Mycobacterium smegmatis acquires extracellular iron using exochelin, mycobactin and carboxymycobactin. The latter two siderophores are synthesized from salicylic acid, which, in turn, is derived from chorismic acid in the shikimic acid pathway. To understand the conversion mechanism of chorismic acid to salicylic acid in M. smegmatis, knockout mutants of the putative key genes, trpE2, entC and entD, were created by targeted mutagenesis. By enzymatic assays with the cell-free extracts of the various knockout mutants, we have shown that TrpE2 converts chorismic acid into isochorismic acid and is thus an isochorismate synthase. The gene products of both entC and entD are involved in the conversion of isochorismic acid into salicylic acid, and hence correspond to salicylate synthase.