PvdD, a nonribosomal peptide synthetase (NRPS) of Pseudomonas aeruginosa PAO1, incorporates two L-threonines into the siderophore pyoverdine. A pvdD mutant did not synthesize pyoverdine and lacked a high Mr iron-regulated cytoplasmic protein (IRCP). Analysis of other IRCPs and the P. aeruginosa genome enabled the remaining pyoverdine NRPSs to be identified. The pvdD mutation could be complemented in trans, enabling design of plasmid-based systems for the generation of novel pyoverdines. Introduction of a truncated pvdD gene resulted in attenuated forms of pyoverdine, and introduction of L-threonine-incorporating NRPSs from other organisms restored pyoverdine production to mutant cells. This is the first successful rational in vivo modification of NRPS modules outside of Bacillus subtilis. The systems employed did not allow incorporation of other residues into pyoverdine, indicating that there are multiple elements contributing toward substrate specificity in NRPSs.