Goranson J, Hovey A K, Frank D W
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
J Bacteriol. 1997 Mar;179(5):1646-54. doi: 10.1128/jb.179.5.1646-1654.1997.
Expression of ExsC, ExsB, and ExsA (the exoenzyme S trans-regulatory locus) of Pseudomonas aeruginosa was analyzed by using complementation, RNase protection, translational fusion, and T7-directed protein expression analyses. T7 expression analyses in E. coli hosts demonstrated that ExsC, ExsA, and a truncated form of ExsD (a partial open reading frame located 3' of ExsA) were translated; however, a product corresponding to ExsB was undetectable. T7-mediated transcription and translation of the antisense strand resulted in production of a 18.5-kDa product, termed ExsB', which overlapped the predicted ExsB product. In complementation experiments, deletion of the region encoding ExsB and most of ExsB' severely reduced exoenzyme S production. Site-specific mutagenesis of the start codons for ExsB and ExsB', however, did not affect exoenzyme S production. RNase protection studies were initiated to examine the hypothesis that RNA encoded within the ExsB/ExsB' region exerted a regulatory effect. RNA encoding ExsB' was not detectable from chromosomal genes or complementation constructs, indicating that ExsB' was not expressed in P. aeruginosa. To determine the pattern of translation, a chloramphenicol acetyltransferase gene (cat) reporter was fused in frame with ExsB and with ExsA in the context of the entire locus or in the absence of the exsB region. These experiments indicated that exsB was not translated but that deletion of the exsB region affected the translation of ExsA-CAT. RNase protection assays further suggested that deletion of exsB resulted in a processing of ExsA mRNA. Our data indicate that the untranslated exsB region of the trans-regulatory locus mRNA mediates either the stability or the translation of exsA. Complementation analysis further suggests that ExsC may play a role in the translation or stability of ExoS.
通过互补分析、核糖核酸酶保护分析、翻译融合分析和T7指导的蛋白质表达分析，对铜绿假单胞菌的ExsC、ExsB和ExsA（外毒素S反式调节基因座）的表达进行了分析。在大肠杆菌宿主中的T7表达分析表明，ExsC、ExsA和ExsD的截短形式（位于ExsA 3'端的部分开放阅读框）能够被翻译；然而，未检测到与ExsB相对应的产物。T7介导的反义链转录和翻译产生了一种18.5 kDa的产物，称为ExsB'，它与预测的ExsB产物重叠。在互补实验中，编码ExsB和大部分ExsB'的区域的缺失严重降低了外毒素S的产生。然而，ExsB和ExsB'起始密码子的位点特异性诱变并不影响外毒素S的产生。启动核糖核酸酶保护研究以检验ExsB/ExsB'区域内编码的RNA发挥调节作用这一假说。从染色体基因或互补构建体中未检测到编码ExsB'的RNA，这表明ExsB'在铜绿假单胞菌中不表达。为了确定翻译模式，在整个基因座的背景下或在没有exsB区域的情况下，将氯霉素乙酰转移酶基因（cat）报告基因与ExsB和ExsA进行读框融合。这些实验表明exsB不被翻译，但exsB区域的缺失影响了ExsA-CAT的翻译。核糖核酸酶保护试验进一步表明exsB的缺失导致ExsA mRNA的加工。我们的数据表明，反式调节基因座mRNA的未翻译exsB区域介导了exsA的稳定性或翻译。互补分析进一步表明，ExsC可能在外毒素S的翻译或稳定性中发挥作用。
