Molecular basis for the reversible ADP-ribosylation of guanosine bases.

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection.

Toxin-antitoxin (TA) systems are widespread in bacteria, but their activation mechanisms and bona fide targets remain largely unknown. Here, we characterize a type III TA system, toxIN, that protects E. coli against multiple bacteriophages, including T4. Using RNA sequencing, we find that the endoribonuclease ToxN is activated following T4 infection and blocks phage development primarily by cleaving viral mRNAs and inhibiting their translation. ToxN activation arises from T4-induced shutoff of host transcription, specifically of toxIN, leading to loss of the intrinsically unstable toxI antitoxin. Transcriptional shutoff is necessary and sufficient for ToxN activation. Notably, toxIN does not strongly protect against another phage, T7, which incompletely blocks host transcription. Thus, our results reveal a critical trade-off in blocking host transcription: it helps phage commandeer host resources but can activate potent defense systems. More generally, our results now reveal the native targets of an RNase toxin and activation mechanism of a phage-defensive TA system.

Toxin-Antidote Elements Across the Tree of Life.

In life's constant battle for survival, it takes one to kill but two to conquer. Toxin-antitoxin or toxin-antidote (TA) elements are genetic dyads that cheat the laws of inheritance to guarantee their transmission to the next generation. This seemingly simple genetic arrangement-a toxin linked to its antidote-is capable of quickly spreading and persisting in natural populations. TA elements were first discovered in bacterial plasmids in the 1980s and have recently been characterized in fungi, plants, and animals, where they underlie genetic incompatibilities and sterility in crosses between wild isolates. In this review, we provide a unified view of TA elements in both prokaryotic and eukaryotic organisms and highlight their similarities and differences at the evolutionary, genetic, and molecular levels. Finally, we propose several scenarios that could explain the paradox of the evolutionary origin of TA elements and argue that these elements may be key evolutionary players and that the full scope of their roles is only beginning to be uncovered.

Toxin-Antitoxin Systems as Phage Defense Elements.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacteria that consist of a growth-inhibiting toxin and its cognate antitoxin. These systems are prevalent in bacterial chromosomes, plasmids, and phage genomes, but individual systems are not highly conserved, even among closely related strains. The biological functions of TA systems have been controversial and enigmatic, although a handful of these systems have been shown to defend bacteria against their viral predators, bacteriophages. Additionally, their patterns of conservation-ubiquitous, but rapidly acquired and lost from genomes-as well as the co-occurrence of some TA systems with known phage defense elements are suggestive of a broader role in mediating phage defense. Here, we review the existing evidence for phage defense mediated by TA systems, highlighting how toxins are activated by phage infection and how toxins disrupt phage replication. We also discuss phage-encoded systems that counteract TA systems, underscoring the ongoing coevolutionary battle between bacteria and phage. We anticipate that TA systems will continue to emerge as central players in the innate immunity of bacteria against phage.

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

An RNA pseudoknot mediates toxin translation and antitoxin inhibition.

Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth-inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation-competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full-length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which "structural processing" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion.

Global Analysis of the E. coli Toxin MazF Reveals Widespread Cleavage of mRNA and the Inhibition of rRNA Maturation and Ribosome Biogenesis.

Toxin-antitoxin systems are widely distributed genetic modules that regulate growth and persistence in bacteria. Many systems, including E. coli MazEF, include toxins that are endoribonucleases, but the full set of targets for these toxins remains poorly defined. Previous studies on a limited set of transcripts suggested that MazF creates a pool of leaderless mRNAs that are preferentially translated by specialized ribosomes created through MazF cleavage of mature 16S rRNA. Here, using paired-end RNA sequencing (RNA-seq) and ribosome profiling, we provide a comprehensive, global analysis of MazF cleavage specificity and its targets. We find that MazF cleaves most transcripts at multiple sites within their coding regions, with very few full-length, leaderless mRNAs created. Additionally, our results demonstrate that MazF does not create a large pool of specialized ribosomes but instead rapidly disrupts ribosome biogenesis by targeting both ribosomal protein transcripts and rRNA precursors, helping to inhibit cell growth.

Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology.

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. Antitoxins are proteins or RNAs that often control their cognate toxins through direct interactions and, in conjunction with other signaling elements, through transcriptional and translational regulation of TA module expression. Three major biological functions of TA modules have been discovered, post-segregational killing ("plasmid addiction"), abortive infection (bacteriophage immunity through altruistic suicide), and persister formation (antibiotic tolerance through dormancy). In this review, we summarize the current state of the field and highlight how multiple levels of regulation shape the conditions of toxin activation to achieve the different biological functions of TA modules.

Toxic antiphage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing α helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.

A type II toxin-antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R.

RNase R (encoded by the rnr gene) is a highly processive 3' → 5' exoribonuclease essential for the growth of the psychrotrophic bacterium Pseudomonas syringae Lz4W at low temperature. The cell death of a rnr deletion mutant at low temperature has been previously attributed to processing defects in 16S rRNA, defective ribosomal assembly, and inefficient protein synthesis. We recently showed that RNase R is required to protect P. syringae Lz4W from DNA damage and oxidative stress, independent of its exoribonuclease activity. Here, we show that the processing defect in 16S rRNA does not cause cell death of the rnr mutant of P. syringae at low temperature. Our results demonstrate that the rnr mutant of P. syringae Lz4W, complemented with a RNase R deficient in exoribonuclease function (RNase RD284A), is defective in 16S rRNA processing but can grow at 4 °C. This suggested that the processing defect in ribosomal RNAs is not a cause of the cold sensitivity of the rnr mutant. We further show that the rnr mutant accumulates copies of the indigenous plasmid pLz4W that bears a type II toxin-antitoxin (TA) system (P. syringae antitoxin-P. syringae toxin). This phenotype was rescued by overexpressing antitoxin psA in the rnr mutant, suggesting that activation of the type II TA system leads to cold sensitivity of the rnr mutant of P. syringae Lz4W. Here, we report a previously unknown functional relationship between the cold sensitivity of the rnr mutant and a type II TA system in P. syringae Lz4W.

Unlocking the potential of microbiome editing: A review of conjugation-based delivery.

In recent decades, there has been a rapid increase in the prevalence of multidrug-resistant pathogens, posing a challenge to modern antibiotic-based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co-inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach.

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

Evaluation of different strategies to produce Vibrio cholerae ParE2 toxin.

Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements, bacteriophage protection, stress response and possibly persister formation. A major bottleneck in the study of TA toxins is the production of sufficient amounts of well-folded, functional protein. Here we examine alternative approaches for obtaining the VcParE2 toxin from Vibrio cholerae. VcParE2 can be successfully produced via bacterial expression in presence of its cognate antitoxin VcParD2, followed by on-column unfolding and refolding. Alternatively, the toxin can be expressed in Spodoptera frugiperda (Sf9) insect cells. The latter requires disruption of the VcparE2 gene via introduction of an insect cell intron. Both methods provide protein with similar structural and functional characteristics.

Comparative analysis of genes expression involved in type II toxin-antitoxin system in Staphylococcus aureus following persister cell formation.

BACKGROUND: The formation of persister cells is the main reason for persistent infections. They are associated with antibiotic treatment failure and subsequently chronic infection. The study aimed to assess the expression of type II toxin/antitoxin (TA) system genes in persister cells of Staphylococcus aureus in the presence of the following antibiotics vancomycin, ciprofloxacin, and gentamicin in exponential and stationary phases. METHODS AND RESULTS: The colony count was used to evaluate the effect of different types of antibiotics on S. aureus persister cell formation during exponential and stationary phases. Moreover, the expression level of TA systems and clpP genes in the persister population in exponential and stationary phases were measured by quantitative reverse transcriptase real-time PCR (qRT-PCR). The results of the study showed the presence of persister phenotype of S. aureus strains in the attendance of bactericidal antibiotics in comparison to the control group during the exponential and stationary phases. Moreover, qRT-PCR resulted in the fact that the role of TA systems involved in the persister cell formation depends on the bacterial growth phase and the type of strain and antibiotic. CONCLUSIONS: In total, the present study provides some data on the persister cell formation and the possible role of TA system genes in this process.

Genome wide screening to discover novel toxin-antitoxin modules in Mycobacterium indicus pranii; perspective on gene acquisition during mycobacterial evolution.

Mycobacterium indicus pranii (MIP), a benign saprophyte with potent immunomodulatory attributes, holds a pivotal position in mycobacterial evolution, potentially serving as the precursor to the pathogenic Mycobacterium avium complex (MAC). Despite its established immunotherapeutic efficacy against leprosy and notable outcomes in gram-negative sepsis and COVID-19 cases, the genomic and biochemical features of MIP remain largely elusive. This study explores the uncharted territory of toxin-antitoxin (TA) systems within MIP, hypothesizing their role in mycobacterial pathogenicity regulation. Genome-wide screening, employing diverse databases, unveils putative TA modules in MIP, setting the stage for a comparative analysis with known modules in Mycobacterium tuberculosis, Mycobacterium smegmatis, Escherichia coli, and Vibrio cholerae. The study further delves into the TA network of MAC and Mycobacterium intracellulare, unraveling interactive properties and family characteristics of identified TA modules in MIP. This comprehensive exploration seeks to illuminate the contribution of TA modules in regulating virulence, habitat diversification, and the evolutionary pathogenicity of mycobacteria. The insights garnered from this investigation not only enhance our understanding of MIP's potential as a vaccine candidate but also hold promise in optimizing tuberculosis drug regimens for expedited recovery.

Back to the Roots: Deep View into the Evolutionary History of ADP-Ribosylation Opened by the DNA-Targeting Toxin-Antitoxin Module DarTG.

In this issue of Molecular Cell, Jankevicius et al. (2016) characterize the DarTG toxin-antitoxin module in which the DarT toxin ADP-ribosylates single-stranded DNA and the DarG antitoxin counteracts DarT by direct binding and by enzymatic removal of the ADP-ribosylation.

Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

RNA-based regulation in bacteria-phage interactions.

Interactions of bacteria with their viruses named bacteriophages or phages shape the bacterial genome evolution and contribute to the diversity of phages. RNAs have emerged as key components of several anti-phage defense systems in bacteria including CRISPR-Cas, toxin-antitoxin and abortive infection. Frequent association with mobile genetic elements and interplay between different anti-phage defense systems are largely discussed. Newly discovered defense systems such as retrons and CBASS include RNA components. RNAs also perform their well-recognized regulatory roles in crossroad of phage-bacteria regulatory networks. Both regulatory and defensive function can be sometimes attributed to the same RNA molecules including CRISPR RNAs. This review presents the recent advances on the role of RNAs in the bacteria-phage interactions with a particular focus on clostridial species including an important human pathogen, Clostridioides difficile.

HEPN RNases - an emerging class of functionally distinct RNA processing and degradation enzymes.

HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) RNases are an emerging class of functionally diverse RNA processing and degradation enzymes. Members are defined by a small α-helical bundle encompassing a short consensus RNase motif. HEPN dimerization is a universal requirement for RNase activation as the conserved RNase motifs are precisely positioned at the dimer interface to form a composite catalytic center. While the core HEPN fold is conserved, the organization surrounding the HEPN dimer can support large structural deviations that contribute to their specialized functions. HEPN RNases are conserved throughout evolution and include bacterial HEPN RNases such as CRISPR-Cas and toxin-antitoxin associated nucleases, as well as eukaryotic HEPN RNases that adopt large multi-component machines. Here we summarize the canonical elements of the growing HEPN RNase family and identify molecular features that influence RNase function and regulation. We explore similarities and differences between members of the HEPN RNase family and describe the current mechanisms for HEPN RNase activation and inhibition.