Tl. Raivio et al., LINKER INSERTION SCANNING OF REGA, AN ACTIVATOR OF EXOTOXIN-A PRODUCTION IN PSEUDOMONAS-AERUGINOSA, Molecular microbiology, 22(2), 1996, pp. 239-254
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.