Pathogenic Yersinia species have been shown to synthesize a siderophore molecule, yersiniabactin, as a virulence factor during iron starvation. Here we provide the first biochemical evidence for the role of the Yersinia pestis high molecular weight protein 2 (HMWP2), a nonribosomal peptide synthetase homologue, and YbtE in the initiation of yersiniabactin biosynthesis. YbtE catalyzes the adenylation of salicylate and the transfer of this activated salicyl group to the N-terminal aryl carrier protein domain (ArCP; residues 1−100) of HMWP2. A fragment of HMWP2, residues 1−1491, can adenylate cysteine and with the resulting cysteinyl-AMP autoaminoacylate the peptidyl carrier protein domain (PCP1; residues 1383−1491) either in cis or in trans. Catalytic release of hydroxyphenylthiazoline carboxylic acid (HPT-COOH) and/or N-(hydroxyphenylthiazolinylcarbonyl)cysteine (HPT-cys) is observed upon incubation of YbtE, HMWP2 1−1491, l-cysteine, salicylate, and ATP. These products presumably arise from nucleophilic attack by water or cysteine of a stoichiometric hydroxyphenylthiazolinylcarbonyl-S-PCP1-HMWP2 intermediate. Detection of the heterocyclization capacity of HMWP2 1−1491 implies salicyl-transferring and thiazoline-forming activity for the HMWP2 condensation domain (residues 101−544) and is the first demonstration of such heterocyclization ability in a nonribosomal peptide synthetase enzyme.