固氮施氏假单胞菌A1501四碳二羧酸结合蛋白DctP的功能及表达特性

Characterization and expression of C4-dicarboxylate binding protein coding gene dctP of the nitrogen fixing bacterium Pseudomonas stutzeri A1501

摘要:

【目的】研究施氏假单胞菌(Pseudomonas stutzeri) A1501四碳二羧酸结合蛋白DctP的生物学功能和自身表达特性。【方法】构建结合蛋白编码基因dctP的非极性突变株，测定dctP突变株以不同四碳二羧酸(琥珀酸延、胡索酸、苹果酸)为唯一碳源时的生长情况和固氮酶活性；构建dctP基因启动子的融合表达载体dctP-lacZ，将其分别转入野生型A1501和ntrBC、rpoN和dctB突变株中，测定在不同四碳二羧酸为唯一碳源诱导条件下重组菌株中的β-半乳糖苷酶活性。【结果】dctP基因的突变使菌株丧失了四碳二羧酸的利用能力，影响了菌株的固氮酶活性；苹果酸、延胡索酸、琥珀酸对dctP-lacZ具有明显的诱导作用；在rpoN、ntrBC和dctB突变株中，dctP的表达量均显著降低。【结论】DctP蛋白在四碳二羧酸的利用过程中起重要的作用，dctP基因的表达是RpoN依赖型，可被四碳二羧酸诱导，受到调控蛋白NtrBC/DctB的协同调控。

Abstract:

[Objective] Current study was designed to determine the functional characterization and expression pattern of C4-dicarboxylate binding protein coding gene dctP in nitrogen fixing bacterium Pseudomonas stutzeri A1501. [Methods] We constructed the nonpolar dctP mutant. The nitrogenase activity and growth curve between the wild type and mutants were assayed in minimal medium supplemented with different C4-dicarboxylates (succinate, malate or fumarate) as a sole carbon source were measured. The dctP-lacZ fusion vector was constructed and transformed to wild type A1501, dctB mutant, rpoN mutant and ntrBC mutant, respectively. The β-galactosidase activity was detected to analyze the expression of dctP gene in recombinant strains grown with different C4-dicarboxylates as a sole carbon source. [Results] It was found that dctP mutant lost the ability to utilise C4-dicarboxylates and its nitrogenase activity was much lower than the wild type. The analysis of β-galactosidase activity indicated that the succinate, malate or fumarate induced expression of the dctP gene. In comparison with wild type, the expression level of dctP gene was significantly decreased in the deletion mutants for dctB, rpoN or ntrBC. [Conclusion] The DctP protein played an important role in the C4-dicarboxylate utilization of P. stutzeri A1501. C4-dicarboxylates (succinate, malate or fumarate) induced the expression of dctP gene. The DctP transcription was RpoN-dependent and under the control of DctB and NtrBC.