°C for 2 days. From the number of colonies formed on YBNM agar, the number of zoospores released from sporangia on one HAT plate was estimated.

RNA-Seq and in silico analyses.

Total RNAs were extracted from the wt and ΔbldC strains as described previously (18). The qualities and quantities of the total RNAs were assessed with a Bioanalyzer DNA1000 (Agilent Technologies, CA, USA). Sequencing libraries were prepared with 3 μg of RNA as the starting material, and the sequencing was performed with a HiSeq 2500 sequencer (Illumina, CA, USA) to generate nondirectional paired-end 150-nucleotide reads. At least 1.2 Gb of sequencing data was obtained from each cDNA library. Library construction and sequencing were performed by Novogene (Beijing, China). The reads were filtered by sequence quality using a CLC Genomics Workbench 6.05 (Qiagen, Venlo, Netherlands) and mapped to the A. missouriensis genome sequence.

Production and purification of the recombinant protein.

A 300-bp DNA fragment containing the bldC-coding sequence was amplified by PCR. The amplified fragment was cloned into pUC19 and sequenced to confirm that no PCR-derived error had been introduced. The cloned fragment was digested and cloned into pET26b, generating pET26b-bldC. The plasmid was introduced into E. coli BL21(DE3). The transformant was cultivated in LB broth (100 mL) at 37°C for 2.5 h. Then, isopropyl-β-d-1-thiogalactopyranoside was added to the culture with a final concentration of 1 mM. After a further cultivation at 30°C for 5 h, the cells were collected by centrifugation at 3,000 × g for 10 min and suspended in 3 mL of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), followed by disruption by sonication. After removal of cell debris by centrifugation at 10,000 × g for 30 min, BldC-His was purified from the cell extract using nickel-nitrilotriacetic acid Superflow resin (Qiagen) according to the manufacturer’s instructions. BldC-His was eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0). The quality of the purified protein was assessed by SDS-PAGE using a 15% polyacrylamide gel.

EMSAs.

DNA fragments were prepared by PCR and ³²P-labeled at the 5′ ends using [γ-³²P]ATP (220 TBq/mmol; PerkinElmer, MA, USA) and T4 polynucleotide kinase (TaKaRa Biochemicals, Shiga, Japan). Recombinant BldC-His protein was incubated with the labeled DNA probes at 30°C for 30 min in a binding buffer (25 mM NaH₂PO₄, 7.5% glycerol, 100 ng/μL bovine serum albumin, 25 ng/μL poly[dI-dC]), and the mixtures were subjected to 6% native PAGE at 34 mA for 2 h. The mutated probes, in which the direct repeat sequences were deleted, were prepared by overlap extension PCR using the wild-type DNA as a template.

DNase I footprinting.

DNA fragments were prepared by PCR and ³²P-labeled at both 5′ ends using [γ-³²P]ATP (PerkinElmer) and T4 polynucloetide kinase (TaKaRa Biochemicals). Labeling at one of the 5′ ends was eliminated by digestion with EcoRI. The reaction mixture contained 10,000 cpm ³²P-labeled DNA probe, 0 to 10 μM BldC-His, 25 mM HEPES, 0.5 mM EDTA, 50 mM KCl, and 10% glycerol (pH 7.9). After incubation of the mixture at 30°C for 30 min, DNase I was added at a final concentration of 20 μg/mL, and the mixture was further incubated for 1 min. The digestion was stopped by adding 100 μL of stop solution (100 mM Tris, 100 mM NaCl, 1% sodium N-lauroylsarcosinate, 10 mM EDTA, 25 μg/mL of salmon sperm DNA, pH 8.0) and 300 μL of phenol-CHCl₃ (1:1). After ethanol precipitation, the pellet was washed with 70% ethanol, dissolved in 4 μL of the formamide-dye mixture (28), and separated on 6% polyacrylamide gel.

Introduction of tcrA into the ΔbldC strain.

A 1.5-kb DNA fragment containing the promoter and coding sequences of tcrA was amplified by PCR. The amplified fragment was cloned into pUC19 and sequenced to confirm that no PCR-derived error had been introduced. The cloned fragment was digested and cloned into the E. coli-A. missouriensis shuttle vector pCAM2 (22). The generated plasmid was introduced into the ΔbldC strain by conjugation. Plasmid pCAM2 was also introduced into the wt and ΔbldC strains to generate vector control strains. Then, apramycin-resistant colonies were obtained.

Data availability.

The nucleotide sequence data of the RNA-Seq analysis have been deposited in the DDBJ Sequence Read Archive under the accession number DRA014123.