The Study On SCO1979 As A DNA-binding Regulatory Protein In Streptomyces Coelicolor

Streptomyces is a gram-positive bacterium that synthesizes various biologically active secondary metabolites,such as antibiotics,antitumor substances,and so on.Streptomyces coelicolor is a model strain of Streptomyces and its genome was sequenced in 2002.Sequence analysis revealed that the genome contains multiple biosynthetic gene clusters of secondary metabolites,such as actinorhodin,undecylprodigiosin and yCPK.The biosynthesis of secondary metabolites is often regulated by global regulator.This study on SCO 1979 as a DNA-binding regulatory protein in Streptomyces coelicolor,to investigate the regulatory effect on the physiological process of Streptomyces coelicolor.First,homologous arm knockout plasmid were constructed,and the mutant strain?1979 was obtained by conjugal transfer.Phenotypic analysis of M145 and ?1979 revealed that the growth rate of ?1979 was significantly slower,the development of aerial hyphae lags behind and spore production is reduced;and the production of actinorhodin,undecylprodigycin,and calcium dependence antibiotic were produced.The level of antibiotics has increased to varying degrees.The difference in the yield of these secondary metabolites has been verified by the relative quantitative determination of ACT,RED,and CDA.In order to verify that the phenotypic variation of the mutant strain ?1979 was caused by the deletion of sco1979,the integrated vector pMS82 carrying the complete coding sequence of sco1979 and its promoter region was complemented to ?1979,to obtain the complementing strain C-?1979.Phenotypic analysis revealed that the growth development and the secondary metabolites biosynthesis of the complementing strain were similar to those of M145,demonstrating that the phenotypic variation of ?1979 was caused by the absence of sco1979.In order to search for genes regulated by the regulatory protein SCO1979,transcriptome analyses were performed on M145 and ?1979.The results showed that there were 477 gene expression differences between M145 and ?1979.The transcript levels of ram,chp and rdl genes related to morphological development were down-regulated,while the expression of ACT,RED and CDA biosynthesis related genes was significantly upregulated.Realtime-PCR was used to quantitatively analyze the expression of some genes.The results were consistent with the transcriptome results.To further investigate the conserved binding sequence of SCO1979,we expressed and purified the His-SCO1979 fusion protein.In vitro binding assays showed that SCO1979 binds directly to the promoters of actII-ORF4,redZ,cdaR,suggesting that sco1979 may negatively regulate the biosynthesis of actinorhodin,undecylriborubicin,and calcium-dependent antibiotics.The regulation of act?-ORF4,redZ,cdaR was achieved.Based on the above experimental results,this study found that the regulatory protein SCO1979 globally regulates the growth development and the biosynthesis of secondary metabolites of S.coelicolor.