生防假单胞菌2P24中氧硫还蛋白DsbA1对抗生素2,4-DAPG产生的调控作用

Pseudomonas fluorescens 2P24 protects various crop plants against soil-borne diseases caused by plant pathogens. Among a range of exoproducts excreted by strain 2P24, the polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG) is particularly relevant to its biocontrol potential. Previously study showed that oxidoreductase DsbA1 was a novel regulator of biocontrol activity in strain 2P24. To dissect the role of DsbA1 on the production of 2,4-DAPG, we designed a molecular trapping assay using the 2P24 DsbA1 CXXS mutant to form inter-molecular DsbA1-substrate disulfide-bonded complex. The DsbA1-substrates will be purified and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The candidates that may influence the production of 2,4-DAPG will be identified by bioinformatics analysis, and the corresponding genes will be further mutated. The effects on antibiotic production will be confirmed by HPLC. Our findings of this project will illustrate the mechanism of oxidoreductase DsbA1 as a novel regulatory element in the complex network controlling expression of biocontrol traits in plant-beneficial pseudomonads. It also provides new ideas for the study of the mechanism of DsbA in plant and human pathogenic bacteria.

荧光假单胞菌（Pseudomonas fluorescens）2P24可防治多种由植物病原菌引起的土传病害。次生代谢物2,4-二乙酰基间苯三酚（2,4-diacetylphloroglucinol，2,4-DAPG）是其防治病害的主要生防因子。前期利用Tn5随机突变获得了dsbA1突变菌株，该突变菌株可影响2,4-DAPG在内的多个生防相关性状表达。本项目通过构建DsbA1 CXXS活性位点突变菌株，诱捕DsbA1-底物复合物。利用液相色谱串联质谱解析底物蛋白，生物信息学方法分析底物蛋白的定位，获得影响2,4-DAPG产生的候选DsbA1底物；构建相应内缺失突变菌株，检测其对2,4-DAPG产生的影响。本项目研究结果将明确菌株2P24硫氧还蛋白DsbA1生防相关性状调控中的作用机制，为生防菌遗传学改造奠定基础；同时本项目研究成果也可为植物及人体病原菌中DsbA蛋白作用机制的研究提供借鉴。

荧光假单胞菌（Pseudomonas fluorescens）2P24可防治多种由植物病原菌引起的土传病害。次生代谢产物2,4-二乙酰基间苯三酚（2,4-diacetylphhloroglucinol, 2,4-DAPG）是其防治病害的主要生防因子。前期利用Tn5随机突变获得了dsbA1突变菌株，该突变菌株可影响2,4-DAPG在内的多个生防相关性状的表达。.本项目通过构建DsbA1 CXXS活性位点突变菌株，诱捕DsbA1-底物复合物，利用液相色谱串联质谱解析底物蛋白。本研究获得了多个与DsbA1蛋白互作的靶标蛋白，其中Gcd蛋白编码葡萄糖水解酶。该蛋白含有6个Cys残基；且该蛋白定位于膜上的蛋白。细菌双杂交实验也表明DsbA1蛋白可与Gcd蛋白互作，而突变Gcd蛋白中C235，C275和C578残基可抑制这种互作，但可以提高Gcd酶活性，从而提高2,4-DAPG的产生。.除Gcd蛋白外，本研究还获得了多个参与环二鸟苷酸（cyclic-di-GMP，c-di-GMP）代谢的蛋白质底物。本研究进一步研究了c-di-GMP对2,4-DAPG产生的影响。研究表明菌株2P24中c-di-GMP可通过影响阻遏蛋白RsmA和RsmE的水平从而调控包括2,4-DAPG和群体感应系统在内的多个生防相关性状的表达。其中二鸟苷酸环化酶C0J56_12915、C0J56_13325和C0J56_27925特异性参与到这一调控过程中。.上述结果表明，菌株2P24中氧硫还蛋白DsbA1可影响多个蛋白功能，从而影响该菌株生防相关性状表达。