CLONING AND CHARACTERIZATION OF THE A-FACTOR RECEPTOR GENE FROM STREPTOMYCES-GRISEUS

A-factor isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its sp ecific receptor protein control streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S . griseus IFO 13350. The NH2-terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpo se of preparing oligonucleotide primers for cloning arpA by the PCR me thod. The arpA gene cloned in this way directed the synthesis of a pro tein having A-factor-specific binding activity when expressed in Esche richia coli under the control of the T7 promoter. The arpA gene was th us concluded to encode a 276-amino-acid protein with a calculated mole cular mass of 29.1 kDa, as determined by nucleotide sequencing. The A- factor-binding activity was observed with a homodimer of ArpA. The NH2 -terminal portion of ArpA contained an alpha-helix-turn-alpha-helix DN A-binding motif that showed great similarity to those of many DNA-bind ing proteins, which suggests that it exerts its regulatory function fo r the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-fac tor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like be havior of ArpA, introduction of arpA into this mutant abolished simult aneously its streptomycin production and aerial mycelium formation. Al l of these data are consistent with the idea that ArpA acts as a repre ssor-type regulator for secondary metabolite formation and morphogenes is during the early growth phase and A-factor at a certain critical in tracellular concentration releases the derepression, thus leading to t he onset of secondary metabolism and aerial mycelium formation. The pr esence of ArpA-like proteins among Streptomyces spp., as revealed by P CR, together with the presence of A-factor-like compounds, suggests th at a hormonal control similar to the A-factor system exists in many sp ecies of this genus.