Regulatory Mechanisms Of ?-butyrolactone Receptor Homologues AvaR2 And AvaR1 In Streptomyces Avermitilis

Streptomyces avermitilis is important industrial microorganism known for producing avermectins,which are anthelmintic agents with high potency,broad-spectrum and low toxicity activities.Avermectins are widely applied in agricultural,veterinary and medical fields.In S.avermitilis,the autoregulatory signalling molecule that triggers avermectin biosynthesis is a novel butenolide-type molecule,avenolide,rather than common ?-butyrolactone(GBL).To elucidate the regulatory mechanisms of avermectin biosynthesis,the regulatory functions and mechanisms of two GBL receptor homologues AvaR2 and AvaRl in S.avermitilis were studied in this work.S.avermitilis contains three GBL receptor homologues:AvaRl(SAV3705),AvaR2(SAV3702)and AvaR3(SAV3703),which all belong to TetR-family transcriptional regulators.Of these,AvaR2 is a close homologue of pseudo GBL receptors.Shake-flask fermentation and morphological observation of avaR2 gene deletion,complementation,and overexpression strains indicated that AvaR2 negatively regulates avermectin biosynthesis and cell growth,but has no effect on morphological differentiation.qRT-PCR,EMSA,DNase I footprinting,5' RACE and ChIP-qPCR assays revealed that AvaR2 directly represses transcription of aveR(the ave cluster-situated activator gene),aco(a key gene for avenolide biosynthesis),its own gene(avaR2)and two other GBL receptor homologous genes(avaRl and avaR3)by binding to their promoter regions.The aveR promoter had the highest affinity for AvaR2.A consensus 18 bp imperfect palindromic sequence(AWWCCRBBHDDNMSGTWT,W:A or T;R:A or G;B:G,C or T;H:A,C or T;D:A,G or T;M:A or C;S:G or C;N:A,G,C or T)was found in the AvaR2-binding regions of these five target genes.This consensus binding sequence was used to predict potential AvaR2 targets,and eleven novel targets were identified by EMSA and qRT-PCR assays,including genes involved in primary metabolism,ribosomal protein synthesis,and stress responses.These findings indicate that AvaR2 is a pleiotropic transcriptional factor.AvaR2 bound and responded to endogenous avenolide and exogenous antibiotics jadomycin B(JadB).apramycin(Apr)and hygromycin B(HygB)to modulate its DNA-binding activity,indicating the important role of AvaR2-mediated signalling transduction system in facilitating inter-and intra-species communication.AvaRl is a close homologue of genuine GBL receptors.Fermentation and morphological observation of avaRl gene deletion,complementation,and overexpression strains indicated that AvaRl negatively regulates avermectin biosynthesis,but has no effect on cell growth and morphological differentiation.qRT-PCR,EMSA,DNase I footprinting,5' RACE and ChIP-qPCR assays revealed that AvaRl directly represses transcription of aveR,aco,its own gene(avaRl)and two other GBL receptor homologous genes(avaR2 and avaR3)by binding to their promoter regions.avaR3p had moderate higher affinity for AvaRl.AvaRl and AvaR2 bound to the same sites on the promoter regions of aveR,aco,avaRl,avaR2 and avaR3,leading to the same consensus binding sequence.Ten novel AvaRl targets were identified by EMSA and qRT-PCR assays,including genes involved in primary metabolism,ribosomal protein synthesis,stress responses and nucleotide metabolism,indicating that AvaRl is also a pleiotropic transcriptional factor.AvaRl and AvaR2 have same and different target genes,indicating that they cross regulate different physiological process.