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Toxin-antitoxin (TA) systems are important for stress adaptation in prokaryotes, including persistence, antibiotic resistance, pathogenicity, and biofilm formation. Toxins can cause cell death, reversible growth stasis, and direct inhibition of crucial cellular processes through various mechanisms, while antitoxins neutralize the effects of toxins. In bacteria, these systems have been studied in detail, whereas their function in archaea remains elusive. During heat stress, the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibited an increase in the expression of several bicistronic type II vapBC TA systems, with the highest expression observed in the vapBC4 system. In the current study, we performed a comprehensive biochemical characterization of the VapBC4 TA system, establishing it as a bonafide type II toxin-antitoxin system. The VapC4 toxin is shown to have high-temperature catalyzed RNase activity specific for mRNA and rRNA, while the VapB4 antitoxin inhibits the toxic activity of VapC4 by interacting with it. VapC4 toxin expression led to heat-induced persister-like cell formation, allowing the cell to cope with the stress. Furthermore, this study explored the impact of vapBC4 deletion on biofilm formation, whereby deletion of vapC4 led to increased biofilm formation, suggesting its role in regulating biofilm formation. Thus, during heat stress, the liberated VapC4 toxin in cells could potentially signal a preference for persister cell formation over biofilm growth. Thus, our findings shed light on the diverse roles of the VapC4 toxin in inhibiting translation, inducing persister cell formation, and regulating biofilm formation in S. acidocaldarius , enhancing our understanding of TA systems in archaea., Importance: This research enhances our knowledge of toxin-antitoxin (TA) systems in archaea, specifically in the thermoacidophilic archaeon Sulfolobus acidocaldarius . TA systems are widespread in both bacterial and archaeal genomes, indicating their evolutionary importance. However, their exact functions in archaeal cellular physiology are still not well understood. This study sheds light on the complex roles of TA systems and their critical involvement in archaeal stress adaptation, including persistence and biofilm formation. By focusing on S. acidocaldarius , which lives in habitats with fluctuating temperatures that can reach up to 90°C, the study reveals the unique challenges and survival mechanisms of this organism. The detailed biochemical analysis of the VapBC4 TA system, and its crucial role during heat stress, provides insights into how extremophiles can survive in harsh conditions. The findings of this study show the various functions of the VapC4 toxin, including inhibiting translation, inducing persister-like cell formation, and regulating biofilm formation. This knowledge improves our understanding of TA systems in thermoacidophiles and has broader implications for understanding how microorganisms adapt to extreme environments.

The Type VI secretory system (T6SS) is a key regulatory network in the bacterial system, which plays an important role in host-pathogen interactions and maintains cell homeostasis by regulating the release of effector proteins in specific competition. T6SS causes cell lysis or competitive inhibition by delivering effector molecules, such as toxic proteins and nucleic acids, directly from donor bacterial cells to eukaryotic or prokaryotic targets. Additionally, it orchestrates synthesis of immune effectors that counteract toxins thus preventing self-intoxication or antagonistic actions by competing microbes. Even so, the mechanism of toxin-antitoxin regulation in bacteria remains unclear. In response, this review discusses the bacterial T6SS's structure and function and the mechanism behind toxin-antitoxin secretion and the T6SS's expression in order to guide the further exploration of the pathogenic mechanism of the T6SS and the development of novel preparations for reducing and replacing toxins and antitoxins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46 , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection., Competing Interests: The authors declare no conflict of interest.

Aims: Klebsiella michiganensis is a medically important bacterium that has been subject to relatively little attention in the literature. Interrogation of sequence data from K. michiganensis strains in our collection has revealed the presence of multiple large plasmids encoding type II toxin-antitoxin (TA) systems. Such TA systems are responsible for mediating a range of phenotypes, including plasmid stability ('addiction') and antibiotic persistence. In this work, we characterize the hipBA TA locus found within the Klebsiella oxytoca species complex (KoSC)., Methods and Results: The HipBA TA system is encoded on a plasmid carried by K. michiganensis PS_Koxy4, isolated from an infection outbreak. Employing viability and plasmid stability assays, we demonstrate that PS_Koxy4 HipA is a potent antibacterial toxin and that HipBA is a functional TA module contributing substantially to plasmid maintenance. Further, we provide in silico data comparing HipBA modules across the entire KoSC., Conclusions: We provide the first evidence of the role of a plasmid-encoded HipBA system in stability of mobile genetic elements and analyse the presence of HipBA across the KoSC. These results expand our knowledge of both a common enterobacterial TA system and a highly medically relevant group of bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Mycobacterium phage Adephagia is a cluster K phage that infects Mycobacterium smegmatis and some strains of Mycobacterium pathogens. Adephagia has a siphoviral virion morphology and is temperate. Its genome is 59,646 bp long and codes for one tRNA gene and 94 predicted protein-coding genes; most genes not associated with virion structure and assembly are functionally ill-defined. Here, we determined the Adephagia gene expression patterns in lytic and lysogenic growth and used structural predictions to assign additional gene functions. We characterized 66 nonstructural genes for their toxic phenotypes when expressed in M. smegmatis, and we show that 25 of these (38%) are either toxic or strongly inhibit growth, resulting in either reduced viability or small colony sizes. Some of these genes are predicted to be involved in DNA metabolism or regulation, but others are of unknown function. We also characterize the HicAB-like toxin-antitoxin (TA) system encoded by Adephagia (gp91 and gp90, respectively) and show that the gp90 antitoxin is lysogenically expressed, abrogates gp91 toxicity, and is required for normal lytic and lysogenic growth., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in Leptospira interrogans serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene vapC in E. coli did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of E. coli co-expressing vapB-vapC genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from L. interrogans . Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium.

Burkholderia pseudomallei ( Bpm ) is a Gram-negative intracellular pathogen that causes melioidosis in humans, a neglected, underreported, and lethal disease that can reach a fatal outcome in over 50% of the cases. It can produce both acute and chronic infections, the latter being particularly challenging to eliminate because of the intracellular life cycle of the bacteria and its ability to generate a "persister" dormant state. The molecular mechanism that allows the switch between growing and persister phenotypes is not well understood but it is hypothesized to be due at least in part to the participation of toxin-antitoxin (TA) systems. We have previously studied the link between one of those systems (defined as HigBA) with specific expression patterns associated with levofloxacin antibiotic exposure. Through in silico methods, we predicted the presence of another three pairs of genes encoding for additional putative HigBA systems. Therefore, our main goal was to establish which mechanisms are conserved as well as which pathways are specific among different Bpm TA systems from the same family. We hypothesize that the high prevalence, and sometimes even redundancy of these systems in the Bpm chromosomes indicates that they can interact with each other and not function as only individual systems, as it was traditionally thought, and might be playing an undefined role in Bpm lifecycle. Here, we show that both the toxin and the antitoxin of the different systems contribute to bacterial survival and that toxins from the same family can have a cumulative effect under environmental stressful conditions., Importance: Toxin-antitoxin (TA) systems play a significant role in bacterial persistence, a phenomenon where bacterial cells enter a dormant or slow-growing state to survive adverse conditions such as nutrient deprivation, antibiotic exposure, or host immune responses. By studying TA systems in Burkholderia pseudomallei , we can gain insights into how this pathogen survives and persists in the host environment, contributing to its virulence and ability to cause melioidosis chronic infections., Competing Interests: The authors declare no conflict of interest.

Bacterial chromosomal type I toxin-antitoxin systems consist of a small protein, typically under 60 amino acids, and a small RNA (sRNA) that represses toxin translation. These gene pairs have gained attention over the last decade for their contribution to antibiotic persistence and phage tolerance in bacteria. However, biological functions for many remain elusive as gene deletions often fail to produce an observable phenotype. For many pairs, it is still unknown when the toxin and/or antitoxin gene are natively expressed within the bacterium. We examined sequence conservation of three type I toxin-antitoxin systems, tisB/istR-1, shoB/ohsC, and zor/orz , in over 2,000 Escherichia coli strains, including pathogenic and commensal isolates. Using our custom database, we found that these gene pairs are widespread across E. coli and have expression potential via BLASTn. We identified an alternative, dominant sequence variant of TisB and confirmed that it is toxic upon overproduction. Additionally, analyses revealed a highly conserved sequence in the zorO mRNA untranslated region that is required for full toxicity. We further noted that over 30% of E. coli genomes contain an orz antitoxin gene only and confirmed its expression in a representative strain: the first confirmed report of a type I antitoxin without its cognate toxin. Our results add to our understanding of these systems, and our methodology is applicable for other type I loci to identify critical regulatory and functional features.IMPORTANCEChromosomal type I toxin-antitoxins are a class of genes that have gained increasing attention over the last decade for their roles in antibiotic persistence which may contribute to therapeutic failures. However, the control of many of these genes and when they function have remained elusive. We demonstrate that a simple genetic conservation-based approach utilizing free, publicly available data yields known and novel insights into the regulation and function of three chromosomal type I toxin-antitoxins in Escherichia coli . This study also provides a framework for how this approach could be applied to other genes of interest., Competing Interests: The authors declare no conflict of interest.

Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Toxin-antitoxin (TA) systems are the major mechanism for persister formation in Mycobacterium tuberculosis ( Mtb ). Previous studies found that HigBA2 (Rv2022c-Rv2021c), a predicted type II TA system of Mtb , could be activated for transcription in response to multiple stresses such as anti-tuberculosis drugs, nutrient starvation, endure hypoxia, acidic pH, etc. In this study, we determined the binding site of HigA2 (Rv2021c), which is located in the coding region of the upstream gene higB2 ( Rv2022c ), and the conserved recognition motif of HigA2 was characterized via oligonucleotide mutation. Eight binding sites of HigA2 were further found in the Mtb genome according to the conserved motif. RT-PCR showed that HigA2 can regulate the transcription level of all eight of these genes and three adjacent downstream genes. DNA pull-down experiments showed that twelve functional regulators sense external regulatory signals and may regulate the transcription of the HigBA2 system. Of these, Rv0903c, Rv0744c, Rv0474, Rv3124, Rv2603c, and Rv3583c may be involved in the regulation of external stress signals. In general, we identified the downstream target genes and possible upstream regulatory genes of HigA2, which paved the way for the illustration of the persistence establishment mechanism in Mtb .

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins also bind to promoter region sequences and repress expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB - vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46 , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation, potentially in response to extracytoplasmic as well as intracellular signals.

In the ongoing battle against antimicrobial resistance, phenotypic drug tolerance poses a formidable challenge. This adaptive ability of microorganisms to withstand drug pressure without genetic alterations further complicating global healthcare challenges. Microbial populations employ an array of persistence mechanisms, including dormancy, biofilm formation, adaptation to intracellular environments, and the adoption of L-forms, to develop drug tolerance. Moreover, molecular mechanisms like toxin-antitoxin modules, oxidative stress responses, energy metabolism, and (p)ppGpp signaling contribute to this phenomenon. Understanding these persistence mechanisms is crucial for predicting drug efficacy, developing strategies for chronic bacterial infections, and exploring innovative therapies for refractory infections. In this comprehensive review, we dissect the intricacies of drug tolerance and persister formation, explore their role in acquired drug resistance, and highlight emerging therapeutic approaches to combat phenotypic drug tolerance. Furthermore, we outline the future landscape of interventions for persistent bacterial infections., (Published by Elsevier B.V.)

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with Δ vapC12 induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with Δ vapC12 succumbed early to the disease. Finally, we reported that the above phenotype of Δ vapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the Δ vapC12 infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tyagi, Sadhu, Sharma, Paul, Pandey, Nain, Rathore, Chatterjee, Awasthi and Pandey.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 β-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., Competing Interests: The authors declare no conflict of interest.

Importance: Microbes use protein toxins as important tools to attack neighboring cells, microbial or eukaryotic, and for self-killing when attacked by viruses. These toxins work through different mechanisms to inhibit cell growth or kill cells. Microbes also use antitoxin proteins to neutralize the toxin activities. Here, we developed a comprehensive database called Toxinome of nearly two million toxins and antitoxins that are encoded in 59,475 bacterial genomes. We described the distribution of bacterial toxins and identified that they are depleted by bacteria that live in hot and cold temperatures. We found 5,161 cases in which toxins and antitoxins are densely clustered in bacterial genomes and termed these areas "Toxin Islands." The Toxinome database is a useful resource for anyone interested in toxin biology and evolution, and it can guide the discovery of new toxins., Competing Interests: The authors declare no conflict of interest.

Arbitrium-coding phages use peptides to communicate and coordinate the decision between lysis and lysogeny. However, the mechanism by which these phages establish lysogeny remains unknown. Here, focusing on the SPbeta phage family's model phages phi3T and SPβ, we report that a six-gene operon called the "SPbeta phages repressor operon" (sro) expresses not one but two master repressors, SroE and SroF, the latter of which folds like a classical phage integrase. To promote lysogeny, these repressors bind to multiple sites in the phage genome. SroD serves as an auxiliary repressor that, with SroEF, forms the repression module necessary for lysogeny establishment and maintenance. Additionally, the proteins SroABC within the operon are proposed to constitute the transducer module, connecting the arbitrium communication system to the activity of the repression module. Overall, this research sheds light on the intricate and specialized repression system employed by arbitrium SPβ-like phages in making lysis-lysogeny decisions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Importance: Bacterial fruit blotch (BFB), which is caused by the seed-borne bacterium Acidovorax citrulli , is a devastating disease affecting cucurbit crops throughout the world. Although seed fermentation and treatment with disinfectants can provide effective management of BFB, they cannot completely guarantee pathogen-free seedstock, which suggests that A. citrulli is a highly stress-resistant pathogen. Toxin-antitoxin (TA) systems are common among a diverse range of bacteria and have been reported to play a role in bacterial stress response. However, there is currently much debate about the relationship between TA systems and stress response in bacteria. The current study characterized a novel TA system (Aave_1720-Aave_1719) from A. citrulli that affects both biofilm formation and survival in response to sodium hypochlorite stress. The mechanism of neutralization differed from typical TA systems as two separate mechanisms were associated with the antitoxin, which exhibited characteristics of both type II and type V TA systems. The Aave_1720-Aave_1719 system described here also constitutes the first known report of a double-ribonuclease TA system in bacteria, which expands our understanding of the range of regulatory mechanisms utilized by bacterial TA systems, providing new insight into the survival of A. citrulli in response to stress., Competing Interests: The authors declare no conflict of interest.

MazEF Toxin-antitoxin (TA) systems are associated with the persistent phenotype of the pathogen, Mycobacterium tuberculosis (Mtb), aiding their survival. Though extensively studied, the mode of action between the antitoxin-toxin and DNA of this family remains largely unclear. Here, the important interactions between MazF7 toxin and MazE7 antitoxin, and how MazE7 binds its promoter/operator region have been studied. To elucidate this, molecular dynamics (MD) simulation has been performed on MazE7, MazF7, MazEF7, MazEF7-DNA, and MazE7-DNA complexes to investigate how MazF7 and DNA affect the conformational change and dynamics of MazE7 antitoxin. This study demonstrated that the MazE7 dimer is disordered and one monomer (Chain C) attains stability after binding to the MazF7 toxin. Both the monomers (Chain C and Chain D) however are stabilized when MazE7 binds to DNA. MazE7 is also observed to sterically inhibit tRNA from binding to MazF7, thus suppressing its toxic activity. Comparative structural analysis performed on all the available antitoxins/antitoxin-toxin-DNA structures revealed MazEF7-DNA mechanism was similar to another TA system, AtaRT_ E.coli. Simulation performed on the crystal structures of AtaR, AtaT, AtaRT, AtaRT-DNA, and AtaR-DNA showed that the disordered AtaR antitoxin attains stability by AtaT and DNA binding similar to MazE7. Based on these analyses it can thus be hypothesized that the disordered antitoxins enable tighter toxin and DNA binding thus preventing accidental toxin activation. Overall, this study provides crucial structural and dynamic insights into the MazEF7 toxin-antitoxin system and should provide a basis for targeting this TA system in combating Mycobacterium tuberculosis .Communicated by Ramaswamy H. Sarma.

Sulfolobus acidocaldarius, a thermoacidophilic crenarchaeon, frequently encounters temperature fluctuations, oxidative stress, and nutrient limitations in its environment. Here, we employed a high-throughput transcriptomic analysis to examine how the gene expression of S. acidocaldarius changes when exposed to high temperatures (92 °C). The data obtained was subsequently validated using quantitative reverse transcription-PCR (qRT-PCR) analysis. Our particular focus was on genes that are involved in the heat shock response, type-II Toxin-Antitoxin systems, and putative transcription factors. To investigate how S. acidocaldarius adapts to multiple stressors, we assessed the expression of these selected genes under oxidative and nutrient stresses using qRT-PCR analysis. The results demonstrated that the gene thβ encoding the β subunit of the thermosome, as well as hsp14 and hsp20, play crucial roles in the majority of stress conditions. Furthermore, we observed overexpression of at least eight different TA pairs belonging to the type II TA systems under all stress conditions. Additionally, four common transcription factors: FadR, TFEβ, CRISPR loci binding protein, and HTH family protein were consistently overexpressed across all stress conditions, indicating their significant role in managing stress. Overall, this work provides the first insight into molecular players involved in the cross-stress adaptation of S. acidocaldarius., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Introduction: Non-tuberculous mycobacteria (NTM) is a major category of environmental bacteria in nature that can be divided into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM) based on their distinct growth rates. To explore differential molecular mechanisms between RGM and SGM is crucial to understand their survival state, environmental/host adaptation and pathogenicity. Comparative genomic analysis provides a powerful tool for deeply investigating differential molecular mechanisms between them. However, large-scale comparative genomic analysis between RGM and SGM is still uncovered., Methods: In this study, we screened 335 high-quality, non-redundant NTM genome sequences covering 187 species from 3,478 online NTM genomes, and then performed a comprehensive comparative genomic analysis to identify differential genomic characteristics and featured genes/protein domains between RGM and SGM., Results: Our findings reveal that RGM has a larger genome size, more genes, lower GC content, and more featured genes/protein domains in metabolism of some main substances (e.g. carbohydrates, amino acids, nucleotides, ions, and coenzymes), energy metabolism, signal transduction, replication, transcription, and translation processes, which are essential for its rapid growth requirements. On the other hand, SGM has a smaller genome size, fewer genes, higher GC content, and more featured genes/protein domains in lipid and secondary metabolite metabolisms and cellular defense mechanisms, which help enhance its genome stability and environmental adaptability. Additionally, orthogroup analysis revealed the important roles of bacterial division and bacteriophage associated genes in RGM and secretion system related genes for better environmental adaptation in SGM. Notably, PCoA analysis of the top 20 genes/protein domains showed precision classification between RGM and SGM, indicating the credibility of our screening/classification strategies., Discussion: Overall, our findings shed light on differential underlying molecular mechanisms in survival state, adaptation and pathogenicity between RGM and SGM, show the potential for our comparative genomic pipeline to investigate differential genes/protein domains at whole genomic level across different bacterial species on a large scale, and provide an important reference and improved understanding of NTM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Wang, Li, Segev, Wang, Wang, Yue, Jiang, Sheng, Levy, Jiang and Chen.)

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 (Rpn S ), which are translated separately from the full-length proteins (Rpn L ), directly block the activities of the toxic Rpn L . The crystal structure of RpnA S 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 RpnP2 L 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.

The most common source of foodborne Salmonella infection in humans is poultry eggs and meat, such that prevention of human infection is mostly achieved by vaccination of farm animals. While inactivated and attenuated vaccines are available, both present drawbacks. This study aimed to develop a novel vaccination strategy, which combines the effectiveness of live-attenuated and safety of inactivated vaccines by construction of inducible self-destructing bacteria utilizing toxin-antitoxin (TA) systems. Hok-Sok and CeaB-CeiB toxin-antitoxin systems were coupled with three induction systems aimed for activating cell killing upon lack of arabinose, anaerobic conditions or low concentration of metallic di-cations. The constructs were transformed into a pathogenic Salmonella enterica serovar Enteritidis strain and bacteria elimination was evaluated in vitro under specific activating conditions and in vivo following administration to chickens. Four constructs induced bacterial killing under the specified conditions, both in growth media and within macrophages. Cloacal swabs of all chicks orally administered transformed bacteria had no detectable levels of bacteria within 9 days of inoculation. By day ten, no bacteria were identified in the spleen and liver of most birds. Antibody immune response was raised toward TA carrying Salmonella which resembled response toward the wildtype bacteria. The constructs described in this study led to self-destruction of virulent Salmonella enteritidis both in vitro and in inoculated animals within a period which is sufficient for the induction of a protective immune response. This system may serve as a safe and effective live vaccine platform against Salmonella as well as other pathogenic bacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Inga vera and Lysiloma tree legumes form nodules with Bradyrhizobium spp. from the japonicum group that represent novel genomospecies, for which we describe here using genome data, symbiovars lysilomae, lysilomaefficiens and ingae. Genes encoding Type three secretion system (TTSS) that could affect host specificity were found in ingae but not in lysilomae nor in lysilomaefficiens symbiovars and uptake hydrogenase hup genes (that affect nitrogen fixation) were observed in bradyrhizobia from the symbiovars ingae and lysilomaefficiens. nolA gene was found in the symbiovar lysilomaefficiens but not in strains from lysilomae. We discuss that multiple genes may dictate symbiosis specificity. Besides, toxin-antitoxin genes were found in the symbiosis islands in bradyrhizobia from symbiovars ingae and lysilomaefficiens. A limit (95%) to define symbiovars with nifH gene sequences was proposed here., (Copyright © 2023. Published by Elsevier GmbH.)

Aim: Persistence cells comprise a subpopulation of bacteria that is resistant to treatment. In this study, the role of the toxin-antitoxin (TA) system in the formation of persistence cells of Acinetobacter baumannii isolates was investigated. Methods: After confirming all isolates, TA systems abkBA, mqsRA and higBA were identified. Persister cells were confirmed using the standard method. Real-time PCR was used to compare the expression of TA systems in isolates in persistence and normal states. Results: The abkAB system was present in all isolates; 4% of isolates formed persister cells. The expression level of the  abkB gene in persistent isolates showed a sevenfold increase compared with nonpersistent isolates. Conclusion:  The abkBA system is proposed as an antipersistence target in A. baumannii isolates.

The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog p -chlorophenylalanine (4-CP) as a negative selection mechanism and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible gene expression cassette (Xyl-S) and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type fst-sm open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3- to 4-log reductions in cell number observed when plating on xylose-supplemented medium. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with Streptococcus gordonii and Streptococcus sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria. IMPORTANCE Multiple medically significant Streptococcus species, such as S. mutans, have highly sophisticated genetic systems available, largely as a consequence of their amenability to genetic manipulation via natural competence. Despite this, few options are available for the creation of markerless mutations in streptococci, especially within wild-type strains. Markerless mutagenesis is a critical tool for genetic studies, as it allows the user to explore many fundamental questions that are not easily addressable using marked mutagenesis. Here, we describe a new approach for streptococcal markerless mutagenesis that offers a variety of advantages over the current approach, which employs induced sensitivity to the toxic substrate 4-CP. The approach employed here should be readily adaptable for the creation of similar markerless mutagenesis systems in other organisms., Competing Interests: The authors declare no conflict of interest.

Toxin-antitoxin (TA) systems are widely present in bacterial genomes. They consist of stable toxins and unstable antitoxins that are classified into distinct groups based on their structure and biological activity. TA systems are mostly related to mobile genetic elements and can be easily acquired through horizontal gene transfer. The ubiquity of different homologous and non-homologous TA systems within a single bacterial genome raises questions about their potential cross-interactions. Unspecific cross-talk between toxins and antitoxins of non-cognate modules may unbalance the ratio of the interacting partners and cause an increase in the free toxin level, which can be deleterious to the cell. Moreover, TA systems can be involved in broadly understood molecular networks as transcriptional regulators of other genes' expression or modulators of cellular mRNA stability. In nature, multiple copies of highly similar or identical TA systems are rather infrequent and probably represent a transition stage during evolution to complete insulation or decay of one of them. Nevertheless, several types of cross-interactions have been described in the literature to date. This implies a question of the possibility and consequences of the TA system cross-interactions, especially in the context of the practical application of the TA-based biotechnological and medical strategies, in which such TAs will be used outside their natural context, will be artificially introduced and induced in the new hosts. Thus, in this review, we discuss the prospective challenges of system cross-talks in the safety and effectiveness of TA system usage.

Introduction. Helicobacter pylori is highly polymorphic, and some strains are much more likely to cause disease than others. Biofilm formation can help bacteria to survive antibiotic treatment, immune attack and other stresses, promoting persistent infection. Hypothesis/Gap Statement. We hypothesized that H. pylori isolates from patients with more severe H. pylori- associated disease would be better at forming biofilms than isolates from patients with less severe disease. Aim. We initially aimed to determine whether or not the biofilm-forming ability of H. pylori isolates was associated with disease in the UK-based patients from whom the bacteria were isolated. Methodology. Biofilm-forming ability of H. pylori isolates was determined using a crystal violet assay on glass coverslips. The complete genome sequence of strain 444A was generated by hybrid assembly of Nanopore MinION and Illumina MiSeq data. Results. Although we found no associations between biofilm-forming ability of H. pylori and disease severity in patients, we discovered that strain 444A had particularly high biofilm-forming ability. This strain had been isolated from a patient with gastric ulcer disease and moderate to severe scores for H. pylori- strain 444A revealed that it possesses numerous biofilm- and virulence-associated genes and a small cryptic plasmid encoding a type II toxin-antitoxin system. H. pylori strain 444A revealed that it possesses numerous biofilm- and virulence-associated genes and a small cryptic plasmid encoding a type II toxin-antitoxin system. Conclusion. There is substantial variation in biofilm-forming ability in H. pylori, but this was not significantly associated with disease severity in our study. We identified and characterized an interesting strain with high biofilm-forming ability, including generation and analysis of the complete genome.

The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including galK- based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the galK gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the hicA gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length hicA gene is not toxic in F. nucleatum, but a truncated hicA gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an rpsJ promoter and is controlled at its translational level by using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin radD gene within the F. nucleatum rad operon and the tnaA gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This hicA -based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria. IMPORTANCE Fusobacterium nucleatum is an indole-producing human oral anaerobe associated with periodontal diseases, preterm birth, and several cancers. Little is known about the mechanisms of fusobacterial pathogenesis and associated factors, mainly due to the lack of robust genetic tools for this organism. Here, we showed that a mutated hicA gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This hicA -based in-frame deletion system efficiently creates in-frame deletion mutations in the wild-type background of F. nucleatum. This is the first report to use the hicA gene as a counterselection marker in a bacterial genetic study.

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea plasmids and genomes to regulate DNA replication, gene transcription, or protein translation. Higher eukaryotic and prokaryotic nucleotide-binding (HEPN) and minimal nucleotidyltransferase (MNT) domains are prevalent in prokaryotic genomes and constitute TA pairs. However, three gene pairs (MTH304/305, 408/409, and 463/464) of Methanothermobacter thermautotropicus ΔH HEPN-MNT family have not been studied as TA systems. Among these candidates, our study characterizes the MTH463/MTH464 TA system. MTH463 expression inhibited Escherichia coli growth, whereas MTH464 did not and blocked MTH463 instead. Using site-directed MTH463 mutagenesis, we determined that amino acids R99G, H104A, and Y106A from the R[ɸX]4-6H motif are involved with MTH463 cell toxicity. Furthermore, we established that purified MTH463 could degrade MS2 phage RNA, whereas purified MTH464 neutralized MTH463 activity in vitro. Our results indicate that the endonuclease toxin MTH463 (encoding a HEPN domain) and its cognate antitoxin MTH464 (encoding the MNT domain) may act as a type II TA system in M. thermautotropicus ΔH. This study provides initial and essential information studying TA system functions, primarily archaea HEPN-MNT family., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Toxin-Antitoxin (TA) systems are abundant in prokaryotes and play an important role in various biological processes such as plasmid maintenance, phage inhibition, stress response, biofilm formation, and dormant persister cell generation. TA loci are abundant in pathogenic intracellular micro-organisms and help in their adaptation to the harsh host environment such as nutrient deprivation, oxidation, immune response, and antimicrobials. Several studies have reported the involvement of TA loci in establishing successful infection, intracellular survival, better colonization, adaptation to host stresses, and chronic infection. Overall, the TA loci play a crucial role in bacterial virulence and pathogenesis. Nonetheless, there are some controversies about the role of TA system in stress response, biofilm and persister formation. In this review, we describe the role of the TA systems in bacterial virulence. We discuss the important features of each type of TA system and the recent discoveries identifying key contributions of TA loci in bacterial pathogenesis., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors.)

YdaT is a functional equivalent of the CII repressor in certain lambdoid phages and prophages. YdaT from the cryptic prophage CP-933P in the genome of Escherichia coli O157:H7 is functional as a DNA-binding protein and recognizes a 5'-TTGATTN 6 AATCAA-3' inverted repeat. The DNA-binding domain is a helix-turn-helix (HTH)-containing POU domain and is followed by a long α-helix (α6) that forms an antiparallel four-helix bundle, creating a tetramer. The loop between helix α2 and the recognition helix α3 in the HTH motif is unusually long compared with typical HTH motifs, and is highly variable in sequence and length within the YdaT family. The POU domains have a large degree of freedom to move relative to the helix bundle in the free structure, but their orientation becomes fixed upon DNA binding., (open access.)

The chemical modification of cellular macromolecules by the transfer of ADP-ribose unit(s), known as ADP-ribosylation, is an ancient homeostatic and stress response control system. Highly conserved across the evolution, ADP-ribosyltransferases and ADP-ribosylhydrolases control ADP-ribosylation signalling and cellular responses. In addition to proteins, both prokaryotic and eukaryotic transferases can covalently link ADP-ribosylation to different conformations of nucleic acids, thus highlighting the evolutionary conservation of archaic stress response mechanisms. Here, we report several structural and functional aspects of DNA ADP-ribosylation modification controlled by the prototype DarT and DarG pair, which show ADP-ribosyltransferase and hydrolase activity, respectively. DarT/DarG is a toxin-antitoxin system conserved in many bacterial pathogens, for example in Mycobacterium tuberculosis , which regulates two clinically important processes for human health, namely, growth control and the anti-phage response. The chemical modulation of the DarT/DarG system by selective inhibitors may thus represent an exciting strategy to tackle resistance to current antimicrobial therapies.

Bacterial cell filamentation is a morphological change wherein cell division is blocked, which can improve bacterial survival under unfavorable conditions (e.g., antibiotic stress that causes DNA damage). As an extrachromosomal DNA molecule, plasmids can confer additionally advantageous traits including antibiotic resistance on the host. However, little is known about whether plasmids could shift bacterial morphological responses to antibiotic stress. Here, it is reported that plasmid-free cells, rather than plasmid-bearing cells, exhibit filamentation and asymmetrical cell division under exposure to sub-inhibitory concentrations of antibiotics (ciprofloxacin and cephalexin). The underlying mechanism is revealed by investigating DNA damage, cell division inhibitor sulA, the SOS response, toxin-antitoxin module (parDE) located on plasmids, and efflux pumps. Significantly higher expression of sulA is observed in plasmid-free cells, compared to plasmid-bearing cells. Plasmid carriage enables the hosts to suffer less DNA damage, exhibit stronger efflux pump activities, and thus have a higher antibiotic tolerance. These benefits are attributed to the parDE module that mediates stress responses from plasmid-bearing cells and mainly contributes to cell morphological changes. Collectively, the findings demonstrate that plasmids can confer additional innate defenses on the host to antibiotics, thus advancing the understanding of how plasmids affect bacterial evolution in hostile environments., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Temperate phages dynamically switch between lysis and lysogeny in their full life cycle. Some Bacillus-infecting phages utilize a quorum-sensing-like intercellular communication system, the "arbitrium," to mediate lysis-lysogeny decisions. However, whether additional factors participate in the arbitrium signaling pathway remains largely elusive. Here, we find that the arbitrium signal induces the expression of a functionally conserved operon downstream of the arbitrium module in SPbeta-like phages. SPbeta yopM and yopR (as well as phi3T phi3T_93 and phi3T_97) in the operon play roles in suppressing phage lytic propagation and promoting lysogeny, respectively. We further focus on phi3T_93 and demonstrate that it directly binds antitoxin MazE in the host MazF/MazE toxin-antitoxin (TA) module and facilitates the activation of MazF's toxicity, which is required for phage suppression. These findings show events regulated by the arbitrium system and shed light on how the interplay between phages and the host TA module affects phage-host co-survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Antimicrobial resistance (AMR) in bacteria is an important global health problem affecting humans, animals, and the environment. AMR is considered as one of the major components in the "global one health". Misuse/overuse of antibiotics in any one of the segments can impact the integrity of the others. In the presence of antibiotic selective pressure, bacteria tend to develop several defense mechanisms, which include structural changes of the bacterial outer membrane, enzymatic processes, gene upregulation, mutations, adaptive resistance, and biofilm formation. Several components of mobile genetic elements (MGEs) play an important role in the dissemination of AMR. Each one of these components has a specific function that lasts long, irrespective of any antibiotic pressure. Integrative and conjugative elements (ICEs), insertion sequence elements (ISs), and transposons carry the antimicrobial resistance genes (ARGs) on different genetic backbones. Successful transfer of ARGs depends on the class of plasmids, regulons, ISs proximity, and type of recombination systems. Additionally, phage-bacterial networks play a major role in the transmission of ARGs, especially in bacteria from the environment and foods of animal origin. Several other functional attributes of bacteria also get successfully modified to acquire ARGs. These include efflux pumps, toxin-antitoxin systems, regulatory small RNAs, guanosine pentaphosphate signaling, quorum sensing, two-component system, and clustered regularly interspaced short palindromic repeats (CRISPR) systems. The metabolic and virulence state of bacteria is also associated with a range of genetic and phenotypic resistance mechanisms. In spite of the availability of a considerable information on AMR, the network associations between selection pressures and several of the components mentioned above are poorly understood. Understanding how a pathogen resists and regulates the ARGs in response to antimicrobials can help in controlling the development of resistance. Here, we provide an overview of the importance of genetic network and regulation of AMR in bacterial pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ramamurthy, Ghosh, Chowdhury, Mukhopadhyay, Dutta and Miyoshi.)

Kill switches provide a biocontainment strategy in which unwanted growth of an engineered microorganism is prevented by expression of a toxin gene. A major challenge in kill switch engineering is balancing evolutionary stability with robust cell killing activity in application relevant host strains. Understanding host-specific containment dynamics and modes of failure helps to develop potent yet stable kill switches. To guide the design of robust kill switches in the agriculturally relevant strain Pseudomonas fluorescens SBW25, we present a comparison of lethality, stability, and genetic escape of eight different toxic effectors in the presence of their cognate inactivators (i.e., toxin-antitoxin modules, polymorphic exotoxin-immunity systems, restriction endonuclease-methyltransferase pair). We find that cell killing capacity and evolutionary stability are inversely correlated and dependent on the level of protection provided by the inactivator gene. Decreasing the proteolytic stability of the inactivator protein can increase cell killing capacity, but at the cost of long-term circuit stability. By comparing toxins within the same genetic context, we determine that modes of genetic escape increase with circuit complexity and are driven by toxin activity, the protective capacity of the inactivator, and the presence of mutation-prone sequences within the circuit. Collectively, the results of our study reveal that circuit complexity, toxin choice, inactivator stability, and DNA sequence design are powerful drivers of kill switch stability and valuable targets for optimization of biocontainment systems.

DinJ-YafQ is a bacterial type II TA system formed by the toxin RNase YafQ and the antitoxin protein DinJ. The activity of YafQ and DinJ has been rigorously studied in Escherichia coli, but little has been reported about orthologous systems identified in different microorganisms. In this work, we report an in vitro and in vivo functional characterization of YafQ and DinJ identified in two different strains of Lacticaseibacillus paracasei and isolated as recombinant proteins. While DinJ is identical in both strains, the two YafQ orthologs differ only for the D72G substitution in the catalytic site. Both YafQ orthologs digest ribosomal RNA, albeit with different catalytic efficiencies, and their RNase activity is neutralized by DinJ. We further show that DinJ alone or in complex with YafQ can bind cooperatively to a 28-nt inverted repeat overlapping the -35 element of the TA operon promoter. Atomic force microscopy imaging of DinJ-YafQ in complex with DNA harboring the cognate site reveals the formation of different oligomeric states that prevent the binding of RNA polymerase to the promoter. A single amino acid substitution (R13A) within the RHH DNA-binding motif of DinJ is sufficient to abolish DinJ and DinJ-YafQ DNA binding in vitro. In vivo experiments confirm the negative regulation of the TA promoter by DinJ and DinJ-YafQ and unveil an unexpected high expression-related toxicity of the gfp reporter gene. A model for the binding of two YafQ-(DinJ) 2 -YafQ tetramers to the promoter inverted repeat showing the absence of protein-protein steric clash is also presented. KEY POINTS: • The RNase activity of L. paracasei YafQ toxin is neutralized by DinJ antitoxin. • DinJ and DinJ-YafQ bind to an inverted repeat to repress their own promoter. • The R13A mutation of DinJ abolishes DNA binding of both DinJ and DinJ-YafQ., (© 2022. The Author(s).)

The MqsRA toxin-antitoxin system is a component of the Escherichia coli stress response. Free MqsR, a ribonuclease, cleaves mRNAs containing a 5'-GC-3' sequence causing a global shutdown of translation and the cell to enter a state of dormancy. Despite a general understanding of MqsR function, the molecular mechanism(s) by which MqsR binds and cleaves RNA and how one or more of these activities is inhibited by its cognate antitoxin MqsA is still poorly understood. Here, we used NMR spectroscopy coupled with mRNA cleavage assays to identify the molecular mechanism of MqsR substrate recognition and the MqsR residues that are essential for its catalytic activity. We show that MqsR preferentially binds substrates that contain purines in the -2 and -1 position relative to the MqsR consensus cleavage sequence and that two residues of MqsR, Tyr81, and Lys56 are strictly required for mRNA cleavage. We also show that MqsA inhibits MqsR activity by sterically blocking mRNA substrates from binding while leaving the active site fully accessible to mononucleotides. Together, these data identify the residues of MqsR that mediate RNA cleavage and reveal a novel mechanism that regulates MqsR substrate specificity., Competing Interests: Conflict of interest The authors declare no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The RelEB3 toxin-antitoxin (TA) system of Salmonella enterica subsp. enterica serovar Typhimurium consists of a RelE3 toxin which suppresses bacterial growth, but its RelB3 antitoxin does not neutralise the toxin. The relEB3 operon is widespread in Proteobacteria and is related to higBA2 from Vibrio cholerae. In contrast to the ribosome-dependent HigB2 toxin, however, the RelE3 toxin degraded free RNA independently of the ribosome. A basic loop possibly involved in HigB2's binding to the ribosome is shortened in RelE3, which instead contains a uniquely conserved R51 important for RelE3's toxicity. The RelB3 antitoxin, meanwhile, specifically recognised the CACCTGGTG palindromic motif in the promoter site. RelB3 contains a unique P14 which is conserved as Ala in most homologues, and mutating P14 to Ala enabled the antitoxin to bind to RelE3 and restored bacterial growth. The P14 RelB3 variant, which most likely arose by a point mutation in a recent ancestor of S. Typhimurium and closely related serovars, could have possibly provided the bacteria with a faster response to stress, and might have spread to other serovars through homologous recombination., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bacteria use quorum sensing (QS) to communicate with each other via secreted small autoinducers produced by individuals. QS allows bacteria to display a unified response that benefits the species during adaptation to environment, colonization, and defense against competitors. In oral streptococci, the CSP-ComDE QS is an inducible DNA damage repair system that is pivotal for bacterial survival. In the oral pathogen Streptococcus mutans, the QS system positively influences the formation of antibiotic persisters, cells that can survive antibiotic attack by entering a non-proliferative state. We recently identified a novel gene, pep299, that is activated in the persister cell fraction induced by QS. In this study, we focused our investigation on the role of pep299, a gene encoding a bacteriocin-like peptide, in the formation of antibiotic persisters. Mutant Δ299, unable to produce Pep299, showed a dramatic reduction in the number of stress-induced persisters. Using a co-culture assay, we showed that cells overproducing pep299 induced the formation of persisters in the mutant, suggesting that Pep299 was actively secreted and detected by neighboring cells. Cells exposed to DNA damage conditions activated the gene expression of pep299. Interestingly, our results suggested that the pep299 gene was also involved in the regulation of a QS-inducible toxin−antitoxin system. Our study suggests that the pep299 gene is at the core of the triggered persistence phenotype in S. mutans, allowing cells to transition into a state of reduced metabolic activity and antibiotic tolerance.

Bacterial resistance to various drugs and antibiotics has become a significant issue in the fight against infectious diseases. Due to the presence of diverse toxin-antitoxin (TA) systems, bacteria undergo adaptive metabolic alterations and can tolerate the effects of drugs and antibiotics. Bacterial TA systems are unique and can be therapeutic targets for developing new antimicrobial agents, owing to their ability to influence bacterial fate. With this background, our study aims to identify novel drug targets against Escherichia coli K12 MG1655 antitoxin using homology modelling approach. In this study, the protein-protein interaction network of 87 E. coli K12 MG1655 TA systems identified through literature mining was screened for the identification of hub proteins. The model evaluation, assessment, and homology modelling of the hub proteins were evaluated. Furthermore, computer-aided mathematical models of selected phytochemicals have been tested against the identified hub proteins. The TA system was functionally enriched in regulation of cell growth, negative regulation of cell growth, regulation of mRNA stability, mRNA catabolic process and RNA phosphodiester bond hydrolysis. RelE, RelB, MazE, MazF, MqsR, MqsA, and YoeB were identified as hub proteins. The robustness and superior quality of the RelB and MazE modelled structure were discovered by model evaluation, quality assessment criteria, and homology modelling of hub proteins. Clorobiocin was found to be a strong inhibitor by docking these modelled structures. Clorobiocin could be utilized as an antibacterial agent against multidrug resistant E. coli which may inactivate antitoxins and cause programmed cell death., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Melioidosis is an underreported human disease caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei ( Bpm ). Both the treatment and the clearance of the pathogen are challenging, with high relapse rates leading to latent infections. This has been linked to the bacterial persistence phenomenon, a growth arrest strategy that allows bacteria to survive under stressful conditions, as in the case of antibiotic treatment, within a susceptible clonal population. At a molecular level, this phenomenon has been associated with the presence of toxin-antitoxin (TA) systems. We annotated the Bpm K96243 genome and selected 11 pairs of genes encoding for these TA systems, and their expression was evaluated under different conditions (supralethal antibiotic conditions; intracellular survival bacteria). The predicted HigB toxin (BPSL3343) and its predicted antitoxin HigA (BPS_RS18025) were further studied using mutant construction. The phenotypes of two mutants (Δ higB and Δ higB Δ higA ) were evaluated under different conditions compared to the wild-type (WT) strain. The Δ higB toxin mutant showed a defect in intracellular survival on macrophages, a phenotype that was eliminated after levofloxacin treatment. We found that the absence of the toxin provides an advantage over the WT strain, in both in vitro and in vivo models, during persister conditions induced by levofloxacin. The lack of the antitoxin also resulted in differential responses to the conditions evaluated, and under some conditions, it restored the WT phenotype, overall suggesting that both toxin and antitoxin components play a role in the persister-induced phenotype in Bpm .

Toxin-antitoxin (TA) systems are genetic modules that consist of a stable protein-toxin and an unstable antitoxin that neutralizes the toxic effect. In type II TA systems, the antitoxin is a protein that inhibits the toxin by direct binding. Type II TA systems, whose roles and functions are under intensive study, are highly distributed among bacterial chromosomes. Here, we identified and characterized a novel type II TA system PrrT/A encoded in the chromosome of the clinical isolate 39016 of the opportunistic pathogen Pseudomonas aeruginosa. We have shown that the PrrT/A system exhibits classical type II TA characteristics and novel regulatory properties. Following deletion of the prrA antitoxin, we discovered that the system is involved in a range of processes including (i) biofilm and motility, (ii) reduced prophage induction and bacteriophage production, and (iii) increased fitness for aminoglycosides. Taken together, these results highlight the importance of this toxin-antitoxin system to key physiological traits in P. aeruginosa. IMPORTANCE The functions attributed to bacterial TA systems are controversial and remain largely unknown. Our study suggests new insights into the potential functions of bacterial TA systems. We reveal that a chromosome-encoded TA system can regulate biofilm and motility, antibiotic resistance, prophage gene expression, and phage production. The latter presents a thus far unreported function of bacterial TA systems. In addition, with the emergence of antimicrobial-resistant bacteria, especially with the rising of P. aeruginosa resistant strains, the investigation of TA systems is critical as it may account for potential new targets against the resistant strains.

Background: Curiosity on toxin-antitoxin modules has increased intensely over recent years as it is ubiquitously present in many bacterial genomes, including pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). Several cellular functions of TA systems have been proposed however, their exact role in cellular physiology remains unresolved., Methods: This study aims to find out the impact of the mazEF toxin-antitoxin module on biofilm formation, pathogenesis, and antibiotic resistance in an isolated clinical ST239 MRSA strain, by constructing mazE and mazF mutants using CRISPR-cas9 base-editing plasmid (pnCasSA-BEC). Transcriptome analysis (RNA-seq) was performed for the mazE antitoxin mutant in order to identify the differentially regulated genes. The biofilm formation was also assessed for the mutant strains. Antibiogram profiling was carried out for both the generated mutants followed by murine experiment to determine the pathogenicity of the constructed strains., Results: For the first time our work showed, that MazF promotes cidA mediated cell death and lysis for biofilm formation without playing any significant role in host virulence as suggested by the murine experiment. Interestingly, the susceptibility to oxacillin, daptomycin and vancomycin was reduced significantly by the activated MazF toxin in the mazE mutant strain., Conclusions: Our study reveals that activated MazF toxin leads to resistance to antibiotics like oxacillin, daptomycin and vancomycin. Therefore, in the future, any potential antibacterial drug can be designed to target MazF toxin against the problematic multi-drug resistant bug., (© 2022. The Author(s).)

Toxin-antitoxin systems are genetic elements that are widespread in prokaryotes. Although molecular mode of action of many of these toxins has been identified, their biological functions are mostly unknown. We investigated the functional integration of the TisB/IstR toxin-antitoxin system in the Escherichia coli SOS genotoxic stress response network. We showed that the tisB gene is induced in cells exposed to high doses of the genotoxic antibiotic trimethoprim. However, we also found that TisB contributes to trimethoprim-induced lethality. This is a consequence of the TisB-induced drop in the proton motive force (PMF), which results in blocking the thymine import and therefore the functioning of the pyrimidine salvage pathway. Conversely, a TisB-induced PMF drop protects cells by preventing the import of some other toxic compounds, like the aminoglycoside antibiotic gentamicin and colicin M, in the SOS-induced cells. Colicins are cytotoxic molecules produced by Enterobacterales when they are exposed to strong genotoxic stresses in order to compete with other microbiota members. We indeed found that TisB contributes to E. coli 's fitness during mouse gut colonization. Based on the results obtained here, we propose that the primary biological role of the TisB toxin is to increase the probability of survival and maintenance in the mammalian gut of their bacterial hosts when they have to simultaneously deal with massive DNA damages and a fierce chemical warfare with other microbiota members. IMPORTANCE The contribution of toxin-antitoxin systems to the persistence of bacteria to antibiotics has been intensively studied. This is also the case with the E. coli TisB/IstR toxin-antitoxin system, but the contribution of TisB to the persistence to antibiotics turned out to be not as straightforward as anticipated. In this study, we show that TisB can decrease, but also increase, cytotoxicity of different antibiotics. This inconsistency has a common origin, i.e., TisB-induced collapse of the PMF, which impacts the import and the action of different antibiotics. By taking into account the natural habitat of TisB bacterial hosts, the facts that this toxin-antitoxin system is integrated into the genotoxic stress response regulon SOS and that both SOS regulon and TisB are required for E. coli to colonize the host intestine, and the phenotypic consequences of the collapse of the PMF, we propose that TisB protects its hosts from cytotoxic molecules produced by competing intestinal bacteria.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella , first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K . pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.