Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.Nonribosomal peptide synthetase (NRPS) enzymology is involved in the biosynthesis of many natural products with diverse biological activities, such as antifungal, antibacterial, anticancer, immunosuppressant, and metal chelation (reviewed in references 18 and 55). While the chemical structures of the natural products biosynthesized by NRPSs are diverse and explain their wide-ranging biological activities, the core enzymology used to synthesize these molecules is conserved. This conservation comes from NRPSs consisting of a set of repeating core protein domains grouped into enzymatic modules, with each module typically controlling the incorporation of one amino acid precursor into the nonribosomal peptide. The structural diversity of the NRPS-synthesized natural products comes from variations in the number and order of the modules, alterations in the substrate incorporated by the modules, and the potential addition of catalytic domains into modules that result in modifications to the growing peptide chain.The core domains that are repeated for each NRPS module are the adenylation (A) domain, peptidyl carrier protein (PCP) domain, and condensation (C) domain. The A domains are commonly referred to as the "gatekeepers" of a module, because they recognize the substrate that is incorporated into the growing peptide chain. In a significant breakthrough in understanding NRPS enzymology, an amino acid substrate specificity code was identified that enables one to deduce the amino acid that is likely recognized by an A domain, given the protein sequence of the A domain (8,9,35,60). This offers the ability to predict which module incorporates each amino acid of a nonribosomal peptide of known structure, but it also provides a means for proposing what amino acid is recognized by an NRPS module of unknown function. Once it recognizes the amino acid su...