Multiple Toxin-antitoxin Systems Research Articles

Landscape of the metaplasmidome of deep-sea hydrothermal vents located at Arctic Mid-Ocean Ridges in the Norwegian-Greenland Sea: ecological insights from comparative analysis of plasmid identification tools.

Plasmids are one of the key drivers of microbial adaptation and evolution. However, their diversity and role in adaptation, especially in extreme environments, remains largely unexplored. In this study, we aimed to identify, characterize, and compare plasmid sequences originating from samples collected from deep-sea hydrothermal vents located in Arctic Mid-Ocean Ridges. To achieve this, we employed, and benchmarked three recently developed plasmid identification tools-PlasX, GeNomad, and PLASMe-on metagenomic data from this unique ecosystem. To date, this is the first direct comparison of these computational methods in the context of data from extreme environments. Upon recovery of plasmid contigs, we performed a multiapproach analysis, focusing on identifying taxonomic and functional biases within datasets originating from each tool. Next, we implemented a majority voting system to identify high-confidence plasmid contigs, enhancing the reliability of our findings. By analysing the consensus plasmid sequences, we gained insights into their diversity, ecological roles, and adaptive significance. Within the high-confidence sequences, we identified a high abundance of Pseudomonadota and Campylobacterota, as well as multiple toxin-antitoxin systems. Our findings ensure a deeper understanding of how plasmids contribute to shaping microbial communities living under extreme conditions of hydrothermal vents, potentially uncovering novel adaptive mechanisms.

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli.

Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA , from a phylogenetically distant bacterium, Salmonella typhimurium, which drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tuneable transient genetically modified organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.

GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes.

Carbapenem and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter , but many plasmid types in this genus have not been well-characterized. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains bla NDM-1 and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by pdif sites recognized by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1186 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin–antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus.

Control of Toxin-Antitoxin Systems by Proteases in Mycobacterium Tuberculosis.

Toxin-antitoxin (TA) systems are small genetic elements composed of a noxious toxin and a counteracting cognate antitoxin. Although they are widespread in bacterial chromosomes and in mobile genetic elements, their cellular functions and activation mechanisms remain largely unknown. It has been proposed that toxin activation or expression of the TA operon could rely on the degradation of generally less stable antitoxins by cellular proteases. The resulting active toxin would then target essential cellular processes and inhibit bacterial growth. Although interplay between proteases and TA systems has been observed, evidences for such activation cycle are very limited. Herein, we present an overview of the current knowledge on TA recognition by proteases with a main focus on the major human pathogen Mycobacterium tuberculosis, which harbours multiple TA systems (over 80), the essential AAA + stress proteases, ClpC1P1P2 and ClpXP1P2, and the Pup-proteasome system.

Composition and potential functions of the dominant microbiota in deep-sea hagfish gut from the South China Sea

Hagfishes, the most ancient jawless fishes, have a pivotal biological status. Gut bacterial communities are considered to be another organ that supports host survival and reproduction, while most gut microbiota cannot be cultured in vitro. In this study, four deep-sea hagfish individuals were collected from the South China Sea (>900 m). 16S rRNA and metagenomics approaches were used to analyze the gut microbiota assembly and the functions of the dominant microbes. The results revealed 16 phyla and 21 genera (relative abundance ≥ 0.5%) of microbes in total. Ureaplasma and Arcobacter were the dominant genera across all the gut samples, and the microbiota diversity showed no significant differences among the different gut regions. To investigate the potential functions of the dominant microbial taxa, the draft genomes of Ureaplasma (Hg1 and Hg2) and Arcobacter (LC1) from the midgut and hindgut were successfully recovered. The Hg1 and Hg2 genomes were enriched in genes encoding phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) and sodium-driven symporters responsible for the uptake of sugars and other carbohydrates. The functional repertoire of Ureaplasma was likely to help the host in the metabolism of multiple carbohydrates, especially chitin degradation. The LC1 genome harbored genes encoding a restriction-methylation system, two CRISPR-Cas systems and multiple toxin-antitoxin systems. The enrichment of genes related to bacterial defense indicated that the functions of LC1 may involve protection against harmful or foreign microorganisms and balancing the host intestinal flora. These results hint that genetic and functional diversification might be present among Ureaplasma and Arcobacter. Though the exact functions of the dominant microbes remain to be further identified, our results allow us a glimpse into hagfish-microbe alliances in deep-sea ecosystems.

Chromosomal toxin-antitoxin systems in Pseudomonas putida are rather selfish than beneficial

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.

The toxic guardians \u2014 multiple toxin-antitoxin systems provide stability, avoid deletions and maintain virulence genes of Pseudomonas syringae virulence plasmids

BackgroundPseudomonas syringae is a γ-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems.ResultsThe three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10− 3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10− 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo.ConclusionsVirulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.

Fic Proteins of Campylobacter fetus subsp. venerealis Form a Network of Functional Toxin-Antitoxin Systems.

Enzymes containing the FIC (filamentation induced by cyclic AMP) domain catalyze post-translational modifications of target proteins. In bacteria the activity of some Fic proteins resembles classical toxin–antitoxin (TA) systems. An excess of toxin over neutralizing antitoxin can enable bacteria to survive some stress conditions by slowing metabolic processes and promoting dormancy. The cell can return to normal growth when sufficient antitoxin is present to block toxin activity. Fic genes of the human and animal pathogen Campylobacter fetus are significantly associated with just one subspecies, which is specifically adapted to the urogenital tract. Here, we demonstrate that the fic genes of virulent isolate C. fetus subsp. venerealis 84-112 form multiple TA systems. Expression of the toxins in Escherichia coli caused filamentation and growth inhibition phenotypes reversible by concomitant antitoxin expression. Key active site residues involved in adenylylation by Fic proteins are conserved in Fic1, Fic3 and Fic4, but degenerated in Fic2. We show that both Fic3 and the non-canonical Fic2 disrupt assembly and function of E. coli ribosomes when expressed independently of a trans-acting antitoxin. Toxicity of the Fic proteins is controlled by different mechanisms. The first involves intramolecular regulation by an inhibitory helix typical for Fic proteins. The second is an unusual neutralization by heterologous Fic–Fic protein interactions. Moreover, a small interacting antitoxin called Fic inhibitory protein 3, which appears unrelated to known Fic antitoxins, has the novel capacity to bind and neutralize Fic toxins encoded in cis and at distant sites. These findings reveal a remarkable system of functional crosstalk occurring between Fic proteins expressed from chromosomal and extrachromosomal modules. Conservation of fic genes in other bacteria that either inhabit or establish pathology in the urogenital tract of humans and animals underscores the significance of these factors for niche-specific adaptation and virulence.

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance.

One of the first identified and best-studied toxin-antitoxin (TA) systems in Escherichia coli is the F-plasmid-based CcdAB system. This system is involved in plasmid maintenance through postsegregational killing. More recently, ccdAB homologs have been found on the chromosome, including in pathogenic strains of E. coli and other bacteria. However, the functional role of chromosomal ccdAB genes, if any, has remained unclear. We show that both the native ccd operon of the E. coli O157 strain (ccdO157) and the ccd operon from the F plasmid (ccdF), when inserted on the E. coli chromosome, lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters, with the O157 operon showing higher protection. While the plasmid-encoded CcdB toxin is a potent gyrase inhibitor and leads to bacterial cell death even under fully repressed conditions, the chromosomally encoded toxin leads to growth inhibition, except at high expression levels, where some cell death is seen. This was further confirmed by transiently activating the chromosomal ccd operon through overexpression of an active-site inactive mutant of F-plasmid-encoded CcdB. Both the ccdF and ccdO157 operons may share common mechanisms for activation under stress conditions, eventually leading to multidrug-tolerant persister cells. This study clearly demonstrates an important role for chromosomal ccd systems in bacterial persistence.IMPORTANCE A large number of free-living and pathogenic bacteria are known to harbor multiple toxin-antitoxin systems, on plasmids as well as on chromosomes. The F-plasmid CcdAB system has been extensively studied and is known to be involved in plasmid maintenance. However, little is known about the function of its chromosomal counterpart, found in several pathogenic E. coli strains. We show that the native chromosomal ccd operon of the E. coli O157 strain is involved in drug tolerance and confers protection from cell death under multiple antibiotic stress conditions. This has implications for generation of potential therapeutics that target these TA systems and has clinical significance because the presence of persisters in an antibiotic-treated population can lead to resuscitation of chronic infection and may contribute to failure of antibiotic treatment.

Synchronized switching of multiple toxin-antitoxin modules by (p)ppGpp fluctuation.

Toxin–antitoxin (TA) loci are widespread in bacteria including important pathogenic species. Recent studies suggest that TA systems play a key role in persister formation. However, the persistence phenotype shows only weak dependence on the number of TA systems, i.e. they are functionally redundant. We use a mathematical model to investigate the interaction of multiple TA systems in the switching between growth and persistence. We explore two scenarios: (i) TA systems are bistable and each TA system experiences its own noise and (ii) the noise in the level of common stress signal (e.g. (p)ppGpp) coordinates all TA systems simultaneously. We find that in the first scenario the exit from the persister state strongly depends on the number of TA systems. However in the second case, we could reproduce the weak dependence. The duration of the high (p)ppGpp state was found to be the key parameter for persistence. The (p)ppGpp-driven synchronized transition of all TA systems results in the redundancy.

Unrelated toxin-antitoxin systems cooperate to induce persistence.

Persisters are drug-tolerant bacteria that account for the majority of bacterial infections. They are not mutants, rather, they are slow-growing cells in an otherwise normally growing population. It is known that the frequency of persisters in a population is correlated with the number of toxin–antitoxin systems in the organism. Our previous work provided a mechanistic link between the two by showing how multiple toxin–antitoxin systems, which are present in nearly all bacteria, can cooperate to induce bistable toxin concentrations that result in a heterogeneous population of slow- and fast-growing cells. As such, the slow-growing persisters are a bet-hedging subpopulation maintained under normal conditions. For technical reasons, the model assumed that the kinetic parameters of the various toxin–antitoxin systems in the cell are identical, but experimental data indicate that they differ, sometimes dramatically. Thus, a critical question remains: whether toxin–antitoxin systems from the diverse families, often found together in a cell, with significantly different kinetics, can cooperate in a similar manner. Here, we characterize the interaction of toxin–antitoxin systems from many families that are unrelated and kinetically diverse, and identify the essential determinant for their cooperation. The generic architecture of toxin–antitoxin systems provides the potential for bistability, and our results show that even when they do not exhibit bistability alone, unrelated systems can be coupled by the growth rate to create a strongly bistable, hysteretic switch between normal (fast-growing) and persistent (slow-growing) states. Different combinations of kinetic parameters can produce similar toxic switching thresholds, and the proximity of the thresholds is the primary determinant of bistability. Stochastic fluctuations can spontaneously switch all of the toxin–antitoxin systems in a cell at once. The spontaneous switch creates a heterogeneous population of growing and non-growing cells, typical of persisters, that exist under normal conditions, rather than only as an induced response. The frequency of persisters in the population can be tuned for a particular environmental niche by mixing and matching unrelated systems via mutation, horizontal gene transfer and selection.

Toxin-antitoxin systems mqsR/ygiT and dinJ/relE of Xylella fastidiosa

Xylella fastidiosa encodes multiple toxin-antitoxin systems, including dinJ/relE and mqsR/ygiT. Phylogenetic analyses indicate these two toxin-antitoxin systems have distinct evolutionary histories. Comparisons among X. fastidiosa subspecies/strains reveal toxin-antitoxin systems are often embedded in prophage sequences, suggesting acquisition via horizontal transfer. Tagged proteins of both toxin-antitoxin systems were over-expressed, purified, and evaluated for activity. Toxins MqsR and RelE are ribonucleases. MqsR cleaved at G▲CU and G▲CC; RelE lacked specificity in vitro. YgiT and DinJ antitoxins inhibit activity of the cognate toxin by direct binding. Single (toxin or antitoxin) and double (toxin and antitoxin) knock-out mutants were constructed in X. fastidiosa strain Temecula. Both antitoxin mutants displayed reduced planktonic cell density relative to wild type. The dinJ− mutant produced less biofilm, whereas the ygiT− mutant produced more biofilm, relative to wild type. Planktonic cell density of both toxin mutants was unaffected, with biofilm reduced slightly. Planktonic cell density and biofilm formation of double mutants were similar to wild type. However, viable cell counts for planktonic cells and biofilm of double mutants were more than one order of magnitude greater than wild type. Collectively, the results indicate that dinJ/relE and mqsR/ygiT are functional toxin-antitoxin systems and likely play different roles in regulation of X. fastidiosa population levels.

Multiple Toxin-Antitoxin Systems in Mycobacterium tuberculosis

The hallmark of Mycobacterium tuberculosis is its ability to persist for a long-term in host granulomas, in a non-replicating and drug-tolerant state, and later awaken to cause disease. To date, the cellular factors and the molecular mechanisms that mediate entry into the persistence phase are poorly understood. Remarkably, M. tuberculosis possesses a very high number of toxin-antitoxin (TA) systems in its chromosome, 79 in total, regrouping both well-known (68) and novel (11) families, with some of them being strongly induced in drug-tolerant persisters. In agreement with the capacity of stress-responsive TA systems to generate persisters in other bacteria, it has been proposed that activation of TA systems in M. tuberculosis could contribute to its pathogenesis. Herein, we review the current knowledge on the multiple TA families present in this bacterium, their mechanism, and their potential role in physiology and virulence.

Molecular mechanisms of multiple toxin–antitoxin systems are coordinated to govern the persister phenotype

Toxin-antitoxin systems are ubiquitous and have been implicated in persistence, the multidrug tolerance of bacteria, biofilms, and, by extension, most chronic infections. However, their purpose, apparent redundancy, and coordination remain topics of debate. Our model relates molecular mechanisms to population dynamics for a large class of toxin-antitoxin systems and suggests answers to several of the open questions. The generic architecture of toxin-antitoxin systems provides the potential for bistability, and even when the systems do not exhibit bistability alone, they can be coupled to create a strongly bistable, hysteretic switch between normal and toxic states. Stochastic fluctuations can spontaneously switch the system to the toxic state, creating a heterogeneous population of growing and nongrowing cells, or persisters, that exist under normal conditions, rather than as an induced response. Multiple toxin-antitoxin systems can be cooperatively marshaled for greater effect, with the dilution determined by growth rate serving as the coordinating signal. The model predicts and elucidates experimental results that show a characteristic correlation between persister frequency and the number of toxin-antitoxin systems.

Toxin-Antitoxin Systems in Escherichia coli Influence Biofilm Formation through YjgK (TabA) and Fimbriae

The roles of toxin-antitoxin (TA) systems in bacteria have been debated. Here, the role of five TA systems in regard to biofilm development was investigated (listed as toxin/antitoxin: MazF/MazE, RelE/RelB, ChpB, YoeB/YefM, and YafQ/DinJ). Although these multiple TA systems were reported previously to not impact bacterial fitness, we found that deletion of the five TA systems decreased biofilm formation initially (8 h) on three different surfaces and then increased biofilm formation (24 h) by decreasing biofilm dispersal. Whole-transcriptome profiling revealed that the deletion of the five TA systems induced expression of a single gene, yjgK, which encodes an uncharacterized protein; quantitative real-time PCR (qRT-PCR) confirmed consistent induction of this gene (at 8, 15, and 24 h). Corroborating the complex phenotype seen upon deleting the TA systems, overexpression of YjgK decreased biofilm formation at 8 h and increased biofilm formation at 24 h; deletion of yjgK also affected biofilm formation in the expected manner by increasing biofilm formation after 8 h and decreasing biofilm formation after 24 h. In addition, YjgK significantly reduced biofilm dispersal. Whole-transcriptome profiling revealed YjgK represses fimbria genes at 8 h (corroborated by qRT-PCR and a yeast agglutination assay), which agrees with the decrease in biofilm formation upon deleting the five TA systems at 8 h, as well as that seen upon overexpressing YjgK. Sand column assays confirmed that deleting the five TA systems reduced cell attachment. Furthermore, deletion of each of the five toxins increased biofilm formation at 8 h, and overexpression of the five toxins repressed biofilm formation at 8 h, a result that is opposite that of deleting all five TA systems; this suggests that complex regulation occurs involving the antitoxins. Also, the ability of the global regulator Hha to reduce biofilm formation was dependent on the presence of these TA systems. Hence, we suggest that one role of TA systems is to influence biofilm formation.

What Is the Benefit to Escherichia coli of Having Multiple Toxin-Antitoxin Systems in Its Genome?

The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.