The monobactams, exemplified by the natural product Sulfazecin, are the only class of ��-lactam antibiotics not inactivated by metallo-��-lactamases, which confer bacteria with extended-spectrum ��-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a Sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the ��-lactam ring of Sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for Sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of Sulfazecin biosynthesis and ��-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic ��-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.
