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Title: The globins of Campylobacter jejuni : a functional study in a heterologous host
Author: Tinajero Trejo, Mariana
ISNI:       0000 0004 2744 7398
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Campylobacter jejuni, a human pathogen, is exposed to NO and reactive nitrogen species (RNS) derived from the host during colonisation of the gut. As a response, C. jejuni expresses a single-domain globin (Cgb) and a truncated globin (Ctb) under control of the NssR regulator. Function of Cgb as an NO and RNS detoxification system and the involvement of Ctb in O2 chemistry have been deduced from gene mutagenesis in vivo and spectroscopic and kinetic characterisation in vitro. However, confirmation of the Cgb activity and further exploration of the Ctb function(s) are restricted in Campylobacter by difficulties in complementation by transformation of plasmids, and the lack of the reductase domain that, in the flavohaemoglobins, reconstitutes the haem ferrous state required for ligand-binding activity. This limits additional insight into the molecular mechanisms of these globins. In the present work, a functional study in the heterologous host E. coli was performed by cloning the cgb and ctb genes under control of arabinose-inducible promoters and expressing the globins in NO-sensitive strains. In this way, it was found that Cgb, but not Ctb, complements the E. coli NO and RNS resistance phenotype of an E. coli hmp mutant aerobically, confirming the function of Cgb as a NO and RNS resistance system. Interestingly, both Cgb- and Ctb-expressing cells consumed NO in an O2-independent manner. However, Cgb failed to protect E. coli anaerobically. Spectroscopic changes of the Cgb and Ctb haems in cellular milieus were evaluated, showing that the haems are reduced in E. coli and C. jejuni soluble extracts even after oxidation by NO. Nevertheless, exploration of candidates for the Cgb electron donor revealed only a minor role for the E. coli flavorubredoxin reductase (NorW), and Cgb reduction was independent of the respiratory chain of E. coli and the lactate dehydrogenase (Cj1585) from C. jejuni, arguing in favour of a non-specific reductase system. Additionally, preliminary tests showed an increased NO evolution from Ctb-expressing E. coli presented with nitrite, suggesting that the globin functions as a NO reductase; NO production by purified Ctb from nitrite supports this hypothesis. Finally, the suitability of Ctb-expressing E. coli cells as a tool to measure CO-release from CO-releasing molecules (CO-RMs) is also presented. Unravelling the molecular mechanisms of these globins constitutes a key step in the understanding of the NO resistance ability of C. jejuni.
Supervisor: Poole, Robert K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available