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Title: The responses of Campylobacter jejuni to oxygen and toxic haem ligands
Author: Smith, Holly K.
ISNI:       0000 0004 2724 9412
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2010
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C. jejuni is a microaerophilic organism, the respiration and growth of which is sensitive to NO, a classical respiratory inhibitor. In response to such stress, expression is induced from a small regulon under the control of a NO-sensitive regulator, NssR. Two haemoglobins are expressed: Cgb is a single domain globin that takes part in the NO detoxification reaction, and the second globin, Ctb, is a truncated protein, the role of which is unclear. It does not demonstrate NO-detoxifying properties but has a high affinity for oxygen, suggesting that it may be involved in oxygen transfer processes. It has also been hypothesised to take part in detoxification of cyanide and part of the work described herein has examined the role of Ctb in reactions with NO and cyanide. This also led to the study of the response of C. jejuni to another classical inhibitor of respiration, CO, which was delivered to cells using a CO-releasing molecule, CORM-3. Microarray and RT-PCR analysis of the ctb mutant cultured under high and low aeration conditions and stressed with NO was undertaken to assess whether the ctb mutation elicits global transcriptional changes when compared to a wild type strain cultured under the same conditions. The results showed no major changes in the transcriptional profile; the most up-regulated genes in the ctb mutant were involved in fumarate metabolism, perhaps indicating a switch to this molecule as terminal electron acceptor of the electron transport chain. The ctb mutation had no effect on the expression of Cgb or other members of the NssR regulon and suggests that Ctb is not intimately involved in the NO detoxification reaction. The phenotype of the ctb mutant was not hypersensitive to cyanide, and expression of neither the gene or protein was significantly induced by cyanide, indicating that the globin is not involved in cyanide detoxification. In comparison to NO and cyanide, CORM-3 had no effect on growth; however in vitro, CO was readily released from the ruthenium molecule and the medium used to culture C. jejuni did not significantly affect the kinetics of this release. The CO released entered cells and bound to cytochromes, and non-growing cells were significantly inhibited by CORM-3, more so than by equivalent concentrations of cyanide or NO. It has been demonstrated for the first time that reactive oxygen species are produced as a consequence of inhibition of the bacterial electron transport chain by CORM-3. A surprising phenotype of the cioAB mutant has been characterised, whereby respiration is significantly faster in the presence of CORM-3 than in the untreated cioAB control, perhaps indicating an uncoupling effect of the molecule. Finally, a preliminary investigation of the enzymatic turnover of Cgb found that formate-stimulated cell-free extracts of C. jejuni reduced the ferric haem of the purified protein gradually over 40 min, thus indicating that endogenous electron donors may contribute towards re-reduction of the haemoglobin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available