Purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of AdpA, the central transcription factor in the A-factor regulatory cascade in the filamentous bacterium Streptomyces griseus, in complex with a duplex DNA.

Streptomyces griseus AdpA is the central transcription factor in the A-factor regulatory cascade and activates a number of genes that are required for both secondary metabolism and morphological differentiation, leading to the onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. The DNA-binding domain of AdpA consists of two helix-turn-helix DNA-binding motifs and shows low nucleotide-sequence specificity. To reveal the molecular basis of the low nucleotide-sequence specificity, an attempt was made to obtain cocrystals of the DNA-binding domain of AdpA and several kinds of duplex DNA. The best diffracting crystal was obtained using a 14-mer duplex DNA with two-nucleotide overhangs at the 5'-ends. The crystal diffracted X-rays to 2.8 Šresolution and belonged to space group C222(1), with unit-cell parameters a = 76.86, b = 100.96, c = 101.25 Å. The Matthews coefficient (V(M) = 3.71 Å(3) Da(-1)) indicated that the crystal was most likely to contain one DNA-binding domain of AdpA and one duplex DNA in the asymmetric unit, with a solvent content of 66.8%.

Conditionally positive effect of the TetR-family transcriptional regulator AtrA on streptomycin production by Streptomyces griseus.

AtrA, a transcriptional activator for actII-ORF4, encoding the pathway-specific transcriptional activator of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2), has been shown to bind the region upstream from the promoter of strR, encoding the pathway-specific transcriptional activator of the streptomycin biosynthetic gene cluster in Streptomyces griseus [Uguru et al. (2005) Mol Microbiol 58, 131-150]. The atrA orthologue (atrA-g) in S. griseus was constitutively transcribed throughout growth from a promoter located about 250 nt upstream of the translational start codon, as determined by S1 nuclease mapping. DNase I footprinting showed that histidine-tagged AtrA-g bound an inverted repeat located upstream of strR at positions -117 to -142 relative to the transcriptional start point of strR as +1. This AtrA-g-binding site was between two AdpA-binding sites at approximately nucleotide positions -270 and -50. AdpA is a central transcriptional activator in the A-factor regulatory cascade and essential for the transcription of strR. AtrA-g and AdpA simultaneously bound the respective binding sites. In contrast to AdpA, AtrA-g was non-essential for strR transcription; an atrA-g-disrupted strain produced streptomycin on routine agar media to the same extent as the wild-type strain. However, the atrA-g-disrupted strain tended to produce a smaller amount of streptomycin than the wild-type strain under some conditions, for example, on Bennett agar containing 1 % maltose and on a minimal medium. Therefore, AtrA-g had a conditionally positive effect on streptomycin production, as a tuner, probably by enhancing the AdpA-dependent transcriptional activation of strR in a still unknown manner.

Control of the Streptomyces Subtilisin inhibitor gene by AdpA in the A-factor regulatory cascade in Streptomyces griseus.

AdpA in the A-factor regulatory cascade in Streptomyces griseus activates a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. The Streptomyces subtilisin inhibitor (SSI) gene, sgiA, in S. griseus was transcribed in response to AdpA, showing that sgiA is a member of the AdpA regulon. AdpA bound a single site upstream of the sgiA promoter at approximately position -70 with respect to its transcriptional start point. Mutational analysis of the AdpA-binding site showed that the AdpA-binding site was essential for transcriptional activation. Mutants in which sgiA was disrupted had higher trypsin, chymotrypsin, metalloendopeptidase, and total protease activities than the wild-type strain, which showed that SgiA modulated the activities of these extracellularly produced proteases. Because a number of genes encoding chymotrypsins, trypsins, and metalloendopeptidases, most of which are SSI-sensitive proteases, are also under the control of AdpA, the A-factor regulatory cascade was thought to play a crucial role in modulating the extracellular protease activities by triggering simultaneous production of the proteases and their inhibitor at a specific timing during growth. Mutants in which sgiA was disrupted grew normally and formed aerial hyphae and spores with the same time course as the wild-type strain. However, exogenous addition of purified SgiA to substrate mycelium grown on agar medium resulted in a delay in aerial mycelium formation, indicating that SgiA is involved in aerial hypha formation in conjunction with proteases.

The Streptomyces subtilisin inhibitor (SSI) gene in Streptomyces coelicolor A3(2).

Streptomyces subtilisin inhibitors (SSIs) are produced by a wide variety of Streptomyces species. Streptomyces coelicolor A3(2) contains two genes, SCO0762 and SCO4010, encoding an SSI-like protein. Of these two genes, SCO0762 was transcribed actively throughout growth. Gene disruption of SCO0762 (mutant DeltaSCO0762) resulted in overproduction of extracellular protease activity, showing that SCO0762 serves as a modulator of extracellular protease activities. Mutant DeltaSCO0762 showed no apparent phenotypic changes in morphological differentiation, forming aerial hyphae and spores in the same time course as the parental strain. SCO4010 appeared to be a pseudogene, because mutant DeltaSCO4010 showed the same protease activity as the parental strain, probably due to amino acid replacement of one (Arg-60) of the important residues for SSI activity, and because the transcription of this gene was extremely low.