Authors: | Panmanee, W; Charoenlap, N; Atichartpongkul, S; Mahavihakanont, A; Whiteside, MD; Winsor, G; Brinkman, FSL; Mongkolsuk, S; Hassett, DJ |
Year: | 2017 |
Journal: | PLoS One 12 Article Link (DOI) |
Title: | The OxyR-regulated phnW gene encoding 2-aminoethylphosphonate: pyruvate aminotransferase helps protect Pseudomonas aeruginosa from tert-butyl hydroperoxide |
Abstract: | The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by < 2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N-7-CTAT-N-7-ATAG-N-7-CTAT). Of > 100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by beta-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate: pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant. |
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