A novel bioassay for the detection of siderophores containing keto-hydroxy bidentate ligands

Abstract A convenient and sensitive pour-plate Petri dish bioassay for the detection of siderophores containing monoprotic keto-hydroxy bidentate ligands (KHBL) has been developed. The bioassay is based on the fact that bacteria of the Proteus-Providencia-Morganella group (Proteeae) utilize various ferric α-hydroxy- or α-ketocarboxylate complexes very efficiently. While P. vulgaris and P. rettgeri were able to utilize virtually all iron complexes supplied, Morganella morganii SBK3 was unable to utilize trihydroxamate type siderophores and was therefore selected as an indicator strain for iron complexes containing keto-hydroxy bidentate ligands (KHBL-siderophores). Filter paper disks containing the ferric complexes of siderophores were tested on tryptone or Luria broth agar, seeded with the indicator strains and supplemented with the ferrous iron chelator 2,2-dipyridyl (300 μM) to reduce the bioavailable iron. In the presence of siderophores, growth inhibition was reversed to provide a zone of growth stimulation. Ferric complexes of α-hydroxycarboxylates, α-ketocarboxylates, salicylic acid, tropolonederivatives, α-hydroxypyridinones, cepabactin, citrate, rhizoferrin and even epihydroxymugineic acid showed significant growth stimulation. From the results with the trihydroxamate-non-utilizing strain, M. morganii SBK3 , it may be inferred that the Proteeae prossess an iron transport system which recognizes ferric α-hydroxycarboxylates, α-ketocarboxylates as well as aromatic and heteroaromatic keto-hydroxy compounds, collectively named keto-hydroxy bidentate ligands. The bioassay is especially suited for detection of new siderophores from low-iron cultures of fungi and bacteria.