Regulatory Mechanisms Of Transcriptional Regulator SAV742 And Alternative ? Factor ?~8 In Streptomyces Avermitilis

Streptomyces avermitilis produces avermectins,a series of 16-membered macrocyclic anthelmintic antibiotics that are widely utilized in medicine,agriculture and animal husbandry.However,the regulatory mechanisms of avermectin biosynthesis remain to be clarified.In this work,the function and regulatory mechanisms of a novel AraC-family transcriptional regulator SAV742 and an alternative ?factor ?8?SAV741?were studied.sav₇42 encodes an AraC-family transcriptional regulator.Morphological observation and shake-flask fermentation of sav₇42 deletion,complementation and overexpression strains implied that SAV742 negatively regulates avermectin biosynthesis and cell growth,while promotes morphological differentiation.EMSA and qRT-PCR assays revealed that SAV742 directly inhibits avermectin production by repressing transcription of ave structural genes aveF,aveA1,aveD and aveA4.SAV742 also directly regulated transcription of its own gene and upstream gene sig8.The precise SAV742-binding sequence on its own promoter region was determined by DNase I footprinting assay coupled with site-directed DNA mutagenesis,and similar sequence were found in the promoter regions of target ave structural genes and sig8.The SAV742 consensus binding sequence RCCGM-N0-15-YHGSC?R:A or G,M:A or C,Y:C or T,H:A,C or T,S:C or G,N:any nucleotide?was thus identified,leading to prediction of the SAV742 targets.Twenty-six new SAV742 targets were identified and experimentally confirmed,including genes involved in primary metabolism,secondary metabolism and morphological development.These findings indicate that SAV742 is a novel global regulator and plays crucial roles in not only avermectin biosynthesis but also coordination of complex physiological process in S.avermitilis.?8 is the first identified alternative ? factor in S.avermitilis and is highly homologous to ?B in S.coelicolor.Shake-flask fermentation and phenotype observation of sig8 gene deletion,complementation and overexpression strains implied that ?8 negatively regulates avermectin production,but has no effect on cell growth or morphological differentiation under normal conditions.EMSA,in vitro transcription and qRT-PCR assays revealed that ?8 inhibits avermectin biosynthesis by indirectly repressing expression of pathway-specific activator gene aveR,and by directly initiating transcription of its downstream gene sav₇42.Relative to growth of wild-type strain,that of a sig8-deletion mutant was more sensitive to NaCl,KCl,H2O2,diamide or heat shock.sig8 transcription was strongly induced in a?8-dependent manner by these environmental stresses.A series of ?8-dependent genes responsive to heat,oxidative and osmotic stress were identified by EMSAs,in vitro transcription and qRT-PCR experiments,indicating that a8 plays an important role in mediating protective responses to various stress conditions by activating transcription of its target genes.Six promoter structures of ?8 target genes were determined by 5'-RACE assay,and a8 consensus binding sequence BGVNBH-N,5-GSNNHH?B:C,G or T,V:A,C or G,S:C or G,H:A,C or T,N:any nucleotide?was identified,leading to prediction of the ?8 targets.The list consists of 940 putative ?8 target genes assignable to 17 functional groups,suggesting the wide range of cellular functions controlled by ?8 in S.avermitilis.In this work,an AraC-family transcriptional regulator,SAV742,which inhibits avermectin biosynthesis by directly repressing transcription of ave structural genes,and an alternative ? factor,?8,which indirectly inhibits avermectin production at transcription level,were identified.These investigations will help to elucidate the complicated regulatory network of avermectin biosynthesis,and provide the basis for genetic construction of avermectin overproducers.