铜绿假单胞菌M18 rsmY突变株的构建及其对PCA和Plt的区别性调控

Construction of Pseudomonas aeruginosa M18 rsmY mutant and its different regulation on PCA and Plt biosynthesis

摘要:

【目的】在假单胞菌中, 小RNA (sRNA)参与初级和次级代谢产物、多种毒素因子以及菌群传感系统的调控, 通过在植物根际促生铜绿假单胞菌M18中研究RsmY对吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的调控作用, 深入了解假单胞菌中次级代谢的途径并为构建高产抗生素工程菌株提供了一定的理论基础。【方法】运用同源重组技术, 构建了铜绿假单胞菌M18株的rsmY突变菌株M18RY, 通过基因过表达、lacZ报告基因融合分析实验, 进一步验证了RsmY对抗生素合成基因的调控作用。【结果】比较野生型M18和突变株M18RY中PCA和Plt在同一培养条件下的生物合成量, 突变菌株M18RY中PCA的产量显著增加, 为野生型菌株的5倍左右, 而Plt的产量降为野生型的1/8。LacZ报告基因融合分析进一步证明了RsmY对PCA的负调控作用主要是通过phz2基因簇来实现的。【结论】结果表明, rsmY基因区别性调控PCA和Plt的生物合成。

Abstract:

[Objective] Small RNAs (sRNAs) regulate various genes expression, which involved in primary and secondary metabolism, quorum sensing system and a variety of toxins factors in various Pseudomonas spp.. Through studying the different role of RsmY on regulation of the two antibiotics phenazine-1-carboxylic acid (PCA) and Pyoluteorin (Plt) in the plant rhizosphere growth-promoting Pseudomonas aeruginosa M18, the results were to shed light on the pathways involved in secondary metabolism and provided some theoretical basis for building high-yield engineering strains. [Methods] The rsmY inactivated mutant M18RY was constructed by homologous recombination, and gene over-expression experiment and lacZ gene fusion report analysis were carried out to investigate the regulatory mechanisms involved in RsmY. [Results] PCA and Plt production were respectively assayed in wild type M18 and mutant M18RY strains. Compared with that in wild type M18, PCA production in rsmY mutant had a 5-fold increase, but Plt production in M18RY had an 8-fold decrease. The results could be confirmed identified by Gene rsmY over-expression experiment. Moreover, lacZ gene fusion report analysis further clarified that RsmY strongly up-regulated PCA biosynthesis via phzA2-G2 gene cluster. [Conclusion] Small RNA RsmY distinctively regulates the PCA and Plt biosynthesis.