Molecular characterization of proteins included in Pseudomonas Aeruginosa iron uptake system

Abstract

Pyoverdine I is the major siderophore produced by Pseudomonas aeruginosa PAO1 to import iron. Its biosynthesis requires the coordinated action of cytoplasmic, periplasmic, and membrane proteins. The individual enzymatic activities of these proteins are well known. However, their subcellular distribution in particular areas of the cytoplasm, periplasm, or within the membrane has never been investigated. For better understanding of the pyoverdine I biosynthetic pathway, chromosomal replacement was used to generate P. aeruginosa strains producing fluorescent fusions with PvdA, one of the initial enzymes in the biosynthetic pathway of pyoverdine I in the cytoplasm. Cellular fractionation indicated that a substantial amount of PvdA-YFP was located in the membrane fraction and this mutant can be a useful tool monitoring PvdA expression. Also, the same technique was used to generate double mutated strains in order to detect protein-protein interaction using FRET.