The Study On SCO3008 As A Novel Regulatory Protein In Streptomyces Coelicolor

Streptomyces is a Gram-positive bacterium with a complex life cycle.They are well known for the remarkable ability of producing a variety of biologically active secondary metabolites.Streptomyces coelicolor is the most representative model strain,and its whole genome was sequenced in 2002.The genome sequence analysis indicated that genome can encode 68 pairs of two-component signal transduction system,13 orphan regulator proteins,and 17 orphan sensor kinases.Orphan regulator proteins exist alone on the chromosome,and there are no genes paired with them in the adjacent position.Most of these orphan regulators play an important role in Streptomyces response to environmental stimuli and regulation of metabolite production.In this study,a preliminary study was carried out on the function of orphan regulator protein SC03008 in the morphogenesis and secondary metabolism of Streptomyces coelicolor.We selected the positive clone 22G7 in Streptomyces coelicolor M145 gene library in our laboratory.We constructed a mutant cosmid with in-frame deletion of sco3008 which replaced by apramycin resistance cassette through PCR-targeting technology.Then the sco3008 mutation was obtained by the intergeneric conjugation between Escherichia coli ET12567/pUZ8002 and Streptomyces,named it A3008.A variety of solid media were selected to analyze the phenotypic defects caused by the deletion of sco3008.It was found that on MS-G,MS-M,R2YE solid medium,A3008 showed a certain degree of damage of aerial hyphae,and overproduction of actinorhodin(Act)and undecylprodigiosin(Red).On YBP solid medium,A3008 showed more obvious phenotypic defects,which can't produce aerial hyphae throughout the observation period and shows a "bald" phenotype;A3008 showed higher overproduction of actinorhodin(Act)and undecylprodigiosin(Red)compared with M145.In order to determine that the phenotypic defect of A3008 was caused by the deletion of sco3008,we constructed a ?3008 complementary strain.Phenotypic changes on differentiation development and secondary metabolism,which caused by mutant strain,were completely restored in the complementary strain.We analyzed the expression differences of wild-type M145 and mutant ?3008 genes by RNA-Seq,and defined the genes regulated by SCO3008 in vivo,Among them,135 genes were overexpressed,including genes for actinorhodin synthesis gene cluster and the undecylprodigiosin synthesis gene cluster;and 568 genes were underexpressed,including genes associated with aerial hyphal growth and spore maturation,such as chaplin gene and rodlin gene,bld gene,whi gene,wbl gene,and membrane protein encoding genes,and some unknown functions structural genes.It indicated that the orphan regulator gene sco3008 plays an important role in regulating the morphogenesis and secondary metabolism of Streptomyces coelicolor.In order to find the target gene directly regulated by SCO3008 in Streptomyces,we constructed a SCO3008-Flag fusion protein complementary strain,which can completely complement the phenotypic defects caused by sco3008 deletion,and can effectively express SCO3008-Flag fusion protein.We used this strain to carry out ChIP-Seq experiments and assisted ChIP-qPCR and DNA-Pull Down experiments to successfully find two target genes directly regulated by SCO3008 in vivo,which are wblA and sco1375.It is indicated that the regulation of SCO3008 on the morphogenesis and secondary metabolism of Streptomyces is achieved by regulating the expression of related genes.In order to further explore the effect of the regulatory protein SCO3008 on the morphogenesis and secondary metabolism to Streptomyces coelicolor,this study used the exogenous strong promoter to construct the SCO3008 overexpression strain.The phenotypic analysis showed that the strain produced less aerial hyphae and the actinorhodin overproduced than the wild strain M145,indicating that the regulation function of the orphan regulatory protein SC03008 is likely to be related to level of protein expression,the regulation function is to promote the growth of aerial hyphae and inhibit the production of Act when the expression is in low level;when the expression level is high,it is shown to inhibit the aerial hyphae growth and facilitate the Act overexpression.In summary,we can clarify the regulation mechanism of orphan regulator protein SCO3008 in differentiation development and secondary metabolism,which is an important global regulator factor.