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A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-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.

AdpA is a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus, activating a number of genes required for secondary metabolism and morphological differentiation. Of the five chymotrypsin-type serine protease genes, sprA, sprB, and sprD were transcribed in response to AdpA, showing that these protease genes are members of the AdpA regulon. These proteases were predicted to play the same physiological role, since these protease genes were transcribed in a similar time course during growth and the matured enzymes showed high end-to-end similarity to one another. AdpA bound two sites upstream of the sprA promoter approximately at positions ؊375 and ؊50 with respect to the transcriptional start point of sprA. Mutational analysis of the AdpA-binding sites showed that both AdpA-binding sites were essential for transcriptional activation. AdpA bound a single site at position ؊50 in front of the sprB promoter and greatly enhanced the transcription of sprB. The AdpA-binding site at position ؊40 was essential for transcription of sprD, although there was an additional AdpA-binding site at position ؊180. Most chymotrypsin activity excreted by S. griseus was attributed to SprA and SprB, because mutant ⌬sprAB, having a deletion in both sprA and sprB, lost almost all chymotrypsin activity, as did mutant ⌬adpA. Even the double mutant ⌬sprAB and triple mutant ⌬sprABD grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) is a chemical signaling molecule, or a microbial hormone, that triggers secondary metabolism and cell differentiation in Streptomyces griseus (7,8). We have revealed the A-factor regulatory cascade, through which production of almost all the secondary metabolites produced by S. griseus and formation of aerial mycelium and spores are triggered. A-factor is gradually accumulated in a growth-dependent manner, with its maximum quantity, about 25 ng/ml, at or near the decision point (1). The decision point is at the middle of the exponential growth phase (20). When the concentration of A-factor reaches a critical level, it binds ArpA, which has bound the promoter of adpA, and dissociates the ArpA from the DNA, thus inducing the transcription of adpA (7,22). The transcriptional factor AdpA activates a number of genes required for secondary metabolism and morphological differentiation. Members of the AdpA regulon include strR, the pathwayspecific transcriptional activator for streptomycin biosynthesis (30); an open reading frame encoding a probable pathwayspecific regulator for a polyketide compound (35); adsA encoding an extracytoplasmic function sigma factor of RNA polymerase essential for aerial mycelium formation (32); ssgA encoding a small acidic protein essential for spore septum formation (33); and amfR encoding a regulatory protein essential for aerial mycelium formation (34). In addition to these genes, sgmA encoding a metalloendopeptidase (12) and sprT and sprU, both e...

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