Haruka Yamazaki

AdpA, a Central Transcriptional Regulator in the A-Factor Regulatory Cascade That Leads to Morphological Development and Secondary Metabolism in Streptomyces griseus

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a microbial hormone that triggers aerial mycelium formation and secondary metabolism in Streptomyces griseus. A-factor produced in a growth-dependent manner switches on the transcription of adpA encoding a transcriptional activator by binding to the A-factor receptor protein (ArpA), which has bound the adpA promoter, and dissociating the DNA-bound ArpA from the DNA. AdpA then activates a number of genes with various functions required for morphological development and secondary metabolism, forming an AdpA regulon. AdpA, which contains a ThiJ/PfpI/DJ-1-like dimerization domain at its N-terminal portion and an AraC/XylS-type DNA-binding domain at its C-terminal portion, is a representative of a large subfamily of the AraC/XylS family. AdpA binds various positions with respect to the transcriptional start points of the target genes and recruits RNA polymerase to the specific promoter region, and facilitates the isomerization of the RNA polymerase-DNA complex into an open complex competent for transcriptional initiation. The AdpA-binding consensus sequence is 5′-TGGCSNGWWY-3′ (S: G or C; W: A or T; Y: T or C; N: any nucleotide). The DNA-binding specificity of AdpA in conjunction with that of other AraC/XylS family members is also discussed.

An A-factor-dependent extracytoplasmic function sigma factor (sigma(AdsA)) that is essential for morphological development in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function sigma factor belonging to a subgroup of the primary sigma(70) family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named sigma(AdsA). Transcription of adsA depended on A-factor and AdpA, since adsA was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that sigma(AdsA) is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomyces species examined by Southern hybridization suggests a common role in morphogenesis in this genus.

DNA‐binding specificity of AdpA, a transcriptional activator in the A‐factor regulatory cascade in Streptomyces griseus

AdpA, belonging to the AraC/XylS family, is the key transcriptional activator for a number of genes of various functions in the A‐factor regulatory cascade in Streptomyces griseus. It consists of a ThiJ/PfpI/DJ‐1‐like dimerization domain at its N‐terminal portion and a DNA‐binding domain with two helix–turn–helix motifs at its C‐terminal portion, representing a large subgroup of the AraC/XylS family. Uracil interference assay and missing T and GA interference assays on several AdpA binding sites, followed by gel mobility shift assays on systematically mutated binding sites, revealed a consensus AdpA‐binding sequence, 5′‐TGGCSNGWWY‐3′ (S: G or C; W: A or T; Y: T or C; N: any nucleotide). A dimer of AdpA bound a site including the two consensus sequences, with a space of 13–14 bp, as an inverted repeat (type I) at various positions, for example more than 200 bp upstream (−200) and 25 bp downstream (+25) from the transcriptional start point of the target gene. In addition, AdpA also bound a site including the consensus sequence in a single copy (type II) at positions, in most cases, from −40 to −50 and from −50 to −60. For transcriptional activation, some genes required simultaneous binding of a dimer of AdpA to type I and II sites, but others required only a single type I or type II site. AdpA bound mutated type I sites with various distances between the two consensus sequences with significant affinities, although the optimal distances for AdpA to bind were 13–14 bp and 2 bp. The DNA‐binding domain is therefore connected to the ThiJ/PfpI/DJ‐1‐like dimerization domain with a flexible linker. The DNA‐binding specificity of AdpA in conjunction with that of other AraC/XylS family members is discussed.

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

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-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, sigma(AdsA), in addition to AdpA. However, it was unlikely that sigma(AdsA) recognized the two ssgA promoters, since their -35 and -10 sequences were not similar to the promoter sequence motifs recognized by sigma(BldN), a sigma(AdsA) 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.

Control by A-Factor of a Metalloendopeptidase Gene Involved in Aerial Mycelium Formation in Streptomyces griseus

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis. An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner. A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA-binding sites. The primary product of sgmA consisted of N-terminal pre, N-terminal pro, mature, and C-terminal pro regions. sgmA was transcribed in an AdpA-dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation. AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position -60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position -260 (B site), as determined by DNase I footprinting. Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation. Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day. SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation.

Transcriptional control by A-factor of strR, the pathway-specific transcriptional activator for streptomycin biosynthesis in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions -270 and -50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin.

amfR, an essential gene for aerial mycelium formation, is a member of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus

In Streptomyces griseus, A‐factor (2‐isocapryloyl‐3R‐hydroxymethyl‐γ‐butyrolactone) acts as a chemical signalling molecule that triggers morphological differentiation and secondary metabolism. A transcriptional activator, AdpA, in the A‐factor regulatory cascade switches on a number of genes required for both processes, thus forming an AdpA regulon. amfR encoding a regulatory protein similar to response regulators of bacterial two‐component regulatory systems and essential for aerial mycelium formation was found to be a member of the AdpA regulon. AdpA bound two sites at nucleotide positions approximately −200 (site 1) and −60 (site 2), with respect to the major transcriptional start point of amfR, and accelerated the transcription of amfR by assisting RNA polymerase in forming an open complex at an appropriate region including the transcriptional start point. Site 2 contributed more to the transcriptional activation of amfR by AdpA than site 1, although AdpA showed a much lower affinity to site 2 than to site 1. The amfR transcription enhanced by AdpA subsequently ceased at day 2 when aerial hyphae began to be formed in the wild‐type strain, whereas in an adsA null mutant amfR was continuously transcribed even until day 3. This implied that amfR was repressed growth dependently by a gene product under the control of σ‐AdsA. Transcription of the promoter upstream of amfT depended on amfR, which is consistent with the idea that AmfR serves as an activator for amfTSBA in the amf operon. The observations that the amfR gene contains a TTA codon, a potential target for bldA‐mediated regulation, and a conserved Asp‐54 residue, which might be phosphorylated by a sensor kinase, suggest that the amf operon is under transcriptional, translational and post‐translational control systems.

Genetic transformation of a Rhizomucor pusillus mutant defective in asparagine-linked glycosylation: production of a milk-clotting enzyme in a less-glycosylated form.

Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura+ transformants with 1 μg pRPPyr4 DNA and 1 × 107 viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0∼1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.

Characterization of alg2 encoding a mannosyltransferase in the zygomycete fungus Rhizomucor pusillus

ALG2 of Saccharomyces cerevisiae encodes the glycosyltransferase that mannosylates Man2GlcNAc2-dolichol diphosphate (PP-Dol) and Man1GlcNAc2-PP-Dol to form Man3GlcNAc2-PP-Dol. The genomic DNA and cDNA encoding an ALG2 homologue were cloned from the zygomycete fungus, Rhizomucor pusillus, and their nucleotide sequences were determined. The cloned cDNA under the control of the yeast GAL1 promoter complemented the temperature-sensitive (ts) growth of the alg2-1 mutant of S. cerevisiae, indicating that it represented a functional ALG2 homologue of R. pusillus. Five introns intervened the R. pusillus alg2 encoding a 455-amino-acid (aa) protein that showed end-to-end similarity in aa sequence to yeast Alg2 and contained a dolichol-binding consensus sequence (Val/Ile-x-Phe-x-x-Ile, where x is any aa) very near its C-terminus. The yeast alg2-1 gene had two mutation points at 377Gly to Arg and 386Gln to Lys. alg2-2 also contained two mutations at 54Glu to Lys and 377Gly to Arg. Site-directed mutagenesis of the fungal Alg2 and determination of their phenotypes in the yeast alg2-1 mutant showed that a mutation at 368Gly (equivalent to 377Gly of yeast Alg2) to Arg resulted in generation of a ts enzyme. The fungal Alg2 containing a mutation at the position corresponding to 54Glu or 386Gln of yeast Alg2 still complemented the ts growth of yeast alg2-1.