ECF-σ5因子参与阿维链霉菌中阿维菌素合成和环境胁迫的研究

ECF-σ5 in Sreptomyces avermitilis is involved in regulation of avermectin biosynthesis and stress response

摘要:

[目的]研究阿维链霉菌中ECF-σ⁵因子对阿维菌素合成、形态分化和环境胁迫的调控,为揭示阿维菌素生物合成的调控机制和ECF-σ因子的调控网络提供依据。[方法]构建sig5基因缺失、回补和过表达菌株,通过形态观察和摇瓶发酵实验初步确定σ⁵对形态分化、菌体生长和阿维菌素合成的影响。进一步通过RT-qPCR、EMSA和ChIP实验寻找确定σ⁵的靶基因,再通过胁迫实验揭示σ⁵可能参与的胁迫反应。[结果]对sig5相关突变株的摇瓶发酵和形态观察结果表明,σ⁵对阿维菌素合成具有抑制作用,但不影响菌体生长和形态分化。sig5基因缺失导致阿维菌素生物合成途径特异性正调控基因aveR和结构基因aveA1的转录水平提高,但σ⁵并不与aveR和aveA1的启动子区结合。σ⁵能结合在自身基因及附近基因SAV612、SAV615、SAV618的启动子区,正调控这些基因及所在操纵子的表达。胁迫实验暗示σ⁵可能参与渗透压引起的胁迫反应。[结论]ECF-σ⁵因子在转录水平间接负调控阿维菌素的合成。

关键词:阿维链霉菌;阿维菌素;ECF-σ因子;σ⁵

Abstract:

[Objective] We investegated the role of extracytoplasmic function (ECF) σ factor, σ⁵, in avermectin biosynthesis, morphological differentiation and stress response in S. avermitilis. [Methods] We constructed sig5 gene deletion, complementation and overexpression strains and determined the role of σ⁵ in avermectin production and morphological differentiation by shaking flask fermentation and morphological observation of these strains. We used RT-qPCR, EMSA and ChIP assays to identify the target genes of σ⁵. We used stress tests to reveal the stress response that σ⁵ may be involved in. [Results] Determination of avermectin production and morphological observation in sig5 related strains implied that σ⁵ inhibits avermectin production, but has no effect on growth or morphology. Deletion of sig5 increased transcription levels of pathway-specific activator gene aveR and structural gene aveA1, but σ⁵ did not bind to the promoter regions of aveR and aveA1. RT-qPCR and ChIP assays showed that σ⁵ positively regulates the transcription of itself and adjacent genes by binding to the promoter regions of sig5, SAV612, SAV615 and SAV618. Stress tests suggested that σ⁵ is involved in responding to osmotic stress. [Conclusion] Our findings indicated that σ⁵ indirectly inhibits avermectin production by affecting transcription of ave genes.

Key words:Streptomyces avermitilis;avermectin;ECF-σ factor;σ⁵