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The ability to form persister cells by Mycobacterium tuberculosis (Mtb) is a prime cause for the emergence of drug‐resistant strains. A large number of toxin–antitoxin systems in the Mtb genome are postulated to promote bacterial persistence. The largest family of toxin–antitoxin systems encoded in the genome of Mtb is VapBC, with 47 VapBC toxin–antitoxin systems regulated by VapB antitoxins. In this study, we characterized the structure of VapB46 antitoxin and determined its interaction with its cognate DNA sequence. Using electrophoretic mobility shift assay and DNase I footprinting we showed that VapB46 binds to two sites in the upstream promoter–operator region. Using nuclear magnetic resonance (NMR)‐based structural studies we found that VapB46 has a well‐folded dimeric N‐terminal domain, which contains a Phd/YefM motif and is involved in DNA binding. The remaining C‐terminal residues are disordered but promote higher order oligomerization of VapB46. We propose a DNA‐binding model in which tetrameric VapB46 binds to the two sites in its promoter–operator region, with each site bound by its dimeric N‐terminal domain. This study characterizes the DNA binding specificity of mycobacterial VapB46 antitoxin from the VapBC46 toxin–antitoxin (TA) operon. VapB46 binds to the promoter–operator region in the upstream DNA sequence through its dimeric N‐terminal domain and regulates the VapBC TA operon, thus providing an insight into the transcriptional regulation of the VapBC46 TA operon. [ABSTRACT FROM AUTHOR]

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (Phhp) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing a counterselectable marker in which a putative toxin-encoding gene virulence-associated protein C (PF0776 (VapC)), controlled by the HHP-inducible promoter, was used in conjunction with the antitoxin-encoding gene PF0775 (VapB), which was fused to a constitutive promoter (PhmtB), and a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive 34 promoter Pgdh. The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369- 39 mutant decreased dramatically compared with that in the wild-type strain. The inducible toxin-antitoxin (TA) system developed in this study greatly increases the genetic tools available for use in hyperthermophiles. Importance Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared with sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible TA system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles. [ABSTRACT FROM AUTHOR]

The oral pathogen Streptococcus mutans communicates using a canonical Gram-positive quorum sensing system, CSP-ComDE. The CSP pheromone already known to be involved in the development of genetic competence positively influences the formation of persisters, dormant variants of regular cells that are highly tolerant to antimicrobial therapy. It is now believed that the persistence phenotype is the end result of a stochastic switch in the expression of toxin-antitoxin (TA) modules. TAs consist of a pair of genes that encode two components, a stable toxin and its cognate labile antitoxin. Transcription analyses revealed that three core genes encoding a putative TA system, called SmuATR, were members of the S. mutans CSP regulon. We hypothesized that S. mutans is using its CSP-ComDE system as a deterministic mechanism for persister formation through the activation of smuATR locus. We showed here that the SmuATR system constitutes a novel tripartite type II TA system in which the smuA and smuT genes encode an antitoxin and a toxin, respectively, while SmuR is a transcriptional repressor involved in the autoregulation of the operon. Ectopic expression of SmuA - SmuT is associated with the CSP-inducible persistence phenotype. In contrast, overexpression of SmuT alone is bactericidal and causes membrane permeabilization. To our knowledge, SmuATR is the first functional chromosomal tripartite TA system shown to be induced by the bacterial quorum sensing system and involved in persister formation. [ABSTRACT FROM AUTHOR]

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

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. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis genome encodes over 80 toxin–antitoxin (TA) systems. While each toxin interacts with its cognate antitoxin, the abundance of TA systems presents an opportunity for potential non-cognate interactions. TA systems mediate manifold interactions to manage pathogenicity and stress response network of the cell and non-cognate interactions may play vital roles as well. To address if non-cognate and heterologous interactions are feasible and to understand the structural basis of their interactions, we have performed comprehensive computational analyses on the available 3D structures and generated structural models of paralogous M. tuberculosis VapBC and MazEF TA systems. For a majority of the TA systems, we show that non-cognate toxin–antitoxin interactions are structurally incompatible except for complexes like VapBC15 and VapBC11, which show similar interfaces and potential for cross-reactivity. For TA systems which have been experimentally shown earlier to disfavor non-cognate interactions, we demonstrate that they are structurally and stereo-chemically incompatible. For selected TA systems, our detailed structural analysis identifies specificity conferring residues. Thus, our work improves the current understanding of TA interfaces and generates a hypothesis based on congenial binding site, geometric complementarity, and chemical nature of interfaces. Overall, our work offers a structure-based explanation for non-cognate toxin-antitoxin interactions in M. tuberculosis.

The xenobiotic response element (XRE) transcription factors belong to a regulator family frequently found in Streptomyces that are often followed by small proteins with a DUF397 domain. In fact, the pair XRE-DUF397 has been proposed to comprise toxin–antitoxin (TA) type II systems. In this work, we demonstrate that one of these putative TA-systems, encoded by the genes SCO4441 and SCO4442 of Streptomyces coelicolor, and denominated Scr1/Scr2 (which stands for S. coelicolorregulator), does not behave as a toxin–antitoxin system under the conditions used as was originally expected. Instead the pair Scr1/Scr2 acts as a strong positive regulator of endogenous antibiotic production in S. coelicolor. The analysis of the 19 Streptomyces strains tested determined that overexpression of the pair Scr1/Scr2 drastically induces the production of antibiotics not only in S. coelicolor, but also in Streptomyces lividans, Streptomyces peucetius, Streptomyces steffisburgensis and Streptomyces sp. CA-240608. Our work also shows that Scr1 needs Scr2 to exert positive regulation on antibiotic production.

Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.

Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.

ABSTRACT Horizontal gene transfer is a major driving force behind the genomic diversity seen in prokaryotes. The cryptic rac prophage in Escherichia coli K-12 carries the gene for a putative transcription factor RacR, whose deletion is lethal. We have shown that the essentiality of racR in E. coli K-12 is attributed to its role in transcriptionally repressing toxin gene(s) called ydaS and ydaT, which are adjacent to and coded divergently to racR. IMPORTANCE Transcription factors in the bacterium E. coli are rarely essential, and when they are essential, they are largely toxin-antitoxin systems. While studying transcription factors encoded in horizontally acquired regions in E. coli, we realized that the protein RacR, a putative transcription factor encoded by a gene on the rac prophage, is an essential protein. Here, using genetics, biochemistry, and bioinformatics, we show that its essentiality derives from its role as a transcriptional repressor of the ydaS and ydaT genes, whose products are toxic to the cell. Unlike type II toxin-antitoxin systems in which transcriptional regulation involves complexes of the toxin and antitoxin, repression by RacR is sufficient to keep ydaS transcriptionally silent.

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.

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.)

Most small noncoding RNAs (sRNAs) are known to base pair with target mRNAs and regulate mRNA stability or translation to trigger various changes in the cell metabolism of Escherichia coli. The SdsR sRNA is expressed specifically during the stationary phase and represses tolC and mutS expression. However, it was not previously known whether the growth-phase-dependent regulation of SdsR is important for cell growth. Here, we ectopically expressed SdsR during the exponential phase and examined cell growth and survival. We found that ectopic expression of SdsR led to a significant and Hfq-dependent cell death with accompanying cell filamentation. This SdsR-driven cell death was alleviated by overexpression of RyeA, an sRNA transcribed on the opposite DNA strand, suggesting that SdsR/RyeA is a novel type of toxin-antitoxin (T/A) system in which both the toxin and the antitoxin are sRNAs. We defined the minimal region required for the SdsR-driven cell death. We also performed RNA-seq analysis and identified 209 genes whose expression levels were altered by more than two-fold following pulse expression of ectopic SdsR at exponential phase. Finally, we found that that the observed SdsR-driven cell death was mainly caused by the SdsR-mediated repression of yhcB, which encodes an inner membrane protein. [ABSTRACT FROM AUTHOR]

The ataRT operon from enteropathogenic Escherichia coli encodes a toxin–antitoxin (TA) module with a recently discovered novel toxin activity. This new type II TA module targets translation initiation for cell‐growth arrest. Virtually nothing is known regarding the molecular mechanisms of neutralization, toxin catalytic action or translation autoregulation. Here, the production, biochemical analysis and crystallization of the intrinsically disordered antitoxin AtaR, the toxin AtaT, the AtaR–AtaT complex and the complex of AtaR–AtaT with a double‐stranded DNA fragment of the operator region of the promoter are reported. Because they contain large regions that are intrinsically disordered, TA antitoxins are notoriously difficult to crystallize. AtaR forms a homodimer in solution and crystallizes in space group P6122, with unit‐cell parameters a = b = 56.3, c = 160.8 Å. The crystals are likely to contain an AtaR monomer in the asymmetric unit and diffracted to 3.8 Å resolution. The Y144F catalytic mutant of AtaT (AtaTY144F) bound to the cofactor acetyl coenzyme A (AcCoA) and the C‐terminal neutralization domain of AtaR (AtaR44–86) were also crystallized. The crystals of the AtaTY144F–AcCoA complex diffracted to 2.5 Å resolution and the crystals of AtaR44–86 diffracted to 2.2 Å resolution. Analysis of these structures should reveal the full scope of the neutralization of the toxin AtaT by AtaR. The crystals belonged to space groups P6522 and P3121, with unit‐cell parameters a = b = 58.1, c = 216.7 Å and a = b = 87.6, c = 125.5 Å, respectively. The AtaR–AtaT–DNA complex contains a 22 bp DNA duplex that was optimized to obtain high‐resolution data based on the sequence of two inverted repeats detected in the operator region. It crystallizes in space group C2221, with unit‐cell parameters a = 75.6, b = 87.9, c = 190.5 Å. These crystals diffracted to 3.5 Å resolution. [ABSTRACT FROM AUTHOR]

DNA harbors the blueprint for life. However, the instructions stored in the DNA could be altered at the RNA level before they are executed. One of these processes is RNA editing, which was shown to modify RNA sequences in many organisms. The most abundant modification is the deamination of adenosine (A) into inosine (I). In turn, inosine can be identified as a guanosine (G) by the ribosome and other cellular machineries such as reverse transcriptase. In multicellular organisms, enzymes from the ADAR (adenosine deaminase acting on RNA) family mediate RNA editing in mRNA, whereas enzymes from the ADAT family mediate A-to-I editing on tRNAs. In bacteria however, until recently, only one editing site was described, in tRNAArg, but never in mRNA. The tRNA site was shown to be modified by tadA (tRNA specific adenosine deaminase) which is believed to be the ancestral enzyme for the RNA editing family of enzymes. In our recent work, we have shown for the first time, editing on multiple sites in bacterial mRNAs and identified tadA as the enzyme responsible for this editing activity. Focusing on one of the identified targets - the self-killing toxin hokB, we found that editing is physiologically regulated and that it increases protein activity. Here we discuss possible modes of regulation on hokB editing, potential roles of RNA editing in bacteria, possible implications, and future research directions. [ABSTRACT FROM AUTHOR]

Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) systems are two component genetic modules widespread in many bacterial genomes, including Mycobacterium tuberculosis ( Mtb ). The TA systems play a significant role in biofilm formation, antibiotic tolerance and persistence of pathogen inside the host cells. Deciphering regulatory motifs of Mtb TA systems is the first essential step to understand their transcriptional regulation. In this study, in silico approaches, that is, the knowledge based motif discovery and de novo motif discovery were used to identify the regulatory motifs of 79 Mtb TA systems. The knowledge based motif discovery approach was used to design a Perl based bio-tool Mtb-sig-miner available at ( https://github.com/zoozeal/Mtb-sig-miner ), which could successfully detect sigma (σ) factor specific regulatory motifs in the promoter region of Mtb TA modules. The manual curation of Mtb-sig-miner output hits revealed that the majority of them possessed σ B regulatory motif in their promoter region. On the other hand, de novo approach resulted in the identification of a novel conserved motif [(T/A)(G/T)NTA(G/C)(C/A)AT(C/A)] within the promoter region of 14 Mtb TA systems. The identified conserved motif was also validated for its activity as conserved core region of operator sequence of corresponding TA system by molecular docking studies. The strong binding of respective antitoxin/toxin with the identified novel conserved motif reflected the validation of identified motif as the core region of operator sequence of respective TA systems. These findings provide computational insight to understand the transcriptional regulation of Mtb TA systems. [ABSTRACT FROM AUTHOR]

Vap BCs, virulence-associated proteins, are the most abundant type II toxin-antitoxin ( TA) systems in prokaryotes. Under normal conditions, toxin and antitoxin interact to form a heterooctameric complex, which upon binding to operator sites, inhibits their own expression. Under stress conditions, the VapB antitoxin is degraded by cellular proteases to release a free VapC toxin, which in turn inhibits cell growth mainly by targeting protein translation. However, the intermediate steps involved in the assembly of the heterooctameric complex have not been resolved. Here, we report a 1.75 Å resolution crystal structure of VapC20, a Sarcin-Ricin loop cleaving toxin from type II TA system of Mycobacterium tuberculosis. Using analytical ultracentrifugation ( AUC) studies, we show that VapC20 exists as a homodimer in solution. The structural analysis of VapC homologs further suggests that VapCs form homodimers. We demonstrate that VapC20 is an obligate homodimer, and its self-association is critical for its folding and activity. Surface plasmon resonance experiments suggest that VapC20 interacts with its cognate antitoxin VapB20 to form a stable complex with nanomolar affinity. A high association rate coupled with a very slow dissociation rate ensures minimal toxicity under normal growth conditions. AUC studies reveal that VapB20 also exists as a homodimer in solution and further associates with VapC20 dimers to form heterotetramers and heterooctamers in a concentration-dependent manner. The results presented here provide valuable insights into the assembly of Vap BC family of toxins which is essential for their function and regulation. Database Structural data are available in the PDB under the accession numbers and . [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) in bacteria In bacteria, cell death occurs under certain stressful conditions, and this process has been designated as programmed cell death (PCD). The biological basis of the PCD are two molecules, a stable toxin protein and an unstable antitoxin being either a short RNA molecule or a protein. The antitoxin has to be synthesized permanently to neutralize the toxin. Both components form a TA module. When the synthesis of the antitoxin is blocked or when it is degraded completely, the free toxin acts either bacteriostatic or bactericide. So far, five different mechanisms have been described of how the antitoxin neutralizes the toxin in the absence of stress. Sporulating Bacillus subtilis cells exert cannibalism that means they kill and lyse non-sporulating cells to take up their nutrients. Streptococcus pneumoniae cells can carry out fratricide. They kill and lyse neighbouring cells, take up fragments of their chromosomal DNA and recombine them with their own DNA. This can result in the uptake of new genes. At the end, two examples of application of TA modules in biotechnology are described. [ABSTRACT FROM AUTHOR]

Bacterial dormancy is a valuable survival strategy upon challenging environmental conditions. Dormant cells tolerate the consequences of high stress levels and may re-populate the environment upon return to favorable conditions. Antibiotic-tolerant bacteria-termed persisters-regularly cause relapsing infections, increase the likelihood of antibiotic resistance, and, therefore, earn increasing attention. Their generation often depends on toxins from chromosomal toxin-antitoxin systems. Here, we review recent insights concerning RNA-based control of toxin synthesis, and discuss possible implications for persister generation. [ABSTRACT FROM AUTHOR]

[Display omitted] 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. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) secretes numerous toxins for bacteria-bacteria competition. TplE is a newly identified trans-kingdom toxin secreted by the T6SS in Pseudomonas aeruginosa, while TplEi neutralizes the toxic effect of TplE to protect bacteria autointoxication. Blocking the interaction of TplE-TplEi could unleash the toxin, causing bacterial cell death. In this study, we applied a crystallographic approach to design a structural-based antimicrobial peptides targeting the interaction of TplE and TplEi. We found that a peptide (designed as “L” peptide based on its shape) derived from TplE can form a crystal complex with TplEi after subtilisin treatment and the crystal structure was solved at 2.2Å. The “L” peptide displays strong binding affinity to TplEi in vitro and can release the TplE toxin to induce bacteria death in vivo. Our findings suggest that as a toxin activator, the “L” peptide could be a possible drug lead for treating P. aeruginosa infection. Our findings provide an example that the T6SS effector and immunity protein could be a potential drug target against bacteria infection.

Toxin–antitoxin (TA) genes were first reported in plasmids and were considered expendable genetic cassettes involved in the stable maintenance of the plasmid replicon by interfering with growth and/or viability of bacteria in which the plasmid was lost. TAs were later found in bacterial chromosomes and also in integrated mobile genetic elements; they were proposed to be involved in the bacterial response to stressful situations. At present, 100s of TAs have been identified and classified in up to six families (I to VI), with those belonging to the type II (constituted by two protein components) being the most studied. Based on well-characterized examples of several type II TAs, we discuss in this review that irrespective of their locations in plasmids or chromosomes, TAs functionally overlap as indicated by: (i) in both locations they can mediate the maintenance of genetic elements to which they are physical linked, and (ii) they can induce persistence or virulence in response to stress situations. Examples of functional confluences in homologous TA systems with different locations are also given. We also consider whether the physiological role of TAs is due to their genetic organization as operons or to their inherent properties, like the short lifespan of the antitoxin component.

Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an “altruistic suicide.” Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa consisting of an endoribonuclease toxin and RNA antitoxin. ToxINPa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa. To try to address this issue we first introduced ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxINPa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxINPa-mediated abortive infection but produced spontaneous “escape” mutants that were insensitive to ToxINPa. Analysis of these resistant mutants revealed three different routes of escaping ToxINPa, namely by mutating asiA (the product of which is a phage transcriptional co-activator); by mutating a conserved, yet functionally unknown, orf84; or by deleting a 6.5–10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxINPa.

Among the great number of addictive modules which have been discovered, only a few have been characterized. However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine. In our study, we tested two different toxins derived from class II addictive modules, pasAB from plasmid pTF-FC2 (Thiobacillus ferrooxidans) and vapBC 2829Rv (Mycobacterium tuberculosis), in terms of their usefulness as growth inhibitors of human cancer cell lines, namely KYSE 30, MCF-7 and HCT 116. Transfection of the pasB and vapC genes into the cells was conducted with the use of two different expression systems. Cellular effects, such as apoptosis, necrosis and changes in the cell cycle, were tested by applying flow cytometry with immunofluorescence staining. Our findings demonstrated that toxins VapC and PasB demonstrate proapoptotic activity in the human cancer cells, regardless of the expression system used. As for the toxin PasB, observed changes were more subtle than for the VapC. The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line.

Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within.

Pathogenic Leptospira spp. is the etiological agent of leptospirosis. The high diversity among Leptospira species provides an array to look for important mediators involved in pathogenesis. Toxin-antitoxin (TA) systems represent an important survival mechanism on stress conditions. vapBC modules have been found in nearly one thousand genomes corresponding to about 40% of known TAs. In the present study, we investigated TA profiles of some strains of Leptospira using a TA database and compared them through protein alignment of VapC toxin sequences among Leptospira spp. genomes. Our analysis identified significant differences in the number of putative vapBC modules distributed in pathogenic, saprophytic, and intermediate strains: four in L. interrogans, three in L. borgpetersenii, eight in L. biflexa, and 15 in L. licerasiae. The VapC toxins show low identity among amino acid sequences within the species. Some VapC toxins appear to be exclusively conserved in unique species, others appear to be conserved among pathogenic or saprophytic strains, and some appear to be distributed randomly. The data shown here indicate that these modules evolved in a very complex manner, which highlights the strong need to identify and characterize new TAs as well as to understand their regulation networks and the possible roles of TA systems in pathogenic bacteria.

A toxin-antitoxin system,vp1842/vp1843, locates within a superintegron on theVibrio parahaemolyticusgenome chromosome I whose toxin genevp1843encodes a DNA nicking endonuclease. We found that thevp1843expression inEscherichia colicells strongly induced chromosomal DNA degradation. On the basis of these observations, we discuss a possible physiological role ofvp1842/vp1843inV. parahaemolyticus. [ABSTRACT FROM PUBLISHER]

Background: The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. Results: In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. Conclusions: The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. [ABSTRACT FROM AUTHOR]

Type II toxin-antitoxin (TA) systems are widespread in bacterial and archeal genomes. These modules are very dynamic and participate in bacterial genome evolution through horizontal gene transfer. TA systems are commonly composed of a labile antitoxin and a stable toxin. Toxins appear to preferentially inhibit the protein synthesis process. Toxins use a variety of molecular mechanisms and target nearly every step of translation to achieve their inhibitory function. This review focuses on a recently identified TA family that includes acetyltransferase toxins. The AtaT and TacT toxins are the best-characterized to date in this family. AtaT and TacT both inhibit translation by acetylating the amino acid charged on tRNAs. However, the specificities of these 2 toxins are different as AtaT inhibits translation initiation by acetylation of the initiator tRNA whereas TacT acetylates elongator tRNAs. The molecular mechanisms of these toxins are discussed, as well as the functions and possible evolutionary origins of this diverse toxin family. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has become a serious healthcare concern within a span of two decades due to its ability to rapidly acquire resistance to all classes of antimicrobial compounds. One of the key features of the A. baumannii genome is an open pan genome with a plethora of plasmids, transposons, integrons, and genomic islands, all of which play important roles in the evolution and success of this clinical pathogen, particularly in the acquisition of multidrug resistance determinants. An interesting genetic feature seen in majority of A. baumannii genomes analyzed is the presence of small plasmids that usually ranged from 2 to 10 kb in size, some of which harbor antibiotic resistance genes and homologs of plasmid mobilization genes. These plasmids are often overlooked when compared to their larger, conjugative counterparts that harbor multiple antibiotic resistance genes and transposable elements. In this mini-review, we will examine our current knowledge of these small A. baumannii plasmids and look into their genetic diversity and phylogenetic relationships. Some of these plasmids, such as the Rep-3 superfamily group and the pRAY-type, which has no recognizable replicase genes, are quite widespread among diverse A. baumannii clinical isolates worldwide, hinting at their usefulness to the lifestyle of this pathogen. Other small plasmids especially those from the Rep-1 superfamily are truly enigmatic, encoding only hypothetical proteins of unknown function, leading to the question of whether these small plasmids are "good" or "bad" to their host A. baumannii. [ABSTRACT FROM AUTHOR]

The PIN (PilT N-terminus) domain is a compact RNA-binding protein domain present in all domains of life. This 120-residue domain consists of a central and parallel β sheet surrounded by α helices, which together organize 4-5 acidic residues in an active site that binds one or more divalent metal ions and in many cases has endoribonuclease activity. In bacteria and archaea, the PIN domain is primarily associated with toxin-antitoxin loci, consisting of a toxin (the PIN domain nuclease) and an antitoxin that inhibits the function of the toxin under normal growth conditions. During nutritional or antibiotic stress, the antitoxin is proteolytically degraded causing activation of the PIN domain toxin leading to a dramatic reprogramming of cellular metabolism to cope with the new situation. In eukaryotes, PIN domains are commonly found as parts of larger proteins and are involved in a range of processes involving RNA cleavage, including ribosomal RNA biogenesis and nonsense-mediated mRNA decay. In this review, we provide a comprehensive overview of the structural characteristics of the PIN domain and compare PIN domains from all domains of life in terms of structure, active site architecture, and activity. [ABSTRACT FROM AUTHOR]

Type II toxins in toxin-antitoxin (TA) systems fold into a similar fold and belong to the RelE/ParE superfamily. However, they display two distinct biochemical activities: RelE toxins are mRNA interferases, while ParE toxins are DNA gyrase (Gyr) inhibitors. Previously, we found a TA system, vp1842/vp1843 , on the Vibrio parahaemolyticus genome whose toxin Vp1843 belongs to the RelE/ParE toxin superfamily. Vp1843, unlike RelE toxins, has neither protein synthesis inhibitory activity nor ribonuclease activity. In this study, we examined the inhibitory potency of Vp1843 with Escherichia coli Gyr. The result showed that Vp1843, unlike other ParE toxins, had little Gyr inhibitory activity, but rather converted supercoiled DNA to open-circular DNA. Analysis showed further that Vp1843 cleaves a single strand in DNA, and that the antitoxin Vp1842 neutralized the nicking endonuclease activity of Vp1843. Mutations of Lys37 and Pro45 in Vp1843 abolished its nicking activity, suggesting that they play a crucial role in nicking endonuclease activity. To our knowledge, Vp1843 is the first toxin with DNA nicking endonuclease activity among the RelE/ParE toxin superfamily. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) systems are small genetic elements that are widely prevalent in the genomes of bacteria and archaea. These modules have been identified in various bacteria and proposed to play an important role in bacterial physiology and virulence. However, their presence in the genomes of Actinobacillus species has received no attention. In this study, we describe the identification of four type II TA systems in Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Reverse transcription PCR analysis revealed that the genes encoding the toxin and antitoxin are co-transcribed. Overexpression of each toxin inhibited the growth of Escherichia coli, and the toxic effect could be counteracted by its cognate antitoxin. The pull-down experiments demonstrated that each toxin interacts with its cognate antitoxin in vivo. The promoter activity assays showed that each antitoxin could autoregulate either positively or negatively the TA operon transcription. In addition, the APJL 0660/0659 TA system is present in half of the detected serovars of A. pleuropneumoniae, while the others are present in all. Collectively, we identified four type II TA systems in A. pleuropneumoniae, and this study has laid the foundation for further functional study of these TA systems. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a severe disease caused by Mycobacterium tuberculosis ( M. tb ) and the well-characterized M. tb MazE/F proteins play important roles in stress adaptation. Recently, the MazF-mt9 toxin has been found to display endonuclease activities towards tRNAs but the mechanism is unknown. We hereby present the crystal structure of apo-MazF-mt9. The enzyme recognizes tRNA Lys with a central UUU motif within the anticodon loop, but is insensitive to the sequence context outside of the loop. Based on our crystallographic and biochemical studies, we identified key residues for catalysis and proposed the potential tRNA-binding site. [ABSTRACT FROM AUTHOR]

Bacteria export proteins across membranes using a range of transport machineries. Type VII secretion systems (T7SSs), originally described in mycobacteria, are now known to be widespread across diverse bacterial phyla. Recent studies have characterized secretion components and mechanisms of type VII secretion in pathogenic and environmental bacteria. A variety of functions have been attributed to T7SS substrates, including interactions with eukaryotes and with other bacteria. Here, we evaluate the growing body of knowledge on T7SSs, with focus on the nonmycobacterial systems, reviewing their phylogenetic distribution, structure and function in diverse settings. [ABSTRACT FROM AUTHOR]

Selfishness is pervasive and manifests at all scales of biology, from societies, to individuals, to genetic elements within a genome. The relentless struggle to seek evolutionary advantages drives perpetual cycles of adaptation and counter-adaptation, commonly referred to as Red Queen interactions. In this review, we explore insights gleaned from molecular and genetic studies of such genetic conflicts, both extrinsic (between genomes) and intrinsic (within genomes or cells). We argue that many different characteristics of selfish genetic elements can be distilled into two types of advantages: an overreplication advantage (e.g. mobile genetic elements in genomes) and a transmission distortion advantage (e.g. meiotic drivers in populations). These two general categories may help classify disparate types of selfish genetic elements. [ABSTRACT FROM AUTHOR]

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.

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.