假单胞菌M18 relA 突变株的构建及其对吩嗪-1-羧酸合成的调控

Construction of Pseudomonas sp. M18 relA mutant and its regulation on phenazine-1-carboxylic acid biosynthesis

摘要:

假单胞菌M18 是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素两种抗生素的植物根际分离细菌。RelA 催化合成的效应分子ppGpp 能介导细菌因营养饥饿引起的应激反应。以M18 菌株染色体DNA 为模板, PCR 扩增获得relA 基因, 通过庆大霉素抗性片段插入失活与同源重组技术, 构建假单胞菌M18 的relA 突变菌株M18RAG。在PPM 培养基中进行PCA 发酵分析, 发现突变菌株M18RAG 的PCA 产量显著升高, 约为野生型菌株的1.5?2 倍。relA 基因反式互补实验以及phzA'-'lacZ 翻译融合测定结果, 均进一步证明了RelA 对PCA 生物合成及其基因表达具有抑制作用。

Abstract:

The rhizosphere bacterium Pseudomonas sp. M18 can simultaneously produce two antibiotics: phenazine-1- carboxylic acid (PCA) and pyoluteorin. ppGpp, which is synthesized by RelA, can mediate bacterial stringent response to nutritional starvation. The relA gene was PCR amplified from the M18 strain chromosomal DNA template. The relA mutant of M18 strain (M18RAG) was constructed through inserted inactivation of gentamicin resistance cassette and homologous recombination. PCA production was assayed in PPM media. It was showed that the relA mutation resulted in a significant enhancement of PCA production. PCA production of M18RAG was about 1.5 to 2 times as much as that of the wild-type strain. The negative regulation of RelA on PCA biosynthesis and its gene expression was further confirmed by the trans complementation test of relA gene and the expression analysis of phzA'-'lacZ translational fusion.