Toxin-antitoxin systems: Biology, identification, and application

Abstract

Toxin-antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin. The toxins of all known TA systems are proteins while the antitoxins are either proteins or non-coding RNAs. Based on the molecular nature of the antitoxin and its mode of interaction with the toxin the TA modules are currently grouped into five classes. In general, the toxin is more stable than the antitoxin but the latter is expressed to a higher level. If supply of the antitoxin stops, for instance under special growth conditions or by plasmid loss in case of plasmid encoded TA systems, the antitoxin is rapidly degraded and can no longer counteract the toxin. Consequently, the toxin becomes activated and can act on its cellular targets. Typically, TA toxins act on crucial cellular processes including translation, replication, cytoskeleton formation, membrane integrity, and cell wall biosynthesis. TA systems and their components are also versatile tools for a multitude of purposes in basic research and biotechnology. Currently, TA systems are frequently used for selection in cloning and for single protein expression in living bacterial cells. Since several TA toxins exhibit activity in yeast and mammalian cells they may be useful for applications in eukaryotic systems. TA modules are also considered as promising targets for the development of antibacterial drugs and their potential to combat viral infection may aid in controlling infectious diseases.

Keywords: RNA interferase; antitoxin; cloning; protein expression; toxin; translation.

Figure 1. Types of TA systems. (A) The symR/symE module of E. coli as an example for a type I system regulated by interference of toxin mRNA translation. SD, Shine-Dalgarno sequence. (B) Regulation of the type I system hok/sok of plasmid R1. (C) The ratA/tpxA module from Bacillus subtilis represents a type I system where toxin mRNA degradation is promoted. (D) The relB/relE two module type II system from E. coli. (E) The ω-ε-ζ three module type II systems from Streptococcus pyogenes plasmid pSM19035. (F) The toxI/N type III system from the Erwinia carotovora plasmid pECA1039. (G) The yeeU/yeeV type IV system of E. coli. (H) The ghoS/ghoT type V system of E. coli. In this and all subsequent figures the toxin and its encoding gene are shown in orange while the antitoxin and its encoding gene are shown in green.

Figure 2. Functions of plasmid encoded TA systems. (A) Stabilization of plasmids by post segregational killing. (B) Exclusion of co-existent compatible plasmids.

Figure 3. Experimental approaches for identification of TA systems. (A) Shotgun cloning: the genome to be investigated is randomly fragmented, cloned and transformed into E. coli. Clones comprising the toxin but not the antitoxin do not proliferate and are absent in the assembly. (B) Plasmid stabilization: the fragmented DNA is cloned in a vector that can normally replicate in wild type hosts but that is highly unstable in polA^- strains. After several rounds of replica plating under non selective conditions only colonies with an insert mediating plasmid stability still contain plasmids at a high frequency.

Figure 4. Application of TA systems for DNA cloning. (A) Insertion of the gene of interest destroys the toxin gene and allows the bacteria to growth. (B) Principle of the StabyCloning^TM system. (C) Principle of the selection used in the Gateway cloning^TM system.

Figure 5. Strategies for artificial activation of TA systems. (A) Disruption of TA complexes. (B) Prevention of complex formation. (C) Activation of cellular proteases, for instance Lon or Clp. (D) Inhibition of TA transcription. (E) Overexpression of the TA system and subsequent removal of the activating drug. (F) Induction of plasmid loss (for plasmid encoded TA systems).

Figure 6. TA systems as antiviral tools. (A) CD4⁺ cells were transfected with a construct containing mazF under control of TAR. After infection of CD4⁺ cells with HIV-1 the viral TAT protein is produced, which binds to the TAR sequence and triggers expression of MazF. The active MazF protein cleaves RNA including HIV-1 and prevents thereby its replication. (B) Cells were transformed with a construct containing a part of mazE (mazEp), a linker and mazF as a polyprotein. The polyprotein remains inactive until the hepatitis C virus (HCV) encoded protease NS3 cleaves the linker. The released active MazF protein cleaves RNA and triggers cell death.