title: Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa.
abstract: 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, PA103 delta regAB::Gm. toxA transcription was obliterated in strain PA103 delta 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 PA103 delta 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 alpha-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 alpha-helix in the N-terminus as being involved in sensory transduction.
authors.affiliation: Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada.
authors.forename: T L
authors.initials: TL
authors.lastname: Raivio
authors.forename: D
authors.initials: D
authors.lastname: Hoffer
authors.forename: R W
authors.initials: RW
authors.lastname: Prince
authors.forename: M L
authors.initials: ML
authors.lastname: Vasil
authors.forename: D G
authors.initials: DG
authors.lastname: Storey
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D005098:Exotoxins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009876:Operon; D010957:Plasmids; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005098:Exotoxins; D014157:Transcription Factors; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C072722:RegA protein, Bacteria