Frank D W, Nair G, Schweizer H P
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Infect Immun. 1994 Feb;62(2):554-63. doi: 10.1128/iai.62.2.554-563.1994.
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.
外毒素S是由铜绿假单胞菌产生的一种ADP核糖基转移酶。外毒素S的合成依赖于一个完整的反式调节基因座，该基因座编码三种蛋白质产物，即ExsC、ExsB和ExsA。为了确定ExsC、-B和-A突变体在外毒素S产生中的表型，将携带编码链霉素抗性的ω插入序列的特异性插入突变引入克隆DNA中，并导入铜绿假单胞菌PA103、PAO1和PAK的染色体中。通过Southern印迹分析来确认ω的插入以及载体序列的解析。通过蛋白质免疫印迹（免疫印迹）分析和ADP核糖基转移酶活性测定来检测亲本菌株和突变体衍生物中外毒素S的表达。在PAK和PAO1菌株中获得了完整的一组突变，但在PA103菌株中，仅在exsA编码区发现了一个插入突变。Southern印迹分析表明，当尝试进行exsC::ω或exsB::ω重组事件时，PA103染色体反式调节基因座发生了广泛的重复和重排。通过蛋白质免疫印迹分析，在突变体菌株的上清液或裂解物组分中未检测到外毒素S抗原。在突变体衍生物的裂解物中检测到了ADP核糖基转移酶活性，但在上清液组分中未检测到。一般分泌途径在突变体菌株中似乎正常发挥作用，因为在亲本菌株和突变体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。当将亲本菌株的细胞外蛋白质谱与插入突变体菌株的类似样品进行比较时，发现了一些差异。其中一些差异似乎与外毒素S无关。这些数据表明，外毒素S反式调节基因座的插入失活可能会影响其他细胞外蛋白质的一个子集。
