Chromosomal toxin-antitoxin systems may act as antiaddiction modules

Abstract

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(Ech)) system indeed protects the cell against postsegregational killing mediated by its F-plasmid ccd (ccd(F)) homologue. Moreover, competition experiments have shown that this system confers a fitness advantage under postsegregational conditions mediated by the ccd(F) system. We propose that ccd(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.

FIG. 1.

Characterization of the chromosomally encoded ccd_Ech system. (A) ccd_Ech encodes functional antitoxin and toxin proteins. MG1655 strains carrying either the pKK223-3 vector (/) or its pKK-ccdA_Ech derivative (ccdA_Ech) were transformed with the compatible pBAD33 vector (/) or its pBAD-ccdB_Ech derivative (ccdB_Ech). The efficiency of transformation was calculated as the ratio of the number of transformants obtained on arabinose plates to the number of transformants obtained on plates without arabinose. Values correspond to the mean of three independent experiments. (B) The CcdB_Ech toxin targets the GyrA subunit of DNA gyrase. The SG22622 strain and its gyrA₄₆₂ derivative were transformed with the pBAD33 vector (/) or its pBAD33-ccdB_Ech (ccdB_Ech) and pBAD33-ccdB_F (ccdB_F) derivatives. The efficiency of transformation was calculated as the ratio of the number of transformants obtained on 1% arabinose plates to the number of transformants obtained on plates without arabinose. (C) The ccd_Ech system is unable to mediate PSK. MG1655 strains carrying either pMLO59 (squares) or its derivatives encoding ccd_Ech (pMLO-ccd_Ech; circles) and ccd_F (pMLO-ccd_F; triangles) were grown at 42°C. Viability (CFU/ml) was monitored for 240 min by plating serial dilutions on LB agar plates with (filled symbols) or without (open symbols) spectinomycin. Values correspond to the mean of three independent experiments.

FIG. 2.

CcdA_Ech antagonizes CcdB_F toxic activity. MG1655 strains containing either the pKK223-3 vector (/) or pKK-ccdA_Ech (ccdA_Ech) and pKK-cidA_F (ccdA_F) were transformed with the pBAD33 vector (/) or its pBAD-ccdB_F derivative (ccdB_F). The efficiency of transformation was calculated as the ratio of the number of transformants obtained on 1% arabinose plates to the number of transformants obtained on plates without arabinose. Values correspond to the mean of three independent experiments.

FIG. 3.

ccd_Ech is an antiaddiction module. The MG1655 (A), ccd_O157 (B), and ccd_Ech (C) strains containing either the pMLO59 replication-thermosensitive plasmid (spectinomycin resistance) (circles) or its derivative encoding the ccd_F system (pMLO-ccd_F; triangles) were grown at 42°C. Viability (CFU/ml) was monitored for 240 min by plating serial dilutions on LB agar plates with (filled symbols) or without (open symbols) spectinomycin. Values correspond to the mean of three independent experiments.

FIG. 4.

ccd_Ech increases fitness under the PSK condition. Competition experiments between the MG1655 strain containing either the pMLO59 vector (column 1) or the pMLO-ccd_F plasmid (column 2) and the ccd_Ech Δara strain containing the pMLO-ccd_F plasmid were carried out. As controls, (i) each strain was cocultivated with its Δara derivative, i.e., MG1655/pMLO-ccd_F in competition with MG1655 Δara/pMLO-ccd_F (column 3) and ccd_Ech/pMLO-ccd_F in competition with ccd_EchΔara/pMLO-ccd_F (column 4); and (ii) the “mirror” competition experiment was carried out with the MG1655 Δara strain containing either the pMLO59 vector (column 5) or the pMLO-ccd_F plasmid (column 6) and the ccd_Ech strain containing the pMLO-ccd_F plasmid. Cultures were mixed at a 1:1 ratio and cocultures grown under conditions similar to those used for the PSK experiments (see Materials and Methods). Viability (CFU/ml) was monitored for 240 min by plating serial dilutions on MacConkey arabinose (1%) agar plates. Relative fitness was calculated as described in reference and represents the advantage of the ccd_Ech strain relative to MG1655. Values correspond to the mean of three independent experiments.

FIG. 5.

Phylogenetic analysis of the ccd_F homologues. By use of the TBlastN software, 27 and 23 homologues of CcdA_F and CcdB_F, respectively, were selected in the complete sequenced genomes of gammaproteobacteria. Phylogenetic relationships were inferred by analyzing amino acids sequences of antitoxins and toxins as described in Materials and Methods. Names of bacterial species are abbreviated as follows: E. chrysanthemi 3937 (E.ch, E.ch.1, E.ch.2, and E.ch.3), E. carotovora subsp. atroseptica SCRI1043 (E.ca.1 and E.ca.2), Photorabdus luminescens subsp. laumondii TTO1 (P.lu), E. coli O157:H7 (E.c.O157), E. coli UTI189 (E.c.UTI189), E. coli CFT073 (E.c.CFT073), E. coli 536 (E.c.536), E. coli APEC01 (E.c.APEC01), Shigella dysenteriae Sd197 (S.dy.Sd197), Saccharophagus degradans 2-40 (S.de.2-40), Yersinia enterocolitica subsp. enterocolitica 8081 (Y.e.8081), and Serratia proteamaculans 568 (S.p.568). Naturally coexisting plasmid-encoded TA systems and chromosomally encoded TA systems are indicated by symbols as follows: O157:H7 contains the pO157 plasmid (triangles), UTI189 contains the pUTI89 plasmid (squares), and Sd197 contains the pSD1 197 plasmid (circles). Note that for E.ca.1, E.ca.2, E.c.APEC01, and E.c.UTI189, CcdA ORFs were detected, while cognate CcdB-encoding ORFs either were not (E.ca.1) or contained frameshift mutations (E.ca.2, E.c.APEC01, and E.c.UTI189) that are predicted to inactivate CcdB.