Identification Of Diguanylate Cyclases CdgC And CdgD As Well As Their Regulatory Functions In Streptomyces Coelicolor

Cyclic 3',5'-diguanylate(c-di-GMP)is a kind of common second messenger in bacteria.As a key component of bacterial signal transduction pathways,c-di-GMP regulates numerous important cellular processes,such as motility,biofilm formation and antibiotic biosynthesis,by directly binding to different types of effectors.Diguanylate cyclases(DGCs)and phosphodiesterases(PDEs)are responsible for the synthesis and degradation of c-di-GMP,respectively,thus synergistically controlling intracellular c-di-GMP levels.Generally,the proteins carrying GGDEF motif possess DGC activity that catalyzes GTP to form c-di-GMP.The proteins carrying EAL or HD-GYP motifs possess PDE activity that hydrolyzes c-di-GMP into dinucleotide 5'-phosphoguanylyl-(3'-5')-guanosine(pGpG)or GMP,respectively.Streptomyces are a kind of high-GC,Gram-positive bacteria with complex morphological differentiation and strong secondary metabolism,which can produce a large number of bioactive secondary metabolites,such as antibiotics,immunosuppressants and anti-tumor compounds.It is reported that both secondary metabolism and morphological differentiation are strictly regulated by multi-layered signal transduction networks in streptomycetes,such as two-component systems and c-di-GMP signalling cascade.Previous studies on c-di-GMP-mediating regulation of growth and secondary metabolism in Streptomyces are limited to the model strains Streptomyces venezuelae and Streptomyces coelicolor,and these researches are still in their infancy stage.For example,there are 10 possible c-di-GMP-metabolizing enzymes on the genome of S.coelicolor,but only four enzymes were characterized.Therefore,it will be very significant in theoretical study and potential applications to systematically identify other functionally unknown c-di-GMP metabolizing enzymes as well as their regulatory roles in morphological differentiation and secondary metabolism in S.coelicolor.In this study,six functionally unidentified c-di-GMP-metabolizing enzymes were explored in S.coelicolor by gene deletion and overexpression strategies.Phenotypic analysis showed that morphological differentiation and antibiotic biosynthesis of all deletion mutants were not significantly affected.However,two of all overexpression strains showed significant phenotypic changes.On one hand,overexpression of cdgD(SCO5345)completely blocked spore formation on R2 YE or MS media,as well as inhibited biosynthesis of actinorhodin(ACT)or prodigiosin(RED)on R2 YE medium.On the other hand,overexpression of cdgC(SCO5511)resulted in a significant increase in ACT production on MS medium,but exhibited no obvious effects on morphological differentiation.Then,we conducted an in-depth study of the functions of CdgD.In vivo site-directed mutagenesis analysis revealed that CdgD with mutated GGDEF motif completely lost its regulatory functions.Intracellular c-di-GMP levels in the cdgD-overexpression strain were 13-27-fold higher than those in the wild-type strain.In vitro enzymatic assay demonstrated that CdgD could catalyze the synthesis of c-di-GMP from GTP.Collectively,these results indicated that CdgD is a new diguanylate cyclase in S.coelicolor.Furthermore,cdgD from S.coelicolor was also heterologously expressed in two important industrial strains Streptomyces pristinaespiralis and Streptomyces hygroscopicus,which significantly inhibited morphological differentiation and biosynthesis of pristinamycin I or 5-oxomilbemycin A3/A4.These results indicated that it is conserved for c-di-GMP mediated signalling pathway involved in growth and secondary metabolism in Streptomyces.Finally,in vivo site-directed mutagenesis analysis revealed that overexpression of CdgC with mutated GGDEF motif could not facilitate ACT biosynthesis,preliminarily domonstrating that CdgC is a diguanylate cyclase in S.coelicolor.Interestingly,cdgC overexpression led to obvious increase in ACT production but no significant change in the intracellular c-di-GMP concentrations,whose detailed mechanism requires to be further investigated.In summary,we identified two novel diguanylate cyclases,CdgC and CdgD,and explored their regulatory roles in morphological differentiation and secondary metabolism in S.coelicolor.Furthermore,we also demonstrated that c-di-GMP-mediated signalling pathways play an important role in growth and antibiotic biosynthesis in industrial Streptomyces.Finally,cdgC overexpression significantly increased ACT production in S.coelicolor.This is the first time to observe positive effects of DGCs on antibiotic biosynthesis in Streptomyces,whose detailed mechanism remains to be determined yet.