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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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Xenorhabdus nematophila is an entomopathogenic bacterium that synthesizes numerous toxins and kills its larval insect host. Apart from such toxins, its genome also has a plethora of toxin-antitoxin (TA) systems. The role of TA systems in bacterial physiology is debatable; however, they are associated with maintaining bacterial genomic stability and their survival under adverse environmental conditions. Here, we explored the functionality and transcriptional regulation of the type II hipBA Xn2 TA system. This TA system was identified in the genome of X. nematophila ATCC 19061, which consists of the hipA Xn2 toxin gene encoding 278 amino acid residues and hipB Xn2 encoding antitoxin of 135 amino acid residues. We showed that overexpression of HipA Xn2 toxin reduced the growth of Escherichia coli cells in a bacteriostatic manner, and amino-acids G8, H164, N167, and S169 were key residues for this growth reduction. Promoter activity and expression profiling of the hipBA Xn2 TA system was showed that transcription was induced in both E. coli as well as X. nematophila upon exposure to different stress conditions. Further, we have exhibited the binding features of HipA Xn2 toxin and HipB Xn2 antitoxin to their promoter. This study provides evidence for the presence of a functional and well-regulated hipBA Xn2 TA system in X. nematophila., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria., Objective: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches., Methods: 3D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn- MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system., Results: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells., Conclusion: The study provides insights into structural and functional features of novel toxin, Xn- MazF and provides an initial inference on control of X. nematophila growth regulated by TA systems., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Here, for the first time, we have investigated the hipBA Xn toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipA Xn toxin and HipB Xn antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipA Xn toxin, hipB Xn antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipA Xn toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipA Xn toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157 bp upstream region of the hipBA Xn TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipA Xn toxin, HipB Xn antitoxin protein, and 157 bp promoter region. Results showed that the hipBA Xn TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. KEY POINTS: •Functional characterization of hipBA Xn TA module from Xenorhabdus nematophila. •hipBA Xn TA module is a functional type II TA module. •Transcriptional characterization of hipBA Xn TA module. •hipBA Xn TA module is a well regulated TA module. Graphical abstract.

Here we report the first essentially complete TAome analysis for type II toxin-antitoxin (TA) system, a major class of TA modules found in bacterial system, from entomopathogenic bacterium Xenorhabdus nematophila ATCC 19061 (NCBI RefSeq NC_014228). We summarize this analysis in terms of TA locus, accession identifier, hits in conserved domain database, toxin superfamily, antitoxin superfamily and chromosomal/mobile genome/plasmid occurrences. Moreover, for TA context analyses we use six different specifications namely virulence factors, mobile genetic elements (MGE), antibiotic resistance genes, secretion systems, prophage and a classification of mobile genetic elements (ACLAME); among these hits are found for only MGE, ACLAME and prophage. A total 39 sets of TA have been discovered in which numbers of TA encoded for MGE, ACLAME and prophage are 15, 15 and 5 respectively while the remaining four have no context hit. In addition, a comparative analysis of TAome was also done with closely related bacterium Photorhabdus luminescens subsp. laumondii TTO1 (NCBI RefSeq NC_005126) and results shows that a total 8 sets of TA are conserved. Further, a bootstrap Neighbor-Joining phylogenetic tree was also constructed for major toxin protein superfamily found namely RelE, HigB, GNAT, CcdB and MazF explored in the TAome of X. nematophila. We also characterized, the most abundantly found TA module (relBE) in this TAome, functionally and transcriptionally. This first TAome analysis of type II TA modules provides new insights in multi-drug tolerance in bacterial populations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)