rpoS基因插入突变对铜绿假单胞菌两个吩嗪合成基因簇的调控

Basic analysis of rpoS mutation on two gene clusters of phenazine in Psedomonas aeruginosa PAO

摘要:

摘要：【目的】为了研究铜绿假单胞菌rpoS基因对吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制。【方法】采用抗庆大霉素基因（gentamycin resistance cassette, aacC1）插入失活的策略构建了rpoS基因突变株PA-SG；同时利用lacZ的翻译融合表达载体pME6015，构建了phz1′-′lacZ 和phz2′-′lacZ翻译融合表达载体pMEZ1和pMEZ2。采用电转化法分别将pMEZ1、pMEZ2和pME6015导入铜绿假单胞菌突变株PA-SG和野生株PAO1，用Miller法检测融合β-半乳糖苷酶活性。【结果】在KMB或PPM 培养基中，pMEZ1在突变株PA-SG中的表达均增强，为野生株的4-5倍；而pMEZ2在突变株PA-SG中的表达均降低，野生株是突变株的2-3倍。【结论】由此推测，铜绿假单胞菌rpoS基因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，rpoS负调控phz1，正调控phz2。

Abstract:

Abstract：As an opportunistic pathogen, Pseudomonas aeruginosa PAO1 can produce phenazine and its derivatives, which play a critical role in their pathogenesis. In many bacteria, RpoS, the product of rpoS gene, mediates biosynthesis of a set of secondary metabolites. [Objective] This study aims to elucidate rpoS gene’s function and regulation on two phenazine gene clusters in Pseudomonas aeruginosa PAO1. [Methods] The rpoS gene and its upstream and downstream fragments were cloned from the chromosome of Pseudomonas aeruginosa. With the insertion of gentamycin resistance cassette (aacC1), the mutant PA-SG has been created by homologous recombination. Translational fusion plasmids phz1′-′lacZ(pMEZ1) and phz2′-′lacZ(pMEZ2) were constructed, and then were introduced into the wild type strain PAO1 and the mutant PA-SG, respectively. Activities of beta-galactosidase in them were determined with Miller method. [Results] In KMB or PPM medium, beta-galactosidase activity of phz1′-′lacZ in the mutant PA-SG is much more than that in the wild type strain. However, beta-galactosidase activity of phz2′-′lacZ in the wild type strain is 2-3 folds more than that in the mutant PA-SG. [Conclusion] With these results, it is suggested that regulation mediated by rpoS gene on two phenazine loci is specific and different.