Under control of the Gac regulatory system, Pseudomonas putida RW10S1 produces promysalin to promote its own swarming and biofilm formation, and to selectively inhibit many other pseudomonads, including the opportunistic pathogen Pseudomonas aeruginosa. This amphipathic antibiotic is composed of salicylic acid and 2,8-dihydroxymyristamide bridged by a unique 2-pyrroline-5-carboxyl moiety. In addition to enzymes for salicylic acid synthesis and activation, the biosynthetic gene cluster encodes divergent type II fatty acid biosynthesis components, unusual fatty acid-tailoring enzymes (two Rieske-type oxygenases and an amidotransferase), an enzyme resembling a proline-loading module of nonribosomal peptide synthetases, and the first prokaryotic member of the BAHD family of plant acyltransferases. Identification of biosynthetic intermediates enabled to propose a pathway for synthesis of this bacterial colonization factor.