MazF-mediated cell death in Escherichia coli: a point of no return

Abstract

mazEF is a stress-induced toxin-antitoxin module, located on the chromosome of Escherichia coli, that we have previously described to be responsible for programmed cell death in E. coli. mazF specifies a stable toxin, and mazE specifies a labile antitoxin. Recently, it was reported that inhibition of translation and cell growth by ectopic overexpression of the toxin MazF can be reversed by the action of the antitoxin MazE ectopically overexpressed at a later time. Based on these results, it was suggested that rather than inducing cell death, mazF induces a state of reversible bacteriostasis (K. Pederson, S. K. Christensen, and K. Gerdes, Mol. Microbiol. 45:501-510, 2002). Using a similar ectopic overexpression system, we show here that overexpression of MazE could reverse MazF lethality only over a short window of time. The size of that window depended on the nature of the medium in which MazF was overexpressed. Thus, we found "a point of no return," which occurred sooner in minimal M9 medium than it did in the rich Luria-Bertani medium. We also describe a state in which the effect of MazF on translation could be separated from its effect on cell death: MazE overproduction could completely reverse the inhibitory effect of MazF on translation, while not affecting the bacteriocidic effect of MazF at all. Our results reported here support our view that the mazEF module mediates cell death and is part of a programmed cell death network.

FIG. 1.

Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. E. coli strain MC4100 ΔmazEF relA1 lacI^q was cotransformed with pQE-Δhis-mazE, carrying mazE (without a His tag), and pBAD-mazF, carrying mazF. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to mid-logarithmic phase (OD₆₀₀, 0.5). At time zero, 0.2% arabinose was added to the cultures to induce mazF expression. To determine CFU, samples were withdrawn at various time points and spread on LB plates containing 100 μg of ampicillin per ml, 50 μg of chloramphenicol per ml, and either 0.2% glucose (to represses mazF expression) or 0.2% glucose and 2 mM IPTG (to induce mazE expression). Induced culture spread on plates with IPTG, ▴; induced culture spread on plates without IPTG, •. The percentage of survivors (indicated next to the plotted measurements) was calculated by comparing the CFU of the mazF-induced culture to that of the uninduced culture at time zero. Error bars indicate standard deviations.

FIG. 2.

Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and to form colonies. E. coli strain MC4100 ΔmazEF relA1 lacI^q was cotransformed as described in the legend to Fig. 1. The cotransformed cells were grown, induced, labeled, and plated as described in Materials and Methods. Aa to Ac, Rate of translation. Ba to Bc, Percentage of survivors. To induce mazE expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after mazF induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of mazE expression. mazE-induced culture, ▴; uninduced culture, •. The relative percentage of protein synthesis and viability (indicated next to the plotted measurements) was calculated compared to that at time zero. The values shown are from one out of three similar experiments.

FIG. 3.

A model for the mazF-induced death pathway. For further details, see Discussion.