Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1:: A tool for identifying differentially regulated genes
Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1:: A tool for identifying differentially regulated genes
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DOI：
10.1101/gr.3513905
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发表时间：
2005-04-01
期刊：
影响因子：
7
通讯作者：
Hancock, REW
中科院分区：
文献类型：
--
作者：
Lewenza, S;Falsafi, RK;Hancock, REW
铜绿假单胞菌是医院（医院来源）感染的主要原因，是慢性囊性纤维化肺部感染的主要病原体，并且由于其高内在抗生素耐药性而难以治疗。铜绿假单胞菌PAO 1基因组序列的完成为全基因组研究提供了机会，以增加我们对这一重要病原体的发病机制和生物学的理解。在这份报告中，我们描述了一个mini-Tn 5-luxCDABE突变体库的建设和高通量反向PCR方法扩增DNA侧翼的插入位点测序和插入位点定位。除了在非必需基因中产生极性敲除突变之外，转座子中存在的无启动子luxCDABE报告基因还充当失活基因的基因表达的实时报告基因。共定位了2519个转座子插入位点，其中77%为非冗余插入。在ORF内的插入中，类似于55%的总的和独特的插入位点是转录luxCDABE融合。观察到在预测的复制末端周围的基因组区域中的低插入位点密度的偏倚。为了证明染色体lux融合的实用性，我们进行了广泛的监管屏幕，以确定差异调节下镁或磷酸盐限制的基因。这种方法导致发现了这些环境适应所需的许多已知和新型基因，包括参与阳离子抗菌肽抗性的基因。这个两用突变库允许功能和调控研究，并将作为研究社区的资源，以进一步了解铜绿假单胞菌生物学。
Pseudomonas aeruginosa is a major cause of nosocomial (hospital-derived)infections, is the predominant pathogen in chronic cystic fibrosis lung infections, and remains difficult to treat due to its high intrinsic antibiotic resistance. The completion of the P. aeruginosa PAO1 genome sequence provides the opportunity for genome-wide studies to increase our understanding of the pathogenesis and biology of this important pathogen. In this report, we describe the construction of a mini-Tn5-luxCDABE mutant library and a high-throughput inverse PCR method to amplify DNA flanking the site of insertion for sequencing and insertion site mapping. In addition to producing polar knockout mutations in nonessential genes, the promoterless luxCDABE reporter present in the transposon serves as a real-time reporter of gene expression for the inactivated gene. A total of 2519 transposon insertion sites were mapped, 77% of which were nonredundant insertions. Of the insertions within an ORF, similar to 55% of total and unique insertion sites were transcriptional luxCDABE fusions. A bias toward low insertion-site density in the genome region that surrounds the predicted terminus of replication was observed. To demonstrate the utility of chromosomal lux fusions, we performed extensive regulatory screens to identify genes that were differentially regulated under magnesium or phosphate limitation. This approach led to the discovery of many known and novel genes necessary for these environmental adaptations, including genes involved in resistance to cationic antimicrobial peptides. This dual-purpose mutant library allows for functional and regulation studies and will serve as a resource for the research community to further our understanding of P. aeruginosa biology.