CONSTRUCTION AND CHARACTERIZATION OF CHROMOSOMAL INSERTIONAL MUTATIONS OF THE PSEUDOMONAS-AERUGINOSA EXOENZYME-S TRANSREGULATORY LOCUS
CONSTRUCTION AND CHARACTERIZATION OF CHROMOSOMAL INSERTIONAL MUTATIONS OF THE PSEUDOMONAS-AERUGINOSA EXOENZYME-S TRANSREGULATORY LOCUS
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DOI：
10.1128/iai.62.2.554-563.1994
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发表时间：
1994-02-01
影响因子：
3.1
通讯作者：
SCHWEIZER, HP
中科院分区：
文献类型：
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作者：
FRANK, DW;NAIR, G;SCHWEIZER, HP
外切酶S是由铜绿假单胞菌产生的ADP-核糖基转移酶。外切酶S的合成依赖于编码三种蛋白产物ExsC、ExsB和ExsA的完整的反式调节位点。为了鉴定ExsC、-B和-A突变体在外切酶S产生中的表型，将编码Ω插入子的具有链霉素抗性的特异性插入突变引入克隆的DNA中，并返回到铜绿假单胞菌PA 103、PAO 1和PAK的染色体。Southern印迹分析用于确认Omega的插入和载体序列的解析。外切酶S的表达在亲本和突变体衍生物通过蛋白质印迹（免疫印迹）分析和ADP-核糖基转移酶活性测量。在菌株PAK和PAO 1中获得了一套完整的突变，但在菌株PA 103中，仅鉴定了exsA编码区中的插入。Southern印迹分析表明，广泛的重复和重排的PA 103染色体的反式调节位点发生时，exsC：：欧米茄或exsB：：欧米茄重组事件进行了尝试。外切酶S抗原在突变株的上清液或裂解物组分中通过Western印迹分析检测不到。ADP核糖基转移酶活性检测到的裂解物中，但不是在突变体衍生物的上清液级分。一般分泌途径似乎在突变株中正常发挥作用，因为在亲本和突变株的上清液中测量了弹性蛋白酶、外毒素A和磷脂酶C。当亲本菌株的细胞外蛋白质谱与来自插入突变菌株的类似样品进行比较时，注意到几个差异。其中一些差异似乎与外切酶S无关。这些数据表明，插入失活的外切酶S的反式调节位点可能会影响其他细胞外蛋白的子集。
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.