Degradation of desferrioxamines by Azospirillum irakense: Assignment of metabolites by HPLC/electrospray mass spectrometry

Based on a recent finding that an Azospirillum isolate ASP-1 possessing hig h 16S rDNA similarity to Azospirillum irakense was able to degrade desferri oxamine type siderophores (Winkelmann et al. BioMetals 9, 78-83, 1996), var ious members of the genus Azospirillum were analyzed for their ability to d egrade desferrioxamines. While the desferrioxamine-degrading activity was a bsent or scarcely detectable in strains of A. lipoferum, A. brasilense, A. amazonense, degradation activity seemed to be confined to the species A. ir akense (KBC-1, KA3). Also the identity of strain ASP-1 as A. irakense could be confirmed by species-specific oligonucleotide hybridization, InterLINE PCR fingerprinting and carbon source utilization pattern (BIOLOG) analysis. Products of desferrioxamine B degradation were analyzed by analytical HPLC and HPLC/electrospray mass spectrometry. Using whole cells and purified en zyme it was shown that the trihydroxamate desferrioxamine B (561 amu) is sp lit at the N-terminal amide bond yielding a monohydroxamate (MH1, 219 amu) and a dihydroxamate (DH1, 361 amu) metabolite. A second monohydroxamate (MH 2, 319 amu) resulted from DH1 after splitting the acetylhydroxamate bond. M inor amounts of a further dihydroxamate (DH2, 419 amu) originated from spli tting the second amide bond in desferrioxamine B. In addition to desferriox amine B, several other linear and cyclic desferrioxamines and derivatives w ere degraded, whereas desferricoprogen and desferri-ferrichrome were not de graded, indicating high substrate specificity of the desferrioxamine hydrol ase in A. irakense species. A simple microtiter plate assay was developed w hich can be used to phenotypically discriminate and identify species of A. irakense from other Azospirillum species by their characteristic feature of desferrioxamine degradation.