Functional analysis of exsC and exsB in regulation of exoenzyme S production by Pseudomonas aeruginosa.

Abstract

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.