Aerobic respiration generates unavoidable reactive oxygen species (ROS, detrimental by-products that damage biological molecules. The accumulation of ROS beyond the cellular capacity for detoxification results in oxidative stress. Bacteria have evolved transcription factors that sense ROS and transduce signals into differential gene expression. This study focuses on oxidative stress responses in Escherichia coli, an enteric bacterium and Pseudomonas aeruginosa, an important opportunistic human pathogen. E. coli activates the response to paraquat-induced superoxide stress through the SoxRS system. This study showed that transcription of pgi, a SoxRS regulated gene, and the corresponding phosphoglucose isomerase activity were induced in a SoxRS-dependent manner. A pgi null mutant was hypersensitive to paraquat when grown in rich medium with glucose. We provide evidence that pgi plays a role in maintaining NADPH for cells under oxidative stress by providing glucose-6-phosphate as a substrate for glucose-6-phosphate dehydrogenase. The opportunistic human pathogen P. aeruginosa has evolved under self-generated redox-cycling compounds, in addition to physiological and host-derived sources of oxidative stress. In an effort to identify novel redox sensor and effector proteins, we characterized the pqrCBAR genes. The mutation of the pqrCBAR genes resulted in hypersensitivity to oxidants, and pqrCBA form an operon under negative transcriptional regulation by PqrR. PqrR, a member of MarR family transcription regulators, has 4 conserved cysteines. Purified PqrR was found to exist as a dimer in solution, contain iron, and display characteristic UV spectra common to proteins with iron-sulfur clusters. DNAseI protection assay showed that PqrR bound in vitro at two distinct sites of the pqrA-pqrR intergenic region. PqrR binding activity was reduced by exposure to air, and reconstituted by DTT treatment. The site-directed mutagenesis of PqrR revealed that 3 conserved cysteine residues were essential for redox-sensing activity. To identify other novel antioxidant mechanisms in P. aeruginosa, we screened 15,003 of random transposon insertion mutants, and confirmed 15 mutants for paraquat sensitive phenotype.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-5093 |
| Date | 01 January 2008 |
| Creators | Rungrassamee, Wanilada |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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