An extracytoplasmic function sigma factor, σ25, differentially regulates avermectin and oligomycin biosynthesis in Streptomyces avermitilis

Abstract

σ(25) is an extracytoplasmic function (ECF) σ factor in the bacterium Streptomyces avermitilis that plays a differential regulatory role in avermectin and oligomycin biosynthesis. Gene deletion, complementation, and overexpression experiments showed that σ(25) inhibited avermectin production but promoted oligomycin production. σ(25) indirectly inhibited avermectin production by affecting the transcription of the pathway-specific activator gene aveR, whereas it directly activated oligomycin production by initiating transcription of the pathway-specific activator gene olmRI. The divergently transcribed genes smrAB are located upstream of sig25 and encode a putative two-component system (TCS). σ(25) was found to initiate its own transcription, and its expression was directly activated by SmrA. The precise SmrA-binding sites in the region upstream of sig25 were determined by DNase I footprinting assays and identified two direct repeat sequences CTGTGA-n5-CTGTGA, suggesting that SmrA regulates sig25 transcription by binding to these direct repeats. The deletion of smrAB had the similar effect on avermectin and oligomycin A production to the deletion of sig25, indicating that σ(25) and SmrAB function similarly in the regulation of antibiotic production. These findings helpfully clarify the regulation of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in S. avermitilis.

SAV742, a Novel AraC-Family Regulator from Streptomyces avermitilis, Controls Avermectin Biosynthesis, Cell Growth and Development

“…Among 350 putative SAV742 targets having regulatory function, three have been reported to negatively regulate avermectin production: avaR1 (sav_3705 , encoding a putative gamma-butyrolactone receptor protein)3435, sig25 (sav_3351 , encoding an ECF sigma factor) and smrA (sav_3352 , encoding a two-component system response regulator)36. EMSAs showed that SAV742 bound specifically to the avaR1 promoter region and sig25-smrA bidirectional promoter region (Fig.…”

“…The obtained PCR product was cut with Bam HI/ Hin dIII and ligated into expression vector pET-28a (+) to generate pET-742, which was then transformed into E. coli BL21 (DE3) for overexpression of N-terminal His 6 -tagged SAV742 recombinant protein. Following induction by 0.2 mM IPTG, bacteria containing His 6 -SAV742 were collected, washed, resuspended in a lysis buffer36, and sonicated on ice. Soluble His 6 -SAV742 was purified by Ni-NTA agarose chromatography (Qiagen) according to the manufacturer’s instructions.…”

Increasing Avermectin Production in Streptomyces avermitilis by Manipulating the Expression of a Novel TetR-Family Regulator and Its Target Gene Product

“…3A. AveT and AveM have opposing effects on avermectin production, and the other transcriptional regulators affect aveR expression (27,28,47), resulting jointly in a gradual increase in the level of avermectin production in cells. The present finding that a late intermediate in the avermectin pathway acts as a regulator of its own production suggests a positive-feedback regulatory mechanism that ensures the irreversible production of avermectins and their appropriate concentration in cells.…”

Engineering microbial hosts for production of bacterial natural products

“…157 Interestingly, ECF σ 25 in S. avermitilis differentially regulates production of two macrolides oligomycin and avermectin, where overexpression of sig25 increases oligomycin production but decreases avermectin titer. 159 Given that σ 25 directly binds to promoter regions of oligomycin biosynthetic genes, it is likely that the associated reduction in avermectin production is due to competition for common precursors. 159 From a host engineering perspective, sigma factors function as resource allocators to distribute the transcriptional resources (e.g.…”

“…159 Given that σ 25 directly binds to promoter regions of oligomycin biosynthetic genes, it is likely that the associated reduction in avermectin production is due to competition for common precursors. 159 From a host engineering perspective, sigma factors function as resource allocators to distribute the transcriptional resources (e.g. RNAP) within the cell.…”