Abstract
Avermectins are useful anthelmintic antibiotics produced by Streptomyces avermitilis. We demonstrated that a novel AraC-family transcriptional regulator in this species, SAV742, is a global regulator that negatively controls avermectin biosynthesis and cell growth, but positively controls morphological differentiation. Deletion of its gene, sav_742, increased avermectin production and dry cell weight, but caused delayed formation of aerial hyphae and spores. SAV742 directly inhibited avermectin production by repressing transcription of ave structural genes, and also directly regulated its own gene (sav_742) and adjacent gene sig8 (sav_741). The precise SAV742-binding site on its own promoter region was determined by DNase I footprinting assay coupled with site-directed DNA mutagenesis, and 5-nt inverted repeats (GCCGA-n10/n12-TCGGC) were found to be essential for SAV742 binding. Similar 5-nt inverted repeats separated by 3, 10 or 15 nt were found in the promoter regions of target ave genes and sig8. The SAV742 regulon was predicted based on bioinformatic analysis. Twenty-six new SAV742 targets were identified and experimentally confirmed, including genes involved in primary metabolism, secondary metabolism and development. Our findings indicate that SAV742 plays crucial roles in not only avermectin biosynthesis but also coordination of complex physiological processes in S. avermitilis.
Highlights
Bacterial transcriptional regulators are classified into ~50 families on the basis of sequence alignment and structural and functional criteria[10]
electrophoretic mobility shift assays (EMSAs) and quantitative real-time RT-PCR (qRT-PCR) results indicate that SAV742 plays a direct role in repressing avermectin production, which is mediated by ave structural genes, but not by the CSR gene aveR
Among transcriptional regulators located outside the ave gene cluster in S. avermitilis, SAV742 is the first shown to directly regulate ave structural genes, reflecting the subtlety and complexity of avermectin biosynthesis regulation
Summary
Bacterial transcriptional regulators are classified into ~50 families on the basis of sequence alignment and structural and functional criteria[10]. Among the known transcriptional factors in Streptomyces, AraC family members participate in the control of genes involved in important biological processes such as carbon source utilization, morphological differentiation, secondary metabolism, pathogenesis and stress responses[10]. Transcription of the encoding adpA gene is triggered by A-factor, which activates hundreds of target genes that are involved in morphological differentiation and secondary metabolism[11,12]. The complete genome sequencing of S. avermitilis[27] revealed 26 encoded AraC family transcriptional regulators, of which only AdpA has been reported to be involved in morphogenesis and melanogenesis by our group[28]. SAV742 acts as a direct repressor of avermectin production by directly controlling transcription of several avermectin biosynthetic genes, and affects cell growth and morphological development. We predicted the SAV742 regulon based on the consensus binding motif of SAV742, and identified new target genes involved in primary metabolism, secondary metabolism and morphological differentiation
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