A-factor Depended Protein, AdpA, A Regulator Involved In Morphological Differentiation And Secondary Metabolism In Streptomyces

Dark brown haloes of melanin around colonies are an easily visualized phenotype displayed by many Streptomyces strains harboring plasmid pIJ702 carrying the melC operon of Streptomyces antibioticus IMRU3270. Spontaneous melanin-negative mutants of pIJ702 occur with a frequency of ca. 1%, and often mutation occurs in the melC operon, which removes the BglII site as part of an inverted repeat. Other melanin-negative mutations seem to occur spontaneously in S. lividans, resulting in white colonies from which intact, melanin-producing pIJ702 can be isolated by introduction into a new host. S. lividans ZX66 was found to be such a mutant and to have a secondary mutation influencing expression of the melC operon on the chromosome. A 3.3 kb DNA fragment was isolated from its progenitor strain, JT46, and a gene able to restore melC operon expression was found to encode a member of an AraC family of transcriptional regulators, which was equivalent to AdpAc in S. coelicolor and therefore was designated AdpAl. Lack of melC operon expression was correlated with a single A-to-C transversion, which altered a single key amino acid residue from Thr to Pro. The transcription of the melC operon was found to be greatly reduced in the adpA mutant background. The counterpart gene in the S. antibioticus strain in which the melC operon carried on pIJ702 originated was also isolated and was found to have an identical regulatory role. Thus, we concluded that the melC operon is under general direct positive control by AdpA family proteins, perhaps at the transcriptional level and certainly at the translational level via bldA, in Streptomyces.S. avermitilis is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites such as avermectin and oligomycin. AdpAa, a regulator of the AvaC family encoded by SAV5261 of S. avermitilis genome, is highly similar to AdpAg, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in S. griseus. adpAa was interrupted and the mutant ZD7 fails to produce melanin and to form aerial mycelium. Furthermore adpAa is also involved in production of avermectin and oligomycin. HPLC analysis indicates that AdpA regulates avermectin and oligomycin biosynthesis positively. S. avermitilis can produce siderophore and siderophore production of the mutant of adpAa was blocked. Reverse transcription PCR for transcript analysis also showed the difference of transcription of genes involved in morphological differentiation and secondary metabolites between wild type strain and ZD7. the transcriptional levels of some genes involved in morphological differentiation were lower in ZD7 compared with those in the wild type strain. There are 32 secondary metabolite clusters in S. avermitilis, and the transcriptional levels of 15 clusters of them were lower in ZD7 compared with those in the wild type strain.Gene aveR is the only putative regulatory gene in avermectin biosynthetic gene cluster from S. avermitilis. In order to test if aveR is involved in the regulation of transcription of avermectin biosynthetic genes, aveR mutant ZD10 was obtained. Analyzed by LC-MS, avermectin productivity was abolished, whereas the yield of oligomycin significantly increased in ZD10. Reverse transcriptase PCR analysis indicated that the transcription level of aveA3, an essential PKS gene, was drastically reduced in ZD10. Therefore, AveR was proved as a positive regulator for avermectin biosynthesis by promoting the transcription of structural genes.