铜绿假单胞菌随机启动子库的构建及受铁调控基因的筛选和鉴定

Construction of a Pseudomonas aeruginosa random promoter library and identification of novel iron-regulated genes by high-throughput library screening

摘要:

【目的】针对铁这种几乎所有微生物都必需的，对病原菌尤为重要的营养元素，在全基因组水平上检测铜绿假单胞菌的铁感应基因。【方法】采用含LuxCDABE报道子的质粒pMS402为载体，构建了铜绿假单胞菌的随机启动子库，建立了特定条件下研究铜绿假单胞菌全基因组基因表达变化的高通量平台。【结果】验证了2个已知的受铁调控的基因，并发现了尚未报道的二氢叶酸还原酶，磷酸葡萄糖脱水酶和柠檬酸铁转运蛋白受铁调控的现象，发现与铁相关的四个功能未知的基因和3个假定蛋白的基因。【结论】研究结果对于阐明铁在铜绿假单胞菌中的调控作用，揭示铁对细菌生理及致病性的作用机制提供了理论基础。随机启动子库的构建也为细菌基因组水平研究基因表达提供了一个有用的工具。

Abstract:

[Objective] Iron is an essential element for bacterial survival and pathogenesis. Using a random promoter library we screened for iron-responsive genes in P. aeruginosa genome. [Method] Plasmid pMS402 containing a promoterless luminescence reporter, LuxCDABE, was used as the vector to construct a P. aeruginosa random promoter library with a size of about 5700 clones. It enabled real-time, in vivo high-throughput gene expression profiling at genomic scale. [Results] Two previously reported iron-regulated genes were identified, and ten new iron- regulated genes were uncovered. The genes encoding dihydrolipoamide acetyltransferase, phosphogluconate dehydratase and Fe (Ⅲ) dicitrate transporter were found to be iron-regulated together with four function-unknown genes and three putative protein encoding genes. [Conclusion] These results provide a basis for elucidating the complex iron regulation network in P. aeruginosa and help our understanding of the roles of iron in bacterial physiology and pathogenesis. The random promoter library system also offers a useful tool in bacterial genomic studies.