Salicylic acid, however, was not synthesized to any appreciable extent from chorismic acid by extracts prepared from any of the mutants grown similarly: ∼1 ng by CFEs from knockouts of trpE2, entC and entD as single mutants and 0.25 ng by entDtrpE2 as a double

mutant (Fig. 1). These very low conversions suggest that a combination of the gene products from trpE2, entC and entD or, probably and more likely, that all three genes play a role in the synthesis of salicylic acid from chorismic acid. To evaluate which genes are involved in the conversion of chorismic acid to isochorismic acid and then in the conversion of isochorismic acid to salicylic acid, the above experiment was modified such that isochorismic Birinapant concentration acid was extracted before estimating salicylic acid and hence could confirm the involvement of trpE2, entC and entD in the stepwise conversion. Accordingly, the CFEs of each of the three single mutants were prepared. Each contained approximately 10 mg protein mL−1 and were incubated individually with chorismic acid as a substrate at 37 °C in a total volume of 2.3 mL (Marshall & Ratledge, 1971). After 1 h, the reaction was stopped with HCl and each mixture was extracted with ethyl acetate (see Materials and methods) to remove any isochorismic acid that had been formed. Each of these solvent extracts,

now in an aqueous buffer, was then divided into three equal aliquots IDH inhibitor drugs and each of these was placed in separate test tubes. For each batch of three solvent extracts, one was incubated without addition of CFE (control), and the other two were incubated with a CFE other than the one that had been used originally (Table 1). In other words, this was a cross-over biochemical reaction. The synthesis of salicylic acid occurred when CFEs from mutants of either entC or entD were used in the first reaction with chorismic acid as a substrate and followed by using the CFE of mutant trpE2 in the second reaction. The synthesis of salicylic acid was completely absent when a CFE of mutant trpE2 was used in the first reaction, irrespective

Metalloexopeptidase of which CFE was used in the second reaction (Table 1). As salicylic acid is principally converted to mycobactin, with only about 5–10% being converted into carboxymycobactin (Ratledge & Ewing, 1996), we then studied the production of mycobactin in the knockout mutants. The wild type and the mutants of M. smegmatis were grown for 7 days in minimal medium under iron-deficient conditions (which are needed to maximize mycobactin formation) with and without salicylic acid added at 5 μg mL−1. The production of mycobactin by the mutants was drastically decreased in minimal medium compared with the wild-type strain (Fig. 2). However, when salicylic acid was included in the medium, the mutant cells had considerably more mycobactin than before, although the amounts were well below those in the wild-type strain (Table 2).