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Bacteria and their viral predators (phages) are constantly evolving to subvert one another. Many bacterial immune systems that inhibit phages are encoded on mobile genetic elements that can be horizontally transmitted to diverse bacteria. Despite the pervasive appearance of immune systems in bacteria, it is not often known if these immune systems function against phages that the host encounters in nature. Additionally, there are limited examples demonstrating how these phages counter-adapt to such immune systems. Here, we identify clinical isolates of the global pathogen Vibrio cholerae harboring a novel genetic element encoding the bacterial immune system DarTG and reveal the immune systems impact on the co-circulating lytic phage ICP1. We show that DarTG inhibits ICP1 genome replication, thus preventing ICP1 plaquing. We further characterize the conflict between DarTG-mediated defense and ICP1 by identifying an ICP1-encoded protein that counters DarTG and allows ICP1 progeny production. Finally, we identify this protein, AdfB, as a functional antitoxin that abrogates the toxin DarT likely through direct interactions. Following the detection of the DarTG system in clinical V. cholerae isolates, we observed a rise in ICP1 isolates with the functional antitoxin. These data highlight the use of surveillance of V. cholerae and its lytic phages to understand the co-evolutionary arms race between bacteria and their phages in nature.IMPORTANCEThe global bacterial pathogen Vibrio cholerae causes an estimated 1 to 4 million cases of cholera each year. Thus, studying the factors that influence its persistence as a pathogen is of great importance. One such influence is the lytic phage ICP1, as once infected by ICP1, V. cholerae is destroyed. To date, we have observed that the phage ICP1 shapes V. cholerae evolution through the flux of anti-phage bacterial immune systems. Here, we probe clinical V. cholerae isolates for novel anti-phage immune systems that can inhibit ICP1 and discover the toxin-antitoxin system DarTG as a potent inhibitor. Our results underscore the importance of V. cholerae and ICP1 surveillance to elaborate novel means by which V. cholerae can persist in both the human host and aquatic reservoir in the face of ICP1.

Small genetic elements known as toxin-antitoxin (TA) systems are abundant in bacterial genomes and involved in stress response, phage inhibition, mobile genetic elements maintenance and biofilm formation. Type II TA systems are the most abundant and diverse, and they are organized as bicistronic operons that code for proteins (toxin and antitoxin) able to interact through a nontoxic complex. However, HicAB is one of the type II TA systems that remains understudied. Here, we review the current knowledge of HicAB systems in different bacteria, their main characteristics and the existing evidence to associate them with some biological roles, are described. The accumulative evidence reviewed here, though modest, underscores that HicAB systems are underexplored TA systems with significant potential for future research. [ABSTRACT FROM AUTHOR]

Introduction: Levels of the human epidermal growth factor receptor 2 (HER2) gene are low in normal breast tissue, but half of the patients with breast cancer exhibit higher levels of this receptor. The differential expression of the HER2 gene in normal and malignant cells makes it an excellent biomarker for therapeutic purposes. In this study, we evaluated the degree of HER2 overexpression in patients and its relationship with age and the occurrence of metastases. Materials & Methods: In this retrospective, registry-based, two-center cohort study, information on 1500 breast cancer patients recruited from Shahid Mostafa Khomeini Hospital in Ilam Province was collected from 2020 to 2023. Results: The likelihood of metastasis in cancer patients with HER2 gene expression was three times higher (adjusted OR: 2.82; 95% CI: 1.79-3.29; P=0.001). Additionally, the involvement of lymph nodes (adjusted OR: 2.01; 95% CI: 0.87-3.79; P=0.03) was significantly associated with increased metastasis. Conclusion: This study demonstrates that HER2 gene expression and the number of involved lymph nodes are significant prognostic factors that increase the risk of metastasis. Therefore, implementing comprehensive breast cancer screenings can play an important role in the treatment and prevention of metastasis in breast cancer patients. [ABSTRACT FROM AUTHOR]

Objective: Persister cells are a specific subset of bacteria capable of surviving exposure to lethal doses of antibiotics, leading to antibiotic therapy failures and infection relapses. This research explores the utilization of drug repositioning to target the Lon protease in Salmonella Typhimurium. Method: In this study, FDA-approved drugs sourced from the Drug Bank database were screened to identify existing pharmaceuticals with the potential to combat the Lon protease. The formation of persister cells in the presence of antibiotics, as well as the combination of antibiotics with potential Lon protease inhibitors, was examined. Furthermore, the expression of type II toxin-antitoxin system genes was analyzed to enhance our comprehension of the inhibitors' effects. Result: Molecular docking analysis revealed that Diosmin and Nafcillin exhibited strong binding affinity to the Lon protease. Molecular dynamics simulation trajectories analysis demonstrated that the interaction of these ligands with the enzyme did not induce instability; rather, the enzyme's structure remained stable. Combinations of ceftazidime and ciprofloxacin with either Nafcillin or Diosmin led to significant reductions in bacterial cell counts. Furthermore, the effectiveness of these combinations, when compared to antibiotics alone, highlighted the substantial impact of Nafcillin and Diosmin in reducing type II TA system gene expression. Conclusion: These findings suggest promising prospects for developing novel therapeutic approaches targeting persister cells to mitigate treatment failures in Salmonella infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin–antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores". [ABSTRACT FROM AUTHOR]

ABSTRACT Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy, and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present a moderate expression system for toxin genes based on an artificial 5′ untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin-antitoxin system tisB/istR-1 in Escherichia coli. TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA-sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpAB and spy. The importance of chaperone genes suggested that TisB-producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells.IMPORTANCEProtein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including exposure to antibiotics. In Escherichia coli, the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which, in turn, promotes an extended state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy.

Pathobionts have been implicated in various chronic diseases, including Crohn's disease (CD), a multifactorial chronic inflammatory condition that primarily affects the gastrointestinal tract, causing inflammation and damage to the digestive system. While the exact cause of CD remains unclear, adherent-invasive Escherichia coli (AIEC) strains have emerged as key contributors to its pathogenesis. AIEC are characterized by their ability to adhere to and invade intestinal epithelial cells and survive and replicate inside macrophages. However, the mechanisms underlying the virulence and persistence of AIEC within their host remain the subject of intensive research. Toxin–antitoxin systems (TAs) play a potential role in AIEC pathogenesis and may be therapeutic targets. These systems generally consist of two components: a toxin harmful to the cell and an antitoxin that neutralizes the toxin's effects. They contribute to bacterial survival in adverse conditions and regulate bacterial growth and behavior, affecting various cellular processes in bacterial pathogens. This review focuses on the current information available to determine the roles of TAs in the pathogenicity of AIEC. Their contribution to the AIEC stress response, biofilm formation, phage inhibition, the maintenance of mobile genetic elements, and host lifestyles is discussed. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTIs) represent a significant challenge in clinical practice, with recurrent forms (rUTIs) posing a continual threat to patient health. Escherichia coli (E. coli) is the primary culprit in a vast majority of UTIs, both community-acquired and hospital-acquired, underscoring its clinical importance. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. The type II TA system, prevalent in prokaryotes, emerges as a critical player in stress response, biofilm formation, and cell dormancy. ccdAB, the first identified type II TA module, is renowned for maintaining plasmid stability. This paper aims to unravel the physiological role of the ccdAB in rUTIs caused by E. coli, delving into bacterial characteristics crucial for understanding and managing this disease. We investigated UPEC-induced rUTIs, examining changes in type II TA distribution and number, phylogenetic distribution, and Multi-Locus Sequence Typing (MLST) using polymerase chain reaction (PCR). Furthermore, our findings revealed that the induction of ccdB expression in E. coli BL21 (DE3) inhibited bacterial growth, observed that the expression of both ccdAB and ccdB in E. coli BL21 (DE3) led to an increase in biofilm formation, and confirmed that ccdAB plays a role in the development of persistent bacteria in urinary tract infections. Our findings could pave the way for novel therapeutic approaches targeting these systems, potentially reducing the prevalence of rUTIs. Through this investigation, we hope to contribute significantly to the global effort to combat the persistent challenge of rUTIs. [ABSTRACT FROM AUTHOR]

ObjectivePersister cells are a specific subset of bacteria capable of surviving exposure to lethal doses of antibiotics, leading to antibiotic therapy failures and infection relapses. This research explores the utilization of drug repositioning to target the Lon protease in Salmonella Typhimurium.MethodIn this study, FDA-approved drugs sourced from the Drug Bank database were screened to identify existing pharmaceuticals with the potential to combat the Lon protease. The formation of persister cells in the presence of antibiotics, as well as the combination of antibiotics with potential Lon protease inhibitors, was examined. Furthermore, the expression of type II toxin-antitoxin system genes was analyzed to enhance our comprehension of the inhibitors’ effects.ResultMolecular docking analysis revealed that Diosmin and Nafcillin exhibited strong binding affinity to the Lon protease. Molecular dynamics simulation trajectories analysis demonstrated that the interaction of these ligands with the enzyme did not induce instability; rather, the enzyme’s structure remained stable. Combinations of ceftazidime and ciprofloxacin with either Nafcillin or Diosmin led to significant reductions in bacterial cell counts. Furthermore, the effectiveness of these combinations, when compared to antibiotics alone, highlighted the substantial impact of Nafcillin and Diosmin in reducing type II TA system gene expression.ConclusionThese findings suggest promising prospects for developing novel therapeutic approaches targeting persister cells to mitigate treatment failures in Salmonella infections.

Toxin–antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer. [ABSTRACT FROM AUTHOR]

ABSTRACTWe propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates.IMPORTANCELive vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

Abstract 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 β-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 β-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.

Abstract 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.

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin–antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of “lipid pores”.

Toxin-antitoxin (TA) systems are commonly found in most species of bacteria, including the causative agent of tuberculosis M. tuberculosis. TA systems play a role in the adaptation of the pathogen to stresses during host infection. Here we present the results of our study of the effect of the mutation in the vapC46 (C113G) gene using Mycolicibacterium smegmatis as a model. Expression of wild-type and mutant variants of the vapC46 gene in pKW08-MCS-Int vector was shown to increase resistance to erythromycin as well as the survival of M. smegmatis cells under short-term exposure to 0.5 mM H2O2. The introduction of the wild-type variant of vapC46 gene into M. smegmatis cells leads to a slowdown in growth. Expression of the mutant variant of the vapC46 gene did not affect the growth rate. However, no change in the level of RNase activity in cells carrying the vapC46 C113G mutation was observed in an in vitro model. Thus, the VapC46 toxin is involved in the regulation of the survival of M. smegmatis under oxidative stress, and the C113G mutation does not affect this function. The normal growth rate of M. smegmatis, containing the mutant vapC46 gene, along with the absence of changes in the ribonuclease activity of the VapC46 toxin, may be associated either with a change in the specificity of the mutant toxin for bacterial cell mRNA, or with the use of tRNA or rRNA as a substrate. The study of the effects of introducing a mutation into the vapC46 gene on the specificity of the VapC46 toxin is the subject of our further research. [ABSTRACT FROM AUTHOR]

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 β-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 β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pathobionts have been implicated in various chronic diseases, including Crohn’s disease (CD), a multifactorial chronic inflammatory condition that primarily affects the gastrointestinal tract, causing inflammation and damage to the digestive system. While the exact cause of CD remains unclear, adherent-invasive Escherichia coli (AIEC) strains have emerged as key contributors to its pathogenesis. AIEC are characterized by their ability to adhere to and invade intestinal epithelial cells and survive and replicate inside macrophages. However, the mechanisms underlying the virulence and persistence of AIEC within their host remain the subject of intensive research. Toxin–antitoxin systems (TAs) play a potential role in AIEC pathogenesis and may be therapeutic targets. These systems generally consist of two components: a toxin harmful to the cell and an antitoxin that neutralizes the toxin’s effects. They contribute to bacterial survival in adverse conditions and regulate bacterial growth and behavior, affecting various cellular processes in bacterial pathogens. This review focuses on the current information available to determine the roles of TAs in the pathogenicity of AIEC. Their contribution to the AIEC stress response, biofilm formation, phage inhibition, the maintenance of mobile genetic elements, and host lifestyles is discussed.

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. [ABSTRACT FROM AUTHOR]

Pseudomonas savastanoi pv. glycinea (Psg) causes bacterial blight on soybeans. Among Psg mutants that we previously generated by transposon mutagenesis to identify potential virulence factors, mutant VTP20 had reduced virulence. Genetic analysis of VTP20 revealed a mutation in operon PsgB076_26935-PsgB076_26940 (vt35-vt40). The vt35-vt40 operon is a toxin–antitoxin (TA) system, in which vt35 is functionally considered an antitoxin component. However, the function of this antitoxin in the vt35-vt40 TA system in Psg is unclear. Thus, here we used an overexpression approach to analyze the effects of vt35 on Psg virulence and found that Psg-vt35-OE caused less severe disease than Psg WT did in spray-inoculated and in syringe-infiltrated soybean leaves. Vt35 also regulated gene expression for multiple extracytoplasmic function (ECF) sigma factors during infection. These results suggest that Vt35 plays a central regulatory role in Psg virulence by regulating virulence factors and controlling alternate sigma factors. [ABSTRACT FROM AUTHOR]

Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA1 and HipA2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.

Introduction: Staphylococcus aureus (S. aureus) is a highly pathogenic and adaptable Gram-positive bacterium that exhibits persistence in various environments. The toxin-antitoxin (TA) system plays a crucial role in the defense mechanism of bacterial pathogens, allowing them to survive in stressful conditions. While TA systems in clinical pathogens have been extensively studied, there is limited knowledge regarding the diversity and evolutionary complexities of TA systems in S. aureus. Methods: We conducted a comprehensive in silico survey using 621 publicly available S. aureus isolates. We employed bioinformatic search and prediction tools, including SLING, TADB2.0, and TASmania, to identify TA systems within the genomes of S. aureus. Results: Our analysis revealed a median of seven TA systems per genome, with three type II TA groups (HD, HD_3, and YoeB) being present in over 80% of the strains. Additionally, we observed that TA genes were predominantly encoded in the chromosomal DNA, with some TA systems also found within the Staphylococcal Cassette Chromosomal mec (SCCmec) genomic islands. Discussion: This study provides a comprehensive overview of the diversity and prevalence of TA systems in S. aureus. The findings enhance our understanding of these putative TA genes and their potential implications in S. aureus ecology and disease management. Moreover, this knowledge could guide the development of novel antimicrobial strategies. [ABSTRACT FROM AUTHOR]

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum. [ABSTRACT FROM AUTHOR]

Toxin–antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer.

Contact-dependent growth inhibition (CDI) is a form of interbacterial competition mediated by CdiB-CdiA two-partner secretion systems. CdiA effector proteins carry polymorphic C-terminal toxin domains (CdiA-CT), which are neutralized by specific CdiI immunity proteins to prevent self-inhibition. Here, we present the crystal structures of CdiA-CT⋅CdiI complexes from Klebsiella pneumoniae 342 and Escherichia coli 3006. The toxins adopt related folds that resemble the ribonuclease domain of colicin D, and both are isoacceptor-specific tRNases that cleave the acceptor stem of deacylated tRNAGAUIle. Although the toxins are similar in structure and substrate specificity, CdiA-CTKp342 activity requires translation factors EF-Tu and EF-Ts, whereas CdiA-CTEC3006 is intrinsically active. Furthermore, the corresponding immunity proteins are unrelated in sequence and structure. CdiIKp342 forms a dimeric β sandwich, whereas CdiIEC3006 is an α-solenoid monomer. Given that toxin-immunity genes co-evolve as linked pairs, these observations suggest that the similarities in toxin structure and activity reflect functional convergence.

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. [ABSTRACT FROM AUTHOR]

Toxin/antitoxin (TA) systems are present in nearly every prokaryotic genome and play the important physiological roles of phage inhibition by reducing metabolism (this includes persistence for the extreme case of complete cessation of metabolism), genetic element stabilization, and biofilm formation. TA systems have also been incorporated into other cell systems, such as CRISPR-Cas and phage quorum sensing. For the simplest and best-studied case, proteinaceous toxins and antitoxins (i.e., type II), toxin activity is masked by direct binding of the antitoxin. A long-standing, unresolved question in the TA field is how toxins are activated when bound to antitoxins at nanomolar affinity. The current paradigm envisions preferential degradation of the antitoxin by a protease, but this is highly unlikely in that a protease cannot discriminate between bound toxin and bound antitoxin because both are highly structured. Strikingly, recent results from several studies show one likely mechanism for toxin activation is conformational changes in the TA complex that result in the release or activation of the toxin as a result of a protein trigger, such as that from phages, and as a result of thermally-driven refolding dynamics., Competing Interests: The authors declare no conflict of interest.

Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy, and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present a moderate expression system for toxin genes based on an artificial 5' untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin-antitoxin system tisB/istR-1 in Escherichia coli . TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA-sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpAB and spy . The importance of chaperone genes suggested that TisB-producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells., Importance: Protein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including exposure to antibiotics. In Escherichia coli , the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which, in turn, promotes an extended state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy., Competing Interests: The authors declare no conflict of interest.

Harmful algal blooms caused by cyanobacteria threaten aquatic ecosystems, the economy, and human health. Previous work has tried to identify the mechanisms that allow blooms to form, focusing on the role of nutrients. However, little is known about how introduced nutrients influence gene expression in situ . To address this knowledge gap, we used in situ mesocosms initiated with water experiencing a Microcystis bloom. We added pulses of nutrients that are commonly associated with anthropogenic sources to the mesocosms for 72 hours and collected samples for metatranscriptomics to examine how the physiological function of Microcystis and bloom status changed. The addition of nitrogen (N) as urea, but not the addition of PO 4 , resulted in conspicuous bloom persistence for at least 9 days after the final introduction of nutrients. The addition of urea initially resulted in the upregulation of photosynthesis machinery, as well as phosphate, carbon, and N transport and metabolism. Once Microcystis presumably became N-replete, upregulation of amino acid metabolism, microcystin biosynthesis, and other processes associated with biomass generation occurred. These capacities coincided with the upregulation of toxin-antitoxin systems, CRISPR- cas genes, and transposases suggesting that phage defense and genome rearrangement are critical in bloom persistence. Overall, our results show the stepwise transcriptional response of a Microcystis bloom to the introduction of nutrients, specifically urea, as it is sustained in a natural setting. The transcriptomic shifts observed herein may serve as markers of the longevity of blooms while providing insight into why Microcystis blooms over other cyanobacteria.IMPORTANCEHarmful algal blooms represent a threat to human health and ecosystems. Understanding why blooms persist may help us develop warning indicators of bloom persistence and create novel mitigation strategies. Using mesocosm experiments initiated with water with an active bloom, we measured the stepwise transcription changes of the toxin-producing cyanobacterium Microcystis in response to the addition of nutrients that are important in causing blooms. We found that nitrogen (N), but not phosphorus, promoted bloom longevity. The initial introduction of N resulted in the upregulation of genes involved in photosynthesis and N import. At later times in the bloom, upregulation of genes involved in biomass generation, phage protection, genomic rearrangement, and toxin production was observed. Our results suggest that Microcystis first fulfills nutritional requirements before investing energy in pathways associated with growth and protection against competitors, which allowed bloom persistence more than a week after the final addition of nutrients.

Anti-phage defenses must rapidly sense and respond to diverse viruses. A recent pair of papers in Nature reveal via structural and functional assays how the PARIS defense system, a recently discovered toxin-antitoxin system, senses phage-associated molecular patterns (PhAMPs), thereby activating an endonuclease toxin that cleaves tRNA to block phage replication., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Transcription factors are the primary regulators of gene expression and recognize specific DNA sequences under diverse physiological conditions. Although they are vital for many important cellular processes, it remains unclear when and how transcription factors and DNA interact. The antitoxin from a toxin–antitoxin system is an example of negative transcriptional autoregulation: during expression of the cognate toxin it is suppressed through binding to a specific DNA sequence. In the present study, the antitoxin HigA2 from Mycobacterium tuberculosis M37Rv was structurally examined. The crystal structure of M. tuberculosis HigA2 comprises three sections: an N-terminal autocleavage region, an α-helix bundle which contains an HTH motif, and a C-terminal β-lid. The N-terminal region is responsible for toxin binding, but was shown to cleave spontaneously in its absence. The HTH motif performs a key role in DNA binding, with the C-terminal β-lid influencing the interaction by mediating the distance between the motifs. However, M. tuberculosis HigA2 exhibits a unique coordination of the HTH motif and no DNA-binding activity is detected. Three crystal structures of M. tuberculosis HigA2 show a flexible alignment of the HTH motif, which implies that the motif undergoes structural rearrangement to interact with DNA. This study reveals the molecular mechanisms of how transcription factors interact with partner proteins or DNA.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10−5 and 8.7× 10−7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.

Bacterial toxin–antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a truncated complex exists in heterohexameric form. The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydrophobic and hydrophilic interactions. Compared with structural homologs, K. pneumoniae MazF shows distinct features in loops β1–β2, β3–β4 and β4–β5. It can be inferred that these three loops have the potential to represent the unique characteristics of MazF, especially various substrate recognition sites. In addition, K. pneumoniae MazF shows ribonuclease activity and the catalytic core of MazF lies in an RNA-binding pocket. Mutation experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity. The findings shown here may contribute to the understanding of the bacterial MazEF TA system and the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.

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. [ABSTRACT FROM AUTHOR]

Toxin–antitoxin systems are widespread in bacteria, including pathogens such as Mycobacterium tuberculosis. The possible functions of the toxin–antitoxin systems in different groups of bacteria can be different and have been actively discussed and refined in recent years. The subject of this work is studying the function of the vapBC2 toxin–antitoxin module genes in M. smegmatis. M. smegmatis mutants with a deletion of the vapBC2 module and a strain with an additional copy of the toxin gene were obtained and studied. It was shown that expression of an additional copy of the toxin gene led to a slowdown in the growth rate, but did not completely inhibit it. These data may indicate a weak RNase activity of the toxin. It was found that the introduction of an additional copy of the toxin gene led to an increase in the sensitivity of M. smegmatis to oxidative stress. The inactivation of the vapBC2 module resulted in increased sensitivity to kanamycin. The introduction of an additional copy of the toxin gene using the pKW08-MCS-Int integrative vector resulted in increased sensitivity to kanamycin, tetracycline, and erythromycin. The determination of the exact mechanism of the vapBC2 module involvement in intrinsic drug resistance/sensitivity is the subject of our further studies. [ABSTRACT FROM AUTHOR]

Background and purpose: Burn wounds are a good host for infections. Acinetobacter baumannii is an opportunistic bacterium in patients with burn infections. Toxin-antitoxin systems (TAS) are genetic elements that are essential for antibiotic resistance and biofilm formation in bacteria, including higBA and relBE TA systems. The present study aimed to investigate the frequency of higBA and relBE genes in A. baumannii strains isolated from burn wound infection as well as the association between genetic elements and antibiotic resistance and biofilm formation. Materials and methods: In this descriptive cross-sectional study, 139 A. baumannii isolates were collected from burn wounds of patients hospitalized in a burn center in Isfahan, Iran. The isolates were identified and confirmed through biochemical and molecular tests. Antibiotic susceptibility testing was performed via disk diffusion method and biofilm production was detected by microtiter plate assay. Presence of higBA and relBE TA system genes was investigated by PCR. Results: Out of 139 A. baumannii isolates, 114 were multi-drug resistant (MDR) and able to form biofilm. relBE and higBA genes were observed in 94.73% and 8.77% of the isolates, respectively. Significant correlations were found between the presence of relBE and higBA genes and resistance to imipenem and levofloxacin (P0.05). Conclusion: In this study, the number of MDR A. baumanni strains was significantly higher compared to previous studies and most species had the ability to form biofilms. According to the association between TA genes and antibiotic resistance, further studies are needed on the TA systems.

The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin-antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues. Access to this cavity could be controlled by a gate-latch mechanism. The function of the Msmeg_6760 toxin is unknown, but structure-based predictions revealed that Msmeg_6760 and Msmeg_6762 are homologous to Rv2034 and Rv2035, a predicted novel TA system involved in Mycobacterium tuberculosis latency during macrophage infection. The Msmeg_6760 toxin fold has not been previously described for bacterial toxins and its unique structural features suggest that toxin activation is likely to be mediated by a novel mechanism.

Bacterial persistence is a phenomenon that is founded by the existence of a subpopulation of multidrug-tolerant cells. These so-called persister cells endure otherwise lethal stress situations and enable restoration of bacterial populations upon return to favorable conditions. Persisters are especially notorious for their ability to survive antibiotic treatments without conventional resistance genes and to cause infection relapse. The persister state is typically correlated with reduction or inhibition of cellular activity. Early on, chromosomal toxin-antitoxin (TA) systems were suspected to induce the persister state in response to environmental stress. However, this idea has been challenged during the last years. Especially the involvement of toxins from type II TA systems in persister formation is put into question. For toxins from type I TA systems the debate has just started. Here, we would like to summarize recent knowledge gained for the type I TA system tisB/istR-1 from Escherichia coli. TisB is a small, membrane-targeting toxin, which disrupts the proton motive force (PMF), leading to membrane depolarization. Based on experimental data, we hypothesize that TisB primarily stabilizes the persister state through depolarization and further, secondary effects. We will present a simple model that will provide a framework for future directions. [ABSTRACT FROM AUTHOR]

Bacterial toxin-antitoxin (TA) systems consist of two or more adjacent genes, encoding a toxin and an antitoxin. TA systems are implicated in evolutionary and physiological functions including genome maintenance, antibiotics persistence, phage defense, and virulence. Eight classes of TA systems have been described, based on the mechanism of toxin neutralization by the antitoxin. Although studied well in model species of clinical significance, little is known about the TA system abundance and diversity, and their potential roles in stress tolerance and virulence of plant pathogens. In this study, we screened the genomes of 339 strains representing the genetic and lifestyle diversity of the Pseudomonas syringae species complex for TA systems. Using bioinformatic search and prediction tools, including SLING, BLAST, HMMER, TADB2.0, and T1TAdb, we show that P. syringae strains encode 26 different families of TA systems targeting diverse cellular functions. TA systems in this species are almost exclusively type II. We predicted a median of 15 TA systems per genome, and we identified six type II TA families that are found in more than 80% of strains, while others are more sporadic. The majority of predicted TA genes are chromosomally encoded. Further functional characterization of the predicted TA systems could reveal how these widely prevalent gene modules potentially impact P. syringae ecology, virulence, and disease management practices. [ABSTRACT FROM AUTHOR]

Objectives: Sub-inhibitory concentrations (sub-MICs) of antibiotics reflect the conditions that bacteria encounter in tissues and the natural environment. Sub-MICs of antibiotics can induce stress and alter the expression of different bacterial genes. Bacteria react to stress conditions using different mechanisms, one of which is the toxin-antitoxin (TA) system. This study investigated the expression of the TA system genes under oxidative and antibiotic stresses in Klebsiella pneumoniae (K. pneumoniae). Methods: To determine the effects of sub-MICs of gentamicin, nalidixic acid, ceftazidime, and certain concentrations of H2O2 on bacterial survival and growth, colony forming units were quantitated and turbidity was assessed following the treatment of K. pneumoniae with ½ MICs of antibiotics and 5 mM H2O2 at different time intervals. The expression of TA system genes in K. pneumoniae was evaluated 1 h after treatment using the quantitative real-time PCR (qRT-PCR) method. Results: The results revealed reduced K. pneumoniae growth in the presence of sub-MICs of antibiotics and 5 mM H2O2 compared to the control. Furthermore, according to the results of the qRT-PCR assay, only the presence of gentamicin could increase the expression of TA system genes. Conclusion: Although the exact role of the TA systems in response to stress is still unclear, this study provided information on the effect of the type II TA systems under oxidative and antibiotic stress conditions.

Bacterial persistence is a phenomenon that is founded by the existence of a subpopulation of multidrug-tolerant cells. These so-called persister cells endure otherwise lethal stress situations and enable restoration of bacterial populations upon return to favorable conditions. Persisters are especially notorious for their ability to survive antibiotic treatments without conventional resistance genes and to cause infection relapse. The persister state is typically correlated with reduction or inhibition of cellular activity. Early on, chromosomal toxin-antitoxin (TA) systems were suspected to induce the persister state in response to environmental stress. However, this idea has been challenged during the last years. Especially the involvement of toxins from type II TA systems in persister formation is put into question. For toxins from type I TA systems the debate has just started. Here, we would like to summarize recent knowledge gained for the type I TA system tisB/istR-1 from Escherichia coli. TisB is a small, membrane-targeting toxin, which disrupts the proton motive force (PMF), leading to membrane depolarization. Based on experimental data, we hypothesize that TisB primarily stabilizes the persister state through depolarization and further, secondary effects. We will present a simple model that will provide a framework for future directions.

ABSTRACT Despite their ubiquitous nature, few antisense RNAs have been functionally characterized, and this class of RNAs is considered by some to be transcriptional noise. Here, we report that an antisense RNA (asRNA), aMEF (antisense mazEF), functions as a dual regulator for the type II toxin-antitoxin (TA) system mazEF. Unlike type I TA systems and many other regulatory asRNAs, aMEF stimulates the synthesis and translation of mazEF rather than inhibition and degradation. Our data indicate that a double-stranded RNA intermediate and RNase III are not necessary for aMEF-dependent regulation of mazEF expression. The lack of conservation of asRNA promoters has been used to support the hypothesis that asRNAs are spurious transcriptional noise and nonfunctional. We demonstrate that the aMEF promoter is active and functional in Escherichia coli despite poor sequence conservation, indicating that the lack of promoter sequence conservation should not be correlated with functionality. IMPORTANCE Next-generation RNA sequencing of numerous organisms has revealed that transcription is widespread across the genome, termed pervasive transcription, and does not adhere to annotated gene boundaries. The function of pervasive transcription is enigmatic and has generated considerable controversy as to whether it is transcriptional noise or biologically relevant. Antisense transcription is one class of pervasive transcription that occurs from the DNA strand opposite an annotated gene. Relatively few pervasively transcribed asRNAs have been functionally characterized, and their regulatory roles or lack thereof remains unknown. It is important to study examples of these asRNAs and determine if they are functional regulators. In this study, we elucidate the function of an asRNA (aMEF) demonstrating that pervasive transcripts can be functional.

Bacterial toxin-antitoxin (TA) systems consist of two or more adjacent genes, encoding a toxin and an antitoxin. TA systems are implicated in evolutionary and physiological functions including genome maintenance, antibiotics persistence, phage defense, and virulence. Eight classes of TA systems have been described, based on the mechanism of toxin neutralization by the antitoxin. Although studied well in model species of clinical significance, little is known about the TA system abundance and diversity, and their potential roles in stress tolerance and virulence of plant pathogens. In this study, we screened the genomes of 339 strains representing the genetic and lifestyle diversity of the Pseudomonas syringae species complex for TA systems. Using bioinformatic search and prediction tools, including SLING, BLAST, HMMER, TADB2.0, and T1TAdb, we show that P. syringae strains encode 26 different families of TA systems targeting diverse cellular functions. TA systems in this species are almost exclusively type II. We predicted a median of 15 TA systems per genome, and we identified six type II TA families that are found in more than 80% of strains, while others are more sporadic. The majority of predicted TA genes are chromosomally encoded. Further functional characterization of the predicted TA systems could reveal how these widely prevalent gene modules potentially impact P. syringae ecology, virulence, and disease management practices.

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

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.