Sunday, December 28, 2008

Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

De Bast M. S., Mine N. and Van Melderen L. (2008) J. Bacteriol. 190:4603-4609

Plasmids are known as carriers of pathogenic determinants and antibiotic resistance genes that help bacteria survive in specific circumstances. However, if the circumstances changed, the bacteria would no longer need such genes, so that the plasmid would become just a burden for bacteria. As a result, plasmid-free bacterial cells would increase their number more rapidly than plasmid-carrying cells. Most of naturally occurring plasmids have special systems to prevent such an event from happening. One of the systems is the Toxin-Antitoxin (TA) system, in which one gene encodes a stable toxin protein and the other gene encodes an unstable antitoxin protein that counteracts the toxin activity. If the plasmid carrying the TA system was lost from a cell, the cell would be immediately killed or damaged by the more stable toxins which persist in the cell; this phenomenon is called postsegregational killing [PSK]. Gene pairs comprising TA systems are called addiction modules. Addiction modules were originally discovered on a plasmid (Hiraga et al., 1986), but recently they have also been discovered on chromosomes (reviewed by Kobayashi I., 2004). Here, we have a question: What is the biological function of chromosomally-located addiction modules?

The first hypothesis proposed is the so-called programmed cell death (PCD) hypothesis: the addiction module induces cell death under stress conditions and the dead cells release nutrients for other cells to remain viable (Aizenman et al., 1996). Recently, this hypothesis has been shown to be unlikely by several research groups (Tsilibaris et al., 2007; Szekeres et al., 2007; Dudde et al., 2007; Pedersen et. al., 2002). The second and more reasonable hypothesis is that addiction modules contribute to stabilize a genome: the toxin reduces the number of bacteria that have lost the chromosomal DNA segment containing the addiction modules, which ensures that the DNA in the region of the addiction module is maintained in the bacterial population (Szekeres et al., 2007).

In this paper, the authors propose a third theory: the "anti-addiction module" hypothesis. In this hypothesis, addiction modules on a chromosome protect bacteria against PSK induced by orthologous addiction modules on a plasmid, which confers selective advantage on a host bacterium under PSK conditions.

To test the anti-addiction module hypothesis, the authors used the CcdB(F)/CcdA(F) TA system of F-plasmid (Hiraga et al., 1986) and its homologous system [CcdB(Ech)/CcdA(Ech) TA system] found in the Escherichia chrysanthemi chromosome; CcdB(F) toxin is 61% identical to CcdB(Ech) while CcdA(F) antitoxin is 65% identical to CcdA(Ech). In this article, the authors first showed that the ccdB(Ech) and ccdA(Ech) genes indeed act as toxin and antitoxin genes in E. coli MG1655 where the ccdB(F)/ccdA(F) homologous genes are absent. However, unlike F-plasmid's ccdB(F)-ccdA(F) gene pair, the ccdB(Ech)-ccdA(Ech) gene pair could not mediate PSK when it was cloned on a plasmid. This suggests that the two homologous TA systems have evolved for different purposes. The authors also showed that CcdA(Ech) can antagonize CcdB(F) toxic activity as efficiently as CcdA(F) can. Furthermore, they showed that the E. coli MG1655 derivative that carries the ccdB(Ech)-ccdA(Ech) gene pair on its chromosome (designated MG1655ccdEch) are more viable than the original MG1655 after the induction of PSK mediated by F-plasmid's ccdB(F)-ccdA(F) gene pair on a plasmid. The following competition assays between MG1655 and MG1655ccdEch in PSK conditions indicated that MG1655ccdEch has a 25% fitness advantage over MG1655. Therefore, all experiments performed in this article support anti-addiction module hypothesis. A related idea was also proposed by Takahashi et al. (2002), using a restriction-modification TA system, in which restriction enzymes act as toxins and modification methyltransferases act as antitoxins. They showed that dcm methyltransferase gene located on the E. coli chromosome protected cells against PSK mediated by a restriction enzyme and DNA modification gene pair on a plasmid.

If the anti-addiction module hypothesis is valid, there would be few cases in nature that counteracting addiction modules are found on both plasmid and chromosome in the same cell, because PSK does not happen in such a situation and consequently there would be no advantage for plasmids to carry the addiction module. However, in the genome of E. coli O157:H7, two homologous ccdB-ccdA gene pairs exist. One ccdB-ccdA gene pair is located on plasmid pO157 and the other is located on the chromosome. The ccd genes of pO157 are identical to F-plasmid's counterparts. Chromosomal ccdB and ccdA gene products, CcdB(O157) and CcdA(O157), are 35% and 30% identical to CcdB(F) and CcdA(F), respectively. As we can expect, the chromosomal ccdA-ccdB gene pair of O157:H7 does not counteract CcdB(F) toxicity and O157:H7 is susceptible to PSK mediated by the CcdB(F)/CcdA(F) TA system (Wibaux et al., 2007).

The integration of addiction modules into the chromosome can protect bacteria from plasmids that may have a high cost under some conditions. However, as the authors state in this article, that in turn drives the evolution of plasmid TA systems so as not to be counteracted by chromosomal TA systems. It thus appears to me that the primal role of chromosomal TA systems is maintaining the integrity of chromosomes in bacterial populations and the secondary role may be protecting bacteria against PSK mediated by invader DNA elements such as phages and plasmids. Do you have another hypothesis? If so, please let me know.

Aizenman E, Engelberg-Kulka H, Glaser G. (1996) An Escherichia coli chromosomal "addiction module" regulated by guanosine 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc. Natl. Acad. Sci. 93:6059-6063.

Budde PP, Davis BM, Yuan J, Waldor MK. (2007) Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae. J Bacteriol. 189:491-500

Hiraga S, Jaffé A, Ogura T, Mori H, Takahashi H. (1986) F plasmid ccd mechanism in Escherichia coli. J. Bacteriol. 166:100-104.

Kobayashi I. (2004) Genetic Addition: a principle of Gene symbiosis in a Genome, in Plasmid Biology (Funnell B. E. and Phillips G. J. eds), pp.105-144 ASM press, Washigton D.C.

Pederson K, Christensen SK, and Gerdes K (2002) Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol. Microbiol. 45:501-510.

Szekeres S, Dauti M, Wilde C, Mazel D, Rowe-Magnus DA. (2007) Chromosomal toxin-antitoxin loci can diminish large-scale genome reductions in the absence of selection. Mol. Microbiol. 63:1588-1605.

Tsilibaris V, Maenhaut-Michel G, Mine N, Van Melderen L (2007) What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome? J. Bacteriol. 189:6101-6108.

Takahashi N, Naito Y, Handa N, Kobayashi I. (2002) A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex. J. Bacteriol. 184:6100-6108.

posted by Hirokazu Yano, University of Idaho

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