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

The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus

ABSTRACTSmall RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment.IMPORTANCEThe emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism