Functional characterisation of the multicopper oxidase from Escherichia coli and Salmonella typhimurium

The multicopper oxidase (MCO) of Escherichia coli has been shown to confer copper tolerance under aerobic conditions by oxidation of cuprous copper to the less toxic cupric species. This MCO has also been shown to possess polyphenol oxidase activity and ferroxidase activity (Fe2+ --> Fe3+). Multicopper oxidases with the later activity have been shown to play an important role in ferrous iron uptake in Pseudomonas aeruginosa (Mco), Saccharomyces cerevisiae (Fet3) and humans (hCp), where mutants exhibit diminished growth under iron limited conditions. Growth of an Escherichia coli cueO mutant was not diminished on metal depleted minimal medium supplemented with 2µM ferrous iron as a sole iron source. However, copper sensitivity under these iron limited conditions was significantly increased in the cueO mutant. Copper sensitivity could be suppressed by the reactive oxygen species quencher Tiron, indicating that copper toxicity was due to oxidative stress. Inductively coupled plasma mass spectroscopy was used to assess transition metal accumulation in wild type and cueO cultures. The cueO mutant was found to accumulate almost 1000 fold more copper than wild type E. coli under iron limited conditions. Copper accumulation could be suppressed by the addition of Zn2+, Mn2+, or Fe suggesting accumulation of Cu in the cueO mutant may be through nonspecific divalent metal ion transporters such as ZupT or MntH. Transcriptional profiling of E. coli and an isogenic cueO mutant was undertaken on metal depleted minimal media. No changes in metal ion transporters were observed under these conditions indicating that changes in metal ion uptake do not occur at the transcriptional level in response to metal ion depletion. However, significant changes in expression of the outermembrane protein Ag43 and genes associated with motility were observed. Ag43 confers auotaggregation and clumping of cells in E. coli and resistance to hydrogen peroxide. Real time PCR measurement of sodC in E. coli delta cueO showed a 3.5 fold increase in transcription. Collectively, these results indicate that deletion of cueO leads to oxidative stress even in the absence of significant copper concentrations, plausibly by Fe2+ or Cu+ catalysed oxidative stress. Resistance to metal catalysed oxidative stress is essential for survival during infection. Deletion of cueO in the closely related enteric pathogen Salmonella typhimurium was found to significantly attenuate colonisation of Peyers patches, mesenteric lymph nodes, spleen and liver. Virulence could be partially restored by expressing cueO in trans. Recombinant CueO from S. typhimurium was expressed in E. coli as a fusion to StrepTagII and purified using streptavidin resin. Using an oxygen electrode cuprous oxidase and ferrooxidase activity was demonstrated for recombinant CueO. Km values for both Cu+ and Fe2+ oxidation were found to be significantly lower than those reported for CueO from E. coli. Measurements of kcat/Km indicated that CueO from S. typhimurium was a significantly better ferroxidase and cuprous oxidase than CueO from E. coli. Characteriation of an E. coli cueO mutant with regard to metal ion uptake, and transcriptional and phenotypic changes that occur in response to deletion of cueO are presented in this thesis. Further, characteristion of CueO as a novel virulence determinant in the closely related enteric pathogen S. typhimurium is presented