Figure 6.

Effect of inducible toxin and TA overexpression for CD2517.1-RCd8 TA module near CRISPR 12 on growth in solid (A) and liquid medium (B–D). (A) Growth phenotype of C. difficile strains CDIP369 (630/p), CDIP357 (630/pT) and CDIP332 (630/pTA) on BHI agar plates supplemented with Tm alone (on the left) or with the addition of 500 ng/ml of ATc inducer (on the right) after 24 h of incubation at 37°C. Schematic representations of pT and pTA constructs are shown. The 630 strain carrying an empty vector (p) is used as a control. (B) Detection of RCd8 and CD2517.1 transcripts. For northern blot analysis, RNA samples were extracted from 630/p control strain, from 630/pT strain overexpressing the CD2517.1 toxin and from 630/pTA strain overexpressing the entire TA module grown at late exponential growth phase in the presence of 250 ng/ml ATc (+ATc) or the absence of inducer. As indicated at the top, the blots were hybridized either with antitoxin- or toxin-specific probe. The same 5S control panel is shown when reprobing of the same membrane was performed. (C) Growth of 630/p strain (triangles), 630/pT strain (diamond) and 630/pTA strain (circle) in TY medium at 37°C in the presence (open symbols) or absence (closed symbols) of 250 ng/ml ATc. The time point of ATc addition is indicated by an arrow. (D) Growth curves for 630/p strain, 630/pT strain and 630/pTA strain in TY medium at 37°C in the presence of 250 ng/ml ATc using a GloMax plate reader (Promega). The mean values and standard deviations are shown for three independent experiments.