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J Bacteriol. Feb 2003; 185(4): 1273–1283.
PMCID: PMC142869

Transcriptional Switch On of ssgA by A-Factor, Which Is Essential for Spore Septum Formation in Streptomyces griseus

Abstract

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) triggers morphological development and secondary metabolism in Streptomyces griseus. A transcriptional activator (AdpA) in the A-factor regulatory cascade switches on a number of genes required for both processes. AdBS11 was identified in a library of the DNA fragments that are bound by AdpA and mapped upstream of ssgA, which is essential for septum formation in aerial hyphae. Gel mobility shift assays and DNase I footprinting revealed three AdpA-binding sites at nucleotide positions about −235 (site 1), −110 (site 2), and +60 (site 3) with respect to the transcriptional start point, p1, of ssgA. ssgA had two transcriptional start points, one starting at 124 nucleotides (p1) and the other starting at 79 nucleotides (p2) upstream of the start codon of ssgA. Of the three binding sites, only sites 1 and 2 were required for transcriptional activation of p1 and p2 by AdpA. The transcriptional switch on of ssgA required the extracytoplasmic function sigma factor, σAdsA, in addition to AdpA. However, it was unlikely that σAdsA recognized the two ssgA promoters, since their −35 and −10 sequences were not similar to the promoter sequence motifs recognized by σBldN, a σAdsA homologue of Streptomyces coelicolor A3(2). An ssgA disruptant formed aerial hyphae, but did not form spores, irrespective of the carbon source of the medium, which indicated that ssgA is a member of the whi genes. Transcriptional analysis of ssfR, located just upstream of ssgA and encoding an IclR-type transcriptional regulator, suggested that no read-through from ssfR into ssgA occurred, and ssgA was transcribed in the absence of ssfR. ssgA was thus found to be controlled by AdpA and not by SsfR to a detectable extent. SsfR appeared to regulate spore septum formation independently of SsgA or through interaction with SsgA in some unknown way, because an ssfR disruptant also showed a whi phenotype.

In the gram-positive, filamentous bacterium Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) acts as a chemical signaling molecule, or microbial hormone, that triggers secondary metabolism and cellular differentiation at an extremely low concentration (7-9). It serves as a switch for biosynthesis of streptomycin and almost all secondary metabolites and for formation of aerial mycelium. A-factor is produced in a growth-dependent manner and accumulated to reach a critical concentration at or near the decision point (1). The decision point is approximately at the middle of the exponential growth phase. A-factor binds its specific receptor, ArpA, and dissociates it from DNA, thereby allowing the transcription of adpA (25, 26). adpA encodes a transcriptional activator for a number of genes required for secondary metabolism and aerial mycelium formation (25, 31). AdpA activates the transcription of strR, a pathway-specific transcriptional activator, which then switches on transcription of other streptomycin biosynthetic genes within the gene cluster (25). AdpA also turns on adsA, encoding an extracytoplasmic function (ECF) sigma factor (σAdsA) of RNA polymerase required for aerial mycelium formation (31). Another target we identified is sgmA encoding a metalloendopeptidase that probably plays a role in degradation of proteins in substrate hyphae for reuse in formation of aerial hyphae (15).

adsA and sgmA were found in a library of DNA fragments identified by a gel mobility shift-PCR method as recognized and bound by AdpA (11, 31). The library includes more than 60 DNA fragments, and we are characterizing them individually. In this paper, we describe the transcriptional activation of ssgA (AdBS11 in the library) by AdpA. ssgA was originally isolated by Kawamoto and Ensign (16, 17) as a gene that, when introduced at a high copy number, inhibited sporulation of an S. griseus mutant strain NY5 in a nutrient-rich liquid medium. The wild-type S. griseus did not sporulate in rich liquid medium, while mutant NY5 sporulated in the same medium. ssgA encodes a 136-amino-acid protein of 14.8 kDa with a strong negative charge (17, 18). Western blot analysis with anti-SsgA antibody suggested a close correlation between SsgA and the onset of sporulation (18). In addition, SsgA was detected only in the presence of A-factor (18), which is in agreement with the present study. The decisive role of ssgA was revealed by gene disruption experiments; ssgA disruptants showed a “white” (whi) phenotype, which suggests that ssgA plays a role in septum formation in aerial hyphae (13). Involvement of ssgA in septation in aerial hyphae was also observed in Streptomyces coelicolor A3(2) (30).

This paper deals with the transcriptional switch on of ssgA by AdpA, which binds three sites in front of the ssgA coding region. We also studied a hypothesized correlation of transcription between ssgA and a putative regulatory gene [ssfR in S. griseus and ssgR in S. coelicolor A3(2)] located upstream of ssgA in the same orientation, since these genes were reported to be phenotypically related (13, 30). Our detailed transcriptional analyses, together with the observed phenotypes of ssgA and ssfR disruptants, excluded the anticipated tight correlation, showing that ssgA was transcribed from its own promoter in the absence of ssfR. We describe here (i) transcription of ssgA in response to A-factor and AdpA, as determined by S1 nuclease mapping; (ii) determination of the three AdpA-binding sites by DNase I footprinting; (iii) the requirement of two of the three AdpA-binding sites for transcriptional activation, as determined by measuring transcription of the ssgA promoter with mutated AdpA-binding sites; and (iv) the phenotypes of ssgA and ssfR disruptants.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

S. griseus IFO13350 was obtained from the Institute of Fermentation (IFO), Osaka, Japan. S. griseus HH1 (10), ΔadpA (25), and ΔadsA (31) mutant strains were described previously. S. griseus strains were grown in YMPD medium (yeast extract [Difco], 0.2%; meat extract [Kyokuto], 0.2%; Bacto-Peptone [Difco], 0.4%; NaCl, 0.5%; MgSO4 · 7H2O, 0.2%; and glucose, 1% [pH 7.2]). For scanning electron microscopy analysis, YMP-mannitol medium (1% mannitol used instead of glucose in YMPD) and R2YE medium (6) were also used. For regeneration of protoplasts, R2YE medium was used. Agar medium contained 2.2% agar. Streptomyces lividans TK21 was used for plasmid construction. Thiostrepton (25 μg/ml) and neomycin (10 μg/ml) were added when necessary. As a convenient Streptomyces plasmid, a low-copy-number shuttle vector pKUM20, with a multicloning site of XhoI-EcoRI-PstI-BamHI-PmaCI-HindIII at the original PstI site of pKU209, was constructed as follows. The original unique sites of BamHI, EcoRI, HindIII, and XhoI on pKU209 containing the ampicillin and thiostrepton resistance genes (14), with its copy number of 1 to 2 per genome, were deleted one by one by the combination of Klenow fragment and self-ligation. An XhoI site was generated at the original PstI site of pKU209 by digestion of pKU209 with PstI, trimming the ends with T4 DNA polymerase, and ligation with an 8-mer XhoI linker. Annealed multi1 and multi2 primers (Table (Table1)1) were inserted into the created XhoI site by standard DNA manipulation. Plasmid pADP10H carrying adpA on the high-copy-number vector pIJ487 was previously described (25). Escherichia coli JM109 and vector pUC19 for DNA manipulation were purchased from Takara Shuzo. E. coli JM110 containing dam and dcm mutations was used to prepare nonmethylated Streptomyces DNA used for gene disruption and plasmid transformation. Histidine-tagged AdpA (AdpA-H) was purified from E. coli BL21 (DE3) harboring pET-adpA, as described previously (31). The media and growth conditions for E. coli were those described by Maniatis et al. (21). Ampicillin (50 μg/ml) and kanamycin (50 μg/ml) were used for E. coli when necessary.

TABLE 1.
Primers used in this study

General recombinant DNA studies.

Restriction enzymes, T4 DNA ligase, and other DNA-modifying enzymes were purchased from Takara Shuzo. [α-32P]dCTP (110 TBq/mmol) for DNA labeling with the Takara BcaBest DNA labeling system and [γ-32P]ATP (220 TBq/mmol) for end labeling at 5′ ends with T4 polynucleotide kinase were purchased from Amersham Biosciences. DNA was manipulated in Streptomyces (6) and E. coli (2, 21), as described earlier. Nucleotide sequences were determined by the dideoxy chain termination method with the Thermo Sequenase fluorescence-labeled primer cycle sequencing kit (Amersham) or the CEQ DTCS (Beckman Coulter) on an automated DNA sequencer.

Cloning a DNA fragment containing AdBS11.

We chose one (AdBS11) of the DNA fragments that had been isolated as targets of AdpA by the gel mobility shift-PCR procedure (11, 31). The DNA fragment was 32P labeled with the BcaBest DNA labeling system and used as the probe in Southern hybridization for cloning a 4.6-kb SalI fragment in pUC19, yielding pS5k. The nucleotide sequence of the 4.6-kb fragment in pS5k was determined, and open reading frames (ORFs) were predicted by the Frame Plot analysis (12).

Gel mobility shift assay.

Purification of histidine-tagged AdpA from E. coli BL21 (DE3) and the gel mobility shift assay were described previously (31). AdBS11 of 375 bp (nucleotide positions −306 to +69, with respect to the transcriptional start point p1 of ssgA [see Fig. Fig.2B2B and and4B])4B]) was cloned in the EcoRI site of pUC19. This fragment was amplified with the commercial primers, M4 and RV (Takara), 32P labeled with T4 polynucleotide kinase, and used as a probe. Probe dw (positions −25 to +198) (Fig. (Fig.4B),4B), was amplified with primers sg-F1 and sg-R1 (Table (Table1)1) and used for the gel mobility shift assay (31).

FIG. 2.
ORFs in the cloned 4.6-kb SalI fragment (A), nucleotide sequence of the region covering the ssgA promoter and a 3′ portion of ssfR (B), and low-resolution S1 nuclease mapping of ssgA in S. griseus (C and D). (A) The positions and directions of ...
FIG. 4.
Three AdpA-binding sites located upstream of ssgA, as determined by DNase I footprinting. (A) DNase I footprinting assays were performed on the sense strand (+) and the antisense strand (−) probes. The amounts of AdpA-H used in lanes 1, ...

S1 nuclease mapping.

RNA was prepared from cells grown on cellophane on the surface of YMPD agar medium and used for S1 nuclease mapping as described by Kelemen et al. (19). Hybridization probes were prepared by PCR with a pair of 32P-labeled and nonlabeled primers. For low-resolution S1 mapping, f1-sF and S1-R1* (Table (Table1)1) were used in Fig. Fig.2C2C and D and Fig. Fig.8D,8D, and S1-F1 and S1-R2* were used in Fig. Fig.6D.6D. Primers indicated with an asterisk were labeled at their 5′ ends with [γ-32P]ATP by using T4 polynucleotide kinase before PCR. For the adpA probe, 5′-AGCCCCCGCATCCCTCCGCGGCGA-3′ (positions −223 to −200 with respect to the transcriptional start point of adpA) (25) and 5′-ACTCGCGAAGCGCACAGGGAAGTG-3′ (positions +54 to +31) were used. For high-resolution S1 mapping, S1-F1 and S1-R1* were used. hrdB encoding a principal σ factor of RNA polymerase was used to check the purity and amount of the RNA used, as described previously (31). Protected fragments were analyzed on 6% polyacrylamide DNA sequencing gels by the method of Maxam and Gilbert (23).

FIG. 6.
Requirement of both sites 1 and 2 for the transcriptional activation of ssgA by AdpA. (A) Construction of mutant ΔssgA. The restriction sites of SmaI, PstI, and SalI are abbreviated as Sm, Pt, and Sl, respectively. The DNA fragment on pKgA used ...
FIG. 8.
Transcription of ssgA in the absence of ssfR. (A) Construction of the mutant ΔssfR strain. The restriction sites of SmaI, SalI, PmaCI, and EcoT14I are abbreviated Sm, Sl, Pm, and EcT, respectively. The DNA fragment on pKfR used for complementation ...

RT-PCR.

Total RNA was isolated from cells grown in YMPD liquid or on agar medium with RNAqueous-Midi (Ambion). Chromosomal DNA purified together with RNA was digested with RNase-free DNase I (Takara). The concentration of RNA was determined by measuring A260 by spectrophotometry. Reverse transcription (RT)-PCR was done with 3 μg of RNA, 2 pmol of gA-RT primer (Table (Table1),1), SuperScript II reverse transcriptase (Invitrogen), and RNase H (Invitrogen) according to the manufacturer's instructions. The PCR primers used were S1-F1 and S1-R2 for amplification of the 470-bp fragment in Fig. Fig.3B,3B, rt-F1 and S1-R2 for the 260-bp fragment, and rt-F2 and S1-R2 for the 200-bp fragment. The PCR conditions were 98°C for 20 s, 60°C for 30 s, and 72°C for 45 s in a total of 25 cycles. In every RT-PCR, no amplification from the genome DNA occurred without reverse transcriptase, indicative of no contamination of DNA.

FIG. 3.
Transcriptional start point of ssgA. (A) Two transcriptional start points of ssgA, as determined by high-resolution S1 mapping. RNA prepared from the wild-type cells grown at 28°C for 3 days on YMPD agar medium was used. The arrowheads indicate ...

DNase I footprinting.

The DNase I footprinting method used in this study was described previously (31). For determination of the AdpA-binding sites in front of the ssgA coding sequence, 32P-labeled fragments were prepared by PCR with f1-sF* and f1-sR for the sense strand of site 1, f1-aF and f1-aR* for the antisense strand of site 1, f2-sF* and f2-sR for the sense strand of site 2, f2-aF and f2-aR* for the antisense strand of site 2, sg-F1* and sg-R1 for the sense strand of site 3, and sg-F1 and sg-R1* for the antisense strand of site 3. 32P-labeled primers are indicated by asterisks.

Mutational analysis of the three AdpA-binding sites.

Mutations were introduced in the three AdpA-binding sites by using the Takara in vitro mutagenesis kit (Mutan-Super Express Km). The SacI fragment, positions −427 to +154, was cloned into pKF19k, resulting in pKF1 (see Fig. Fig.5B),5B), and using pKF1 as the template, sites 1, 2, and 3 were mutated with the s1-mt, s2-mt, and s3-mt primers (Table (Table1),1), respectively, resulting in pKF2, pKF3, and pKF4. A series of double mutations and a triple mutation were also introduced to generate pKF5 to pKF8 (Fig. (Fig.5B).5B). For determination of the binding of AdpA-H to the mutated sites by gel mobility shift assays, 32P-labeled probes of about 150 bp were prepared by PCR with the following primer pairs: s1-F and s1-R for probe pb11 (see Fig. Fig.5B),5B), s2-F and s2-R for probe pb22, and s3-F and s3-R for probe pb33. Probe pb12 amplified with s1-F and s2-R and probe pb23 amplified with s2-F and s3-R were also used for gel mobility shift assays.

FIG. 5.
Mutational analysis of the AdpA-binding sites. (A) Mutations introduced in the AdpA-binding sites. An XhoI site generated in sites 1 and 2 and a BamHI site generated in site 3 are indicated by italic letters. m1, m2, and m3 represent the mutations introduced ...

For construction of plasmids containing ssgA with a series of mutated promoters, ssgA was excised with SmaI and PstI from pS5k and cloned in pUC19. With this plasmid as the template, the SacI site at position +176 was disrupted by PCR with primers 5′-CCGAGGAGCTCTCGTTCCGTATTCCGGTGgagctgCGATACGAGG-3′ (positions +144 to +188; the underline indicates a SacI site, and the lowercase letters indicate a disrupted SacI site) and RV. The amplified fragment was digested with SacI and PstI and cloned in pUC19, resulting in pSG5. After the correct sequence of the PCR fragment was confirmed, the wild type and a series of mutated promoters were excised from pKF1 to pKF8 with SacI and ligated into the SacI site of pSG5. The ssgA sequences with various mutated promoters were excised with EcoRI and HindIII and ligated between the same sites of the shuttle vector pKUM20, resulting in pKGA1 to pKGA8. The plasmids, prepared first from E. coli JM110 and then from S. griseus HH1, were introduced into the ΔssgA strain. Transcription of ssgA with a series of mutated promoters on the plasmids was determined by S1 nuclease mapping with 32P-probe prepared by PCR with S1-F1 and S1-R2.

Gene disruption.

The 3.4-kb neomycin resistance gene aphII, obtained by digestion of Tn5 (3) with HindIII, was cloned in pUC19, resulting in pAPH3k. The region corresponding to the sequence upstream from Pro-35 of SsgA in pS5k was amplified by PCR with primers M4 (Takara) and EB-sgF (5′-cctgaattcagatctCCGGGAAGGTGGAACGTCATCC-3′ [positions +231 to +210; the underline indicates EcoRI plus BglII sites]), digested with XbaI and EcoRI, and ligated between the same sites of pUC19, resulting in pSG1. The region corresponding to the sequence downstream from Glu-122 of SsgA in pS5k was amplified with primers EB-sgR (5′-cctgaattcagatctGGAGGACGCACTGGGCCGCATC-3′ [positions +487 to +508; the underline indicates EcoRI plus BglII sites]) and RV (Takara), digested with EcoRI and SphI, and cloned between the same sites of pUC19, resulting in pSG2. After confirmation of the correct sequence of the PCR fragment, the XbaI-BglII insert from pSG1 and the BglII-SphI insert from pSG2 were excised and ligated together between the XbaI and SphI sites of pUC19. In the BglII site thus generated, a 1.9-kb BamHI fragment of aphII excised from pAPH3k was ligated, resulting in pSG3, in which the sequence encoding from Gly-36 to Leu-121 of SsgA was replaced by the aphII sequence. The ssgA::aphII construct was excised from pSG3 with DraI and ligated in the blunt-ended HindIII site of pIJ486 in S. lividans TK21, resulting in pSG4. pSG4 was propagated in S. griseus HH1 and linearized with EcoRI, alkali denatured with 0.1 M NaOH, and introduced by protoplast transformation into S. griseus IFO13350, as described previously (24). Correct replacement of the disrupted ssgA sequence, as a result of double crossover, was checked by Southern hybridization with a 1.8-kb SmaI-PstI fragment excised from pS5k and a 1.3-kb SmaI fragment of aphII excised from pAPH3k (probes 1 and 3, respectively, in Fig. Fig.6A)6A) against the SmaI-digested chromosomal DNA. For complementation of the ssgA disruption, a 1.8-kb SmaI-PstI fragment containing the whole ssgA gene with an about 660-bp upstream region of the SsgA coding sequence was excised from pS5k, cloned in pUC19, excised as an EcoRI-HindIII fragment, and finally inserted between the EcoRI and HindIII sites of pKUM20, resulting in pKgA.

For construction of the ssfR null mutant, the 1.3-kb aphII determinant excised from pAPH3k with SmaI was ligated into the PmaCI site of pS5k, resulting in pFR1. The ssfR::aphII construct was excised from pFR1 with DraI, cloned into pIJ486, digested with EcoRI to produce a linear form, denatured, and introduced into S. griseus IFO13350 as described above for ssgA::aphII. Correct replacement of the disrupted ssfR sequence was checked by Southern hybridization with a 1.0-kb SmaI fragment excised from pS5k and the 1.3-kb SmaI fragment of aphII (probes 2 and 3, respectively, in Fig. Fig.8A)8A) against the SmaI-digested chromosomal DNA. For complementation of the ssfR disruption, a 1.6-kb EcoT14I fragment containing the whole ssfR gene, including an about 530-bp upstream region of the SsfR-coding sequence, was excised from pS5k, blunt ended with Klenow fragment, cloned in the SmaI site of pUC19, excised as an EcoRI-HindIII fragment, and finally inserted between the EcoRI and HindIII sites of pKUM20, resulting in pKfR.

Scanning electron microscopy.

S. griseus strains were grown on YMPD, YMP-mannitol, and R2YE agar medium at 28°C for 3 days, and agar blocks containing the spores and hyphae were cut out. For preparation of the specimens, the agar blocks were fixed with 2% osmium tetroxide for 40 h and then dehydrated by air drying for 1 h. Each specimen was sputter coated with platinum-gold and examined with a Hitachi S4000 scanning electron microscope.

Nucleotide sequence accession number.

The nucleotide sequence reported in this paper has been deposited in the DDBJ, EMBL, and GenBank DNA databases under accession no. AB091268.

RESULTS

AdpA binding to AdBS11.

AdBS11 was identified in a library of DNA fragments that were bound by AdpA (11, 31). The AdBS11 fragment, cloned in the EcoRI site of pUC19, was amplified by PCR, 32P labeled, and used as the probe for the gel mobility shift assay (Fig. (Fig.1A).1A). AdpA-H bound the probe, giving a smeared, retarded signal. In addition, considerable radioactivity aggregated in the gel well. This binding was apparently specific; an excess amount of nonlabeled probe competed for binding. The reason for the smeared signal and aggregated probes in the well was later clear; AdBS11 contained two complete AdpA-binding sites and part of a third binding site (see below).

FIG. 1.
Binding of AdpA-H to AdBS11 (A) and to a region downstream of AdBS11 (B). (A) AdBS11 positioned at −306 to +69 (see Fig. Fig.4B)4B) was 32P labeled and used in the gel mobility shift assay. The amounts of AdpA-H used in lanes 1, ...

We cloned a 4.6-kb DNA fragment by using AdBS11 as the probe in Southern hybridization and determined the nucleotide sequence (Fig. (Fig.2A).2A). AdBS11, consisting of 375 bp, covered a 3′-terminal portion of ssfR and a region upstream of ssgA (Fig. (Fig.2B),2B), both of which were reported to be involved in spore septum formation in S. griseus (13, 18) and S. coelicolor A3(2) (30). The gene organization from orf1 to orf4 in Fig. Fig.2A2A is the same as that in S. coelicolor A3(2). Orf1, Orf3, and Orf4 are putative membrane proteins, and Orf2 is a protein of unknown function.

During gel mobility shift assays with various portions as probes, we noticed that an additional region downstream of AdBS11 at nucleotide positions −25 to +198 with respect to the transcriptional point of ssgA was also bound by AdpA-H (Fig. (Fig.1B).1B). This binding was also specific, because an excess amount of nonlabeled probe competed the binding (data not shown).

Dependence of ssgA transcription on adpA and A-factor.

Since the AdpA-binding sites were located in front of ssgA, we analyzed the ssgA transcription in an adpA null (ΔadpA) mutant and an A-factor-deficient (HH1) mutant by using RNA prepared from cells grown on YMPD agar medium (Fig. (Fig.2C).2C). Under these conditions, the wild-type S. griseus strain IFO13350 grew as substrate mycelium at day 1, as a mixture of substrate and aerial hyphae at day 2, and as a mixture of aerial hyphae and spores at day 3. ssgA was transcribed from two start points, p1 and p2, both of which were enhanced at days 2 and 3. Although the ssgA transcription is enhanced at day 2 in this particular experiment, our repeated S1 mapping of ssgA with different batches of RNA showed that the ssgA transcription was enhanced at day 2 and much enhanced at day 3 (Fig. (Fig.2D2D and and8D).8D). hrdB encoding σHrdB was used as a constitutively expressed internal control to measure the amount and purity of the RNA. In the ΔadpA and HH1 mutants, very little p1 and p2 transcript was detected, indicating that the ssgA transcription depended on adpA and A-factor. Consistent with this, overexpression of adpA enhanced the ssgA transcription starting at both p1 and p2 (Fig. (Fig.2D).2D). The dependence of ssgA transcription on A-factor coincides with the observation that SsgA was produced in response to A-factor, when assayed by Western blot analysis with anti-SsgA antibody (18).

The transcriptional start points of ssgA were determined by high-resolution S1 mapping to be 124 nucleotides (p1) and 79 nucleotides (p2) upstream of the translational start codon (Fig. (Fig.3A).3A). We also examined possible transcriptional read-through from ssfR located upstream of ssgA in the same orientation, since in S. coelicolor A3(2) containing ssgR-ssgA in the same gene organization as in S. griseus, ssgA was reported to be probably transcribed by read-through from ssgR (30). As shown in Fig. Fig.3B,3B, no PCR product of 470 bp, representing a read-through transcript, was detected after 25 cycles of amplification with the RNA prepared from either liquid or agar medium, whereas the two PCR products representing the ssgA transcripts, as positive controls, were clearly detected at the expected positions of 260 bp and 200 bp. PCR with these probes against the chromosomal DNA amplified the corresponding DNAs at almost the same efficiency (data not shown). Although an increase in the number of PCR cycles up to 30 led to slight detection of the 470-bp product, these data show that very little transcriptional read-through from ssfR into ssgA occurs.

Very little ssgA transcript was detected in the ΔadsA mutant strain, as in the ΔadpA and HH1 mutants (Fig. (Fig.2C).2C). Transcription of adsA, encoding an ECF σ factor of RNA polymerase, depends on AdpA and is essential for aerial mycelium formation (31). This means that ssgA is controlled doubly by AdpA. Two explanations for the dependence of ssgA transcription on σAdsA are possible. RNA polymerase containing σAdsA would recognize the p1 and p2 promoters of ssgA. Or, alternatively, some gene product under the control of σAdsA would facilitate transcription of ssgA in conjunction with AdpA. The putative gene product might be a σ factor responsible for ssgA transcription. The −10 and −35 promoter sequences of bldM recognized by σBldN, a σAdsA homologue in S. coelicolor A3(2) (4), are distinctly different from the probable −10 and −35 sequences of p1 and p2 of ssgA (Fig. (Fig.3C).3C). It is therefore unlikely that σAdsA recognizes the p1 and p2 promoters, and a certain gene product under the control of σAdsA is involved in the switch on of ssgA transcription by AdpA in an as-yet-unknown manner.

Determination of AdpA-binding sequences by DNase I footprinting.

During our DNase I footprinting experiments to determine the AdpA-binding sequence in AdBS11, we unexpectedly found two AdpA-binding sites in the 375-bp fragment. By using AdBS11 as a 32P-labeled probe, we determined the nucleotides of the two sites that were protected by DNase I digestion. Nucleotide positions −216 to −250 of the sense strand and positions −220 to −255 of the antisense strand at site 1 were protected (Fig. (Fig.4).4). At site 2, nucleotide positions −95 to −119 of the sense strand and positions −100 to −127 of the antisense strand were protected. The nucleotide sequence of site 3, which is located downstream of AdBS11, was similarly determined with probe dw. Nucleotide positions +35 to +79 of the sense strand and positions +42 to +87 of the antisense strand were protected. The protected region at site 3 was longer than those at sites 1 and 2, than the two AdpA sites in front of sgmA (15), and than the AdpA-binding site for adsA (31). It is unlikely, however, that a multimeric form of AdpA binds site 3, because, as shown below, the positions of the retarded signal for sites 1, 2, and 3 in the gel mobility shift assays with AdpA-H (Fig. (Fig.5C)5C) are almost the same.

We have so far determined more than 10 AdpA-binding sequences (15, 31; our unpublished data). Alignment of these AdpA-binding sequences does not give a definitive consensus sequence. Our preliminary data obtained from the study of the adsA promoter suggest that the sequence 5′-(A/T)(A/T)X3G-3′ (where X is any nucleotide), located in the vicinity of one of the 5′ ends of the binding site, is important for AdpA binding. All three AdpA-binding sites contain this sequence.

Requirement of two of the three AdpA-binding sites for ssgA activation.

The presence of three AdpA-binding sites in front of ssgA prompted us to determine which one or two or all of the sites are required for transcriptional activation of ssgA. For this purpose, we introduced a mutation in the 5′-(A/T)(A/T)X3G-3′ sequence described above in each of the three binding sites by site-directed mutagenesis with the intact SacI fragment on pKF1 as the template (Fig. (Fig.5A),5A), generating pKF2 to pKF4, which contained a SacI fragment carrying each of the mutated binding sites, as shown in Fig. Fig.5B.5B. For example, the SacI fragment in pKF2 contained the mutated site 1 and the intact sites 2 and 3. None of the mutated sites bound AdpA-H (Fig. (Fig.5C,5C, top). When the DNA, amplified by PCR with pKF2 as the template and with the primers for amplification of the region corresponding to pb11, was used for the gel mobility shift assay, no retarded signal was observed, whereas the pb11 region similarly prepared with pKF1 was retarded by AdpA-H. The combination of pKF3 as the template and probe pb22 showed no binding of AdpA-H to the mutated site 2. The combination of pKF4 and pb33 also showed no binding of AdpA-H to the mutated site 3.

We also generated pKF5 to -7, which contained a SacI fragment carrying the mutations at two sites. The SacI fragment in pKF5, for example, contained the mutated sites 1 and 2 and the intact site 3. When the pb12 region amplified with pKF5 was used as the probe, no retardation occurred (Fig. (Fig.5C,5C, middle), indicative of no AdpA binding to the mutated sites 1 and 2. When pKF1 was used as the template and the pb12 region was used as the probe, two apparent retarded signals were observed. This means that AdpA-H binds the two intact sites 1 and 2. This is consistent with the observations that the pb12 regions prepared with pKF2 and pKF3, each containing one intact AdpA-binding site, yielded a single retarded signal. The experiments with the probe 23 region (Fig. (Fig.5C,5C, bottom) also supported this idea: the probe prepared with pKF1 yielded two retarded signals, whereas those with pKF3 and pKF4 gave a single signal.

We also constructed pKF8 containing a SacI fragment that carried the mutated sites 1, 2, and 3 and no intact AdpA-binding sites. This SacI fragment gave no retarded signals when the pb12 or pb23 region was used as the probe (data not shown).

We tried to assess the promoter activity of the SacI fragments on a low-copy-number plasmid in the ΔssgA genetic background. For this purpose, we generated the ΔssgA mutant strain by replacing more than half of the ssgA coding region with the neomycin resistance gene (Fig. (Fig.6A).6A). Correct disruption was checked by Southern hybridization with probes 1 and 3 (Fig. (Fig.6B).6B). The SacI fragments in pKF1 to pKF8 were separately placed in front of the ssgA coding sequence on the low-copy-number plasmid pKUM20, resulting in pKGA1 to pKGA8 (Fig. (Fig.6C).6C). We introduced each of the plasmids into the ΔssgA strain and measured ssgA transcription by S1 nuclease mapping in the ΔssgA genetic background. Because the 32P-labeled 5′ end of the probe was located in the region that was deleted from the chromosome, the mRNA detected was derived from the ssgA promoters on the pKGA series of plasmids. Low-resolution S1 mapping with RNA prepared from the wild-type S. griseus strain revealed two transcripts representing p1 and p2 in Fig. Fig.2,2, whereas no transcripts were detected when RNA from the ΔssgA strain was used, as expected (Fig. (Fig.6D).6D). Of the transformants harboring pKGA-series plasmids, mRNA signals were detected only in those harboring pKGA1, as a positive control, and pKGA4. The sizes of these ssgA-specific transcripts corresponded to p1 and p2. No other transformants yielded detectable ssgA transcripts. These results indicated that both site 1 and site 2 are required for transcriptional switch on of ssgA by AdpA and that site 3 was not essential.

Phenotypes of the ΔssgA mutant and complementation of the defect in spore septum formation of the ΔssgA strain by pKGA4.

S griseus strain NRRL B2682, which was used for isolation of ssgA (17, 20) and for determination of the phenotype of ssgA disruptants (13), produces abundant submerged spores in chemically defined medium. The phenotypes were studied in submerged culture. In this strain, ΔssgA mutants show carbon source-dependent morphogenesis; they never produce submerged spores in glucose-containing minimal medium but produce a few spores in mannitol-containing minimal medium (13). However, the S. griseus strain IFO13350, which we have long studied, shows poor growth on minimal medium, irrespective of the carbon source, and forms few spores either on agar or in liquid medium. Because of the different phenotypes between strains NRRL B2682 and IFO13350, we had to determine, in detail, the phenotypes of the ΔssgA mutant derived from strain IFO13350 on solid medium.

S. griseus IFO13350 forms abundant spores on a nutrient rich medium, such as YMPD and R2YE. Figure Figure77 shows scanning electron micrographs of the IFO13350 wild-type strain and the ΔssgA mutant strain grown at 28°C for 3 days on R2YE medium. The wild type forms chains of spores, whereas the ΔssgA strain forms only aerial hyphae. The ΔssgA strain did not form spores even after prolonged cultivation. The same phenotypes were also observed on YMPD and YMP-mannitol medium when examined by scanning electron microscopy. Thus, ssgA is a member of the whi genes and is essential for sporulation, probably for septum formation in aerial hyphae, as reported previously (13, 30). The ssgA mutation did not affect production of streptomycin or yellow pigment, consistent with the idea that ssgA is involved in a late step in morphological differentiation.

FIG. 7.
Scanning electron micrographs of ΔssgA transformants. wt, wild type. Strains were grown on R2YE agar medium at 28°C for 3 days. The defect in spore formation in the ΔssgA strains was complemented by pKGA1 and pKGA4, but not by ...

The defect in sporulation of the ΔssgA mutant was recovered by pKGA1 and pKGA4 (Fig. (Fig.7),7), which is in agreement with the observations that the transcription of ssgA on these plasmids was active (Fig. (Fig.6D).6D). pKGA2 and the vector pKUM20, as negative controls, did not restore the defect. pKGA3, -5, -6, -7, or -8 did not allow the ΔssgA strain to form spores (data not shown). The transcriptional analysis of ssgA with the mutated AdpA-binding sites and the phenotypic restoration by the transcriptionally active ssgA genes clearly show that ssgA is essential for sporulation and is activated by AdpA binding to sites 1 and 2.

Transcription of ssgA in the absence of ssfR and phenotypes of the ΔssfR mutant.

ssfR, encoding a putative regulator belonging to the family of IclR-type transcriptional factors, is located upstream of ssgA. Jiang and Kendrick (13) pointed out the possibility that ssgA is regulated by ssfR, although no mRNA work was reported. van Wezel et al. (30) also referred to a possible transcriptional correlation between ssgA and ssgR, a counterpart of ssfR, in S. coelicolor A3(2). If this is the case, ssgA would be switched on by AdpA and controlled by SsfR. We tested this hypothesis by measuring transcription of ssgA in the ΔssfR mutant. For this purpose, we disrupted ssfR by inserting the neomycin resistance gene into the PmaCI site within the ssfR coding region (Fig. (Fig.8A).8A). Correct disruption was confirmed by Southern hybridization with probes 2 and 3 against the SmaI-digested chromosomal DNA (Fig. (Fig.8B8B).

As was reported for S. griseus NRRL B2682 (13), the ΔssfR mutant derived from S. griseus IFO13350 grew as aerial hyphae at day 3 and did not form spores even by prolonged cultivation (Fig. (Fig.8C).8C). Introduction of the whole ssfR gene on a low-copy-number plasmid, pKUM20 (plasmid pKfR), into the ΔssfR strain restored sporulation. Although Jiang and Kendrick (13) also reported that the ΔssfR strain when grown in a minimal liquid medium with mannitol formed abundant submerged spores, the ΔssfR mutants derived from strain IFO13350 did not form spores on YMP-mannitol medium or on YMPD medium. All of these observations indicate that ssfR is involved in sporulation, perhaps in septation in aerial hyphae, but do not show detectable carbon source dependence.

Transcription of ssgA in the wild-type strain IFO13350 was determined by low-resolution S1 mapping with RNA prepared from YMPD medium and YMP-mannitol medium (Fig. (Fig.8D).8D). The mRNA levels of ssgA were similar, irrespective of glucose or mannitol as a carbon source, which is consistent with the observation that the ΔssgA strain failed to form spores on YMPD or YMP-mannitol medium. We also examined ssgA transcription in the absence of ssfR and found that ssgA was transcribed in the ΔssfR mutant with the same kinetics (Fig. (Fig.8D).8D). The amount in the p1 transcript at day 3 was notably increased, and that in the p2 transcript was somewhat distinguishably decreased, the reason for which is unclear. However, these results exclude the hypothesized possibility that SsfR is a required transcriptional regulator for ssgA in S. griseus IFO13350 under the growth conditions we examined.

DISCUSSION

The study of AdBS11 in the library of DNA fragments that are bound by AdpA has led to the identification of ssgA, which was reported to be essential for sporulation, perhaps in formation of septa in aerial hyphae, in both S. griseus NRRL B2682 (13) and S. coelicolor A3(2) (30). The ΔssgA mutants derived from strain IFO13350 formed aerial hyphae but failed to form spores, irrespective of the carbon source. Although some differences between strains IFO13350 and NRRL B2682 in the ability to form submerged spores and in carbon source dependence of morphogenesis are recognized, we can safely say that ssgA is essential for sporulation at a step after aerial hyphae have formed. ssgA is thus a true member of the whi genes. We had thought that A-factor, and accordingly AdpA, switched on the genes that were required for the onset of aerial mycelium formation and secondary metabolism. The present study, however, demonstrates the direct regulation of ssgA by AdpA at a rather late step in the sporulation process.

AdpA bound three sites upstream of the ssgA coding sequence, and the simultaneous presence of sites 1 and 2 (site 1, located at nucleotide position about −235, with respect to the transcriptional start point, and site 2, located at position about −110) was essential for transcriptional activation by AdpA. The necessity of the two AdpA-binding sites was confirmed by phenotypic complementation of ΔssgA mutants with mutated ssgA sequences. For transcriptional activation by AdpA of adsA encoding an ECF σ factor, a single AdpA-binding site, located at nucleotide positions +7 to +47, is sufficient (31). Transcriptional activation by AdpA of sgmA encoding a metalloendopeptidase is also accomplished by AdpA binding to a single site at positions −49 to −71 (15). This would allow AdpA to recruit RNA polymerase to the target promoter or interact with RNA polymerase that has been placed at the promoter, as is found for many transcriptional activators (5, 27). However, the distances of the two AdpA-binding sites from the transcriptional start point of ssgA seem to be too far for the DNA-bound AdpA to interact with the promoter-bound RNA polymerase without DNA bending. DNA bending in the transcriptional regulation is found for the arabinose operon in E. coli (22, 28). To elucidate the mechanism by which AdpA activates ssgA transcription apparently requires in vitro transcription analysis.

Site 3, located downstream of p1 and just at p2, was not necessary for ssgA activation by AdpA. Of the three AdpA-binding sites, site 3 was bound by AdpA at a higher affinity than the other two sites, when judged by the intensity of the retarded signal (data not shown). The nucleotide sequence at site 3 protected from DNase I digestion was also unusually long in comparison with those we have previously determined for other AdpA-binding sites. AdpA may bind site 3 in an unusual, but still unknown, manner. The location of site 3 with respect to the transcriptional start points leads us to speculate that AdpA bound at site 3 serves as a repressor to modulate ssgA transcription under some growth conditions, although there has been no evidence for it. The control of ssgA should be very strict, since introduction of ssgA on a low-copy-number plasmid into strain NRRL B2682 caused mycelial fragmentation in liquid culture (18).

Jiang and Kendrick (13) isolated ssgA and ssfR on a DNA fragment that complemented the mutation of S. griseus SKK2600. Strain SKK2600 is a mutant that can form submerged spores in the presence of cefoxitin, which inhibits septum formation during sporulation. The mutation point was a C-to-T exchange at nucleotide position −14 within the ssfR coding region. This mutation point corresponds to the third nucleotide of the Ile codon (ATC) in ssfR and does not result in amino acid replacement. It was therefore a mystery. Our transcriptional study has revealed that this nucleotide corresponds to the 5′ end of the −10 hexamer for p1 (Fig. (Fig.3C).3C). The CAGTAT hexamer is changed to the TAGTAT sequence due to the C-to-T mutation, which we speculate disturbs the recognition of the p1 promoter by a certain σ factor. Although it is unclear what σ factor is responsible for p1 transcription, the resulting TAGTAT sequence becomes more similar to the −10 consensus sequence, TAGRRT (where R = G or A), recognized by σHrdB (29). In fact, mutant SKK2600 forms sporelike cells under conditions in which the parental strain does not sporulate, and this phenotype is similar to that observed for the parental strain overexpressing ssgA (13).

The same gene organization of ssfR-ssgA in both S. griseus and S. coelicolor A3(2) and the spore-negative phenotype of the ΔssfR mutant in the two strains, together with the fact that ssfR encodes a protein belonging to IclR-type transcriptional factors, suggested a correlation between ssgA and ssfR (13, 30). However, the present study shows that ssgA is transcribed at almost the same efficiency in the presence and absence of ssfR. SsfR and SsgA may function independently in different regulatory pathways, or they may function through protein-protein interaction in the same pathway, since the ΔssfR strain shows the same phenotype as the ΔssgA strain. A better picture of ssgA regulation in connection with ssfR and with carbon source dependence needs further and careful study.

Acknowledgments

H. Yamazaki was supported by the Japan Society for the Promotion of Science. This work was supported by the Asahi Glass Foundation and by the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan (BDP-02-VI-2-8).

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