Iron Transport by Pyoverdine Into Pseudomonas Aeruginosa

Royt, Paulette

Abstract

Pseudomonas aeruginosa, an opportunistic pathogen, produces many virulence factors. The production of some of these factors in influenced by the concentration of available iron. Iron also regulates the production of exogenous siderophores, chelators that deliver iron to the cell. Thus the study of iron transport by siderophore is important research because it impacts upon the problem of production of these virulence factors. The primary objective of this research is to delineate the steps of iron transport by the siderophore, pyoverdine, into the gram-negative bacterium, Pseudomonas aeruginosa. This will be accomplished by examining the binding of 55Fe pyoverdine to isolate inner and outer membranes of late logarithmic phase iron-fed and iron- starved cells. In addition, 57Fe, the Mossbauer isotope, will be complexed to pyoverdine and incubated with inner and outer membranes of late logarithmic phase iron-fed and iron-starved cells and the Mossbauer spectra determined. Together these methodologies will show 1) whether ferripyoverdine binds to both membranes, 2) the quantity that binds, and 3) whether any of the iron is transferred to other membrane bound molecules either as Fe3+ or reduce as Fe2+. Complementing these binding studies, transport studies using both methodologies will also be performed. Experiments using 55Fepyoverdine will determine the optimum conditions of iron uptake by whole cells. Then whole cells will be incubated with 57 Fepyoverdine, and at designated time intervals, aliquots withdrawn, the inner and outer membranes and cytoplasm collected. The Mossbauer spectra of these samples will reveal: (1) the oxidation state of the iron on both membranes and in the cytoplasm (2) whether or not the iron has remained complexed to the siderophore and (3) if ferripyoverdine entered the cytoplasm. Thus, using these two methodologies the steps of iron transport by pyoverdine across the cell envelope will be defined.