铜绿假单胞菌全局调控因子RsmA的缺失影响2个吩嗪基因簇的表达

Differential expression of two phenazine-producing loci mediated by deficiency of the global regulator RsmA in Psedomonas aeruginosa PAO1

摘要:

摘要:【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。

Abstract:

Abstract:In many Pseudomonas，RsmA mediates the production of a set of secondary metabolites or virulence factors．［Objective］Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1．［Methods］ We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA． With the insertion of gentamycin resistance cassette ( aacC1)，the deletion mutant PA-RG was created and verified with PCR． To complement and overexpress the rsmA gene，pME10R and pME32R were also constructed． By constructing the translational fusion plasmids phz1'-'lacZ pMEZ1and phz2'-'lacZ pMEZ2，we introduced them into the wild type strain PAO1 and the mutant PA-RG，respectively． Activities of beta-galactosidase were determined with Miller method． ［Results］In glycerol-alanine medium，overexpression of the rsmA gene results in dramatical decrease of pyocyanin production in PA-RG and PAO1 strain． In addition，beta-galactosidase activity of phz1'-'lacZ in the mutant PA-RG was much higher than that in the wild type strain． However，beta-galactosidase activity of phz2'-'lacZ in the wild type strain was 2fold more than that in the mutant PA-RG． ［Conclusion］ The regulation mediated by RsmA on two phenazine loci is specific and differential．