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Linker insertion scanning of regA , an activator of exotoxin A production in Pseudomonas aeruginosa
Author(s) -
Raivio Tracy L.,
Hoffer Danita,
Prince Robert W.,
Vasil Michael L.,
Storey Douglas G.
Publication year - 1996
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1996.00102.x
Subject(s) - biology , linker , activator (genetics) , exotoxin , microbiology and biotechnology , pseudomonas aeruginosa , biochemistry , bacteria , genetics , gene , toxin , computer science , operating system
RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa . To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA. In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription. First, we constructed a strain with the regAB locus deleted from the chromosome, PA103Δ regAB ::Gm. toxA transcription was obliterated in strain PA103Δ regAB ::Gm, demonstrating that the regAB locus is essential for ETA production. Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA . These regA linker insertion mutants were sequenced and screened in PA103Δ regAB ::Gm for their effects on regulation of ETA production. Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mutations (pTR53), resulted in a lack of iron‐regulated ETA production and occurred directly upstream from a predicted transmembrane α‐helix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132‐2 and pTR163) occurred within or flanked a region of RegA between aa 87–142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87–142 and implicate the predicted transmembrane α‐helix in the N‐terminus as being involved in sensory transduction.