Figure 6.

ccdAB_ST TA system influences the transcription of the polycistronic operon. rsdB of pSLT plasmid was tagged with a 3xFLAG epitope in its 3′-end. (A,B) RsdB-3xFLAG levels were analyzed in pSLT plasmid lacking the PccdAB_ST promoter or the entire ccdAB_ST locus (including toxin, antitoxin and the promoter of the system). Bands intensity in (A) were quantified taking into account the number of cells loaded in the acrylamide gels and the band intensity of the loading control DnaK (B) Deletion of either the ccdAB_ST operon or the promoter of the system, PccdAB_ST, reduced RsdB 2.3 and 2.1 fold, respectively. (C) Conditional cooperativity phenomenon was analyzed in a wild type pSLT carrying the tagged 3xFLAG rsdB. CcdB_ST or VapC_ST (negative control) proteins were supplement in trans from pCcdB and pVapC plasmids. Due to the conditional cooperativity phenomenom, overexpression of CcdB_ST results in derepression of PccdAB_ST and increased amount of RsdB. Quantification was carried out as described for A and B. Protein expression of CcdB_ST and VapC_ST was confirmed in SDS-acrylamide gels stained with Coomassie blue (Supplementary Figure 1). Histograms represent the quantification of at least four independent experiments. ^***p < 0.001 by one-way ANOVA.