Toxin-antitoxin Research Articles

Unraveling the role of toxin-antitoxin systems in Burkholderia pseudomallei: exploring bacterial pathogenesis and interactions within the HigBA families.

Toxin-antitoxin (TA) systems play a significant role in bacterial persistence, a phenomenon where bacterial cells enter a dormant or slow-growing state to survive adverse conditions such as nutrient deprivation, antibiotic exposure, or host immune responses. By studying TA systems in Burkholderia pseudomallei, we can gain insights into how this pathogen survives and persists in the host environment, contributing to its virulence and ability to cause melioidosis chronic infections.

Toxin-antitoxin system gene mutations driving Mycobacterium tuberculosis transmission revealed by whole genome sequencing.

The toxin-antitoxin (TA) system plays a vital role in the virulence and pathogenicity of Mycobacterium tuberculosis (M. tuberculosis). However, the regulatory mechanisms and the impact of gene mutations on M. tuberculosis transmission remain poorly understood. To investigate the influence of gene mutations in the toxin-antitoxin system on M. tuberculosis transmission dynamics. We performed whole-genome sequencing on the analyzed strains of M. tuberculosis. The genes associated with the toxin-antitoxin system were obtained from the National Center for Biotechnology Information (NCBI) Gene database. Mutations correlating with enhanced transmission within the genes were identified by using random forest, gradient boosting decision tree, and generalized linear mixed models. A total of 13,518 M. tuberculosis isolates were analyzed, with 42.29% (n = 5,717) found to be part of genomic clusters. Lineage 4 accounted for the majority of isolates (n = 6488, 48%), followed by lineage 2 (n = 5133, 37.97%). 23 single nucleotide polymorphisms (SNPs) showed a positive correlation with clustering, including vapB1 G34A, vapB24 A76C, vapB2 T171C, mazF2 C85T, mazE2 G104A, vapB31 T112C, relB T226A, vapB11 C54T, mazE5 T344C, vapB14 A29G, parE1 (C103T, C88T), and parD1 C134T. Six SNPs, including vapB6 A29C, vapB31 T112C, parD1 C134T, vapB37 G205C, Rv2653c A80C, and vapB22 C167T, were associated with transmission clades across different countries. Notably, our findings highlighted the positive association of vapB6 A29C, vapB31 T112C, parD1 C134T, vapB37 G205C, vapB19 C188T, and Rv2653c A80C with transmission clades across diverse regions. Furthermore, our analysis identified 32 SNPs that exhibited significant associations with clade size. Our study presents potential associations between mutations in genes related to the toxin-antitoxin system and the transmission dynamics of M. tuberculosis. However, it is important to acknowledge the presence of confounding factors and limitations in our study. Further research is required to establish causation and assess the functional significance of these mutations. These findings provide a foundation for future investigations and the formulation of strategies aimed at controlling TB transmission.

Decoding the TAome and computational insights into parDE toxin-antitoxin systems in Pseudomonas aeruginosa.

Toxin-antitoxin (TA) modules are widely found in the genomes of pathogenic bacteria. They regulate vital cellular functions like transcription, translation, and DNA replication, and are therefore essential to the survival of bacteria under stress. With a focus on the type II parDE modules, this study thoroughly examines TAome in Pseudomonas aeruginosa, a bacterium well-known for its adaptability and antibiotic resistance. We explored the TAome in three P. aeruginosa strains: ATCC 27,853, PAO1, and PA14, and found 15 type II TAs in ATCC 27,853, 12 in PAO1, and 13 in PA14, with significant variation in the associated mobile genetic elements. Five different parDE homologs were found by further TAome analysis in ATCC 27,853, and their relationships were confirmed by sequence alignments and precise genomic positions. After comparing these ParDE modules' sequences to those of other pathogenic bacteria, it was discovered that they were conserved throughout many taxa, especially Proteobacteria. Nucleic acids were predicted as potential ligands for ParD antitoxins, whereas ParE toxins interacted with a wide range of small molecules, indicating a diverse functional repertoire. The interaction interfaces between ParDE TAs were clarified by protein-protein interaction networks and docking studies, which also highlighted important residues involved in binding. This thorough examination improves our understanding of the diversity, evolutionary dynamics, and functional significance of TA systems in P. aeruginosa, providing insights into their roles in bacterial physiology and pathogenicity.

A type II toxin–antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R

RNase R (encoded by the rnr gene) is a highly processive 3’ → 5’ exoribonuclease essential for the growth of the psychrotrophic bacterium P. syringae Lz4W at low temperature. The cell death of a rnr deletion mutant at low temperature has been previously attributed to the processing defects in 16S rRNA, defective ribosomal assembly and inefficient protein synthesis. We recently showed that RNase R is required to protect P. syringae Lz4W from DNA damage and oxidative stress, independent of its exoribonuclease activity. Here, we show that the processing defect in 16S rRNA does not cause cell death of the rnr mutant of P. syringae at low temperature. Our results demonstrate that the rnr mutant of P. syringae Lz4W, complemented with a RNase R deficient in exoribonuclease function (RNase RD284A) is defective in 16S rRNA processing but can grow at 4 oC. This suggested that the processing defect in ribosomal RNAs is not a cause of the cold sensitivity of the rnr mutant. We further show that the rnr mutant accumulates copies of the indigenous plasmid of P. syringae Lz4W, pLz4W, that bears a type II toxin-antitoxin system (psA-psT). This phenotype was rescued by over-expressing antitoxin psA in the rnr mutant, suggesting that activation of the type II toxin-antitoxin system leads to cold sensitivity of the rnr mutant of P. syringae Lz4W. Here, we report a previously unknown functional relationship between the cold sensitivity of the rnr mutant and a type II toxin-antitoxin system in P. syringae Lz4W.

An RNA pseudoknot mediates toxin translation and antitoxin inhibition

Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth-inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation-competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full-length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which "structural processing" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion.

Evaluating the antibacterial effect of meropenem-loaded chitosan/sodium tripolyphosphate (TPP) nanoparticles on Acinetobacter baumannii isolated from hospitalized patients

BackgroundAcinetobacter baumannii is a health threat due to its antibiotic resistance. Herein, antibiotic susceptibility and its association with the Toxin-antitoxin (TA) system genes in A. baumannii clinical isolates from Iran were investigated. Next, we prepared meropenem-loaded chitosan nanoparticles (MP-CS) and investigated their antibacterial effects against meropenem-susceptible bacterial isolates.MethodsOut of 240 clinical specimens, 60 A. baumannii isolates were assessed. Antibiotic resistance of the isolates against conventional antibiotics was determined alongside investigating the presence of three TA system genes (mazEF, relBE, and higBA). Chitosan nanoparticles were characterized in terms of size, zeta potential, encapsulation efficiency, and meropenem release activity. Their antibacterial effects were assessed using the well diffusion method, minimum inhibitory concentration (MIC), and colony-forming unit (CFU) counting. Their cytotoxic effects and biocompatibility index were determined via the MTT, LDH, and ROS formation assays.ResultsAmpicillin, ceftazidime, and colistin were the least effective, and amikacin and tobramycin were the most effective antibiotics. Out of the 60 isolates, 10 (16.7%), 5 (8.3%), and 45 (75%) were multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR), respectively. TA system genes had no significant effect on antibiotic resistance. MP-CS nanoparticles demonstrated an average size of 191.5 and zeta potential of 27.3 mV alongside a maximum encapsulation efficiency of 88.32% and release rate of 69.57%. MP-CS nanoparticles mediated similar antibacterial effects, as compared with free meropenem, against the A. baumannii isolates with significantly lower levels of meropenem. MP-CS nanoparticles remarkably prevented A549 and NCI-H292 cell infection by the A. baumannii isolates alongside demonstrating a favorable biocompatibility index.ConclusionAntibiotic-loaded nanoparticles should be further designed and investigated to increase their antibacterial effect against A. baumannii and assess their safety and applicability in vivo settings.

HigA2 (Rv2021c) Is a Transcriptional Regulator with Multiple Regulatory Targets in Mycobacterium tuberculosis.

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

Gene Regulatory Mechanism of Mycobacterium Tuberculosis during Dormancy.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex, is a zoonotic disease that remains one of the leading causes of death worldwide. Latent tuberculosis infection reactivation is a challenging obstacle to eradicating TB globally. Understanding the gene regulatory network of Mtb during dormancy is important. This review discusses up-to-date information about TB gene regulatory networks during dormancy, focusing on the regulation of lipid and energy metabolism, dormancy survival regulator (DosR), White B-like (Wbl) family, Toxin-Antitoxin (TA) systems, sigma factors, and MprAB. We outline the progress in vaccine and drug development associated with Mtb dormancy.

Longitudinal multi-omics analysis of host microbiome architecture and immune responses during short-term spaceflight

Maintenance of astronaut health during spaceflight will require monitoring and potentially modulating their microbiomes. However, documenting microbial shifts during spaceflight has been difficult due to mission constraints that lead to limited sampling and profiling. Here we executed a six-month longitudinal study to quantify the high-resolution human microbiome response to three days in orbit for four individuals. Using paired metagenomics and metatranscriptomics alongside single-nuclei immune cell profiling, we characterized time-dependent, multikingdom microbiome changes across 750 samples and 10 body sites before, during and after spaceflight at eight timepoints. We found that most alterations were transient across body sites; for example, viruses increased in skin sites mostly during flight. However, longer-term shifts were observed in the oral microbiome, including increased plaque-associated bacteria (for example, Fusobacteriota), which correlated with immune cell gene expression. Further, microbial genes associated with phage activity, toxin–antitoxin systems and stress response were enriched across multiple body sites. In total, this study reveals in-depth characterization of microbiome and immune response shifts experienced by astronauts during short-term spaceflight and the associated changes to the living environment, which can help guide future missions, spacecraft design and space habitat planning.

Discovery of Antimicrobial Agents Based on Structural and Functional Study of the Klebsiella pneumoniae MazEF Toxin-Antitoxin System.

Klebsiella pneumoniae causes severe human diseases, but its resistance to current antibiotics is increasing. Therefore, new antibiotics to eradicate K. pneumoniae are urgently needed. Bacterial toxin-antitoxin (TA) systems are strongly correlated with physiological processes in pathogenic bacteria, such as growth arrest, survival, and apoptosis. By using structural information, we could design the peptides and small-molecule compounds that can disrupt the binding between K. pneumoniae MazE and MazF, which release free MazF toxin. Because the MazEF system is closely implicated in programmed cell death, artificial activation of MazF can promote cell death of K. pneumoniae. The effectiveness of a discovered small-molecule compound in bacterial cell killing was confirmed through flow cytometry analysis. Our findings can contribute to understanding the bacterial MazEF TA system and developing antimicrobial agents for treating drug-resistant K. pneumoniae.

Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis.

Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.

The biological function of the type II toxin-antitoxin system ccdAB in recurrent urinary tract infections.

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.

DNA binding reveals hidden interdomain allostery of a MazE antitoxin from Mycobacterium tuberculosis

Type II toxin−antitoxin (TA) systems are ubiquitously distributed genetic elements in prokaryotes and are crucial for cell maintenance and survival under environmental stresses. The antitoxin is a modular protein consisting of the disordered C-terminal region that physically contacts and neutralizes the cognate toxin and the well-folded N-terminal DNA binding domain responsible for autorepression of TA transcription. However, how the two functional domains communicate is largely unknown. Herein, we determined the crystal structure of the N-terminal domain of the type II antitoxin MazE-mt10 from Mycobacterium tuberculosis, revealing a homodimer of the ribbon-helix-helix (RHH) fold with distinct DNA binding specificity. NMR studies demonstrated that full-length MazE-mt10 forms the helical and coiled states in equilibrium within the C-terminal region, and that helical propensity is allosterically enhanced by the N-terminal binding to the cognate operator DNA. This coil-to-helix transition may promote toxin binding/neutralization of MazE-mt10 and further stabilize the TA–DNA transcription repressor. This is supported by many crystal structures of type II TA complexes in which antitoxins form an α-helical structure at the TA interface. The hidden helical state of free MazE-mt10 in solution, favored by DNA binding, adds a new dimension to the regulatory mechanism of type II TA systems. Furthermore, complementary approaches using X-ray crystallography and NMR allow us to study the allosteric interdomain interplay of many other full-length antitoxins of type II TA systems.

Molecular stripping underpins derepression of a toxin-antitoxin system.

Transcription factors control gene expression; among these, transcriptional repressors must liberate the promoter for derepression to occur. Toxin-antitoxin (TA) modules are bacterial elements that autoregulate their transcription by binding the promoter in a T:A ratio-dependent manner, known as conditional cooperativity. The molecular basis of how excess toxin triggers derepression has remained elusive, largely because monitoring the rearrangement of promoter-repressor complexes, which underpin derepression, is challenging. Here, we dissect the autoregulation of the Salmonella enterica tacAT3 module. Using a combination of assays targeting DNA binding and promoter activity, as well as structural characterization, we determine the essential TA and DNA elements required to control transcription, and we reconstitute a repression-to-derepression path. We demonstrate that excess toxin triggers molecular stripping of the repressor complex off the DNA through multiple allosteric changes causing DNA distortion and ultimately leading to derepression. Thus, our work provides important insight into the mechanisms underlying conditional cooperativity.

Bioinformatics analysis of type II toxin-antitoxin systems and regulatory functional assessment of HigBA and SS-ATA in Streptococcus suis1

Toxin-antitoxin (TA) systems, which are prevalent in bacteria and archaea, play diverse roles in bacterial physiology and have been proposed to be significant in stress adaptation. Despite the extensive characterization of numerous TA systems in various bacteria, the investigation of these systems within Streptococcus suis (S. suis) is still limited. Here, we systematically analyzed the type II TA systems of 95 S. suis genomes available in the GenBank database using TAfinder. A total of 612 putative type II TA systems were retrieved and classified into 10 categories by phylogenetic analysis. Notably, an elevated occurrence of these TA systems was observed among the important prevalent serotypes 2, 4, 5, 9, 14, Chz, NCL1, and NCL3 strains. The following study identified the activities of TA systems using two strategies and confirmed the regulatory effect of HigBA on the type VII secretion system in S. suis by measuring β-galactosidase activity and transcriptional changes. Moreover, we unveiled a hitherto uncharacterized, highly prevalent novel TA system, with the composition of antitoxin-toxin-antitoxin (SS-ATA), which regulates the downstream two-component signaling system. Altogether, this study systematically analyzed the type II TA systems within S. suis, highlighting the widespread distribution of HigBA and SS-ATA as important regulatory elements in S. suis.

Comparative analysis of genes expression involved in type II toxin-antitoxin system in Staphylococcus aureus following persister cell formation.

The formation of persister cells is the main reason for persistent infections. They are associated with antibiotic treatment failure and subsequently chronic infection. The study aimed to assess the expression of type II toxin/antitoxin (TA) system genes in persister cells of Staphylococcus aureus in the presence of the following antibiotics vancomycin, ciprofloxacin, and gentamicin in exponential and stationary phases. The colony count was used to evaluate the effect of different types of antibiotics on S. aureus persister cell formation during exponential and stationary phases. Moreover, the expression level of TA systems and clpP genes in the persister population in exponential and stationary phases were measured by quantitative reverse transcriptase real-time PCR (qRT-PCR). The results of the study showed the presence of persister phenotype of S. aureus strains in the attendance of bactericidal antibiotics in comparison to the control group during the exponential and stationary phases. Moreover, qRT-PCR resulted in the fact that the role of TA systems involved in the persister cell formation depends on the bacterial growth phase and the type of strain and antibiotic. In total, the present study provides some data on the persister cell formation and the possible role of TA system genes in this process.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae

ABSTRACT Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid- carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

PCR Development for Analysis of Some Type II Toxin-Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis.

Toxin-Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64M. tuberculosis and 26M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

Effect of Divalent Metal Ions on the Ribonuclease Activity of the Toxin Molecule HP0894 from Helicobacter pylori.

Bacteria and archaea respond and adapt to environmental stress conditions by modulating the toxin-antitoxin (TA) system for survival. Within the bacterium Helicobacter pylori, the protein HP0894 is a key player in the HP0894-HP0895 TA system, in which HP0894 serves as a toxin and HP0895 as an antitoxin. HP0894 has intrinsic ribonuclease (RNase) activity that regulates gene expression and translation, significantly influencing bacterial physiology and survival. This activity is influenced by the presence of metal ions such as Mg2+. In this study, we explore the metal-dependent RNase activity of HP0894. Surprisingly, all tested metal ions lead to a reduction in RNase activity, with zinc ions (Zn2+) causing the most significant decrease. The secondary structure of HP0894 remained largely unaffected by Zn2+ binding, whereas structural rigidity was notably increased, as revealed using CD analysis. NMR characterized the Zn2+ binding, implicating numerous His, Asp, and Glu residues in HP0894. In summary, these results suggest that metal ions play a regulatory role in the RNase activity of HP0894, contributing to maintaining the toxin molecule in an inactive state under normal conditions.

MazEF Homologs in Symbiobacterium thermophilum Exhibit Cross-Neutralization with Non-Cognate MazEFs.

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.