What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome?

Abstract

The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.

FIG. 1.

The five TA systems are not involved in the growth inhibition mediated by amino acid starvation and do not advantage poststress recovery. Individual cultures of the wild-type (WT) (square) and Δ5 (triangle) strains (A and B) and mixed cultures of the wild-type strain and the nalidixic acid-resistant derivative of the Δ5 strain (Δ5 Nal^r) (circle) (C and D) were challenged with SHT (2.5 mg/ml) (see Materials and Methods). The viability of treated (open symbols) and untreated (filled symbols) cells was measured for 120 min upon SHT addition (A and C). After 120 min of starvation, SHT was removed and the viability of individual (B) and mixed (D) cultures was measured for 180 min (until early stationary phase). For each graph, the number of CFU/ml at each time point (tx) was normalized to that at time zero (t₀). The graphs represent the averages from three (A and B) and two (C and D) independent experiments. Similar results were obtained in experiments in which the wild-type Nal^r strain was mixed with the Δ5 strain (data not shown).

FIG. 2.

The five TA systems are not involved in the adaptation to a nutritional downshift. Individual cultures of the wild-type (WT) (square) and Δ5 (triangle) strains (A) and mixed cultures of the wild-type and Δ5 Nal^r (circle) strains (B) were challenged by a nutritional downshift (see Materials and Methods). The number of CFU/ml at each time point (tx) was normalized to that at time zero (t₀) for each strain. The graphs represent the averages from three independent experiments. Similar results were obtained when a wild-type Nal^r strain was mixed with the Δ5 strain (data not shown).

FIG. 3.

The five TA systems are not involved in the growth inhibition mediated by an acidic shock and do not advantage poststress recovery. Cultures of the wild-type (WT) (square) and Δ5 (triangle) strains were shifted from pH 6.8 to pH 4 medium (see Materials and Methods). The viability of treated (open symbols) and untreated (filled symbols) cells was measured for 120 min (A). After 120 min, the wild-type and Δ5 cultures were shifted back to pH 6.8. Viability was measured for 120 min (until early stationary phase) (B). The number of CFU/ml at each time point (tx) was normalized to that at time zero (t₀) for each strain. The graphs represent the averages from three independent experiments.

FIG. 4.

The five TA systems are not involved in the growth inhibition mediated by rifampin (rif) and do not advantage poststress recovery. Individual cultures of the wild-type (WT) (square) and Δ5 (triangle) strains were challenged with rifampin (100 μg/ml) (see Materials and Methods). The viability of treated (open symbols) and untreated (filled symbols) cells was measured for 120 min (A). After 120 min of treatment, rifampin was removed and the viability of both strains was measured for 240 min (until early stationary phase) (B). The graphs represent the averages for three independent experiments. The number of CFU/ml at each time point (tx) was normalized to that at time zero (t₀) for each strain.

FIG. 5.

The five TA systems do not enhance competitiveness in long-term stationary-phase cultures. The viability of individual cultures of the wild-type (WT) (open square) and the Δ5 (open triangle) strains (A) and of mixed cultures of the wild-type and Δ5 Nal^r (open circle) cultures (B) was measured daily for 7 days (see Materials and Methods). The graphs represent the averages from three independent experiments. The number of CFU/ml at each time point (dx) was normalized to that at day 1 (d₁) for each strain. Similar results were obtained with the symmetrical mixed culture of the wild-type Nal^r and Δ5 strains and with a mixed culture of wild-type and Δ5 yefM-yoeB::Kan strains (data not shown; see Materials and Methods).