Pseudobactin_B10, the fluorescent siderophore produced by the rhizobacterium Pseudomonas strain B10, contains the hydroxamate ligand D-N⁵-hydroxyornithine (D-N⁵-OH-Orn). We cloned the L-Orn N⁵-oxygenase (psbA) gene from a genomic library of Pseudomonas strain B10 and demonstrated that PsbA is involved in the conversion of L-Orn to its N⁵-OH derivative. PsbA shows significant similarity to microbial -amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. The psbA gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific psbA mutant of Pseudomonas strain B10 was biochemically complemented with the precursor L-N⁵-OH-Orn, suggesting that L-Orn is hydroxylated before conversion to the D isomer. The L-Orn N⁵-hydroxylase-defective mutants of Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen Erwinia carotovora in iron-poor medium. The extent of in vitro inhibition of E. carotovora was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms.