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-N7-CTAT-N7-ATAG-N7-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 ß-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.
Highlights
Pseudomonas aeruginosa (PA) is an important human pathogen that is currently ranked fifth by the World Health Organization in overall infections [1]
We were interested in the promoter regions of potentially OxyR-regulated genes that were not predicted to be involved in the classical response to oxidative stress (such as genes encoding catalases A [5] and B [6], alkyl hydroperoxide reductases (Ahps, [6]), organic hydroperoxide reductase (Ohr), overlapping regulon genes (Ssp), and membrane proteins involved is the Ssp response
In contrast to a recent study by Wei et al [19] who identified genes under OxyR control when organisms were exposed to H2O2, we found that only 8/40 of the genes matched from bioinformatic studies in the aforementioned study
Summary
Pseudomonas aeruginosa (PA) is an important human pathogen that is currently ranked fifth by the World Health Organization in overall infections [1]. Burn and cancer chemotherapy patients, chronic alcoholics, the immunocompromised (e.g., HIV infection) and patients suffering from cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lung disease are particular prone to highly problematic or fatal infections by this organism [2,3]. The response to such infections, especially in the airway, is mediated by macrophages and subsequently neutrophils, both of which are capable of triggering an oxygen-dependent antimicrobial respiratory burst. Ohr is inducible by organic hydroperoxide but is not part of the OxyR regulon [7]
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