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【目的】为了研究铜绿假单胞菌全局调控因子RsmA对两个吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制。【方法】采用基因缺失和抗性基因(gentamycin resistance cassette,aacC1)插入相结合的策略构建了rsmA基因缺失突变株PA-RG;通过构建互补表达载体和过表达载体,进一步确认RsmA对绿脓菌素的调控作用;采用电转化方法将构建的翻译融合表达载体pMEZ1(phz1’-’lacZ)和pMEZ2(phz2’-’lacZ)分别导入铜绿假单胞菌突变株PA-RG和野生株PAO1,采用Miller法测定融合β-半乳糖苷酶活性。【结果】在GA培养基中,互补分析和过表达分析表明,RsmA抑制绿脓菌素的合成。此外,pMEZ1在突变株PA-RG中的表达增强,为野生株的2-3倍;而pMEZ2在突变株PA-RG中的表达降低,野生株是突变株的2倍。【结论】由此初步判定,铜绿假单胞菌全局调控因子RsmA对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上RsmA负调控phz1,正调控phz2。
【Objective】 The purpose of this study was to investigate the regulatory mechanism and mechanism of phz1 and phz2 in two phenazine synthetic gene clusters in order to study the global regulatory factor of Pseudomonas aeruginosa RsmA. 【Method】 The gene deletion and resistance gene (gentamycin resistance cassette, aacC1) insertion combined to construct the rsmA gene deletion mutant PA-RG. Through the construction of complementary expression vector and overexpression vector, we further confirmed the effect of RsmA on Pseudomonas aeruginosa (Phz1 ’-’ lacZ) and pMEZ2 (phz2 ’-’ lacZ) were transformed into Pseudomonas aeruginosa mutant PA-RG and wild-type PAO1 respectively by electroporation method. The fusion β-galactosidase activity was determined by the Miller method. 【Result】 In GA medium, complementation analysis and overexpression analysis showed that RsmA inhibited pyocyanin synthesis. In addition, the expression of pMEZ1 in the mutant PA-RG was enhanced 2-3 times that of the wild-type strain, while the expression of pMEZ2 in the PA-RG mutant was reduced twice as much as the wild-type strain. 【Conclusion】 From this preliminary result, RsmA, a global regulator of Pseudomonas aeruginosa, is specific to the regulation of two different phenazine synthetic gene clusters. To a certain extent, RsmA negatively regulates phz1 and positively regulates phz2.