On-line capillary liquid chromatography-electrospray mass spectrometry and tandem mass spectrometry, in combination with precursor feeding and acetylation studies, were used to identify and characterize the siderophores of the fire blight pathogen Erwinia amylovoru. Proferrioxamines D, , El G,, G , , X, and X, were found, with D, and E being the major siderophores for all strains of E. amylovoru studied. These proferrioxamines have previously been observed with other microbial species. In addition, eleven additional hydroxamate compounds were found, nine of which occur naturally and two which were induced by directed fermentation. The novel natural proferrioxamines included three cyclic tetrahydroxamates, designated T,-,. The most abundant of these, T,, consists of four 5succinylamino-1-hydroxyaminopentane residues. In T, and T,, one of these residues is substituted by 4 succinylamino-1-hydroxyaminobutane and 3-succinylamino-l-hydroxyaminopropane, respectively. E. amylovora also incorporated Isuccinylamino-1-hydroxyaminopropane into a novel trihydroxamate, which has been designated X, . The novel finding that proferrioxamines may partly be comprised of 1,fdiaminopropane residues is noteworthy because previous feeding studies with Streptomyces olivaceus Tii 2718 suggested that 1,fdiaminopropane is biosynthetically not tolerated. A truncated proferrioxamine G , was also identified, which is referred to as G,,, and, on feeding of diaminobutane, tetrahydroxamates T, and T, , which are derived from T, by substitution of two and three diaminopentane with diaminobutane residues. The structures of five other hydroxamates, with molecular masses of 5-30 (X,), 544 (X9), 622 (T.,), 719 (T,) and 733 (T6), are not yet known. The results prove that the biosynthetic capabilities of E. amylovoru for proferrioxamine production are flexible. E. amylovora may therefore be a good organism for the fermentative production of novel proferrioxamines that may be useful for reversing or avoiding aluminum and iron intoxications in man, for tumor diagnosis and therapy with radiolabeled antibodies and for use as DNA-cleaving reagents. On the other hand, the production of all the different proferrioxamines involves common biosynthetic steps, some of which are not used in higher plants or vertebrates. Interference with the biosynthesis of proferrioxamines may therefore provide an opportunity for the development of alternative fire blight control agents.