FIGURE 5.

Activation of mazEF operon upon induction of an inactive, active-site mutant E24A MazF. Both E. coli strains, MG1655 (contains WT mazEF operon on chromosome) and MGΔmazEF (lacks mazEF operon on chromosome), were induced for the expression of E24A MazF from the pBAD24 plasmid with 0.2% (w/v) arabinose for 1 h, total RNA was prepared and converted to cDNA, and quantitative PCR was performed to detect the mRNA specific to mazE. Expression of the mazEF operon was compared in samples from identical cells grown in either the absence of inducer (arabinose) or presence of repressor (glucose). The y axis represents normalized expression of the mazEF operon under different conditions. The x axis represents the different conditions under which MG1655 cells were grown. As a control for the specificity of E24A MazF for mazEF operon activation, transcription of mazEF operon in the presence of an unrelated protein, Trx was monitored. The overexpression of E24A MazF causes the derepression of the mazEF operon in WT MG1655 strain, and even uninduced cells have slightly higher levels of mazEF operon activation compared with cells repressed for E24A MazF. Lack of activation of mazEF in uninduced and induced Trx cells shows that the effect of E24A MazF is specific to the mazEF operon.