Escherichia coli MazF leads to the simultaneous selective synthesis of both "death proteins" and "survival proteins"

Abstract

The Escherichia coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful.

Figure 1. MazF overproduction in E. coli does not completely inhibit protein synthesis.

E. coli strain MC4100 relA1, hosting plasmid pSA1 that bears an IPTG-inducible mazF gene, was grown to mid-logarithmic phase; the culture was divided into two parts, to one of which IPTG was added. Subsequently, [³⁵S]methionine was added to both the induced and the uninduced cultures. (A) At various times, samples were taken from each culture, and the incorporation of radioactive material into the TCA insoluble fraction was determined. In a separate experiment, MC4100 relA1/pSA1 was grown to mid-logarithmic phase and further incubated for 15 minutes in the presence or absence of IPTG. Then, the cells were labeled for 5 minutes with [³⁵S]methionine, lysed, and (B) run on a 1D-gel for high molecular weight proteins or (C) run on a 1D-gel for low molecular weight proteins. The arrows in B–C indicate the estimated molecular weights (kDa) of proteins synthesized after MazF induction.

Figure 2. 2D-gels showing that MazF induction resulted in a global change in the pattern of protein synthesis.

Samples of MC4100 relA1/pSA1 cultures, which were grown and treated as described in the legends for Figure 1B and 1C, were also run on 2D-gels. Autoradiograms of these gels were computer stained: magenta for cultures in which MazF had been induced, and green for cultures in which MazF was not induced. (A) The two autoradiograms were superimposed to give a dual-channel image. Consequently, proteins whose level of synthesis was increased after mazF induction appear in magenta and those whose level of synthesis was reduced appear in green. Proteins whose level of synthesis did not change after mazF induction appear in black. For each spot, the ratio of the results for the MazF-induced and MazF-uninduced cultures was calculated. (B) A copy of (A) in which the spots whose ratio was more than 2 are circled in magenta. (C) A copy of (A) in which the spots whose ratio was less than 0.5 are circled in green. The horizontal black line in Figure 1A–1C indicates a MW of ∼20 kDa.

Figure 3. Some E. coli genes encoding proteins synthesized after MazF induction are involved in cell death, and some in cell survival.

(A) Identified proteins (circled), which are synthesized after MazF induction. (B) Genes encoding the proteins identified in (A) were individually deleted from the chromosome of E. coli MC4100 relA⁺. These deletion mutants, as well as the WT and its ΔmazEF derivative as controls, were briefly treated with spectinomycin to inhibit translation for only a short period, and then plated to determine viability (Materials and Methods). (C) As In (B), but the cells were treated with nalidixic acid to cause damage to the DNA. Genes whose deletion resulted in increased viability were called “Death Genes”. Genes whose deletion resulted in reduced viability were called “Survival Genes.”

Figure 4. The mazEF-mediated cell death process - an extended model.

Text and arrows in black represent the mazEF-mediated cell death process that occurs following either DNA damage or brief inhibition of protein synthesis. Text in red represents proteins that were found to participate in cell death, only when mazEF was induced by DNA damage. Text in green represents proteins that were found to participate in cell death and survival, only when mazEF was induced by brief inhibition of translation. For a discussion of this model please see the text.