Cloning of the conserved regulatory operon by its aerial mycelium-inducing activity in an amfR mutant of Streptomyces griseus.
We report cloning and characterization of a 2.8 kb DNA fragment that suppressed the aerial mycelium-deficient phenotype of an amfR mutant of Streptomyces griseus when it was introduced on a high-copy-number plasmid. Nucleotide sequencing revealed that the cloned DNA fragment contained a part of a regulatory operon homologous to one of the conserved operons identified in the genome of Streptomyces coelicolor A3(2). The operon appeared to consist of 5 CDSs (rarA-E; restoration of aerial mycelium formation in an amfR mutant): rarA encoded a membrane protein with weak similarity to the histidine kinase of the two-component regulatory system; rarB and rarC products did not show marked similarity to other proteins with known function; rarD encoded a G-protein carrying two GTP-binding consensus sequences conserved in the eukaryotic Ras-like proteins; rarE product showed end-to-end homology to cytochrome P450. The 2.8 kb fragment contained a 5'-end incomplete rarA and complete rarB-D in the downstream from the promoter region of mel operon of the vector plasmid. Subcloning showed that the region containing rarA only is sufficient for the aerial mycelium-inducing activity. The truncation of rarA at its 5' terminus was essential for the restoration activity, which implied that the mutated rarA product causes unusual signaling that directs the onset of morphogenesis without amfR function. Inactivation of both rarA in Streptomyces griseus and cvnD9, a rarD ortholog in S. coelicolor resulted in precocious and glucose-resistant formation of aerial mycelium and secondary metabolites, which suggested that the operon negatively regulates the onset of differentiation. S1 nuclease protection analysis showed that the transcriptional activity of the promoter preceding rarA is developmentally regulated in an amfR- and glucose-dependent manner.
Komatsu
Mamoru
M
Life Science Research Center, College of Bioresource Sciences, Nihon University, Fujisawa, Japan.
Kuwahara
Yoshitaka
Y
Hiroishi
Ayako
A
Hosono
Kuniaki
K
Beppu
Teruhiko
T
Ueda
Kenji
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D023061:Gene Order; D005798:Genes, Bacterial / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics; D013305:Streptomyces griseus / Q000235:genetics* / Q000254:growth & development; D014157:Transcription Factors / Q000235:genetics*; D024363:Transcription Initiation Site
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors; C114795:amfR protein, Streptomyces griseus