Tag Archives: toxin-antitoxin

Beating a Toxin-Antitoxin System; Evading Suicide

Bacteria have evolved many different systems to evade viral predation. One strategy, abortive infection (Abi), involves altruistic suicide. Mediated by a toxin-antitoxin (TA) system, the suicide of the infected cell protects the clonal bacterial population by preventing the spread of replicated bacteriophage. A new paper in Plos Genetics has discovered a molecular mimicry-based strategy that allows phage to escape abortive infection.

Toxin-antitoxin systems are widespread prokaryotic genetic elements found on both plasmids and bacterial chromosomes. Encoding a relatively long-lived toxin and a more labile antitoxin expressed from a single bi-cistronic operon, they were originally characterised as ‘addiction modules’. In the event that a plasmid expressing a TA system fails to be inherited by a daughter cell, the absence of antitoxin allows the persisting toxin to kill the cell – post-segregational killing. These attributes as ‘selfish elements’ made it slightly surprising that so many TA systems have been found encoded on bacterial chromosomes themselves. I’ve previously written about an example of one such TA system’s activity in mediating a stress response in E. coli, and they’ve also been implicated in the formation of antibiotic resisting ‘persister’ cells.

TA systems have been classified into three different classes defined by the level of the molecular interaction between their two components. In type I systems, translation of the toxin is prevented by an antisense RNA antitoxin binding to its’ transcript, whilst in type II systems both partners are proteins. Most recently, type III TA systems have been characterised in which the toxin is neutralised by binding of an RNA antitoxin. Examples of all three varieties have been found to protect bacteria from phage infection via abortive infection; phage replication disrupts the normal cellular transcriptional program, interrupting antitoxin production and hence leading to cell death.

The first type III TA system to be characterised, ToxIN, was found on plasmids in the phytopathogen, Pectobacterium atrosepticum (Pba), and shown to inhibit the propagation of multiple different bacteriophage. ToxN is an endoribonuclease, whilst the antitoxin ToxI, is a 36nt RNA structured as a ‘pseudoknot’. The partners combine into a hetero-hexameric structure composed of 3 ToxN molecules and 3 ToxI pseudoknots.

Blower et al. have discovered that phage can evade the Abi system by producing molecular mimics of the ToxI RNA. The lytic bacteriophage ΦTE, normally fails to infect Pba carrying a toxIN-containing plasmid. At low frequency however, new phage strains emerge capable of evading the Abi system. Upon sequencing the genomes of these ‘escape strains’, the researchers discovered that they all contained sequence expansions at one specific locus. The toxI locus contains 5.5 repeats of the 36nt RNA pseudoknot-encoding sequence. The ‘escape locus’ from the phage normally encoded 1.5 repeats of a pseudo-ToxI sequence. In all the escape strains this repeat had been expanded so that it contained either 4.5 or 5.5 repeats. These expansions had probably arisen due to strand-slippage during phage replication. In one escape strain homologous recombination had occurred between the phage pseudo-ToxI and the endogenous toxI; the phage had effectively hijacked a normal antitoxin- encoding gene.

The 1.5 repeat pseudo-ToxI could not inhibit Abi (as the sequence was out of phase it did not actually encode a functional psudoknot). However, the repeat expansions had allowed the phage to make an antitoxin mimic that protected them from the TA system and hence Abi.

ΦTE is capable of generalised transduction – the ability to package and transfer chromosomal and plasmid DNA from its’ host and transfer it during infection. Blower et al. showed that one of the ΦTE escape strains is able to transduce the plasmid encoded ToxIN – a case of a bacteriophage horizontally transferring an anti-phage defence mechanism. This brings into focus the complex evolutionary dynamics operating between the three different genetic entities being studied; the bacterial cell, the plasmid encoding the TA system, and the bacteriophage evading it and potentially propagating it. From the selfish viewpoint of the TA module what’s best, preventing the spread of the phage or being disseminated by it? These speculations aren’t about to be easily answered, however, it is an interesting way to analyse further examples of similar systems.

Blower, T., Evans, T., Przybilski, R., Fineran, P., & Salmond, G. (2012). Viral Evasion of a Bacterial Suicide System by RNA–Based Molecular Mimicry Enables Infectious Altruism PLoS Genetics, 8 (10) DOI: 10.1371/journal.pgen.1003023

A modified ribosome mediates stress in E.coli.

Vesper, O., Amitai, S., Belitsky, M., Byrgazov, K., Kaberdina, A., Engelberg-Kulka, H., & Moll, I. (2011). Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli Cell, 147 (1), 147-157 DOI: 10.1016/j.cell.2011.07.047

This paper has characterised an interesting new mechanism of stress adaptation in bacteria in which ribosomes are modified to selectively translate a subset of mRNAs that have also been modified by the same enzyme.

Toxin-antitoxin (TA) modules are widespread prokaryotic genetic elements that have generally been characterised as selfish DNA when encoded on plasmids. Chromosomally located TA systems functions are more likely to be integrated into the host cells regulatory networks. mazEF is a well characterised chromosomal TA system in E.coli. The two genes are cotranscribed as an operon; mazE encoding a relatively labile antitoxin that inactivates the more stable endoribonuclease MazF. Under conditions of cell stress mazEF expression is inhibited. As MazE is less stable and degraded by a protease, MazF activity is released. MazF cleaves single stranded mRNAs at ACA sequences hence inhibiting protein synthesis. However this inhibition is not global: about 10% of protein’s synthesis are specifically enabled by MazF action. Some of these protein’s actions are responsible for programmed cell death, others have been shown to permit the survival of a subpopulation of bacterial cells (Amitai et al.2009). This new paper has uncovered the mechanism by which the selective synthesis of this subset of the cell’s proteins is activated by MazF.

Analysing transcripts encoding proteins known to be synthesised in the presence of MazF activity the authors found that they were cleaved at ACA sequences at or closely upstream of their AUG translation start sites. This creates a population of leaderless mRNAs (lmRNAs) that the paper also shows are selectively translated in the presence of MazF activity. Postulating that the selective translation of lmRNAs could  be mediated by MazF modifications to the ribosome itself, the investigators went on to show that MazF also cleaves the 16S rRNA of the 30S ribosomal subunit. This cleavage results in the loss of 43nt from the 3′ end of the 16S rRNA including the anti-Shine Dalgarno sequence (aSD). SD – aSD interactions are important for the initiation of translation of canonical mRNAs with structured 5′ UTRs. However, in this case MazF generates specialised “Stress Ribosomes” lacking the aSD that selectively translate a “leaderless mRNA regulon” also generated by cleavage by MazF.

This paper is important and interesting in that it has discovered an elegant and novel molecular mechanism employed by bacteria during times of environmental stress. It also adds greatly to understanding bacterial programmed cell death and the functions of chromosomally located TA systems both of which are contentious subjects in relation to how their evolution typifies aspects of both altruism and selfishness.