Fig. 3: ClpC2 protects Msm against CymA toxicity.

a A clpC2 knockout (ΔclpC2) strain was prepared in Msm. The ΔclpC2 (red diamonds), parent (WT, black squares) as well as ΔclpC2 strains complemented with clpC2 WT (comclpC2 WT, grey circles) or complemented with clpC2 F99A (comclpC2 F99A, blue triangles) were grown to exponential phase, then diluted to an OD₆₀₀ of 0.005 before monitoring the OD₆₀₀ of the cultures grown in either the absence or presence of 150 nM CymA. Error bars depict the standard deviation from three biological replicates. b The effect of CymA on cellular ClpC2 protein levels was analysed by Western blot. Msm culture was grown to an OD₆₀₀ of 0.5 in 7H9 medium at 37 °C before adding 150 nM CymA. Cells were harvested, then lysed at the indicated time points following addition of CymA. RpoB is included as a loading control. c The counteractive effect of ClpC2 in a CymA-stimulated FITC-casein degradation assay. The proteolytic activity of 0.5 µM ClpC1 hexamer/0.8 µM ClpP1P2 tetradecamer was stimulated by 2 µM CymA and the rate of degradation was determined following the addition of increasing concentrations of ClpC2. The rate of degradation is normalised so that 0% stimulation is the proteolytic activity in the absence of CymA and 100% stimulation is equivalent to the maximum CymA-induced stimulation above basal activity. Error bars represent standard deviation from three independent experiments. d Quantitative Western blot was employed to analyse the ratio of ClpC1 (open circles) to ClpC2 (filled squares) following addition of CymA. Msm culture was grown to an OD₆₀₀ of 0.5 in 7H9 medium at 37 °C before adding 150 nM CymA. Samples were harvested at the indicated time points following addition of CymA and ClpC1 or ClpC2 proteins quantified by immunoblotting in reference to a protein standard curve using purified ClpC1 and ClpC2 protein. ClpC1 and ClpC2 protein levels are normalised relative to ClpC1 at timepoint 0 h. Representative blots are shown in Supplementary Fig. 3. Error bars depict standard deviation from biological triplicates. e Expression levels of clpC2 in the absence and presence of 150 nM CymA as analysed by RT-qPCR. Msm cultures were grown to an OD₆₀₀ of 0.5 followed by a one-hour incubation in either the absence or presence of 150 nM CymA. clpC2 gene expression was normalised to the housekeeping gene rpoB. Error bars represent standard deviation of technical triplicates from three biological replicates. Statistical analysis by two-tailed unpaired Student’s t test; P value = 0.0015.