铜绿假单胞菌水解弹性蛋白能力相关基因的筛选与鉴定

Mutational analysis of genes involved in elastin hydrolysis in Pseudomonas aeruginosa

【目的】研究铜绿假单胞菌弹性蛋白水解能力相关基因。【方法】应用人工Mu转座技术构建铜绿假单胞菌野生型菌株PA68的转座突变文库,从2000多个突变子中筛选得到4株弹性蛋白水解能力改变的突变子,并通过克隆及测序获得转座子插入位点侧翼的序列。将铜绿假单胞菌弹性蛋白酶结构基因lasB的转录启始区序列整合入载体pDN19lacΩ并将该重组质粒电转化入野生型菌株PA68及4个突变株中,对报告基因在不同菌株中的表达水平进行测定。【结果】发现4个突变株中Mu转座子分别插入lasA、galU、xcpZ和ptsP 4个基因。ptsP基因失活的突变株中,lasB基因的转录水平是野生型菌株的7%,xcpZ和lasA基因的失活使lasB基因的转录水平分别降低为野生株的54%和75%,galU基因的插入失活使lasB基因的转录上升了1倍。【结论】推测ptsP和galU基因很可能直接或间接地调控着弹性蛋白酶的生物合成。

[Objective] Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of acute and chronic infections. When growing in the host, it secrets a lot of virulence factors including elastase. This work aimed to explore the genes involved in hydrolyzing ability of Pseudomonas aeruginosa towards elastin. [Methods] We performed a transposon mutagenesis analysis of P. aeruginosa PA68 to identify candidate genes involved in elastin hydrolysis. We also monitored the promoter activity of the lasB, a gene encoding the elastase, in the mutants and the wild-type by introducing a PlasB-lacZ transcriptional fusion. [Results] Four mutants with altered levels of elastase production were isolated （elastase activity relative to wild-type was shown in parenthesis）： 10 （51%）, 17 （131%）, 27 （8%） and 84 （13%）. Locations of the transposon were mapped to the genome lasA, gaIU, xcpZ and ptsP, respectively. The results of the lasB promoter＇s activity were consistent with the elastase activity data （β-galactosidase activity relative to wild-type was shown in parenthesis）： 10（75%）, 17（201%）, 27（54%） and 84（7%）. [Conclusion] Taken together, the data build up a connection of these four genes with elastase production. This is the first report that gene galU and ptsP may be employed in the regulation of the biosynthesis of elastase.