Klebba, Philip E2019-06-032019-06-032009https://hdl.handle.net/11244/320212Iron is an essential metal element for the metabolism pathway of most microorganisms. For pathogens, iron is also an important factor for virulence. But in the host body, the availability of free iron is very low. Iron mainly associates with heme in hemoglobin or is tightly bound to iron-associated proteins like transferrin, lactoferrin and ferritin. Both Gram-negative and Gram-positive pathogens have iron transporters in their cell envelopes to acquire iron from the environment, and one of the target iron compound for these transport systems is heme in the red blood cells.Listeria monocytogenes is a Gram-positive pathogen, which can infect human and animals and causes listeriosis. L. monocytogenes can utilize multiple iron sources including siderophores, transferrin, lactoferrin, heme and hemoglobin. In the genome of L. monocytogenes EGD-e, the hup operon encodes an ABC transporter for the uptake of heme. Site-directed deletion of hupC and hupG impaired the uptake of heme and hemoglobin in L. monocytogenes EGD-e. The function of these two genes can be complemented via a Listeria phage integration vector.[59Fe]-heme was used to measure the thermodynamic and kinetic parameters of heme transport by L. monocytohenes. The [59Fe]-heme binding experiments showed that EGD-e wild type bound heme with an affinity about 4.7nM. The deletion of genes in hup operon did not change the binding affinity very much, and also the deletions did not completely eliminate the uptake of heme in the [59Fe]-heme transport experiments. This data indicates there are other transporters available in L. monocytogenes to transport heme other than the Hup transporter.Binding experiments revealed that the sortase B-dependent surface protein (formerly called "SvpA") encoded by lmo2185, has a role in heme binding. Deletion of these proteins resulted in the increase of KD and decrease of capacity. Deletion of lmo2185 also reduced the uptake of hemoglobin in nutrition tests.The second part of this study is about the function of TonB in E. coli. TonB is an inner-membrane-associated protein in Gram-negative species. In the outer membrane of Gram-negative bacteria, there are ligand-gated receptors for the active transport of nutrients, such as siderophores and vitamins. These receptors are TonB-dependent, probably because TonB facilities the transport by transducing the proton motive force energy from the inner membrane to the outer membrane.The C-terminus of TonB contains LysM motifs, whose main function is binding to bacterial peptidoglycan. The TonB C-terminus has affinity for peptidoglycan. Site-directed mutagenesis and fluorescence quenching methods were used in this study to investigate the binding. Peptidoglycan quenched the fluorescence of FM labeled cysteine mutants of a MalE-TonB69C fusion protein. After changing two residues, E205 and D189, which may be part of the binding site, to alanine, the affinity of the C-terminus of TonB for peptidoglycan still remains.203 pagesapplication.pdfGram-positive bacteriaIron--MetabolismListeria monocytogenesEscherichia coliBacterial proteinsBiological transport, Activetext