Fig. 2

Point mutations introduced with the stuffer-assisted genome site-directed mutagenesis method lead to detectable phenotypic changes. a Simplified representation of glutathione synthesis and recycling. Condensation of glutamate and cysteine by Gsh1p is followed by the addition of a glycine by Gsh2p, yielding reduced glutathione. Glutathione oxidized by reactive oxygen species (ROS) is recycled to its reduced form by the NADPH-dependent Glr1p enzyme. b Sequencing shows successful insertion of the stuffer and subsequent introduction of a point mutation in the GSH1 promoter sequence c ROS accumulation induced by exposure to SSL was compared between a gsh1(A(−73)T) point mutant generated with the method, and in its parent wildtype strain (WT). ROS accumulation was assessed using flow cytometry, measuring the mean fluorescence of cells treated with CellROX Deep Red reagent. ROS were measured 16 h after inoculation in minimal medium (Mid-log), after overnight incubation in undiluted SSL (acute stress), or after 24 and 48 h in minimal medium containing 70 % SSL