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Toxin-antitoxin (TA) systems are widespread among bacterial chromosomes and mobile genetic elements. Although in plasmids TA systems have a clear role in their vertical inheritance by selectively killing plasmid-free daughter cells (postsegregational killing or addiction phenomenon), the physiological role of chromosomally encoded ones remains under debate. The assumption that chromosomally encoded TA systems are part of stress response networks and/or programmed cell death machinery has been called into question recently by the observation that none of the five canonical chromosomally encoded TA systems in the Escherichia coli chromosome seem to confer any selective advantage under stressful conditions (V. Tsilibaris, G. Maenhaut-Michel, N. Mine, and L. Van Melderen, J. Bacteriol. 189:6101-6108, 2007). Their prevalence in bacterial chromosomes indicates that they might have been acquired through horizontal gene transfer. Once integrated in chromosomes, they might in turn interfere with their homologues encoded by mobile genetic elements. In this work, we show that the chromosomally encoded Erwinia chrysanthemi ccd (control of cell death) ([ccd.sub.Ech]) system indeed protects the cell against postsegregational killing mediated by its F-plasmid ccd ([ccd.sub.F]) homoiogue. Moreover, competition experiments have shown that this system confers a fitness advantage under postsegregational conditions mediated by the [ccd.sub.F] system. We propose that [ccd.sub.Ech] acts as an antiaddiction module and, more generally, that the integration of TA systems in bacterial chromosomes could drive the evolution of plasmid-encoded ones and select toxins that are no longer recognized by the antiaddiction module.

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.

Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid ([ccd.sub.F]) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain ([ccd.sub.O157]). The [ccd.sub.F] and the [ccd.sub.O157] systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the [ccd.sub.F] system. We have shown that the chromosomal [ccd.sub.O157] system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB[O.sub.157] toxin targets the DNA gyrase, and the CcdA[O.sub.157] antitoxin is degraded by the Lon protease. The [ccd.sub.O157] chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of [ccd.sub.F] [ccd.sub.O157] is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccdr system is able to mediate postsegregational killing in an E. coli strain harboring the [ccd.sub.O157] system in its chromosome. This shows that the plasmidic [ccd.sub.F] system is functional in the presence of its chromosomal counterpart.