Iron transport in two pathogenic Gram-negative bacteria

Campylobacter jejuni and Escherichia coli strain F11 are two Gram-negative pathogens with a versatile armament of iron uptake systems to cope with the fluctuating host nutrient environment. Our current understanding of Gram-negative iron uptake systems focuses heavily on a prototypical scheme involving a TonB-dependent outer membrane receptor and an ABC transporter, with little knowledge on systems that do not fall neatly into this paradigm. The primary focus of this thesis is the characterization of three such atypical iron uptake proteins from C. jejuni (ChaN and P19) and pathogenic E. coli (FetP). C. jejuni ChaN is a 30 kDa, iron-regulated lipoprotein hypothesized to be involved in iron uptake. The crystal structure of ChaN reveals that it can bind two cofacial heme groups in a pocket formed by a ChaN dimer. Each heme iron is coordinated by a single tyrosine from one monomer and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Analytical ultracentrifugation studies demonstrate heme-dependent dimerization in solution. Cell fractionation of C. jejuni shows that ChaN is localized to the outer membrane. Based on these findings, the predicted in vivo role of ChaN in iron uptake is discussed. C. jejuni cFtr1-P19 and E. coli FetMP are homologous iron-regulated systems also proposed to be iron transporters. Through growth studies in both organisms, we show that P19 and FetMP are required for optimal growth under iron-limited conditions. Furthermore, metal binding analysis demonstrates that recombinant P19 and FetP bind both copper and iron. Dimerization of P19 is shown to be metal dependent in vitro and is detected in vivo by cross-linking. Through x-ray crystallography, we have determined the structures of P19 and FetP with various metals bound, thus revealing the locations of the highly conserved copper and iron binding sites. Additionally, the crystal structure of FetP reveals two copper positions in each binding site that is likely functionally important. Through mutagenesis, residues contributing to the alternative copper positions were identified. Together, these studies provide insight into the mechanism of iron transport by the two systems and allow for the development of functional models.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat