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Abstract Toxin–antitoxin systems (TAs) are ubiquitous among bacteria and play a crucial role in the dissemination and evolution of antibiotic resistance, such as maintaining multi-resistant plasmids and inducing persistence formation. Generally, activities of the toxins are neutralised by their conjugate antitoxins. In contrast, antitoxins are more liable to degrade under specific conditions such as stress, and free active toxins interfere with essential cellular processes including replication, translation and cell-wall synthesis. TAs have also been shown to be responsible for plasmid maintenance, stress management, bacterial persistence and biofilm formation. We discuss here the recent findings of these multifaceted TAs (type I–VI) and in particular examine the role of TAs in augmenting the dissemination and maintenance of multi-drug resistance in bacteria. As antimicrobial resistance continues to escalate in Gram-negative bacteria, understanding the role of toxin–antitoxin systems in plasmid maintenance and inducing persistence becomes increasingly important.

Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ϕ80, a notorious laboratory contaminant, apparently caused several of the phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon but no longer support a role of TA modules in E. coli persister formation under unstressed conditions. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field. IMPORTANCE The recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies identified multiple genetic pathways involved in persister formation but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study, we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely differing results based on poorly controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis for future studies on the formation and eradication of antibiotic-tolerant persisters. The recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies identified multiple genetic pathways involved in persister formation but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study, we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely differing results based on poorly controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis for future studies on the formation and eradication of antibiotic-tolerant persisters.

Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. The ability of TA-encoded toxins to affect stress tolerance when overexpressed supports the hypothesis of TA systems being associated with stress adaptation. However, the deletion of TA genes has usually no effects on stress tolerance, supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of chromosomal TA systems to Pseudomonas putida . We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence or biofilm formation. Our results instead show that TA loci are costly and decrease the competitive fitness of P. putida . Still, the cost of multiple TA systems is low and detectable in certain conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or moderately potent proteins. Analysis of P. putida TA systems’ homologs among fully sequenced Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems spread among bacteria by horizontal gene transfer.

Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race 1 – 5 . While the mechanisms of many bacterial antiphage defence systems are known 1 , how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin–antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor. The bacterial phage anti-restriction-induced system, PARIS, operates as a toxin–antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr.

GCN5-related N -acetyl-transferase (GNAT)-like enzymes from toxin–antitoxin modules are strong inhibitors of protein synthesis. Here, we present the bases of the regulatory mechanisms of ataRT , a model GNAT-toxin–antitoxin module, from toxin synthesis to its action as a transcriptional de-repressor. We show the antitoxin (AtaR) traps the toxin (AtaT) in a pre-catalytic monomeric state and precludes the effective binding of ac-CoA and its target Met-transfer RNA fMet . In the repressor complex, AtaR intrinsically disordered region interacts with AtaT at two different sites, folding into different structures, that are involved in two separate functional roles, toxin neutralization and placing the DNA-binding domains of AtaR in a binding-compatible orientation. Our data suggests AtaR neutralizes AtaT as a monomer, right after its synthesis and only the toxin–antitoxin complex formed in this way is an active repressor. Once activated by dimerization, later neutralization of the toxin results in a toxin–antitoxin complex that is not able to repress transcription. Structural and biochemical analysis of the AtaR–AtaT toxin–antioxin system reveals that AtaR traps AtaT in a precatalytic monomeric state and forms a heterohexameric complex to neutralize AtaT and repress transcription of the operon.

Toxin-antitoxin (TA) systems were initially discovered as plasmid addiction systems on low-copy-number plasmids. Thousands of TA loci have since been identified on chromosomes, plasmids and mobile elements in bacteria and archaea with diverse roles in bacterial physiology and in maintenance of genetic elements. Here, we identified and characterised a plasmid mediated type II TA system in Enterobacteriaceae as a member of the ParDE super family. This system (hereafter, ParDE I ) is distributed among IncI and IncF-type antibiotic resistance and virulence plasmids found in avian and human-source Escherichia coli and Salmonella . It is found that ParDE I is a plasmid stability and stress response module that increases tolerance of aminoglycoside, quinolone and β-lactam antibiotics in E. coli by ~100–1,000-fold, and thus to levels beyond those achievable in the course of antibiotic therapy for human infections. ParDE I also confers a clear survival advantage at 42 °C and expression of the ParE I toxin in trans induces the SOS response, inhibits cell division and promotes biofilm formation. This transmissible high-level antibiotic tolerance is likely to be an important factor in the success of the IncI and IncF plasmids which carry it and the important pathogens in which these are resident.

Bacterial toxin-antitoxin (TA) systems, which were discovered in 1985, have since been studied extensively. These studies have focused particularly on the distribution of these bacterial TA systems on either plasmids or on bacterial chromosomes, their functionality, their targets, their relation to virulence, and their mechanisms of action. Bacterial toxin-antitoxin (TA) systems encode a toxin and an antitoxin that counteracts the toxin. Such TA systems are found abundantly on bacterial chromosomes and on extrachromosomal genetic elements. The toxin is always a protein. Based on the nature of the antitoxin (protein or RNA) and on their mode of regulation, they are classified into six groups (I to VI). In the group II TA systems, both the toxin and the antitoxin are proteins, and the gene specifying the antitoxin precedes the gene specifying for the toxin. Here, we studied the intracellular localization in Escherichia coli cells of the proteins specified by the following type II TA modules: mazEF , chpBIK , mqsRA , and rnlAB . We visualized the localization of these proteins by fusing them with the fluorescent protein mCherry using recombinant DNA technology. We used fluorescence microscopy and image analysis software to obtain and quantify protein distribution data. With the exception of the chpBIK TA module, we found that the localization of each toxin-antitoxin complex was different from the localization of the toxin itself. Our results demonstrate clearly that the presence of the antitoxin shifts the localization of its respective toxin toward the middle of the cell, which could contribute to the reduction of cellular toxicity. IMPORTANCE Bacterial toxin-antitoxin (TA) systems, which were discovered in 1985, have since been studied extensively. These studies have focused particularly on the distribution of these bacterial TA systems on either plasmids or on bacterial chromosomes, their functionality, their targets, their relation to virulence, and their mechanisms of action. Our study, reported here, is the first to clarify the intracellular localization of the proteins specified for some type II TA systems. We have shown that, with the exception of the chpBIK module, each toxin-antitoxin complex was localized in a different part of the cell than the toxin itself. Our results revealed clearly that the presence of the antitoxin changes the localization of the toxin by moving the toxin toward the middle of the cell. Until now, the general view has been that the antagonistic effect of the antitoxins over their cognate toxins is based only on their direct structural interactions. Here, we show that this antagonistic effect is also a function of a specific change in the intracellular localization of the toxin.

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin–antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SO x H-containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle. Classical toxin–antitoxin systems in bacteria are based on silencing of a toxin by an antitoxin that, when inactivated, releases the toxin, resulting in a change in metabolism. Here, the authors characterize an oxygen-sensitive toxin–antitoxin system and discuss the implications for the role of the Hha antitoxin.

SecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis , we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB–SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions. SecB homologs can be associated with stress-responsive type II toxin–antitoxin (TA) systems and form tripartite toxin-antitoxin-chaperone systems (TAC). Here the authors provide structural insights into TACs by presenting the crystal structure of the M . tuberculosis TA-associated SecB chaperone in complex with the C-terminal ChAD (chaperone addiction) extension of the antitoxin HigA1.

Burkholderia pseudomallei ( Bpm ) is a bacterial pathogen that causes Melioidosis, a disease with up to 40% mortality and an infection relapse of 15–23% despite antibiotic treatment. Ineffective clearance of Bpm by antibiotics is believed to be due to persistence, a hibernation-like survival mechanism modulated, in part, by toxin–antitoxin systems (TAS). Several organisms possess a repertoire of TASs but defining environmental cues eliciting their activity is hindered by laborious in vitro experiments, especially when there are many toxins with redundant function. Here, we identified which of 103 proteins in Bpm that share features found in toxins of the TAS and repurposed transcriptional data to identify which ones play a role in surviving intracellular host defenses. Putative toxins with the strongest transcriptional response were found to have low conservation between Bpm strains, while toxins that were constitutively expressed were highly conserved. Further examination of highly conserved toxins BPSS0899, BPSS1321, and BPSL1494 showed that they were functional, and their mutation led to reduce survival within macrophages and reduced in vivo persistence-associated pathology (abscesses) during treatment, but did not affect macrophages persistence. These findings highlight the utility of a data-driven approach to select putative toxins and suggests a selective role for some TAS in host survival.