Chromosomal Toxin-antitoxin Systems Research Articles

Isolation and characterization of a phage collection against Pseudomonas putida.

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

Structural Variations and Rearrangements in Bacterial Type II Toxin-Antitoxin Systems.

Bacteria encode a wide range of survival and immunity systems, including CRISPR-Cas, restriction-modification systems, and toxin-antitoxin systems involved in defence against bacteriophages, as well as survival during challenging growth conditions or exposure to antibiotics. Toxin-antitoxin (TA) systems are small two- or three-gene cassettes consisting of a metabolic regulator (the "toxin") and its associated antidote (the "antitoxin"), which also often functions as a transcriptional regulator. TA systems are widespread in the genomes of pathogens but are also present in commensal bacterial species and on plasmids. For mobile elements such as plasmids, TA systems play a role in maintenance, and increasing evidence now points to roles of chromosomal toxin-antitoxin systems in anti-phage defence. Moreover, the widespread occurrence of toxin-antitoxin systems in the genomes of pathogens has been suggested to relate to survival during host infection as well as in persistence during antibiotic treatment. Upon repeated exposure to antibiotics, TA systems have been shown to acquire point mutations as well as more dramatic rearrangements such as in-frame deletions with potential relevance for bacterial survival and pathogenesis. In this review, we present an overview of the known functional and structural consequences of mutations and rearrangements arising in bacterial toxin-antitoxin systems and discuss their relevance for survival and persistence of pathogenic species.

Toxin-Antitoxin System HigBA in Xylella fastidiosa Is Expressed at Low Temperature but Does Not Impact Virulence or Grapevine Recovery in Greenhouse Trials

Xylella fastidiosa has multiple chromosomally encoded type II toxin-antitoxin (TA) systems, several of which have unknown functions in the bacterial lifecycle. In many bacterial species, type II TA systems are associated with bacterial stress survival under various detrimental environmental conditions, and with influencing disease-associated phenotypes such as biofilm development and expression of virulence factors. Chromosomal TA systems are often found in genomic regions with evidence of phage insertion, pointing to potential acquisition of these elements by horizontal gene transfer. One of the TA systems found in X. fastidiosa, higBA, is transcriptionally expressed at higher levels at 15°C than at 28°C. However, a higBA deletion mutant (Δ higBA) in X. fastidiosa subsp. fastidiosa Stag's Leap is not deficient in growth or viability under in vitro cold stress and is able to infect and cause disease in grapevines comparable to the wild-type strain. Grapevines infected with X. fastidiosa Δ higBA recover from infection following cold treatment at 4°C at a similar rate to wild-type infected plants, suggesting that this TA system does not contribute significantly to X. fastidiosa survival at a low temperature in planta. Although a definitive function for HigBA in X. fastidiosa has yet to be determined, this study adds to a growing body of work describing the functions and expression dynamics of TA systems in plant pathogenic bacteria. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Biological Functions of Type II Toxin-Antitoxin Systems in Bacteria.

After the first discovery in the 1980s in F-plasmids as a plasmid maintenance system, a myriad of toxin-antitoxin (TA) systems has been identified in bacterial chromosomes and mobile genetic elements (MGEs), including plasmids and bacteriophages. TA systems are small genetic modules that encode a toxin and its antidote and can be divided into seven types based on the nature of the antitoxin molecules and their mechanism of action to neutralise toxins. Among them, type II TA systems are widely distributed in chromosomes and plasmids and the best studied so far. Maintaining genetic material may be the major function of type II TA systems associated with MGEs, but the chromosomal TA systems contribute largely to functions associated with bacterial physiology, including the management of different stresses, virulence and pathogenesis. Due to growing interest in TA research, extensive work has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules. However, there are still controversies about some of the functions associated with different TA systems. This review will discuss the most current findings and the bona fide functions of bacterial type II TA systems.

A Primary Physiological Role of Toxin/Antitoxin Systems Is Phage Inhibition.

Toxin/antitoxin (TA) systems are present in most prokaryote genomes. Toxins are almost exclusively proteins that reduce metabolism (but do not cause cell death), and antitoxins are either RNA or proteins that counteract the toxin or the RNA that encodes it. Although TA systems clearly stabilize mobile genetic elements, after four decades of research, the physiological roles of chromosomal TA systems are less clear. For example, recent reports have challenged the notion of TA systems as stress-response elements, including a role in creating the dormant state known as persistence. Here, we present evidence that a primary physiological role of chromosomally encoded TA systems is phage inhibition, a role that is also played by some plasmid-based TA systems. This includes results that show some CRISPR-Cas system elements are derived from TA systems and that some CRISPR-Cas systems mimic the host growth inhibition invoked by TA systems to inhibit phage propagation.

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

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

Reassessing the Role of the Type II MqsRA Toxin-Antitoxin System in Stress Response and Biofilm Formation: mqsA Is Transcriptionally Uncoupled from mqsR.

Toxin-antitoxin (TA) systems are broadly distributed modules whose biological roles remain mostly unknown. The mqsRA system is a noncanonical TA system in which the toxin and antitoxins genes are organized in operon but with the particularity that the toxin gene precedes that of the antitoxin. This system was shown to regulate global processes such as resistance to bile salts, motility, and biofilm formation. In addition, the MqsA antitoxin was shown to be a master regulator that represses the transcription of the csgD, cspD, and rpoS global regulator genes, thereby displaying a pleiotropic regulatory role. Here, we identified two promoters located in the toxin sequence driving the constitutive expression of mqsA, allowing thereby excess production of the MqsA antitoxin compared to the MqsR toxin. Our results show that both antitoxin-specific and operon promoters are not regulated by stresses such as amino acid starvation, oxidative shock, or bile salts. Moreover, we show that the MqsA antitoxin is not a global regulator as suggested, since the expression of csgD, cspD and rpoS is similar in wild-type and ΔmqsRA mutant strains. Moreover, these two strains behave similarly in terms of biofilm formation and sensitivity to oxidative stress or bile salts.IMPORTANCE There is growing controversy regarding the role of chromosomal toxin-antitoxin systems in bacterial physiology. mqsRA is a peculiar toxin-antitoxin system, as the gene encoding the toxin precedes that of the antitoxin. This system was previously shown to play a role in stress response and biofilm formation. In this work, we identified two promoters specifically driving the constitutive expression of the antitoxin, thereby decoupling the expression of antitoxin from the toxin. We also showed that mqsRA contributes neither to the regulation of biofilm formation nor to the sensitivity to oxidative stress and bile salts. Finally, we were unable to confirm that the MqsA antitoxin is a global regulator. Altogether, our data are ruling out the involvement of the mqsRA system in Escherichia coli regulatory networks.

Type I toxin-dependent generation of superoxide affects the persister life cycle of Escherichia coli

Induction of growth stasis by bacterial toxins from chromosomal toxin-antitoxin systems is suspected to favor formation of multidrug-tolerant cells, named persisters. Recurrent infections are often attributed to resuscitation and regrowth of persisters upon termination of antibiotic therapy. Several lines of evidence point to oxidative stress as a crucial factor during the persister life cycle. Here, we demonstrate that the membrane-depolarizing type I toxins TisB, DinQ, and HokB have the potential to provoke reactive oxygen species formation in Escherichia coli. More detailed work with TisB revealed that mainly superoxide is formed, leading to activation of the SoxRS regulon. Deletion of the genes encoding the cytoplasmic superoxide dismutases SodA and SodB caused both a decline in TisB-dependent persisters and a delay in persister recovery upon termination of antibiotic treatment. We hypothesize that expression of depolarizing toxins during the persister formation process inflicts an oxidative challenge. The ability to counteract oxidative stress might determine whether cells will survive and how much time they need to recover from dormancy.

Characterization and comparative analysis of toxin-antitoxin systems in Acetobacter pasteurianus.

Bacterial toxin-antitoxin (TA) systems play important roles in diverse cellular regulatory processes. Here, we characterize three putative type II TA candidates from Acetobacter pasteurianus and investigate the profile of type II TA systems in the genus Acetobacter. Based on the gene structure and activity detection, two-pairs loci were identified as the canonical hicAB and higAB TA systems, respectively, and DB34_01190-DB34_01195 as a putative new one without a canonical TA architecture. Physiologically, the expression of the three pairs conferred E. coli with additional plasmid maintenance and survival when under acetic acid stress. Chromosomal TA systems can be horizontally transferred within an ecological vinegar microbiota by co-option, and there was a tendency for toxin module loss. The antitoxin retention in the genome is suggested to have a broad role in bacterial physiology. Furthermore, A. pasteurianus strains, universally domesticated and used for industrial vinegar fermentation, showed a higher number of type II TA loci compared to the host-associated ones. The amount of TA loci per genome showed little positive relationship to insertion sequences, although its prevalence was species-associated, to the extent of even being strain-associated. The TA system is a candidate of studying the resistant mechanistic network, the TAs-dependent translatome affords a real-time profile to explore stress adaptation of A. pasteurianus, promoting industrial development.

Implication on the Function of Novel Xn-relE Toxin Structure of Xenorhabdus nematophila Using Homology Modeling

Background: Bacterial chromosomal toxin-antitoxin systems are involved in various cell functions such as stress response, promoting cell cycle arrest and bringing about the onset of programmed cell death. Unlike RelBE TA module of Escherichia coli, genome of Xenorhabdus nematophila has two separate TA modules for RelB and RelE. Here RelE being the toxin bears its own antitoxin and RelB antitoxin bears its separate toxin counterpart. More interestingly these modules are located distantly in genome. Objective: In this study, Xn-relE toxin model structure from X. nematophila is explored for the first time. Toxic effect of Xn-relE has already been shown by endogenous killing in our earlier report. Methods: Since no crystallographic structure for Xn-relE toxin is available till date. The models of X. nematophila Xn-relE toxin and its antitoxin Xn-relEAT were developed using the I-TASSER server and analyzed to define gene ontology. The models were validated by using VERIFY-3D. Results: Homology models for X. nematophila Xn-relE toxin and its antitoxin Xn-relEAT was obtained and interactions were established. The structural and functional annotation of this TA system designate it as Type II TA module. Conclusion: The present study sheds light on the structure and function of Xn-relE toxin of Xn RelE TA module, whose applicability in the area of agricultural sciences is pronounced. Keywords: X. nematophila, toxin-antitoxin module, Xn-relE toxin, Xn-relEAT antitoxin, homology modeling, GO-term.

Characterization of a Deinococcus radiodurans MazF: A UACA-specific RNA endoribonuclease.

Microbes are known to withstand environmental stresses by using chromosomal toxin–antitoxin systems. MazEF is one of the most extensively studied toxin–antitoxin systems. In stressful environments, MazF toxins modulate translation by cleaving single‐stranded RNAs in a sequence‐specific fashion. Previously, a chromosomal gene located at DR0417 in Deinococcus radiodurans was predicted to code for a MazF endoribonuclease (MazFDR 0417); however, its function remains unclear. In the present study, we characterized the molecular function of MazFDR 0417. Analysis of MazFDR 0417‐cleaved RNA sites using modified massively parallel sequencing revealed a unique 4‐nt motif, UACA, as a potential cleavage pattern. The activity of MazFDR 0417 was also assessed in a real‐time fluorometric assay, which revealed that MazFDR 0417 strictly recognizes the unique tetrad UACA. This sequence specificity may allow D. radiodurans to alter its translation profile and survive under stressful conditions.

Desperate times call for desperate measures: benefits and costs of toxin-antitoxin systems.

Toxin-antitoxin (TA) loci were first described as killing systems for plasmid maintenance. The surprisingly abundant presence of TA loci in bacterial chromosomes has stimulated an extensive research in the recent decade aimed to understand the biological importance of these potentially deadly systems. Accumulating evidence suggests that the evolutionary success of genomic TA systems could be explained by their ability to increase bacterial fitness under stress conditions. While TA systems remain quiescent under favorable growth conditions, the toxins can be activated in response to stress resulting in growth suppression and development of stress-tolerant dormant state. Yet, several studies suggest that the TA-mediated stress protection is costly and traded off against decreased fitness under normal growth conditions. Here, we give an overview of the fitness benefits of the chromosomal TA systems, and discuss the costs of TA-mediated stress protection.

Comprehensive Functional Analysis of the 18 Vibrio cholerae N16961 Toxin-Antitoxin Systems Substantiates Their Role in Stabilizing the Superintegron.

The role of chromosomal toxin-antitoxin (TA) systems, which are ubiquitous within the genomes of free-living bacteria, is still debated. We have scanned the Vibrio cholerae N16961 genome for class 2 TA genes and identified 18 gene pair candidates. Interestingly, all but one are located in the chromosome 2 superintegron (SI). The single TA found outside the SI is located on chromosome 1 and is related to the well-characterized HipAB family, which is known to play a role in antibiotic persistence. We investigated this clustering within the SI and its possible biological consequences by performing a comprehensive functional analysis on all of the putative TA systems. We demonstrate that the 18 TAs identified encode functional toxins and that their cognate antitoxins are able to neutralize their deleterious effects when expressed in Escherichia coli. In addition, we reveal that the 17 predicted TA systems of the SI are transcribed and expressed in their native context from their own promoters, a situation rarely found in integron cassettes. We tested the possibility of interactions between noncognate pairs of all toxins and antitoxins and found no cross-interaction between any of the different TAs. Although these observations do not exclude other roles, they clearly strengthen the role of TA systems in stabilizing the massive SI cassette array of V. cholerae. The chromosomal toxin-antitoxin systems have been shown to play various, sometimes contradictory roles, ranging from genomic stabilization to bacterial survival via persistence. Determining the interactions between TA systems hosted within the same bacteria is essential to understand the hierarchy between these different roles. We identify here the full set of class 2 TAs carried in the Vibrio cholerae N16961 genome and found they are all, with a single exception, located in the chromosome 2 superintegron. Their characterization, in terms of functionality, expression, and possible cross-interactions, supports their main role as being the stabilization of the 176-cassette-long array of the superintegron but does not exclude dual roles, such as stress response elements, persistence, and bacteriophage defense through abortive infection mechanisms.

Toxin-antitoxin systems in the mobile genome of Acidithiobacillus ferrooxidans.

Toxin-antitoxin (TA) systems are genetic modules composed of a pair of genes encoding a stable toxin and an unstable antitoxin that inhibits toxin activity. They are widespread among plasmids and chromosomes of bacteria and archaea. TA systems are known to be involved in the stabilization of plasmids but there is no consensus about the function of chromosomal TA systems. To shed light on the role of chromosomally encoded TA systems we analyzed the distribution and functionality of type II TA systems in the chromosome of two strains from Acidithiobacillus ferrooxidans (ATCC 23270 and 53993), a Gram-negative, acidophilic, environmental bacterium that participates in the bioleaching of minerals. As in other environmental microorganisms, A. ferrooxidans has a high content of TA systems (28-29) and in twenty of them the toxin is a putative ribonuclease. According to the genetic context, some of these systems are encoded near or within mobile genetic elements. Although most TA systems are shared by both strains, four of them, which are encoded in the active mobile element ICEAfe1, are exclusive to the type strain ATCC 23270. We demostrated that two TA systems from ICEAfe1 are functional in E. coli cells, since the toxins inhibit growth and the antitoxins counteract the effect of their cognate toxins. All the toxins from ICEAfe1, including a novel toxin, are RNases with different ion requirements. The data indicate that some of the chromosomally encoded TA systems are actually part of the A. ferrooxidans mobile genome and we propose that could be involved in the maintenance of these integrated mobile genetic elements.

Cryptic Streptococcus mutans 5.6-kb plasmids encode a toxin–antitoxin system for plasmid stabilization

Background: In all Streptococcus mutans strains, 5–13% carry a 5.6-kb plasmid. Despite its frequency, little is known about its mediated functions with most of the information coming from a single study focussing on plasmid pUA140.Objective: Here, we describe the sequence and genetic organization of two S. mutans 5.6-kb plasmids, pDC09 and pNC101.Results: Based on PicoGreen dsDNA quantification and Real-Time quantitative PCR (RTQ-PCR), the plasmid copy number was found to range between 10 and 74, depending on the strain tested. In contrast to literature, we identified six instead of five open reading frames (ORFs). While the putative gene products of ORF1 (as a Rep-protein) and ORF2 (as a Mob-protein) could be confirmed as being identical to those from pUA140, the functions of ORF3 (unknown) and ORF 4 (possibly AtpE homologue) could not be further revealed. However, the product of ORF5 showed a fairly high identity (38–50%) and structural similarity (58–74%) to RelE of Streptococcus pneumoniae, Streptococcus equi, and Streptococcus downei. In addition, we identified a functionally corresponding ORF6 encoding a protein with 61–68% identity (81–86% similarity) to the S. equi and S. downei antitoxin of the RelB family. RelE and RelB together form a plasmid-encoded toxin-antitoxin (TA) system, RelBEplas. Despite its rather limited sequence similarity with chromosomal TA systems in S. mutans (RelBEchro, MazEF, HicBA), we found similar tertiary structures applying I-Tasser protein prediction analysis.Conclusion: Type II-toxins, as the plasmid-encoded RelE, are RNA endonucleases. Depending on their mRNA cleavage activity, they might 1) kill every plasmid-free progeny, thereby stabilizing plasmid transfer at the expense of the host and/or 2) help S. mutans enter a dormant state and survive unfavourable environmental conditions. Whilst a function in plasmid stabilization has been confirmed, a function in persistence under nutritional stress, tested here by inducing amino acid starvation, could not be demonstrated so far.

Toxin-Antitoxin Genes of the Gram-Positive Pathogen Streptococcus pneumoniae: So Few and Yet So Many

Pneumococcal infections cause up to 2 million deaths annually and raise a large economic burden and thus constitute an important threat to mankind. Because of the increase in the antibiotic resistance of Streptococcus pneumoniae clinical isolates, there is an urgent need to find new antimicrobial approaches to triumph over pneumococcal infections. Toxin-antitoxin (TA) systems (TAS), which are present in most living bacteria but not in eukaryotes, have been proposed as an effective strategy to combat bacterial infections. Type II TAS comprise a stable toxin and a labile antitoxin that form an innocuous TA complex under normal conditions. Under stress conditions, TA synthesis will be triggered, resulting in the degradation of the labile antitoxin and the release of the toxin protein, which would poison the host cells. The three functional chromosomal TAS from S. pneumoniae that have been studied as well as their molecular characteristics are discussed in detail in this review. Furthermore, a meticulous bioinformatics search has been performed for 48 pneumococcal genomes that are found in public databases, and more putative TAS, homologous to well-characterized ones, have been revealed. Strikingly, several unusual putative TAS, in terms of components and genetic organizations previously not envisaged, have been discovered and are further discussed. Previously, we reported a novel finding in which a unique pneumococcal DNA signature, the BOX element, affected the regulation of the pneumococcal yefM-yoeB TAS. This BOX element has also been found in some of the other pneumococcal TAS. In this review, we also discuss possible relationships between some of the pneumococcal TAS with pathogenicity, competence, biofilm formation, persistence, and an interesting phenomenon called bistability.

Functional Identification of Toxin-Antitoxin Molecules from Helicobacter pylori 26695 and Structural Elucidation of the Molecular Interactions

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

The role of Escherichia coli YrbB in the lethal action of quinolones

To explore bacterial cellular factors that protect against the lethal effect of antimicrobial stress as potential targets of antimicrobial potentiators, the role of Escherichia coli YrbB in protecting cells from quinolone-mediated cell death was studied. A set of isogenic strains containing different mutations in stress response genes of E. coli was constructed by P1-mediated transduction. The susceptibility of these strains to the lethal action of quinolones was determined by measuring viable colony counts on agar plates after treatment with quinolones under various conditions. A yrbB mutation rendered E. coli cells more susceptible to the lethal action of quinolones under conditions in which bacteriostatic susceptibility was unaffected. YrbB worked in both lethal pathways of quinolone action. Hydroxyl radical accumulation was required for nalidixic acid-mediated killing; however, in the absence of functional YrbB there was an additional mechanism through which nalidixic acid could kill cells independently of hydroxyl radical action. The E. coli chromosomal toxin-antitoxin system ChpB, but not the SOS system, was found to be involved in the hydroxyl radical-independent lethal mechanism. In addition, proteases ClpP and Lon were also involved in the action of YrbB. Besides quinolones, YrbB also played a protective role in cellular responses to other stressors, such as mitomycin C, ultraviolet light and hydrogen peroxide. YrbB played a protective role in the lethal action of quinolones through a hydroxyl radical-independent and toxin-antitoxin-dependent mechanism, which makes it a potential target for antimicrobial enhancement.

The relBE2Spn toxin-antitoxin system of Streptococcus pneumoniae: role in antibiotic tolerance and functional conservation in clinical isolates.

Type II (proteic) chromosomal toxin-antitoxin systems (TAS) are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6). To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, ΔrelB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection.

The communication factor EDF and the toxin-antitoxin module mazEF determine the mode of action of antibiotics.

It was recently reported that the production of Reactive Oxygen Species (ROS) is a common mechanism of cell death induced by bactericidal antibiotics. Here we show that triggering the Escherichia coli chromosomal toxin–antitoxin system mazEF is an additional determinant in the mode of action of some antibiotics. We treated E. coli cultures by antibiotics belonging to one of two groups: (i) Inhibitors of transcription and/or translation, and (ii) DNA damaging. We found that antibiotics of both groups caused: (i) mazEF-mediated cell death, and (ii) the production of ROS through MazF action. However, only antibiotics of the first group caused mazEF-mediated cell death that is ROS-dependent, whereas those of the second group caused mazEF-mediated cell death by an ROS-independent pathway. Furthermore, our results showed that the mode of action of antibiotics was determined by the ability of E. coli cells to communicate through the signaling molecule Extracellular Death Factor (EDF) participating in mazEF induction.