The Study On SCO2832 As An IclR-family Transcriptional 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 Calcium-dependent antibiotic gene clasters.The biosynthesis of secondary metabolites is often regulated by global regulator.but these regulatory mechanisms are currently unclear.The IcIR family is widely distributed in Gram-positive bacteria and has a variety of important regulatory functions.SC02832 is a putative Ic1R family protein in the Streptomyces coelicolor genome whose function has not been elucidated to date.In this paper,the function of the putative regulatory protein SC02832 was explored in order to investigate the regulation mechanism and principle.First,using PCR-targeting knockout plasmid,and the mutant strain ?2832 was obtained by conjugal transfer.Phenotypic analysis of M145 and ?2832 revealed that A 2832 produced a decrease in the production of actinomycin,undecyl polymycin and calcium-dependent antibiotics.The relative quantitative determination of these secondary metabolites has been confirmed by relative quantitative determination of ACT,RED and CDA..In order to verify that the phenotypic variation of the mutant strain A2832 was caused by the deletion of sco2832,the integrated vector pMS82 carrying the complete coding sequence of sco2832 and its promoter region was complemented to ?2832,to obtain the complementing strain C-A2832.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 A2832 w'as caused by the absence of sco2832.In order to search for genes regulated by the regulatory protein SC02832,transcriptome analyses were performed on M145 and A2832 at 72h.The results showed that there were 25 1 gene expression differences between M 145 and A2832.The transcript levels of ACT.RED and CDA biosynthesis related genes was significantly downregulated,The gene cluster of CDA biosynthesis and morphological development such as chp,ram.rdl is also down-regulated.Realtime-PCR was used to quantitatively analyze the expression of some genes.The results were consistent with the transcriptome results.And to further investigate the conserved binding sequence of SC02832,we use pET22b expressed and purified the His-SC02832 fusion protein.Based on the above experimental data,this study found that the IcIR family transcriptional regulatory protein SC02832 globally regulates the growth and development of Streptomyces coelicolor and the transcriptional expression of secondary metabolites ACT,RED,and CDA.