Balancing at survival's edge: the structure and adaptive benefits of prokaryotic toxin–antitoxin partners

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Many prokaryotes express toxin–antitoxin (TA) pairs that are harmful to their hosts if not maintained in delicate balance. The maintenance of potentially lethal toxin–antitoxin pairs could be viewed as a high-risk strategy. However, accumulating evidence suggests that toxin–antitoxin pairs can confer selective evolutionary benefits such as adaptive stress responses, starvation recovery and herd immunity to predation. Many of the known TA pairs interact as proteins, but recent work has identified a new class of antitoxins that are RNA cleavage products. Structural studies have revealed common folds for diverse toxins, highlighting unexpected evolutionary relationships within different toxin classes. TA pairs appear to have diverged in function considerably, to meet the specialised requirements of their varied prokaryotic hosts.

Introduction

Throughout their evolutionary histories, bacteria have experienced intense selective pressure from the physical and chemical environment and from astronomical numbers of niche competitors and predators. They must cope with fluctuations in nutrient availability and the challenges of diverse toxic compounds, including antimicrobials and damaging oxidising agents. Accordingly, bacteria have evolved highly refined response mechanisms to handle a wide spectrum of stress conditions. Toxin–antitoxin (TA) systems provide one such strategy for general and specific stress responses. Originally identified as post-segregational killing systems that ensure the selfish maintenance of plasmids [1], TA systems are now recognised as contributing more selectively beneficial attributes to their host organism. These include responses to antibiotics and nutrient depletion [2], the genesis of subpopulations that survive and persist after antibiotic exposure [3], protection from phage infection [4] and the regulation of pathogenicity [5], amongst others [6, 7].

The fundamental genetic module for a TA system comprises a single promoter controlling expression of bicistronic antitoxin and toxin genes. These genetic loci can be grouped into distinct classes according to the nature of the interacting molecules (Figure 1). In Type I loci, the antitoxin is a transcript that is antisense to the toxin mRNA, and pairing of the two RNAs promotes mutual degradation. Type I systems will not be covered here, but are reviewed elsewhere [8]. In Type II loci, both toxin and antitoxin are proteins and together they form a complex that masks the activity of the toxin. The distinguishing feature of the recently discovered Type III systems is that a proteinaceous toxin is inhibited by an antitoxic RNA [9]. In response to stimuli, the antitoxins are degraded and toxins take effect, often inducing bacteriostasis. Once the stress abates, antitoxin levels are restored, the cognate toxin becomes safely sequestered, and cell growth continues.

TA systems are prevalent throughout the eubacteria and archaea [10, 11, 12, 13], though accruing structural data on representatives of the different TA classes have revealed that toxin proteins belong to only a restricted group of structural families.

Section snippets

Six structural classes of toxin

Toxins of the Type II and Type III TA systems can be sub-divided into six groups, according to toxin structural homology (Table 1). The toxins within a group often show only limited sequence similarity, despite having a common fold [14].

The Kid family members have compact globular folds with twisted anti-parallel beta-sheet cores, enshrouded by alpha-helices [15, 16••]. Many Kid-like toxins cleave mRNAs and have accordingly been dubbed mRNA ‘interferases’ [17]. One exception is CcdB, which

RNA binding and cleavage by Kid family toxins

The Type III system, ToxIN [16••], was identified originally as a phage abortive infection system [4, 31]. The crystal structure of the ToxIN complex reveals that ToxN belongs to the Kid structural family (Table 1). This relationship was not anticipated in advance of the ToxIN crystal structure, because the sequence identity between the ToxN and Kid proteins is very low (∼10% based on the a posteriori structure-based alignment) [16••]. ToxN, however, has more extensive RNA interaction surfaces

Ribosome-dependent endoribonucleases

Great insight into the RelE toxin mechanism has been provided by the structures of Escherichia coli RelE alone and in complex with the Thermus thermophilus ribosome and substrate mRNA [20••]. When bound to the ribosome, RelE mimics A site tRNA facing the P site tRNA. A surface patch of RelE forms extensive interactions with 16S rRNA, but RelE makes few contacts with the 50S subunit or ribosomal proteins [20••]. At the A site, RelE re-orients the 2′OH of the mRNA substrate at the second codon

Antitoxins inactivate toxins in complex

For continued survival, bacterial cells must hold toxins in check, prior to activation. Antitoxins negate cognate toxin activity through a toxin binding domain, which is often natively unstructured until complex formation. Within the Kid family, the C-terminal domain of MazE acts as a single-stranded RNA decoy, wrapping across the surface of MazF, blocking the active site and forcing out the S1–S2 loop that, in Kid, provides the stabilizing H17 of the catalytic triad [38]. The Kid family

Transcriptional autoregulation by toxin–antitoxin complexes

Known Type II antitoxins, though diverse, usually have a second, structured, DNA-binding domain. They can be loosely grouped according to this motif (Table 1). Via this domain, certain antitoxins maintain a balanced stoichiometry of toxin to antitoxin by negatively regulating transcription of their cognate locus. Often, a TA complex will have greater repressive action over the antitoxin alone. Within a specific toxin group, cognate antitoxins will have differing DNA-binding domains, such as for

Concluding remarks

TA systems are ubiquitous throughout prokaryotic lineages, and work from recent years has expanded our knowledge of their structures and illuminated their functions. There are currently few basic toxin folds, which appear to have diverged tremendously in sequence whilst adapting to stresses of varied nature. Much still remains to be learned for these systems regarding their functionality, substrate binding specificity and how particular stresses are sensed and lead to activation. As mechanistic

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The authors are supported by grants from the Biotechnology and Biological Sciences Research Council (UK) and the Wellcome Trust (UK).

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      Meanwhile, the antitoxin molecule, which under normal conditions acts as a potent inhibitor of its cognate toxin, is usually degraded under stress conditions, thus allowing the toxin to function [2,6]. Based on the toxin-neutralizing mechanisms of the antitoxin, TA systems are classified into six different types (type I to VI) [6,7]. Type II system constitutes the most-well characterized group of TAs, and these employ proteins as both the toxin and antitoxin molecules [8].

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