The mammalian host responds to E. coli by producing an arsenal of oxidative and nitrosative stresses, including peroxynitrite, which is formed by the near diffusion- limited reaction of nitric oxide with superoxide. Peroxynitrite, the main subject of this thesis, is regarded as a highly toxic species due to its multiple targets that include lipids, DNA and proteins; however information regarding specific bacterial targets is lacking. Toxicity of peroxynitrite was found to be dependent upon both O2 availability and numbers of target cells. A sublethal concentration of peroxynitrite was selected to stress E. coli and uncover the transcriptional response under tightly controlled chemostat conditions. Up-regulation of cysteine and arginine biosynthetic pathways was observed in addition to the oxidative stress response and systems involved in iron-sul fur cluster assembly/repair, glutathione import and phosphate transport. Expression of karG, encoding a catalase-peroxidase, was particularly elevated. Consequently. KatG was characterised to determine whether it exhibited the capacity to break down peroxynitrite, an activity attributed to the same enzyme from Mycobacterium tuberculosis. However. KatG was an inefficient peroxynitritase. The S-nitrosoproteome of E. coli generated in response to GSNO was examined. Reductive chemiluminescence was used to quantify S-nitrosothiol formation within cells and to establish the contribution of selected systems in protecting the cell from this post-translational modification. An I7srR mutant was more susceptible to GSNO- mediated S-nitrosation than wild type cells, whereas separate mutations in hmp and ytfl: had negligible effects. A mutation in I7IjA however caused a reduction in 5- nitrosothiol production relative to wild type cells. suggesting that cytochrome c nitrite reductase promotes S-nitrosation. SNO capture (SNOCAP). a proteomic method applied to identify S-nitrosated proteins, was also applied to E. coli cells stressed with GSNO. Targets were generally categorised into those involved in the oxidative stress response, protein biosynthesis and intermediary metabolism. The global impact of NO on metalloproteins in E. coli was investigated by electron paramagnetic resonance. Spectral features corresponding to haern-nitrosyls and iron- sulfur nitrosyls were uncovered. Additionally. this technique was applied to cells stressed with peroxynitrite and revealed a diminished signal corresponding to iron- sulfur centres with a concurrent increase in signal attributable to free iron. This thesis represents the first detailed integrated study of ONOO' toxicity in E. coli.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:575076 |
| Date | January 2011 |
| Creators | Bowman, Lesley A. H. |
| Publisher | University of Sheffield |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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