假单胞菌GP72 rpeB突变株的构建及其对吩嗪类抗生素合成的调控

Construction of Pseudomonas sp. GP72 rpeB mutant and its regulation on PCA and 2-OH-PHZ biosynthesis

摘要:

【目的】绿针假单胞菌GP72是一株可生产吩嗪类抗生素吩嗪-1-羧酸 (PCA)及2-羟基吩嗪(2-OH-PHZ)的生防假单胞菌。RpeA/RpeB双元调控系统是其双元调控系统中的一组，本文旨在研究这一系统中的应答调控子(RR)RpeB对于PCA及2-OH-PHZ的生物合成影响。【方法】通过生物信息学分析获得了rpeA/rpeB双元调控系统的序列，并从GP72中扩增出rpeB基因，通过同源重组技术构建卡那霉素抗性片段插入突变rpeB的突变菌株GP72BN。利用发酵实验、rpeB基因回补实验及荧光定量PCR实验，验证rpeB对于吩嗪类抗生素合成及相关基因表达的调控作用。【结果】在KMB培养基中，rpeB突变株的PCA产量下降为野生型的49.5%，2-OH-PHZ产量下降为野生型的67.3%。rpeB基因的回补可以在一定程度上回复PCA及2-OH-PHZ的产量。荧光定量PCR实验结果表明，rpeB突变株中群体感应系统基因phzI/phzR及吩嗪合成基因簇基因phzE转录水平均显著下调，而PCA转化为2-OH-PHZ的修饰基因phzO转录水平变化不显著。【结论】RpeB正调控PCA与2-OH-PHZ合成途径的表达。RpeB很可能是通过调控群体感应基因phzI/phzR和phz基因簇的表达，从而影响PCA的合成，并间接调控其衍生物2-OH-PHZ的合成。

Abstract:

[Objective] Rhizobacterium Pseudomonas chlororaphis GP72 produces two antibiotics, phenazine-1-carboxilic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). RpeB is the response regulator (RR) of RpeA/RpeB two-component signal transduction system. We studied the effect of RpeB on PCA and 2-OH-PZH biosynthesis and its genes expression. [Methods] The rpeB gene was amplified from the GP72 genome. The rpeB mutant (GP72BN) was constructed through inserted inactivation of kanamycin resistance cassette and homologous recombination. Phenazines production assay, gene complement experiment and qRT-PCR analysis were used to assess the influence of RpeB on PCA and 2-OH-PHZ biosynthesis and its gene expression. [Results] In KMB media, PCA and 2-OH-PHZ production of rpeB mutant was decreased by 49.5% and 67.3%. The complementation of rpeB could recover the production of these two phenazine antibiotics. The results of qRT-PCR showed that the transcription of phzI/phzR and phzE strongly decreased in rpeB mutant, but the transcription of phzO only decreased a little. [Conclusion] RpeB positively regulates PCA and 2-OH-PHZ biosynthesis, and it might work for upstream of quorum sensing gene phzI/phzR to influence the expression of phz operon.