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An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan¹

Author for correspondence: Sueharu Horinouchi. Tel: +81 3 5841 5123. Fax: +81 3 5841 8021. e-mail: asuhori{at}hongo.ecc.u-tokyo.ac.jp

In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high end-to-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX–AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST–AfsR-g); the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX–AfsK-g in the presence of [-³²P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX–AfsK-g phosphorylated serine and threonine residues of GST–AfsR-g in the presence of [-³²P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1%, but did form spores on mannitol- and glycerol-containing media; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism.

Keywords: Streptomyces griseus, serine/threonine kinase, signal transduction, protein phosphorylation, aerial mycelium formation

Abbreviations: afsK-c and afsK-g, afsK from S. coelicolor A3(2) and S. griseus, respectively; afsR-c and afsR-g, afsR from S. coelicolor A3(2) and S. griseus, respectively; GST, glutathione S-transferase; TRX, thioredoxin

The GenBank accession numbers for the afsK-g and afsR-g sequences determined in this work are D45246 and AB025225, respectively.

^a Present address: Department of Applied Biological Science, Nihon University, Fujisawa-shi, Kanagawa 252-8510, Japan.

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