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

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

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

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

Addictive manipulation: a perspective on the role of reproductive parasitism in the evolution of bacteria-eukaryote symbioses.

Wolbachia bacteria encompass noteworthy reproductive manipulators of their arthropod hosts. which influence host reproduction to favour their own transmission, also exploiting toxin-antitoxin systems. Recently, multiple other bacterial symbionts of arthropods have been shown to display comparable manipulative capabilities. Here, we wonder whether such phenomena are truly restricted to arthropod hosts. We focused on protists, primary models for evolutionary investigations on eukaryotes due to their diversity and antiquity, but still overall under-investigated. After a thorough re-examination of the literature on bacterial-protist interactions with this question in mind, we conclude that such bacterial 'addictive manipulators' of protists do exist, are probably widespread, and have been overlooked until now as a consequence of the fact that investigations are commonly host-centred, thus ineffective to detect such behaviour. Additionally, we posit that toxin-antitoxin systems are crucial in these phenomena of addictive manipulation of protists, as a result of recurrent evolutionary repurposing. This indicates intriguing functional analogy and molecular homology with plasmid-bacterial interplays. Finally, we remark that multiple addictive manipulators are affiliated with specific bacterial lineages with ancient associations with diverse eukaryotes. This suggests a possible role of addictive manipulation of protists in paving the way to the evolution of bacteria associated with multicellular organisms.

The two paralogous copies of the YoeB-YefM toxin-antitoxin module in Staphylococcus aureus differ in DNA binding and recognition patterns.

Toxin-antitoxin (TA) systems are ubiquitous regulatory modules for bacterial growth and cell survival following stress. YefM-YoeB, the most prevalent type II TA system, is present in a variety of bacterial species. In Staphylococcus aureus, the YefM-YoeB system exists as two independent paralogous copies. Our previous research resolved crystal structures of the two oligomeric states (heterotetramer and heterohexamer-DNA ternary complex) of the first paralog as well as the molecular mechanism of transcriptional autoregulation of this module. However, structural details reflecting molecular diversity in both paralogs have been relatively unexplored. To understand the molecular mechanism of how Sa2YoeB and Sa2YefM regulate their own transcription and how each paralog functions independently, we solved a series of crystal structures of the Sa2YoeB-Sa2YefM. Our structural and biochemical data demonstrated that both paralogous copies adopt similar mechanisms of transcriptional autoregulation. In addition, structural analysis suggested that molecular diversity between the two paralogs might be reflected in the interaction profile of YefM and YoeB and the recognition pattern of promoter DNA by YefM. Interaction analysis revealed unique conformational and activating force effected by the interface between Sa2YoeB and Sa2YefM. In addition, the recognition pattern analysis demonstrated that residues Thr7 and Tyr14 of Sa2YefM specifically recognizes the flanking sequences (G and C) of the promoter DNA. Together, these results provide the structural insights into the molecular diversity and independent function of the paralogous copies of the YoeB-YefM TA system.

Discovery of a novel type IIb RelBE toxin-antitoxin system in Mycobacterium tuberculosis defined by co-regulation with an antisense RNA.

Toxin-antitoxin loci regulate adaptive responses to stresses associated with the host environment and drug exposure. Phylogenomic studies have shown that Mycobacterium tuberculosis encodes a naturally expanded type II toxin-antitoxin system, including ParDE/RelBE superfamily members. Type II toxins are presumably regulated exclusively through protein-protein interactions with type II antitoxins. However, experimental observations in M. tuberculosis indicated that additional control mechanisms regulate RelBE2 type II loci under host-associated stress conditions. Herein, we describe for the first time a novel antisense RNA, termed asRelE2, that co-regulates RelE2 production via targeted processing by the Mtb RNase III, Rnc. We find that convergent expression of this coding-antisense hybrid TA locus, relBE2-asrelE2, is controlled in a cAMP-dependent manner by the essential cAMP receptor protein transcription factor, Crp, in response to the host-associated stresses of low pH and nutrient limitation. Ex vivo survival studies with relE2 and asrelE2 knockout strains showed that RelE2 contributes to Mtb survival in activated macrophages and low pH to nutrient limitation. To our knowledge, this is the first report of a novel tripartite type IIb TA loci and antisense post-transcriptional regulation of a type II TA loci.

Evaluation of antibiotic resistance, toxin-antitoxin systems, virulence factors, biofilm-forming strength and genetic linkage of Escherichia coli strains isolated from bloodstream infections of leukemia patients.

BACKGROUND: One of the most common complications in patients with febrile neutropenia, lymphoma, leukemia, and multiple myeloma is a bloodstream infection (BSI). OBJECTIVE: This study aimed to evaluate the antibiotic resistance patterns, virulence factors, biofilm-forming strength, and genetic linkage of Escherichia coli strains isolated from bloodstream infections (BSIs) of leukemia patients. METHODS: The study conducted in Iran from June 2021 to December 2022, isolated 67 E. coli strains from leukemia patients' bloodstream infections in hospitals in two different areas. Several techniques including disk diffusion and broth microdilution were used to identify patterns of antibiotic resistance, microtiter plate assay to measure biofilm formation, and PCR to evaluate the prevalence of different genes such as virulence factors, toxin-antitoxin systems, resistance to ß-lactams and fluoroquinolone antibiotics of E. coli strains. Additionally, the genetic linkage of the isolates was analyzed using the Enterobacterial Repeat Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method. RESULTS: The results showed that higher frequency of BSI caused by E. coli in man than female patients, and patients with acute leukemia had a higher frequency of BSI. Ampicillin and Amoxicillin-clavulanic acid showed the highest resistance, while Imipenem was identified as a suitable antibiotic for treating BSIs by E. coli. Multidrug-resistant (MDR) phenotypes were present in 22% of the isolates, while 53% of the isolates were ESBL-producing with the blaCTX-M gene as the most frequent ß-lactamase gene. The fluoroquinolone resistance genes qnrB and qnrS were present in 50% and 28% of the isolates, respectively. More than 80% of the isolates showed the ability to form biofilms. The traT gene was more frequent than other virulence genes. The toxin-antitoxin system genes (mazF, ccdAB, and relB) showed a comparable frequency. The genetic diversity was detected in E. coli isolates. CONCLUSION: Our results demonstrate that highly diverse, resistant and pathogenic E. coli clones are circulating among leukemia patients in Iranian hospitals. More attention should be paid to the treatment and management of E. coli bloodstream infections in patients with leukemia.

Effect of ZnO nanoparticles on biofilm formation and gene expression of the toxin-antitoxin system in clinical isolates of Pseudomonas aeruginosa.

BACKGROUND: Biofilm formation by Pseudomonas aeruginosa (P. aeruginosa) is known to be characteristic of this organism. This bacterium is considered one of the most life-threatening bacteria and has been identified as a priority pathogen for research by WHO. Biofilm-producing P. aeruginosa is a concern in many parts of the world due to antibiotic resistance. Alginate also plays an important role in the biofilm formation of P. aeruginosa as well as the emergence of antibiotic resistance in biofilms. In addition, the systems of toxin-antitoxin( TA) play an important role in biofilm formation. Metal nanoparticle(NP) such as zinc oxide (ZnO) also have extensive biological properties, especially anti-biofilm properties. Therefore, this study was conducted in relation to the importance of zinc oxide nanoparticles (ZnO NPs) in biofilm formation and also the correlation of gene expression of TA systems in clinical isolates of P. aeruginosa. METHODS: A total of 52 P. aeruginosa isolates were collected from burns (n = 15), UTI (n = 31), and trachea (n = 6) in hospitals in Ilam between May 2020 and October 2020. Biofilm formation was assessed using a microtiter plate assay. MIC and sub-MIC concentrations of ZnO NPs (10-30 nm with purity greater than 99.8%) in P. aeruginosa were determined. Subsequently, biofilm formation was investigated using sub-MIC concentrations of ZnO NPs. Finally, total RNA was extracted and RT- qPCR was used to determine the expression levels of genes of mazEF, mqsRA, and higBA of TA systems. RESULTS: Six isolates of P. aeruginosa were found to form strong biofilms. The results showed that ZnO NPs were able to inhibit biofilm formation. In our experiments, we found that the sub-MIC concentration of ZnO NPs increased the gene expression of antitoxins mazE and mqsA and toxin higB of TA systems treated with ZnO NPs. CONCLUSIONS: In the present study, ZnO NPs were shown to effectively inhibit biofilm formation in P. aeruginosa. Our results support the relationship between TA systems and ZnO NPs in biofilm formation in P. aeruginosa. Importantly, the expression of antitoxins mazE and mqsA was high after treatment with ZnO NPs, but not that of antitoxin higA.

Zinc oxide nanoparticles impact the expression of the genes involved in toxin-antitoxin systems in multidrug-resistant Acinetobacter baumannii.

The aim of this study was to investigate the effect of zinc oxide nanoparticles (ZnO-NPs) on the expression of genes involved in toxin-antitoxin (TA) systems in multidrug-resistant (MDR) Acinetobacter baumannii. Seventy clinical isolates of A. baumannii were collected from variuos clinical samples. Antimicrobial susceptibility test was determined by disk diffusion. Type II TA system-related genes including GNAT, XRE-like, hipA, hipB, hicA, hicB were screened using polymerase chain reaction (PCR). ZnO-NPs prepared and characterized by field emission scanning electron microscopy and X-ray diffraction. MIC of ZnO-NPs of A. baumannii isolates was performed using the microdilution method. The expression of type II TA systems-related genes were assessed with and without exposure to ZnO-NPs using real-time PCR. The highest rate of resistance and sensitivity was observed against cefepime (77.14%), and ampicillin/sulbactam (42.85%), respectively. All A. baumannii isolates were considered as MDR. In this study, three TA loci were identified for A. baumannii including GNAT/XRE-like, HicA/HicB, and HipA/HipB and their prevalence was 100%, 42%, and 27.1%, respectively. There was no significant relationship between the prevalence of these systems and the origin of A. baumannii. Our data showed significant correlations between the presence of HicA/HicB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05), and the presence of HipA/HipB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05). In presence of ZnO-NPs, the expression of all studied genes decreased. GNAT and hicB showed the highest and lowest expression changes by 2.4 folds (p < 0.001) and 1.3 folds (p < 0.05), respectively. This study demonstrates the promising potential of nanoparticles to impact the expression of the genes involved in TA Systems. So, the application of ZnO-NPs may be helpful to design target-based strategies towards MDRs pathogens for empowered clinical applications by microbiologists and nanotechnologists.

Regulation of Mannitol Metabolism in Enterococcus faecalis and Association with parEF0409 Toxin-Antitoxin Locus Function.

The parEF0409 type I toxin-antitoxin locus is situated between genes for two paralogous mannitol family phosphoenolpyruvate phosphotransferase systems (PTSs). In order to address the possibility that parEF0409 function was associated with sugar metabolism, genetic and phenotypic analyses were performed on the flanking genes. It was found that the genes were transcribed as two operons: the downstream operon essential for mannitol transport and metabolism and the upstream operon performing a regulatory function. In addition to genes for the PTS components, the upstream operon harbors a gene similar to mtlR, the key regulator of mannitol metabolism in other Gram-positive bacteria. We confirmed that this gene is essential for the regulation of the downstream operon and identified putative phosphorylation sites required for carbon catabolite repression and mannitol-specific regulation. Genomic comparisons revealed that this dual-operon organization of mannitol utilization genes is uncommon in enterococci and that the association with a toxin-antitoxin system is unique to Enterococcus faecalis. Finally, we consider possible links between parEF0409 function and mannitol utilization. IMPORTANCE Enterococcus faecalis is both a common member of the human gut microbiota and an opportunistic pathogen. Its evolutionary success is partially due to its metabolic flexibility, in particular its ability to import and metabolize a wide variety of sugars. While a large number of phosphoenolpyruvate phosphotransferase sugar transport systems have been identified in the E. faecalis genome bioinformatically, the specificity and regulation of most of these systems remain undetermined. Here, we characterize a complex system of two operons flanking a type I toxin-antitoxin system required for the transport and metabolism of the common dietary sugar mannitol. We also determine the phylogenetic distribution of mannitol utilization genes in the enterococcal genus and discuss the significance of the association with toxin-antitoxin systems.

Disruption of MenT2 toxin impairs the growth of Mycobacterium tuberculosis in guinea pigs.

Toxin-antitoxin (TA) systems are abundantly present in the genomes of various bacterial pathogens. TA systems have been implicated in either plasmid maintenance or protection against phage infection, stress adaptation or disease pathogenesis. The genome of Mycobacterium tuberculosis encodes for more than 90 TA systems and 4 of these belong to the type IV subfamily (MenAT family). The toxins and antitoxins belonging to type IV TA systems share sequence homology with the AbiEii family of nucleotidyl transferases and the AbiEi family of putative transcriptional regulators, respectively. Here, we have performed experiments to understand the role of MenT2, a toxin from the type IV TA system, in mycobacterial physiology and disease pathogenesis. The ectopic expression of MenT2 using inducible vectors does not inhibit bacterial growth in liquid cultures. Bioinformatic and molecular modelling analysis suggested that the M. tuberculosis genome has an alternative start site upstream of the annotated menT2 gene. The overexpression of the reannotated MenT2 resulted in moderate growth inhibition of Mycobacterium smegmatis. We show that both menT2 and menA2 transcript levels are increased when M. tuberculosis is exposed to nitrosative stress, in vitro. When compared to the survival of the wild-type and the complemented strain, the ΔmenT2 mutant strain of M. tuberculosis was more resistant to being killed by nitrosative stress. However, the survival of both the ΔmenT2 mutant and the wild-type strain was similar in macrophages and when exposed to other stress conditions. Here, we show that MenT2 is required for the establishment of disease in guinea pigs. Gross pathology and histopathology analysis of lung tissues from guinea pigs infected with the ∆menT2 strain revealed significantly reduced tissue damage and inflammation. In summary, these results provide new insights into the role of MenT2 in mycobacterial pathogenesis.

relBE toxin-antitoxin system as a reliable anti-biofilm target in Pseudomonas aeruginosa.

AIMS: The ability of the pathogenic bacterium Pseudomonas aeruginosa to produce biofilms has made it more difficult to treat its infections with current antibiotics. Several genes are involved in biofilm production, and toxin-antitoxin (TA) loci have been reported to be responsible for the regulation of biofilm-associated genes. This study was aimed at evaluating various TA loci in P. aeruginosa to find a reliable target in order to disrupt biofilm formation. METHODS AND RESULTS: Thirty clinical isolates of P. aeruginosa were assessed for biofilm production as well as the presence of various TA loci in their genomes. The relBETA locus was present in all 30 P. aeruginosa isolates but its expression was not detectable in isolates that did not show biofilm production. Quantitative real-time -PCR (q-PCR) also demonstrated that the expression of relBE was higher in isolates with stronger biofilm-producing capability. Knocking out the relBE locus in one biofilm-producing P. aeruginosa isolate led to the cessation of biofilm-producing capacity in that isolate and eliminated the expression of ndvB, which is among the genes involved in biofilm production. CONCLUSIONS: These results inferred the involvement of relBE TA locus in the regulation of biofilm production in P. aeruginosa and indicated the possibility of relBE as an anti-biofilm target for this pathogen.

Nutritional stress induced intraspecies competition revealed by transcriptome analysis in Sphingomonas melonis TY.

Bacteria have developed various mechanisms by which they can compete or cooperate with other bacteria. This study showed that in the cocultures of wild-type Sphingomonas melonis TY and its isogenic mutant TYΔndpD grow with nicotine, the former can outcompete the latter. TYΔndpD undergoes growth arrest after four days when cocultured with wild-type TY, whereas the coculture has just entered a stationary phase and the substrate was nearly depleted, and the interaction between the two related strains was revealed by transcriptomic analysis. Analysis of the differential expression genes indicated that wild-type TY inhibited the growth of TYΔndpD mainly through toxin-antitoxin (TA) systems. The four upregulated antitoxin coding genes belong to type II TA systems in which the bactericidal effect of the cognate toxin was mainly through inhibition of translation or DNA replication, whereas wild-type TY with upregulated antitoxin genes can regenerate cognate immunity protein continuously and thus prevent the lethal action of toxin to itself. In addition, colicin-mediated antibacterial activity against closely related species may also be involved in the competition between wild-type TY and TYΔndpD under nutritional stress. Moreover, upregulation of carbon and nitrogen catabolism related-, stress response related-, DNA repair related-, and DNA replication-related genes in wild-type TY showed that it triggered a series of response mechanisms when facing dual stress of competition from isogenic mutant cells and nutritional limitation. Thus, we proposed that S. melonis TY employed the TA systems and colicin to compete with TYΔndpD under nutritional stress, thereby maximally acquiring and exploiting finite resources. KEY POINTS: • Cross-feeding between isogenic mutants and the wild-type strain. • Nutrition stress caused a shift from cooperation to competition. • TYΔndpD undergo growth arrest by exogenous and endogenous toxins.

HigBA toxin-antitoxin system of Weissella cibaria is involved in response to the bile salt stress.

BACKGROUND: Toxin-antitoxin (TA) systems are prevalent adaptive genetic elements in bacterial genomes, which can respond to environmental stress. While, few studies have addressed TA systems in probiotics and their roles in the adaptation to gastrointestinal transit (GIT) environments. RESULTS: The Weissella cibaria 018 could survive in pH 3.0-5.0 and 0.5-3.0 g L-1 bile salt, and its HigBA system responded to the bile salt stress, but not to acid stress. The toxin protein HigB and its cognate antitoxin protein HigA had 85.1% and 100% similarity with those of Lactobacillus plantarum, respectively, and they formed the stable tetramer HigB-(HigA)2 -HigB structure in W. cibaria 018. When exposed to 1.5-3.0 g L-1 bile salt, the transcriptions of higB and higA were up-regulated with 4.39-19.29 and 5.94-30.91 folds, respectively. Meanwhile, W. cibaria 018 gathered into a mass with 48.07% survival rate and its persister cells were found to increase 8.21% under 3.0 g L-1 bile salt. CONCLUSION: The HigBA TA system of W. cibaria 018 responded to the bile salt stress, but not to acid stress, which might offer novel perspectives to understand the tolerant mechanism of probiotics to GIT environment. © 2022 Society of Chemical Industry.

Depletion of the DarG antitoxin in Mycobacterium tuberculosis triggers the DNA-damage response and leads to cell death.

Of the ~80 putative toxin-antitoxin (TA) modules encoded by the bacterial pathogen Mycobacterium tuberculosis (Mtb), three contain antitoxins essential for bacterial viability. One of these, Rv0060 (DNA ADP-ribosyl glycohydrolase, DarGMtb ), functions along with its cognate toxin Rv0059 (DNA ADP-ribosyl transferase, DarTMtb ), to mediate reversible DNA ADP-ribosylation (Jankevicius et al., 2016). We demonstrate that DarTMtb -DarGMtb form a functional TA pair and essentiality of darGMtb is dependent on the presence of darTMtb , but simultaneous deletion of both darTMtb -darGMtb does not alter viability of Mtb in vitro or in mice. The antitoxin, DarGMtb , forms a cytosolic complex with DNA-repair proteins that assembles independently of either DarTMtb or interaction with DNA. Depletion of DarGMtb alone is bactericidal, a phenotype that is rescued by expression of an orthologous antitoxin, DarGTaq , from Thermus aquaticus. Partial depletion of DarGMtb triggers a DNA-damage response and sensitizes Mtb to drugs targeting DNA metabolism and respiration. Induction of the DNA-damage response is essential for Mtb to survive partial DarGMtb -depletion and leads to a hypermutable phenotype.

Antibiotic tolerance in biofilm persister cells of Staphylococcus aureus and expression of toxin-antitoxin system genes.

The ability of Staphylococcus aureus to form biofilm and persister cells is the main cause of recurrent infections. This study aimed to evaluate the expression of toxin-antitoxin (TA) systems in persister cells within S. aureus biofilms. Time-dependent variation in the persister population present in biofilms of S. aureus was examined after treatment with bactericidal antibiotics. Then, the relative expression level of type II TA system (mazF, relE1, and relE2), type I TA system (sprG), and clpP protease genes in S. aureus strains were assessed by Real _Time PCR. Among the sixteen isolates, two isolates were found to be the strongest biofilm producers. The established biofilm of these isolates showed a comparable biphasic pattern at the lethal dose of the antibiotics. The expression level of TA system genes was increased and strain-specific expression patterns were observed under antibiotics stress conditions. Persisters within a biofilm may establish a reservoir for relapsing infection and could contribute to treatment failures. Hence, the possible role of the TA systems should be considered in biofilm and persister cell formation.

Identification and characterization of the type II toxin-antitoxin systems in the carbapenem-resistant Acinetobacterbaumannii.

Carbapenem -resistant A. baumannii (CRAB) is a major cause of both community-associated and nosocomial infections that are difficult to control and treat worldwide. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. The functional diversity and ubiquitous distribution in bacterial genomes are causing significant attention toward TA systems in bacteria. However, there is no enough information on the prevalence and identity of TA systems in CRAB clinical isolates. This study aimed to identify type II toxin-antitoxin systems in carbapenem-resistant A. baumannii (CRAB) isolates. A total of 80 A. baumannii isolates were collected from different clinical samples. Antibiotic resistance patterns of A. baumannii isolates were evaluated phenotypically and genetically. The frequency of type II TA genes was evaluated in CRAB isolates using PCR. Moreover, the expression level of the most prevalent TA encoding genes in some clinical isolates were evaluated by RT-qPCR. To determine whether the SplT and SplA are functional, the growth of E. coli BL21 cells (DE3/pLysS) harboring pET28a, pET28a-splTA, and pET28a-splT were analyzed by kill-rescue assay. All of the isolates were resistant to third generation of cephalosporins, ciprofloxacin and levofloxacin, whereas, 72%, 81% and 87% were resistant to amikacin, carbapenems and tetracycline, respectively. The cheTA in 47 isolates (72.5%) and splTA in 39 isolates (60%) of 65 isolates were the most common genes encoding type II TA among CRAB isolates. RT-qPCR demonstrated that cheTA and splTA transcripts are produced in the clinical isolates. There was a significant correlation between the presence of splTA genes and blaOXA-24 in CRAB isolates. Over-expression of the splT gene in E. coli results in inhibition of bacterial growth, whereas co-expression of splTA effectively restores the growth. This study presents the first identification of the type II TA systems among the carbapenem -resistant A. baumannii isolates, in Iran.

A systematic search for switch-like behavior in type II toxin-antitoxin systems.

Bistable switch-like behavior is a ubiquitous feature of gene regulatory networks with decision-making capabilities. Type II toxin-antitoxin (TA) systems are hypothesized to facilitate a bistable switch in toxin concentration that influences the dormancy transition in persister cells. However, a series of recent retractions has raised fundamental questions concerning the exact mechanism of toxin propagation in persister cells and the relationship between type II TA systems and cellular dormancy. Through a careful modeling search, we identify how sp: bistablilty can emerge in type II TA systems by systematically modifying a basic model for the RelBE system with other common biological mechanisms. Our systematic search uncovers a new combination of mechanisms influencing bistability in type II TA systems and explores how toxin bistability emerges through synergistic interactions between paired type II TA systems. Our analysis also illustrates how Descartes' rule of signs and the resultant can be used as a powerful delineator of bistability in mathematical systems regardless of application.

Role of Toxin-Antitoxin-Regulated Persister Population and Indole in Bacterial Heat Tolerance.

YafQ is an endoribonuclease toxin that degrades target gene transcripts such as that of tnaA, a gene encoding tryptophanase to synthesize indole from tryptophan. DinJ is the cognate antitoxin of YafQ, and the YafQ-DinJ system was reported to regulate persister formation by controlling indole production in Escherichia coli In this study, we investigated the role of YafQ-DinJ, indole production, and persister population in bacterial heat tolerance. yafQ (ΔyafQ), dinJ (ΔdinJ), and tnaA (ΔtnaA) single-gene knockout mutants showed approximately 10-fold higher heat tolerance than wild-type (WT) E. coli BW25113. Persister fractions of all mutants were slightly larger than that of the WT. Interestingly, these persister cells showed an approximately 100-fold higher heat tolerance than normal cells, but there was no difference among the persister cells of all mutants and the WT in terms of heat tolerance. Indole and its derivatives promoted a drastic reduction of bacterial heat tolerance by just 10 min of pretreatment, which is not sufficient to affect persister formation before heat treatment. Surprisingly, indole and its derivatives also reduced the heat tolerance of persister cells. Among the tested derivatives, 5-iodoindole exhibited the strongest effect on both normal and persister cells.IMPORTANCE Our study demonstrated that a small persister population exhibits significantly higher heat tolerance than normal cells and that this small fraction contributes to the heat tolerance of the total bacterial population. This study also demonstrated that indole, known to inhibit persister formation, and its derivatives are very promising candidates to reduce the heat tolerance of not only normal bacterial cells but also persister cells.

The expression of type II TA system genes following exposure to the sub-inhibitory concentration of gentamicin and acid stress in Brucella spp.

BACKGROUND: Brucellosis is one of the most common diseases that afflicts both humans and animals. Bacteria react to stress conditions using different mechanisms one of which is Toxin-Antitoxin (TA) systems. It is believed that the Toxin-Antitoxin (TA) systems have a key role in the chronicity of the disease. This study investigated the expression of TA system genes under acid and antibiotic stresses in Brucella spp. METHODS: Fifty Brucella isolates (17 isolated from animals and 31 isolated from human specimens, and two standard strains) were analyzed using PCR (using two pairs of primers). Then, to determine the effects of sub-MIC of gentamicin on bacterial survival and growth, colony forming unit was quantitated and turbidity was assessed following the treatment of Brucella spp, with ½ MIC of gentamicin at different time intervals. Furthermore, the colony forming unit of Brucella spp, was assessed under acid stress (pH = 5.5) compared to the control (pH = 7.6). Moreover, the expression of TA system genes in Brucella spp, was evaluated 1 h after treatment using qRT-PCR method. RESULTS: A total of 50 isolates, including 41 (82%) Brucella melitensis and 7 (14%) Brucella abortus with two standard strains Brucella melitensis (16 M) and Brucella abortus (B19) were investigated. Our results revealed the reduced growth of Brucella spp. in the presence of sub-MIC of gentamicin compared to the control. Furthermore, according to the results of qRT-PCR assay, gentamicin could increase the expression of TA system genes. Also, results of qRT-PCR showed that under acid stress, the expression of TA system gene COGT/COGAT decreased compared to the control. CONCLUSION: Although the exact role of the TA systems in response to stress is still unclear, our study provided information on the effect of the type II TA systems under the acid and antibiotic stress conditions. However, further studies are still required.

The interaction of phages and bacteria: the co-evolutionary arms race.

Since the dawn of life, bacteria and phages are locked in a constant battle and both are perpetually changing their tactics to overcome each other. Bacteria use various strategies to overcome the invading phages, including adsorption inhibition, restriction-modification (R/E) systems, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, abortive infection (Abi), etc. To counteract, phages employ intelligent tactics for the nullification of bacterial defense systems, such as accessing host receptors, evading R/E systems, and anti-CRISPR proteins. Intense knowledge about the details of these defense pathways is the basis for their broad utilities in various fields of research from microbiology to biotechnology. Hence, in this review, we discuss some strategies used by bacteria to inhibit phage infections as well as phage tactics to circumvent bacterial defense systems. In addition, the application of these strategies will be described as a lesson learned from bacteria and phage combats. The ecological factors that affect the evolution of bacterial immune systems is the other issue represented in this review.