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. 2017 Sep 5;199(19):e00397-17. doi: 10.1128/JB.00397-17

FIG 5.

FIG 5

Low-level expression of CcdBO157 leads to reversible growth inhibition. (A) Plasmids pBAD24ccdBF, pBAD24ccdBO157, and pBAD24ccdB100T were individually transformed in E. coli strain BW25113 and grown in LB medium containing 0.2% glucose. Cells were induced with various concentrations of arabinose at an OD600 of 0.2. The x axis indicates various concentrations of arabinose used for transient expression of the construct. Induced cells were grown for 2 h, washed twice with LB medium, diluted, and plated on 0.2% glucose-containing medium to repress further expression. CcdBF expression leads to complete cell death, even under highly repressed conditions. CcdBO157 leads to cell death at very high expression levels (0.2% arabinose) but leads to growth inhibition when the expression is low (0.002% to 0.02% arabinose). The y axis is shown on a log scale. Results from two independent experiments are plotted as two different bars. Overexpression of the G100T mutant is nontoxic to E. coli Top10 cells. WT CcdBF is toxic to cells even under highly repressed conditions, and no transformants are obtained under any condition. (B) To score the expression of ccdBO157 from its own promoter, the active-site inactive mutant of CcdBF, G100T, was transformed into the E. coli BW25113 ccdF (having chromosomal CcdF) and BW25113 ccdO157 (having chromosomal CcdF) strains individually. Expression of G100T was induced with various concentrations of arabinose (as described above) at an OD600 of 0.2. Cells were grown for 2 h, washed twice, diluted, and plated on 0.2% glucose-containing medium to repress further expression. Overexpressed G100T will titrate out cellular CcdA, leading to release of WT CcdB as well as derepression of the ccdAB operon. The reduction in cell survival at higher levels of induction (0.2% arabinose) of the mutant in BW25113 ccdO157 cells shows that there is cross activity between the CcdBF and CcdAO157 TA systems. Low expression levels of the mutant (0.002% to 0.02% arabinose) result in reduced toxicity and cause growth inhibition.