LINKER INSERTION SCANNING OF REGA, AN ACTIVATOR OF EXOTOXIN-A PRODUCTION IN PSEUDOMONAS-AERUGINOSA

RegA is a transcriptional activator that controls exotoxin A (ETA) pro duction in Pseudomonas aeruginosa. To date, functional assays performe d with the purified protein have not clearly defined the molecular mec hanism of action of RegA. In this study, we sought to identify importa nt coding regions of regA by analysing the sequences around linker ins ertion mutations in regA that affected toxA transcription. First, we c onstructed a strain with the regAB locus deleted from the chromosome, PA103 Delta regAB::Gm. toxA transcription was obliterated in strain PA 103 Delta regAB::Gm, demonstrating that the regAB locus is essential f or ETA production. Next, we constructed a series of 6 bp linker insert ion mutations distributed throughout regA. These regA linker insertion mutants were sequenced and screened in PA103 Delta regAB::Gm for thei r effects on regulation of ETA production. Six linker insertion mutati ons occurring between amino acids (aa) 53 and 163 of RegA were isolate d that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mu tations (pTR53), resulted in a lack of iron-regulated ETA production a nd occurred directly upstream from a predicted transmembrane alpha-hel ix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132-2 a nd pTR163) occurred within or flanked a region of RegA between aa 87-1 42 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unident ified transcriptional activation domain in RegA between aa 87-142 and implicate the predicted transmembrane alpha-helix in the N-terminus as being involved in sensory transduction.