The Study On SCO5351 As A Novel Regulatory Protein In Streptomyces Coelicolor

Streptomyces is a Gram-positive bacterium belonging to actinomycetes and widely distributed in nature.Its growth and development process is very complicated.During this process,it produces a variety of secondary metabolites,including antibiotics,immunosuppressants,antitumor inhibitors,extracellular enzymes,active enzymes and so on,which are widely used in industry,medicine,agriculture,biological control and other aspects have an important role and significance for human life and production processes.Streptomyces coelicolor is a model strain of Streptomyces.And genome sequencing of Streptomyces coelicolor was completed in 2002,and 965 genes with regulatory functions were discovered.These regulatory genes include 67 pairs of typical two component signal transduction systems,65 sigma factors that control gene transcription-regulated,and 25 DNA binding proteins that do not belong to any protein family.However,these regulatory proteins have a number of global regulatory factors,and their functions are unknown,so it is of great significance to study their regulatory mechanism.This subject mainly studied the function of regulatory protein SCO5351 in Streptomyces coelicolor,the biological significance of this protein in the morphological development and secondary metabolites biosynthesis of Streptomyces coelicolor was explored.Firstly,we constructed the in-frame deletion mutant of the RR-coding gene sco5351 after the construction of recombinant vector and the conjugation between Streptomyces coelicolor and E.Coli.The mutant strain ?sco5351 lacking the sco5351 gene was obtained in S.coelicolor M145.The observed phenotypes of S.coelicolor M145 and the mutant strain ?sco5351,respectively,revealed that the formation of airborne mycelia in ?sco5351 was slightly delayed on YBP solid medium,and the yield of actinorhodin(Act)was significantly reduced.Undecyltrimethoprim(Red)yield declines slightly and the synthesis of calcium-dependent antibiotics is also affected.To determine that the phenotypic variation of Asco5351 was caused by the loss of sco5351.We constructed replenishing strain C-?sco5351 and phenotype of C-?sco5351 was restored,indicating that the phenotypic change of ?sco5351 was caused by the loss of sco5351.The related secondary metabolites Act,Red,and CDA were quantitatively analyzed using spectrophotometry and biological activity assays,respectively.To quantify the differences between M145 and ?sco5351,and it was determined that SCO5351 can affect three antibiotics yield such as Act,Red,and CDA.Scanning electron microscopy confirmed delayed sporulation in ?sco5351.To find the target gene population regulated by SCO5351,we used the RNA-sequence technique to analyze the differential gene expression levels of wild-type M145 and the mutant ?sco5351.The results showed that the expression of 104 genes was significantly up-regulated and the expression of 231 genes was significantly down-regulated in ?sco5351.There are several genes associated with growth and development that are down-regulated,including 8 chaplin genes and 2 rodlin genes encoding hydrophobic sheath components on the surface of aerial hyphae.Several gene clusters related to the synthesis of secondary metabolites are also down-regulated,including actinomycin Act,undecylprodigiosin Red,and calcium-dependent antibiotic CDA.The expression of these related genes was verified by RT-PCR.The results of RT-PCR were consistent with the results of RNA-sequence,indicating that sco5351 not only affected the morphological development of Streptomyces coelicolor,but also regulated the synthesis of secondary metabolites.In order to find the target genes directly controlled by SCO5351 and conserved binding sites,we heterologously expressed and purified SCO5351 in E.coli and performed EMSA.The results showed that SCO5351 could not specifically bind to the promoters of the chp and rdl genes involved in differentiation and development,could not bind the promoter regions of the genes such as actORF4,redD/redZ,and cdaR,which are specific regulators of secondary metabolite synthesis.SCO5351 cannot directly bind to the promoter regions of these genes to regulate the development differentiation or the secondary metabolites synthesis.In vitro experiments can not find the target gene and conserved sequence of SCO5351,we want to achieve this goal through in vivo chip-sequence experiments.Firstly,the trophic strains with 3*Flag tags at the C-terminus and N-terminus of SCO5351 were constructed.It was found that their phenotypes could be completely recovered.SCO5351 has a site of dimer formation.The existence of dimer sites allows us to speculate that SCO5351 may form dimers with itself or with other proteins and directly bind to promoter regions of related genes to exert regulatory functions.In order to verify the role of this dimer site,we performed a point mutation on the dimer site of SCO5351 and found that point mutation SCO5351 could not replenish the?sco5351 phenotype,indicating that the dimer site is very important for SCO5351.Based on the above results,it was determined that SCO5351 can affect the aerial hyphae and spore formation process of Streptomyces coelicolor,and also affect the biosynthesis process of secondary metabolites such as Act,Red,and CDA,and is an important global regulatory factor in S.coelicolor.