Fig. 5: In vitro analysis of key enzymatic activities and glutathione metabolism.
a Enzyme activity ratios were calculated from the specific activity for each of the indicated reactions assessed under H2O2-induced oxidative stress and control (Ctrl.) conditions. Each bar represents the mean value of the corresponding ratio ± standard deviations of triplicate measurements from at least two independent experiments, and the horizontal dashed line indicates an activity ratio = 1 (i.e. no changes in enzymatic activities across experimental conditions). Statistical comparisons between enzyme activity ratios were assessed by the Student’s t test with Welch’s correction. Single (*) and double asterisks (**) identify significant differences at the p < 0.05 and p < 0.01 levels, respectively. Actual p values for the Glk and Gad activity ratios in the glucose conversion routes were p = 0.0019 and 0.0293, respectively. Actual p values for the Zwf and GntZ activity ratios in the PP pathway were p = 0.0087 and 0.0096, respectively. Circled numbers identify the enzymes in the biochemical network of Fig. S1. b Glutathione metabolism in P. putida KT2440. The key activities involved in biosynthesis and recycling of the reduced (GSH) and oxidized (GS–SG) forms of glutathione are indicated along with the corresponding PP identifiers. c Enzymatic determination of total glutathione and the fraction of the oxidized and reduced form. Bars represent the mean value of the corresponding parameter ± standard deviations of duplicate measurements from at least five independent experiments, with individual measurements indicated as empty circles, and the asterisk (*) identifies significant differences between stressed cells and control conditions at P < 0.05 as assessed by the Student’s t test with the Bonferroni correction. Actual p values for the total glutathione content and the GSH/GS–SG ratio between H2O2-treated and control cultures were p = 0.0361 and 0.0117, respectively. CDW, cell dry weight. d Impact of the carbon substrate on the growth of P. putida KT2440 upon an oxidative challenge. Normalized growth coefficients, representing the fraction of the specific growth rate (μ) in the presence of 3 mM H2O2 as compared with that of control (Ctrl.) conditions, were calculated in cultures using glucose, α-ketoglutarate (α-KG) or glycerol as the carbon source. Each bar represents the mean value of the normalized growth coefficients ± standard deviations of triplicate independent experiments, while the arrows and the accompanying percentages indicate the relative reduction in the growth rate under oxidative stress.
