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首页> 外文期刊>Infection and immunity >Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus.
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Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus.

机译：铜绿假单胞菌外切酶S反式调控基因座的染色体插入突变的构建和表征。

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摘要

Exoenzyme S is an ADP-ribosyltransferase produced by Pseudomonas aeruginosa. Synthesis of exoenzyme S depends on an intact trans-regulatory locus encoding three protein products, ExsC, ExsB, and ExsA. To identify the phenotype of ExsC, -B, and -A mutants in exoenzyme S production, specific insertional mutations with the streptomycin resistance-encoding omega interposon were introduced into cloned DNA and returned to the chromosomes of P. aeruginosa PA103, PAO1, and PAK. Southern blot analysis was used to confirm insertion of omega and resolution of vector sequences. Exoenzyme S expression was measured in parental and mutant derivatives by Western blot (immunoblot) analysis and ADP-ribosyltransferase activity measurement. A complete set of mutations were obtained in strains PAK and PAO1, but in strain PA103, only an insertion in the exsA coding region was identified. Southern blot analysis demonstrated that extensive duplication and rearrangement of the PA103 chromosomal trans-regulatory locus occurred when exsC::omega or exsB::omega recombination events were attempted. Exoenzyme S antigen was not detectable in the supernatant or lysate fractions of mutant strains by Western blot analysis. ADP-ribosyltransferase activity was detected in the lysate but not in the supernatant fractions of mutant derivatives. The general secretion pathway appeared to function normally in mutant strains, as elastase, exotoxin A, and phospholipase C were measured in the supernatants of parental and mutant strains. Several differences were noted when the extracellular protein profiles of parental strains were compared with similar samples from the insertional mutant strains. Some of these differences appeared to be unrelated to exoenzyme S. These data suggest that insertional inactivation of the exoenzyme S trans-regulatory locus may affect a subset of other extracellular proteins.
机译：外切酶S是由铜绿假单胞菌产生的ADP-核糖基转移酶。外酶S的合成取决于完整的反式调节基因座，该基因座编码三种蛋白质产物，ExsC，ExsB和ExsA。为了鉴定外酶S生产中的ExsC，-B和-A突变体的表型，将具有链霉素抗性的欧米茄插入子的特定插入突变引入克隆的DNA中，并返回到铜绿假单胞菌PA103，PAO1和PAK的染色体。 Southern印迹分析用于确认Ω的插入和载体序列的分离。通过Western印迹（免疫印迹）分析和ADP-核糖基转移酶活性测量来测量亲本和突变体衍生物中外酶S的表达。在菌株PAK和PAO1中获得了完整的突变集，但在菌株PA103中，仅鉴定到exsA编码区中的插入。 Southern印迹分析表明，当尝试进行exsC :: omega或exsB :: omega重组事件时，发生了PA103染色体反式调控位点的大量复制和重排。通过蛋白质印迹分析在突变菌株的上清液或裂解物级分中未检测到外切酶S抗原。在溶解产物中检测到ADP-核糖基转移酶活性，但在突变衍生物的上清液馏分中未检测到。一般的分泌途径似乎在突变菌株中正常发挥作用，因为在亲本和突变菌株的上清液中检测到了弹性蛋白酶，外毒素A和磷脂酶C。将亲本菌株的胞外蛋白谱与插入突变株的相似样品进行比较时，发现了一些差异。这些差异中的一些似乎与外切酶S无关。这些数据表明外切酶S反式调控位点的插入失活可能影响其他细胞外蛋白的一部分。

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