Pip介导铜绿假单胞菌吩嗪基因簇phz2的表达

Positive regulation in expression of the phenazineproducing operon phz2 mediated by Pip in Pseudomonas aeruginosa PAO1

摘要:

摘要:【目的】为了确定铜绿假单胞菌调控因子Pip对两个不同吩嗪合成基因簇(phz1和phz2)的具体调控方式与可能的调控机制。【方法】根据基因比对结果，采用同源重组技术构建Pip 调控因子缺失突变株PA-PG以及克隆pip 基因作互补分析;再以已构建的吩嗪基因簇缺失突变株PA-Z1G和PA-Z2K为受体菌，构建突变株PA-PD-Z1G和PA-PG-Z2K，测定并比较野生株及相关突变株的吩嗪-1-羧酸和绿脓菌素的合成量，推定Pip对两个不同吩嗪合成基因簇的调控方式。【结果】在GA培养基中，突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都比野生型明显减少;互补分析显示，突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都显著提高并恢复到野生株PAO1水平;突变株PA-Z1G的吩嗪-1-羧酸和绿脓菌素合成量因Pip缺失而显著减少;而突变株PA-Z2K的吩嗪-1-羧酸和绿脓菌素合成量在Pip缺失后仍保持不变。【结论】初步推定，转录调控因子Pip对铜绿假单胞菌吩嗪合成代谢的确具有促进作用;Pip通过正向调控吩嗪基因簇phz2的合成功能实现对吩嗪合成代谢的调控。

Abstract:

Abstract:Pseudomonas aeruginosa PAO1，an opportunistic pathogenic bacterium，produces phenazine and its derivatives which play a critical role in pathogen-host interaction during its infection．In a biological control strain P．chlororaphis PCL1391，Pip positively regulates PCN production．［Objective］Our aim is to identify the function and regulation of an ORF of PA0243 (the homolog of Pip) in Pseudomonas aeruginosa PAO1．［Methods］We first cloned the fragment of the pip gene from the chromosomal DNA of P．aeruginosa PAO1 and constructed the pip-defect mutant PA-PG with the insertion of gentamycin resistance cassette (aacC1)．With construction and introduction of pME10P (containing the whole pip gene region)，complementation of the pip was then carried out． With creation of the mutants PA-PD-Z1G and PA-PGZ2K，phenazine-1-carboxylic acid and pyocyanin were measured in GA medium in relative mutants，respectively．［Results］In GA medium，production of phenazine-1-carboxylic acid and pyocyanin in the mutant PA-PG decreased dramatically in comparison with that produced in the wild type strain PAO1．The amounts of phenazine-1-carboxylic acid and pyocyanin，however，were recovered with complementation of the derivative PA-PG bearing pME10P．The production of phenazine-1-carboxylic acid and pyocyanin in mutant PA-PG-Z2K were same to those in parental strain PA-Z2K．Phenazine-1-carboxylic acid and pyocyanin produced by the mutant PA-PD-Z1G were lower than those in the original strain PA-Z1G．［Conclusion］With these results，it is uggested that Pip exerts positively regulation in phenazine biosynthesis by specifically modulating expression of the phz2 operon，not by mediating expression of the phz1 operon in P．aeruginosa PAO1.