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Twenty microliters of supernatant was transferred into a 96-well plate, and 200 l of 37 C o-dianisidine hydrochloride solution was added immediately before the optical density was read at 450 nm and again 30 s later, as previously described 26

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However, in line with the specificity of CPA, the C-terminal residues of the L585I, L585M, and L585V variants were cleaved in vitro upon exposure to the enzyme for 5 hours at 37 C, as confirmed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of the C-terminal peptides (Supplementary Table 1)