Detection and differentiation of microbial siderophores by isoelectric focusing and chrome azurol S overlay

Abstract

Siderophores are microbial, low molecular weight iron-chelating compounds. Fluorescent Pseudomonads produce different, strain-specific fluorescent siderophores (pyoverdines) as well as non-fluorescent siderophores in response to low iron conditions. We present an isoelectric focusing method applicable to unpurified as well as to purified pyoverdine samples where the fluorescent siderophores are visualized under UV illumination. Siderophores from different Pseudomonas sp., amongst which are P. aeruginosa, P. fluorescens and P. putida, including egg yolk, rhizospheric and clinical isolates as well as some derived Tn5 mutants were separated by this technique. Different patterns could be observed for strains known to produce different siderophores. The application of the chrome azurol S assay as a gel overlay further allows immediate detection of non-fluorescent siderophores or possibly degradation products with residual siderophore activity. The method was also applied to other microbial siderophores such as deferrioxamine B.