[show abstract][hide abstract] ABSTRACT: We cloned a DNA fragment that suppressed the aerial-mycelium-deficient phenotype in an amfS mutant of Streptomyces griseus when it was introduced into the cells via a high-copy-number plasmid. The sasABCDR gene cluster was identified as being responsible for this suppressive activity. The proteins encoded by sasABCD were of unknown function, but the operon structure was found to be conserved in all the strains of Streptomyces spp. and related organisms whose genomes have been sequenced. sasR, the flanking opposite coding sequence, encoded a putative DNA-binding protein. Subcloning revealed that the presence of all five coding sequences was essential for complete suppression. Scanning electron microscopy of Streptomyces griseus strains carrying the sas gene cluster at a high copy-number revealed that bundle-like structures consisting of several aerial hyphae were often formed. S1 nuclease protection analyses were performed to identify the transcriptional start site in the promoters preceding sasA and sasR. The promoter preceding sasA was highly active during vegetative growth. Null mutants for sasABCD among the S. griseus and S. coelicolor A3(2) cells exhibited bald phenotypes; this suggested a positive regulatory role of this gene cluster in the onset of morphogenesis in these two phylogenetically distinct Streptomyces species.