The acquisition of iron is crucial for bacterial pathogenesis. In this thesis I
describe the development and validation of a biosensor for heme in bacteria,
an important host source of iron for human pathogens. I demonstrate that the
co-expression of a phytochrome-based fluorophore (PBF), which utilises the
heme-derived metabolite biliverdin IXα as a chromophore, with a heme
oxygenase (HO) in Gram-negative bacteria results in heme-dependent near
infra-red fluorescence. Using this PBF+HO reporter, I probe heme metabolism
in the opportunistic human pathogen Pseudomonas aeruginosa and for the
first time provide direct evidence linking the previously putative heme receptor
HxuC to heme metabolism. Further, coupling of the heme biosensor to
transposon mutagenesis and Fluorescence-Activated Cell Sorting was used
to screen a P. aeruginosa transposon library leading to the identification of
several putative novel components of heme metabolism. Finally, I
demonstrate the potential of our fluorescent biosensor to be used to study
heme metabolism and host-pathogen interactions in vivo using a live adult
zebrafish model of infection. The PBF+HO heme biosensor allows the highthroughput,
non-invasive study of bacterial heme metabolism in real-time and
proves a useful tool for gaining further insight into iron-heme metabolism in
bacterial pathogens.