Quantifying GCL Degradation in Mycobacteria

For induction assays in which GCL was added to the growth medium, GCL degradation was monitored by a colorimetric method typically used for the analysis of ester molecules (Yang et al., 2006 (link)). The reaction between the ester group of GCL and hydroxylamine leads to the formation of hydroxamic acid in alkaline solution. This hydroxamic acid forms a highly colored complex with ferric ions, which can be quantified by spectrophotometry at 520 nm. GCL was quantified as described by Cirou et al. (2012) (link) and Barbey et al. (2018) (link). Briefly, culture supernatants were centrifuged at 5000 × g for 5 min, and 200 μl of the resulting supernatant was successively mixed with 250 μl of 2 M hydroxylamine (Sigma-Aldrich)/3.5 M NaOH (Merck) (1:1, v/v) and 250 μl of 10% iron chloride (Sigma-Aldrich) prepared in 4 M HCl/95% ethanol (Merck) (1:1, v/v). The absorbance of the mixture at 520 nm was analyzed against a blank consisting of the same mixture but with the culture supernatant s replaced with non-inoculated 7H9 medium without GCL. Non-inoculated 7H9 medium supplemented with GCL was incubated and analyzed under the same conditions as the inoculated media to check the stability of GCL.