1887

Abstract

In 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 showed high end-to-end similarity in amino acid sequence with the respective 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 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 -transferase (GST–AfsR-g); the fusion product in the soluble fraction in was purified. Incubation of AfsK-g or TRX–AfsK-g in the presence of [γ-P]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 [γ-P]ATP. Disruption of chromosomal had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The 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 is essential for morphogenesis in the presence of glucose. Introduction of restored aerial mycelium formation in the disruptants. The phenotype of - disruptants was similar to that of disruptants; introduction of restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in is conditionally needed for morphological differentiation, whereas in A3(2) it is conditionally involved in secondary metabolism.

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1999-09-01
2019-10-16
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