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1
Wilbaux, Myriam, Natacha Mine, Anne-Marie Guérout, Didier Mazel, and Laurence Van Melderen. "Functional Interactions between Coexisting Toxin-Antitoxin Systems of the ccd Family in Escherichia coli O157:H7." Journal of Bacteriology 189, no. 7 (January 26, 2007): 2712–19. http://dx.doi.org/10.1128/jb.01679-06.
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ABSTRACT Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid (ccd F) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain (ccd O157). The ccd F and the ccd O157 systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the ccd F system. We have shown that the chromosomal ccd O157 system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdBO157 toxin targets the DNA gyrase, and the CcdAO157 antitoxin is degraded by the Lon protease. The ccd O157 chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of ccd F. ccd O157 is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccd F system is able to mediate postsegregational killing in an E. coli strain harboring the ccd O157 system in its chromosome. This shows that the plasmidic ccd F system is functional in the presence of its chromosomal counterpart.
2
Singletary, Larissa A., Janet L. Gibson, Elizabeth J. Tanner, Gregory J. McKenzie, Peter L. Lee, Caleb Gonzalez, and Susan M. Rosenberg. "An SOS-Regulated Type 2 Toxin-Antitoxin System." Journal of Bacteriology 191, no. 24 (October 16, 2009): 7456–65. http://dx.doi.org/10.1128/jb.00963-09.
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ABSTRACT The Escherichia coli chromosome encodes seven demonstrated type 2 toxin-antitoxin (TA) systems: cassettes of two or three cotranscribed genes, one encoding a stable toxin protein that can cause cell stasis or death, another encoding a labile antitoxin protein, and sometimes a third regulatory protein. We demonstrate that the yafNO genes constitute an additional chromosomal type 2 TA system that is upregulated during the SOS DNA damage response. The yafNOP genes are part of the dinB operon, of which dinB underlies stress-induced mutagenesis mechanisms. yafN was identified as a putative antitoxin by homology to known antitoxins, implicating yafO (and/or yafP) as a putative toxin. Using phage-mediated cotransduction assays for linkage disruption, we show first that yafN is an essential gene and second that it is essential only when yafO is present. Third, yafP is not a necessary part of either the toxin or the antitoxin. Fourth, although DinB is required, the yafNOP genes are not required for stress-induced mutagenesis in the E scherichia coli Lac assay. These results imply that yafN encodes an antitoxin that protects cells against a yafO-encoded toxin and show a protein-based TA system upregulated by the SOS response.
3
Ni, Songwei, Baiyuan Li, Kaihao Tang, Jianyun Yao, Thomas K. Wood, Pengxia Wang, and Xiaoxue Wang. "Conjugative plasmid-encoded toxin–antitoxin system PrpT/PrpA directly controls plasmid copy number." Proceedings of the National Academy of Sciences 118, no. 4 (January 22, 2021): e2011577118. http://dx.doi.org/10.1073/pnas.2011577118.
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Toxin–antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin–antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.
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Rathore, Jitendra Singh, and Lalit Kumar Gautam. "Expression, Purification, and Functional Analysis of Novel RelE Operon fromX. nematophila." Scientific World Journal 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/428159.
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Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.Escherichia coliRelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin fromXenorhabdus nematophilapossessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter inE. coliTop 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.
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Piscotta, Frank J., Philip D. Jeffrey, and A. James Link. "ParST is a widespread toxin–antitoxin module that targets nucleotide metabolism." Proceedings of the National Academy of Sciences 116, no. 3 (December 31, 2018): 826–34. http://dx.doi.org/10.1073/pnas.1814633116.
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Toxin–antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654–COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.
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Alkhalili, Rawana, Joel Wallenius, and Björn Canbäck. "Towards Exploring Toxin-Antitoxin Systems in Geobacillus: A Screen for Type II Toxin-Antitoxin System Families in a Thermophilic Genus." International Journal of Molecular Sciences 20, no. 23 (November 22, 2019): 5869. http://dx.doi.org/10.3390/ijms20235869.
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The toxin-antitoxin (TA) systems have been attracting attention due to their role in regulating stress responses in prokaryotes and their biotechnological potential. Much recognition has been given to type II TA system of mesophiles, while thermophiles have received merely limited attention. Here, we are presenting the putative type II TA families encoded on the genomes of four Geobacillus strains. We employed the TA finder tool to mine for TA-coding genes and manually curated the results using protein domain analysis tools. We also used the NCBI BLAST, Operon Mapper, ProOpDB, and sequence alignment tools to reveal the geobacilli TA features. We identified 28 putative TA pairs, distributed over eight TA families. Among the identified TAs, 15 represent putative novel toxins and antitoxins, belonging to the MazEF, MNT-HEPN, ParDE, RelBE, and XRE-COG2856 TA families. We also identified a potentially new TA composite, AbrB-ParE. Furthermore, we are suggesting the Geobacillus acetyltransferase TA (GacTA) family, which potentially represents one of the unique TA families with a reverse gene order. Moreover, we are proposing a hypothesis on the xre-cog2856 gene expression regulation, which seems to involve the c-di-AMP. This study aims for highlighting the significance of studying TAs in Geobacillus and facilitating future experimental research.
7
Tu, Chih-Han, Michelle Holt, Shengfeng Ruan, and Christina Bourne. "Evaluating the Potential for Cross-Interactions of Antitoxins in Type II TA Systems." Toxins 12, no. 6 (June 26, 2020): 422. http://dx.doi.org/10.3390/toxins12060422.
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The diversity of Type-II toxin–antitoxin (TA) systems in bacterial genomes requires tightly controlled interaction specificity to ensure protection of the cell, and potentially to limit cross-talk between toxin–antitoxin pairs of the same family of TA systems. Further, there is a redundant use of toxin folds for different cellular targets and complexation with different classes of antitoxins, increasing the apparent requirement for the insulation of interactions. The presence of Type II TA systems has remained enigmatic with respect to potential benefits imparted to the host cells. In some cases, they play clear roles in survival associated with unfavorable growth conditions. More generally, they can also serve as a “cure” against acquisition of highly similar TA systems such as those found on plasmids or invading genetic elements that frequently carry virulence and resistance genes. The latter model is predicated on the ability of these highly specific cognate antitoxin–toxin interactions to form cross-reactions between chromosomal antitoxins and invading toxins. This review summarizes advances in the Type II TA system models with an emphasis on antitoxin cross-reactivity, including with invading genetic elements and cases where toxin proteins share a common fold yet interact with different families of antitoxins.
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Habib, Gul, Qing Zhu, and Baolin Sun. "Bioinformatics and Functional Assessment of Toxin-Antitoxin Systems in Staphylococcus aureus." Toxins 10, no. 11 (November 14, 2018): 473. http://dx.doi.org/10.3390/toxins10110473.
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Staphylococcus aureus is a nosocomial pathogen that can cause chronic to persistent infections. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. These systems are frequently studied in Escherichia coli and Mycobacterial species but rarely explored in S. aureus. In the present study, we thoroughly analyzed the S. aureus genome and screened all possible TA systems using the Rasta bacteria and toxin-antitoxin database. We further searched E. coli and Mycobacterial TA homologs and selected 67 TA loci as putative TA systems in S. aureus. The host inhibition of growth (HigBA) TA family was predominantly detected in S. aureus. In addition, we detected seven pathogenicity islands in the S. aureus genome that are enriched with virulence genes and contain 26 out of 67 TA systems. We ectopically expressed multiple TA genes in E. coli and S. aureus that exhibited bacteriostatic and bactericidal effects on cell growth. The type I Fst toxin created holes in the cell wall while the TxpA toxin reduced cell size and induced cell wall septation. Besides, we identified a new TA system whose antitoxin functions as a transcriptional autoregulator while the toxin functions as an inhibitor of autoregulation. Altogether, this study provides a plethora of new as well as previously known TA systems that will revitalize the research on S. aureus TA systems.
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Pathak, Chinar, Hookang Im, Sun-bok Jang, Yeon-Jin Yang, Hye-Jin Yoon, Hong-Man Kim, Ae-Ran Kwon, and Bong-Jin Lee. "Toxins from TA system of Helicobacter pylori and insight into mRNase activity." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C828. http://dx.doi.org/10.1107/s2053273314091712.
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The toxin-antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. The HP0892-HP0893 and HP0894-HP0895 toxin-antitoxin systems are the only two known TA systems belonging to Helicobacter pylori. In both of these TA systems, the antitoxin binds and inhibits the toxin and regulates the transcription of the TA operon. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remains unclear. Therefore, here an attempt was made to shed some light on the mechanism behind the TA system of HP0892-HP0893 and HP0894-HP0895. Here, we present the crystal structures of apo- and copper-bound HP0894 at 1.28 Å and 1.89 Å, respectively, and the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues involving the mRNase active site was shown. Through the combined approach of structural analysis along with isothermal calorimetry studies and structural homology search, the amino acids involved in mRNase active site were monitored. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself acting as a general acid in an acid-base catalysis reaction, while His47 and His60 stabilize the transition state. Glu58 acts as a general base, and substrate reorientation is caused by Phe88. In the mRNase active site of HP0892 toxin, the most catalytically important residue, His86, reorients itself to exhibit RNase activity while Glu58 acts as a general base. His47 and His60 are considered to be involved in enzymatic activity. Glu58 and Asp64 are involved in substrate binding and specific sequence recognition. The mutational constructs were used for isothermal calorimetric studies to analyze the effect of catalytic residues.
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Choi, Wonho, Yoshihiro Yamaguchi, Ji-Young Park, Sang-Hyun Park, Hyeok-Won Lee, Byung-Kwan Lim, Michael Otto, Masayori Inouye, Min-Ho Yoon, and Jung-Ho Park. "Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens." Microorganisms 9, no. 5 (May 20, 2021): 1107. http://dx.doi.org/10.3390/microorganisms9051107.
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Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.
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Daimon, Yasushi, Shin-ichiro Narita, and Yoshinori Akiyama. "Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σEin Escherichia coli." Journal of Bacteriology 197, no. 14 (April 27, 2015): 2316–24. http://dx.doi.org/10.1128/jb.00079-15.
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ABSTRACTσE, an alternative σ factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth ofEscherichia colinot only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of thehicBgene has been reported to suppress the lethality caused by the loss of σE.hicBencodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of σEand, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption ofhicAabolished suppression of the σEessentiality in the absence ofhicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the σEessentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins,E. colicells with no suppressor mutations grew independently of σE. Taken together, our results indicate that the activation of TA system toxins can suppress the σEessentiality and affect the extracytoplasmic stress responses.IMPORTANCEσEis an alternative σ factor involved in extracytoplasmic stress responses. Unlike other alternative σ factors, σEis indispensable for the survival ofE. colieven under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the σEessentiality, suggesting a connection between TA systems and σEfunction. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.
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Lee, Ki-Young, and Bong-Jin Lee. "Dynamics-Based Regulatory Switches of Type II Antitoxins: Insights into New Antimicrobial Discovery." Antibiotics 12, no. 4 (March 23, 2023): 637. http://dx.doi.org/10.3390/antibiotics12040637.
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Type II toxin-antitoxin (TA) modules are prevalent in prokaryotes and are involved in cell maintenance and survival under harsh environmental conditions, including nutrient deficiency, antibiotic treatment, and human immune responses. Typically, the type II TA system consists of two protein components: a toxin that inhibits an essential cellular process and an antitoxin that neutralizes its toxicity. Antitoxins of type II TA modules typically contain the structured DNA-binding domain responsible for TA transcription repression and an intrinsically disordered region (IDR) at the C-terminus that directly binds to and neutralizes the toxin. Recently accumulated data have suggested that the antitoxin’s IDRs exhibit variable degrees of preexisting helical conformations that stabilize upon binding to the corresponding toxin or operator DNA and function as a central hub in regulatory protein interaction networks of the type II TA system. However, the biological and pathogenic functions of the antitoxin’s IDRs have not been well discussed compared with those of IDRs from the eukaryotic proteome. Here, we focus on the current state of knowledge about the versatile roles of IDRs of type II antitoxins in TA regulation and provide insights into the discovery of new antibiotic candidates that induce toxin activation/reactivation and cell death by modulating the regulatory dynamics or allostery of the antitoxin.
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Kasari, Villu, Kristi Kurg, Tõnu Margus, Tanel Tenson, and Niilo Kaldalu. "The Escherichia coli mqsR and ygiT Genes Encode a New Toxin-Antitoxin Pair." Journal of Bacteriology 192, no. 11 (March 16, 2010): 2908–19. http://dx.doi.org/10.1128/jb.01266-09.
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ABSTRACT Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells.
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Soutourina, Olga. "Type I Toxin-Antitoxin Systems in Clostridia." Toxins 11, no. 5 (May 6, 2019): 253. http://dx.doi.org/10.3390/toxins11050253.
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Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed.
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Sala, Ambre Julie, Patricia Bordes, Sara Ayala, Nawel Slama, Samuel Tranier, Michèle Coddeville, Anne-Marie Cirinesi, Marie-Pierre Castanié-Cornet, Lionel Mourey, and Pierre Genevaux. "Directed evolution of SecB chaperones toward toxin-antitoxin systems." Proceedings of the National Academy of Sciences 114, no. 47 (November 7, 2017): 12584–89. http://dx.doi.org/10.1073/pnas.1710456114.
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SecB chaperones assist protein export in bacteria. However, certain SecB family members have diverged to become specialized toward the control of toxin-antitoxin (TA) systems known to promote bacterial adaptation to stress and persistence. In such tripartite TA-chaperone (TAC) systems, the chaperone was shown to assist folding and to prevent degradation of its cognate antitoxin, thus facilitating inhibition of the toxin. Here, we used both the export chaperone SecB ofEscherichia coliand the tripartite TAC system ofMycobacterium tuberculosisas a model to investigate how generic chaperones can specialize toward the control of TA systems. Through directed evolution of SecB, we have identified and characterized mutations that specifically improve the ability of SecB to control our model TA system without affecting its function in protein export. Such a remarkable plasticity of SecB chaperone function suggests that its substrate binding surface can be readily remodeled to accommodate specific clients.
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Kang, Sung-Min, Do-Hee Kim, Chenglong Jin, and Bong-Jin Lee. "A Systematic Overview of Type II and III Toxin-Antitoxin Systems with a Focus on Druggability." Toxins 10, no. 12 (December 4, 2018): 515. http://dx.doi.org/10.3390/toxins10120515.
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Toxin-antitoxin (TA) systems are known to play various roles in physiological processes, such as gene regulation, growth arrest and survival, in bacteria exposed to environmental stress. Type II TA systems comprise natural complexes consisting of protein toxins and antitoxins. Each toxin and antitoxin participates in distinct regulatory mechanisms depending on the type of TA system. Recently, peptides designed by mimicking the interfaces between TA complexes showed its potential to activate the activity of toxin by competing its binding counterparts. Type II TA systems occur more often in pathogenic bacteria than in their nonpathogenic kin. Therefore, they can be possible drug targets, because of their high abundance in some pathogenic bacteria, such as Mycobacterium tuberculosis. In addition, recent bioinformatic analyses have shown that type III TA systems are highly abundant in the intestinal microbiota, and recent clinical studies have shown that the intestinal microbiota is linked to inflammatory diseases, obesity and even several types of cancer. We therefore focused on exploring the putative relationship between intestinal microbiota-related human diseases and type III TA systems. In this paper, we review and discuss the development of possible druggable materials based on the mechanism of type II and type III TA system.
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Saavedra De Bast, Manuel, Natacha Mine, and Laurence Van Melderen. "Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules." Journal of Bacteriology 190, no. 13 (April 25, 2008): 4603–9. http://dx.doi.org/10.1128/jb.00357-08.
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ABSTRACT Toxin-antitoxin (TA) systems are widespread among bacterial chromosomes and mobile genetic elements. Although in plasmids TA systems have a clear role in their vertical inheritance by selectively killing plasmid-free daughter cells (postsegregational killing or addiction phenomenon), the physiological role of chromosomally encoded ones remains under debate. The assumption that chromosomally encoded TA systems are part of stress response networks and/or programmed cell death machinery has been called into question recently by the observation that none of the five canonical chromosomally encoded TA systems in the Escherichia coli chromosome seem to confer any selective advantage under stressful conditions (V. Tsilibaris, G. Maenhaut-Michel, N. Mine, and L. Van Melderen, J. Bacteriol. 189:6101-6108, 2007). Their prevalence in bacterial chromosomes indicates that they might have been acquired through horizontal gene transfer. Once integrated in chromosomes, they might in turn interfere with their homologues encoded by mobile genetic elements. In this work, we show that the chromosomally encoded Erwinia chrysanthemi ccd (control of cell death) (ccdEch ) system indeed protects the cell against postsegregational killing mediated by its F-plasmid ccd (ccd F) homologue. Moreover, competition experiments have shown that this system confers a fitness advantage under postsegregational conditions mediated by the ccd F system. We propose that ccdEch acts as an antiaddiction module and, more generally, that the integration of TA systems in bacterial chromosomes could drive the evolution of plasmid-encoded ones and select toxins that are no longer recognized by the antiaddiction module.
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Hill, Virginia, Hatice Akarsu, Rubén Sánchez Barbarroja, Valentina L. Cippà, Peter Kuhnert, Martin Heller, Laurent Falquet, et al. "Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems." PLOS Genetics 17, no. 10 (October 21, 2021): e1009365. http://dx.doi.org/10.1371/journal.pgen.1009365.
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Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications.
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Kamruzzaman, Muhammad, Alma Y. Wu, and Jonathan R. Iredell. "Biological Functions of Type II Toxin-Antitoxin Systems in Bacteria." Microorganisms 9, no. 6 (June 11, 2021): 1276. http://dx.doi.org/10.3390/microorganisms9061276.
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After the first discovery in the 1980s in F-plasmids as a plasmid maintenance system, a myriad of toxin-antitoxin (TA) systems has been identified in bacterial chromosomes and mobile genetic elements (MGEs), including plasmids and bacteriophages. TA systems are small genetic modules that encode a toxin and its antidote and can be divided into seven types based on the nature of the antitoxin molecules and their mechanism of action to neutralise toxins. Among them, type II TA systems are widely distributed in chromosomes and plasmids and the best studied so far. Maintaining genetic material may be the major function of type II TA systems associated with MGEs, but the chromosomal TA systems contribute largely to functions associated with bacterial physiology, including the management of different stresses, virulence and pathogenesis. Due to growing interest in TA research, extensive work has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules. However, there are still controversies about some of the functions associated with different TA systems. This review will discuss the most current findings and the bona fide functions of bacterial type II TA systems.
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Lee, Min Woo, Elizabeth E. Rogers, and Drake C. Stenger. "Xylella fastidiosa Plasmid-Encoded PemK Toxin Is an Endoribonuclease." Phytopathology® 102, no. 1 (January 2012): 32–40. http://dx.doi.org/10.1094/phyto-05-11-0150.
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Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK toxin-antitoxin (TA) system. PemK toxin inhibits bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK by direct binding. PemK and PemI were overexpressed in Escherichia coli and activities of each were assessed. Purified PemK toxin specifically degraded single-stranded RNA but not double-stranded RNA, double-stranded DNA, or single-stranded DNA. Addition of PemI antitoxin inhibited nuclease activity of PemK toxin. Purified complexes of PemI bound to PemK exhibited minimal nuclease activity; removal of PemI antitoxin from the complex restored nuclease activity of PemK toxin. Sequencing of 5′ rapid amplification of cDNA ends products of RNA targets digested with PemK revealed a preference for cleavage between U and A residues of the sequence UACU and UACG. Nine single amino-acid substitution mutants of PemK toxin were constructed and evaluated for growth inhibition, ribonuclease activity, and PemI binding. Three PemK point-substitution mutants (R3A, G16E, and D79V) that lacked nuclease activity did not inhibit growth. All nine PemK mutants retained the ability to bind PemI. Collectively, the results indicate that the mechanism of stable inheritance conferred by pXF-RIV11 pemI/pemK is similar to that of the R100 pemI/pemK TA system of E. coli.
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Norouzi, Masoumeh, Abbas Maleki, Elham Aboualigalehdari, and Sobhan Ghafourian. "Type II toxin- antitoxin systems in clinical isolates of antibiotic resistant Acinetobacter baumannii." Genetika 54, no. 2 (2022): 625–32. http://dx.doi.org/10.2298/gensr2202625n.
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The over use of antibiotics to treat infections in humans and animals made a phenomenon of the antibiotic-resistant bacteria. While studies focused to find on new antibiotics but, identification of novel antibacterial targets in bacteria is very important. By Toxin antitoxin systems this hypothesis could be done, whereas by the activation of a toxin or inactivation of an antitoxin, the raised toxin kills the bacterium. These systems are attractive target for antimicrobial therapy. However, the most important step for potency of TA system, as an antibacterial target, is to identify a TA system that is prevalent in all resistant clinical isolates. So, the prevalence of different TA systems among clinical isolates of Acinetobacter baumannii in Emam khomeini hospital, Ilam, Iran was evaluated to determine which TA system is prevalent in all antibiotic resistant A. baumannii. So, one hundred A. baumannii clinical isolates were identified during one-year period in Emam khomeini hospital, Ilam, Iran. A. baumannii clinical isolates were isolated from hospitalized patients in ICU and burn patients. All isolates were resistant to at least one antibiotic. Then, the isolates were subjected to evaluation to find mazEF and higBA TA genes by PCR. The results showed the frequency of mazEF and highBA TA genes in all isolates was 72% and 39%, respectively. mazEF or higBA TA systems are not presented in all isolates. So, the potency of these two TA systems are in challenged. Also, all isolates were not positive for one TA gene. So, more research in different geographical area should be done with functionality of TA genes.
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Chlebicka, Kinga, Emilia Bonar, Piotr Suder, Emeline Ostyn, Brice Felden, Benedykt Wladyka, and Marie-Laure Pinel-Marie. "Impacts of the Type I Toxin–Antitoxin System, SprG1/SprF1, on Staphylococcus aureus Gene Expression." Genes 12, no. 5 (May 18, 2021): 770. http://dx.doi.org/10.3390/genes12050770.
Full textAbstract:
Type I toxin–antitoxin (TA) systems are widespread genetic modules in bacterial genomes. They express toxic peptides whose overexpression leads to growth arrest or cell death, whereas antitoxins regulate the expression of toxins, acting as labile antisense RNAs. The Staphylococcus aureus (S. aureus) genome contains and expresses several functional type I TA systems, but their biological functions remain unclear. Here, we addressed and challenged experimentally, by proteomics, if the type I TA system, the SprG1/SprF1 pair, influences the overall gene expression in S. aureus. Deleted and complemented S. aureus strains were analyzed for their proteomes, both intracellular and extracellular, during growth. Comparison of intracellular proteomes among the strains points to the SprF1 antitoxin as moderately downregulating protein expression. In the strain naturally expressing the SprG1 toxin, cytoplasmic proteins are excreted into the medium, but this is not due to unspecific cell leakages. Such a toxin-driven release of the cytoplasmic proteins may modulate the host inflammatory response that, in turn, could amplify the S. aureus infection spread.
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Agarwal, Sakshi, Arun Sharma, Rania Bouzeyen, Amar Deep, Harsh Sharma, Kiran K. Mangalaparthi, Keshava K. Datta, et al. "VapBC22 toxin-antitoxin system from Mycobacterium tuberculosis is required for pathogenesis and modulation of host immune response." Science Advances 6, no. 23 (June 2020): eaba6944. http://dx.doi.org/10.1126/sciadv.aba6944.
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Virulence-associated protein B and C toxin-antitoxin (TA) systems are widespread in prokaryotes, but their precise role in physiology is poorly understood. We have functionally characterized the VapBC22 TA system from Mycobacterium tuberculosis. Transcriptome analysis revealed that overexpression of VapC22 toxin in M. tuberculosis results in reduced levels of metabolic enzymes and increased levels of ribosomal proteins. Proteomics studies showed reduced expression of virulence-associated proteins and increased levels of cognate antitoxin, VapB22 in the ΔvapC22 mutant strain. Furthermore, both the ΔvapC22 mutant and VapB22 overexpression strains of M. tuberculosis were susceptible to killing upon exposure to oxidative stress and showed attenuated growth in guinea pigs and mice. Host transcriptome analysis suggests upregulation of the transcripts involved in innate immune responses and tissue remodeling in mice infected with the ΔvapC22 mutant strain. Together, we demonstrate that the VapBC22 TA system belongs to a key regulatory network and is essential for M. tuberculosis pathogenesis.
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Yao, Jianyun, Xiangkai Zhen, Kaihao Tang, Tianlang Liu, Xiaolong Xu, Zhe Chen, Yunxue Guo, et al. "Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system." Nucleic Acids Research 48, no. 19 (October 12, 2020): 11054–67. http://dx.doi.org/10.1093/nar/gkaa855.
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Abstract The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.
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Shapira, Shiran, Ilana Boustanai, Dina Kazanov, Marina Ben Shimon, Ahmad Fokra, and Nadir Arber. "Innovative dual system approach for selective eradication of cancer cells using viral-based delivery of natural bacterial toxin–antitoxin system." Oncogene 40, no. 31 (June 25, 2021): 4967–79. http://dx.doi.org/10.1038/s41388-021-01792-8.
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AbstractThe inactivation of p53, a tumor suppressor, and the activation of the RAS oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin–antitoxin (TA) system can be used for selective and effective eradication of RAS-mutated cancer cells. This out of the box strategy holds great promise for effective cancer treatment and management. We provide proof of concept for a novel platform to selectively eradicate cancer cells using an adenoviral delivery system based on the adjusted natural bacterial system. We generated adenoviral vectors carrying the mazF toxin (pAdEasy-Py4-SV40mP-mCherry-MazF) and the antitoxin mazE (pAdEasy-RGC-SV40mP-MazE-IRES-GFP) under the regulation of RAS and p53, resp. The control vector carries the toxin without the RAS-responsive element (pAdEasy-ΔPy4-SV40mP-mCherry-MazF). In vitro, the mazF-mazE TA system (Py4-SV40mP-mCherry-MazF+RGC-SV40mP-MazE-IRES-GFP) induced massive, dose-dependent cell death, at 69% compared to 19% for the control vector, in a co-infected HCT116 cell line. In vivo, the system caused significant tumor growth inhibition of HCT116 (KRASmut/p53mut) tumors at 73 and 65% compared to PBS and ΔPY4 control groups, resp. In addition, we demonstrate 65% tumor growth inhibition in HCT116 (KRASmut/p53wt) cells, compared to the other two control groups, indicating a contribution of the antitoxin in blocking system leakage in WT RAS cells. These data provide evidence of the feasibility of using mutations in the p53 and RAS pathway to efficiently kill cancer cells. The platform, through its combination of the antitoxin (mazE) with the toxin (mazF), provides effective protection of normal cells from basal low activity or leakage of mazF.
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Bodogai, Monica, Szilamér Ferenczi, Denys Bashtovyy, Paul Miclea, Péter Papp, and Ilona Dusha. "The ntrPR Operon of Sinorhizobium meliloti Is Organized and Functions as a Toxin-Antitoxin Module." Molecular Plant-Microbe Interactions® 19, no. 7 (July 2006): 811–22. http://dx.doi.org/10.1094/mpmi-19-0811.
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The chromosomal ntrPR operon of Sinorhizobium meliloti encodes a protein pair that forms a toxin-antitoxin (TA) module, the first characterized functional TA system in Rhizobiaceae. Similarly to other bacterial TA systems, the toxin gene ntrR is preceded by and partially overlaps with the antitoxin gene ntrP. Based on protein homologies, the ntrPR operon belongs to the vapBC family of TA systems. The operon is negatively autoregulated by the NtrPNtrR complex. Promoter binding by NtrP is weak; stable complex formation also requires the presence of NtrR. The N-terminal part of NtrP is responsible for the interaction with promoter DNA, whereas the C-terminal part is required for protein-protein interactions. In the promoter region, a direct repeat sequence was identified as the binding site of the NtrPNtrR complex. NtrR expression resulted in the inhibition of cell growth and colony formation; this effect was counteracted by the presence of the antitoxin NtrP. These results and our earlier observations demonstrating a less effective downregulation of a wide range of symbiotic and metabolic functions in the ntrR mutant under micro-oxic conditions and an increased symbiotic efficiency with the host plant alfalfa suggest that the ntrPR module contributes to adjusting metabolic levels under symbiosis and other stressful conditions.
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Zielenkiewicz, Urszula, Magdalena Kowalewska, Celina Kaczor, and Piotr Cegłowski. "In Vivo Interactions between Toxin-Antitoxin Proteins Epsilon and Zeta of Streptococcal Plasmid pSM19035 in Saccharomyces cerevisiae." Journal of Bacteriology 191, no. 11 (April 3, 2009): 3677–84. http://dx.doi.org/10.1128/jb.01763-08.
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ABSTRACT The widespread prokaryotic toxin-antitoxin (TA) systems involve conditional interaction between two TA proteins. The interaction between the Epsilon and Zeta proteins, constituting the TA system of plasmid pSM19035 from Streptococcus pyogenes, was detected in vivo using a yeast two-hybrid system. As we showed using Saccharomyces cerevisiae, the Zeta toxin hybrid gene also exerts its toxic effects in a dose-dependent manner in eukaryotic cells. Analysis of mutant proteins in the two-hybrid system demonstrated that the N-terminal part of Zeta and the N-terminal region of Epsilon are involved in the interaction. The N-terminal region of the Zeta protein and its ATP/GTP binding motif were found to be responsible for the toxicity.
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Valizadeh, Nasrin, Firuzeh Valian, Nourkhoda Sadeghifard, Shahriar Karami, Iraj Pakzad, Hossein Kazemian, and Sobhan Ghafourian. "The Role of Peganum harmala Ethanolic Extract and Type II Toxin Antitoxin System in Biofilm Formation." Drug Research 67, no. 07 (March 20, 2017): 385–87. http://dx.doi.org/10.1055/s-0043-102060.
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AbstractToxin antitoxin system is a regulatory system that antitoxin inhibits the toxin. We aimed to determine the role of TA loci in biofilm formation in K. pneumoniae clinical and environmental isolates; also inhibition of biofilm formation by Peganum harmala. So, 40 K. pneumoniae clinical and environmental isolates were subjected for PCR to determine the frequency of mazEF, relEB, and mqsRA TA loci. Biofilm formation assay subjected for all isolates. Then, P. harmala was tested against positive biofilm formation strains. Our results demonstrated that relBE TA loci were dominant TA loci; whereas mqsRA TA loci were negative in all isolates. The most environmental isolates showed weak and no biofilm formation while strong and moderate biofilm formation observed in clinical isolates. Biofilm formations by K. pneumoniae in 9 ug/ml concentration were inhibited by P. harmala. In vivo study suggested to be performed to introduce Peganum harmala as anti-biofilm formation in K. pneumoniae.
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Tamman, Hedvig, Andres Ainelo, Mari Tagel, and Rita Hõrak. "Stability of the GraA Antitoxin Depends on Growth Phase, ATP Level, and Global Regulator MexT." Journal of Bacteriology 198, no. 5 (December 14, 2015): 787–96. http://dx.doi.org/10.1128/jb.00684-15.
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ABSTRACTBacterial type II toxin-antitoxin systems consist of a potentially poisonous toxin and an antitoxin that inactivates the toxic protein by binding to it. Most of the toxins regulate stress survival, but their activation depends on the stability of the antitoxin that has to be degraded in order for the toxin to be able to attack its cellular targets. The degradation of antitoxins is usually rapid and carried out by ATP-dependent protease Lon or Clp, which is activated under stress conditions. ThegraTAsystem ofPseudomonas putidaencodes the toxin GraT, which can affect the growth rate and stress tolerance of bacteria but is under most conditions inactivated by the unusually stable antitoxin GraA. Here, we aimed to describe the stability features of the antitoxin GraA by analyzing its degradation rate in total cell lysates ofP. putida. We show that the degradation rate of GraA depends on the growth phase of bacteria being fastest in the transition from exponential to stationary phase. In accordance with this, higher ATP levels were shown to stabilize GraA. Differently from other antitoxins, the main cellular proteases Lon and Clp are not involved in GraA stability. Instead, GraA seems to be degraded through a unique pathway involving an endoprotease that cleaves the antitoxin into two unequal parts. We also identified the global transcriptional regulator MexT as a factor for destabilization of GraA, which indicates that the degradation of GraA may be induced by conditions similar to those that activate MexT.IMPORTANCEToxin-antitoxin (TA) modules are widespread in bacterial chromosomes and have important roles in stress tolerance. As activation of a type II toxin is triggered by proteolytic degradation of the antitoxin, knowledge about the regulation of the antitoxin stability is critical for understanding the activation of a particular TA module. Here, we report on the unusual degradation pathway of the antitoxin GraA of the recently characterized GraTA system. While GraA is uncommonly stable in the exponential and late-stationary phases, its degradation increases in the transition phase. The degradation pathway of GraA involves neither Lon nor Clp, which usually targets antitoxins, but rather an unknown endoprotease and the global regulator MexT, suggesting a new type of regulation of antitoxin stability.
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Xie, Zhoujie, Fengxia Qi, and Justin Merritt. "Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems." Applied and Environmental Microbiology 79, no. 20 (August 9, 2013): 6375–84. http://dx.doi.org/10.1128/aem.02320-13.
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ABSTRACTDespite the plethora of genetic tools that have been developed for use inStreptococcus mutans, theS. mutansgenetic system still lacks an effective gene induction system exhibiting low basal expression and strong inducibility. Consequently, we created two hybrid gene induction cassettes referred to as Xyl-S1 and Xyl-S2. Both Xyl-S cassettes are xylose inducible and controlled by theBacillus megateriumxylose repressor. The Xyl-S cassettes each demonstrated >600-fold-increased reporter activity in the presence of 1.2% (wt/vol) xylose. However, the Xyl-S1 cassette yielded a much higher maximum level of gene expression, whereas the Xyl-S2 cassette exhibited much lower uninduced basal expression. The cassettes also performed similarly inStreptococcus sanguinisandStreptococcus gordonii, which suggests that they are likely to be useful in a variety of streptococci. We demonstrate how both Xyl-S cassettes are particularly useful for the study of toxin-antitoxin (TA) modules using both the previously characterizedS. mutans mazEFTA module and a previously uncharacterized HicAB TA module inS. mutans. HicAB TA modules are widely distributed among bacteria and archaea, but little is known about their function. We show that HicA serves as the toxin component of the module, while HicB serves as the antitoxin. Our results suggest that, in contrast to that of typical TA modules, HicA toxicity inS. mutansis modest at best. The implications of these results for HicAB function are discussed.
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Heaton, Brook E., Julien Herrou, Anne E. Blackwell, Vicki H. Wysocki, and Sean Crosson. "Molecular Structure and Function of the Novel BrnT/BrnA Toxin-Antitoxin System of Brucella abortus." Journal of Biological Chemistry 287, no. 15 (February 14, 2012): 12098–110. http://dx.doi.org/10.1074/jbc.m111.332163.
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Type II toxin-antitoxin (TA) systems are expressed from two-gene operons that encode a cytoplasmic protein toxin and its cognate protein antitoxin. These gene cassettes are often present in multiple copies on bacterial chromosomes, where they have been reported to regulate stress adaptation and persistence during antimicrobial treatment. We have identified a novel type II TA cassette in the intracellular pathogen Brucella abortus that consists of the toxin gene, brnT, and its antitoxin, brnA. BrnT is coexpressed and forms a 2:2 tetrameric complex with BrnA, which neutralizes BrnT toxicity. The BrnT2-BrnA2 tetramer binds its own promoter via BrnA, and autorepresses its expression; its transcription is strongly induced in B. abortus by various stressors encountered by the bacterial cell during infection of a mammalian host. Although highly divergent at the primary sequence level, an atomic resolution (1.1 Å) crystal structure of BrnT reveals a secondary topology related to the RelE family of type II ribonuclease toxins. However, overall tertiary structural homology to other RelE family toxins is low. A functional characterization of BrnT by site-directed mutagenesis demonstrates a correspondence between its in vitro activity as a ribonuclease and control of bacteriostasis in vivo. We further present an analysis of the conserved and variable features of structure required for RNA scission in BrnT and the RelE toxin family. This structural investigation informs a model of the RelE-fold as an evolutionarily flexible scaffold that has been selected to bind structurally disparate antitoxins, and exhibit distinct toxin activities including RNA scission and DNA gyrase inhibition.
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Boss, Lidia, and Barbara Kędzierska. "Bacterial Toxin-Antitoxin Systems’ Cross-Interactions—Implications for Practical Use in Medicine and Biotechnology." Toxins 15, no. 6 (June 4, 2023): 380. http://dx.doi.org/10.3390/toxins15060380.
Full textAbstract:
Toxin-antitoxin (TA) systems are widely present in bacterial genomes. They consist of stable toxins and unstable antitoxins that are classified into distinct groups based on their structure and biological activity. TA systems are mostly related to mobile genetic elements and can be easily acquired through horizontal gene transfer. The ubiquity of different homologous and non-homologous TA systems within a single bacterial genome raises questions about their potential cross-interactions. Unspecific cross-talk between toxins and antitoxins of non-cognate modules may unbalance the ratio of the interacting partners and cause an increase in the free toxin level, which can be deleterious to the cell. Moreover, TA systems can be involved in broadly understood molecular networks as transcriptional regulators of other genes’ expression or modulators of cellular mRNA stability. In nature, multiple copies of highly similar or identical TA systems are rather infrequent and probably represent a transition stage during evolution to complete insulation or decay of one of them. Nevertheless, several types of cross-interactions have been described in the literature to date. This implies a question of the possibility and consequences of the TA system cross-interactions, especially in the context of the practical application of the TA-based biotechnological and medical strategies, in which such TAs will be used outside their natural context, will be artificially introduced and induced in the new hosts. Thus, in this review, we discuss the prospective challenges of system cross-talks in the safety and effectiveness of TA system usage.
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Jørgensen, Mikkel G., Deo P. Pandey, Milena Jaskolska, and Kenn Gerdes. "HicA of Escherichia coli Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea." Journal of Bacteriology 191, no. 4 (December 5, 2008): 1191–99. http://dx.doi.org/10.1128/jb.01013-08.
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ABSTRACT Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.
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Park, Jin-Young, Hyo Jung Kim, Chinar Pathak, Hye-Jin Yoon, Do-Hee Kim, Sung Jean Park, and Bong-Jin Lee. "Induced DNA bending by unique dimerization of HigA antitoxin." IUCrJ 7, no. 4 (June 26, 2020): 748–60. http://dx.doi.org/10.1107/s2052252520006466.
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The bacterial toxin–antitoxin (TA) system regulates cell growth under various environmental stresses. Mycobacterium tuberculosis, the causative pathogen of tuberculosis (TB), has three HigBA type II TA systems with reverse gene organization, consisting of the toxin protein HigB and labile antitoxin protein HigA. Most type II TA modules are transcriptionally autoregulated by the antitoxin itself. In this report, we first present the crystal structure of the M. tuberculosis HigA3 antitoxin (MtHigA3) and MtHigA3 bound to its operator DNA complex. We also investigated the interaction between MtHigA3 and DNA using NMR spectroscopy. The MtHigA3 antitoxin structure is a homodimer that contains a structurally well conserved DNA-binding domain at the N-terminus and a dimerization domain at the C-terminus. Upon comparing the HigA homologue structures, a distinct difference was found in the C-terminal region that possesses the β-lid, and diverse orientations of two helix–turn–helix (HTH) motifs from HigA homologue dimers were observed. The structure of MtHigA3 bound to DNA reveals that the promoter DNA is bound to two HTH motifs of the MtHigA3 dimer presenting 46.5° bending, and the distance between the two HTH motifs of each MtHigA3 monomer was increased in MtHigA3 bound to DNA. The β-lid, which is found only in the tertiary structure of MtHigA3 among the HigA homologues, causes the formation of a tight dimerization network and leads to a unique arrangement for dimer formation that is related to the curvature of the bound DNA. This work could contribute to the understanding of the HigBA system of M. tuberculosis at the atomic level and may contribute to the development of new antibiotics for TB treatment.
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Jin, Chenglong, Sung-Min Kang, Do-Hee Kim, and Bong-Jin Lee. "Structural and functional analysis of the Klebsiella pneumoniae MazEF toxin–antitoxin system." IUCrJ 8, no. 3 (March 5, 2021): 362–71. http://dx.doi.org/10.1107/s2052252521000452.
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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.
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Kędzierska, Barbara, and Katarzyna Potrykus. "Minigene as a Novel Regulatory Element in Toxin-Antitoxin Systems." International Journal of Molecular Sciences 22, no. 24 (December 13, 2021): 13389. http://dx.doi.org/10.3390/ijms222413389.
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The axe-txe type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation is needed for the cell. In this report, we provide evidence that a minigene encoded within the axe-txe operon influences translation of the Txe toxin. This is the first example to date of such a regulatory mechanism identified in the TA modules. Here, in a series of genetic studies, we employed translational reporter gene fusions to establish the molecular basis of this phenomenon. Our results show that translation of the two-codon mini-ORF displays an in cis mode of action, and positively affects the expression of txe, possibly by increasing its mRNA stability through protection from an endonuclease attack. Moreover, we established that the reading frame in which the two cistrons are encoded, as well as the distance between them, are critical parameters that affect the level of such regulation. In addition, by searching for two-codon ORFs we found sequences of several potential minigenes in the leader sequences of several other toxins belonging to the type II TA family. These findings suggest that this type of gene regulation may not only apply for the axe-txe cassette, but could be more widespread among other TA systems.
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Hosseini, Nava, Maryam Pourhajibagher, Nasim Chiniforush, Nazanin Hosseinkhan, Parizad Rezaie, and Abbas Bahador. "Modulation of Toxin-Antitoxin System Rnl AB Type II in Phage-Resistant Gammaproteobacteria Surviving Photodynamic Treatment." Journal of Lasers in Medical Sciences 10, no. 1 (December 15, 2018): 21–28. http://dx.doi.org/10.15171/jlms.2019.03.
Full textAbstract:
Type II toxin-antitoxin (TA) systems are the particular type of TA modules which take part in different kinds of cellular actions, such as biofilm formation, persistence, stress endurance, defense of the bacterial cell against multiple phage attacks, plasmid maintenance, and programmed cell death in favor of bacterial population. Although several bioinformatics and Pet lab studies have already been conducted to understand the functionality of already discovered TA systems, still, more work in this area is required. Rnl AB type II TA module, which is composed of RnlA toxin and RnlB antitoxin, is a newly discovered type II TA module which takes part in the defense mechanism against T4 bacteriophage attack in Escherichia coli K-12 strain MH1 that has not been widely studied in other bacteria. Because of the significant role of class Gammaproteobacteriacea in a diverse range of health problems, we chose here to focus on this class to survey the presence of the Rnl AB TA module. For better categorization and description of the distribution of this module in this class of bacteria, the corresponding phylogenetic trees are illustrated here. Neighbor-joining and the maximum parsimony methods were used in this study to take a look at the distribution of domains present in RnlA and RnlB proteins, among members of Gammaproteobacteria. Also, the possible roles of photodynamic therapy (PDT) in providing a substrate for better phage therapy are herein discussed.
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Zhou, Jingyi, Shouyi Li, Haozhou Li, Yongxin Jin, Fang Bai, Zhihui Cheng, and Weihui Wu. "Identification of a Toxin–Antitoxin System That Contributes to Persister Formation by Reducing NAD in Pseudomonas aeruginosa." Microorganisms 9, no. 4 (April 2, 2021): 753. http://dx.doi.org/10.3390/microorganisms9040753.
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Bacterial persisters are slow-growing or dormant cells that are highly tolerant to bactericidal antibiotics and contribute to recalcitrant and chronic infections. Toxin/antitoxin (TA) systems play important roles in controlling persister formation. Here, we examined the roles of seven predicted type II TA systems in the persister formation of a Pseudomonas aeruginosa wild-type strain PA14. Overexpression of a toxin gene PA14_51010 or deletion of the cognate antitoxin gene PA14_51020 increased the bacterial tolerance to antibiotics. Co-overexpression of PA14_51010 and PA14_51020 or simultaneous deletion of the two genes resulted in a wild-type level survival rate following antibiotic treatment. The two genes were located in the same operon that was repressed by PA14_51020. We further demonstrated the interaction between PA14_51010 and PA14_51020. Sequence analysis revealed that PA14_51010 contained a conserved RES domain. Overexpression of PA14_51010 reduced the intracellular level of nicotinamide adenine dinucleotide (NAD+). Mutation of the RES domain abolished the abilities of PA14_51010 in reducing NAD+ level and promoting persister formation. In addition, overproduction of NAD+ by mutation in an nrtR gene counteracted the effect of PA14_51010 overexpression in promoting persister formation. In combination, our results reveal a novel TA system that contributes to persister formation through reducing the intracellular NAD+ level in P. aeruginosa.
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Nigam, Akanksha, Adi Oron-Gottesman, and Hanna Engelberg-Kulka. "A Bias in the Reading of the Genetic Code of Escherichia coli is a Characteristic for Genes that Specify Stress-induced MazF-mediated Proteins." Current Genomics 21, no. 4 (August 8, 2020): 311–18. http://dx.doi.org/10.2174/1389202921999200606215305.
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Background: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Materials and Methods: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Results: Here it is reported that for most of the E. coli proteins mediated by stress-induced MazF, the ACA threonine codon in their mRNAs is not in-frame but rather out-of-frame; in these same RNAs, the three synonymous threonine codons, ACG, ACU, and ACC, are in-frame. In contrast, for proteins translated by the canonical translation system, in the majority of mRNAs, the ACA codon is located in-frame. Conclusion: The described bias in the genetic code is a characteristic of E. coli genes specifying for stress-induced MazF-mediated proteins.
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Jimmy, Steffi, Chayan Kumar Saha, Tatsuaki Kurata, Constantine Stavropoulos, Sofia Raquel Alves Oliveira, Alan Koh, Albinas Cepauskas, et al. "A widespread toxin−antitoxin system exploiting growth control via alarmone signaling." Proceedings of the National Academy of Sciences 117, no. 19 (April 28, 2020): 10500–10510. http://dx.doi.org/10.1073/pnas.1916617117.
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Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin−antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS–antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.
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Zhou, Juan, Xue-Jian Du, Ying Liu, Zeng-Qiang Gao, Zhi Geng, Yu-Hui Dong, and Heng Zhang. "Insights into the Neutralization and DNA Binding of Toxin–Antitoxin System ParESO-CopASO by Structure-Function Studies." Microorganisms 9, no. 12 (December 3, 2021): 2506. http://dx.doi.org/10.3390/microorganisms9122506.
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ParESO-CopASO is a new type II toxin–antitoxin (TA) system in prophage CP4So that plays an essential role in circular CP4So maintenance after the excision in Shewanella oneidensis. The toxin ParESO severely inhibits cell growth, while CopASO functions as an antitoxin to neutralize ParESO toxicity through direct interactions. However, the molecular mechanism of the neutralization and autoregulation of the TA operon transcription remains elusive. In this study, we determined the crystal structure of a ParESO-CopASO complex that adopted an open V-shaped heterotetramer with the organization of ParESO-(CopASO)2-ParESO. The structure showed that upon ParESO binding, the intrinsically disordered C-terminal domain of CopASO was induced to fold into a partially ordered conformation that bound into a positively charged and hydrophobic groove of ParESO. Thermodynamics analysis showed the DNA-binding affinity of CopASO was remarkably higher than that of the purified TA complex, accompanied by the enthalpy change reversion from an exothermic reaction to an endothermic reaction. These results suggested ParESO acts as a de-repressor of the TA operon transcription at the toxin:antitoxin level of 1:1. Site-directed mutagenesis of ParESO identified His91 as the essential residue for its toxicity by cell toxicity assays. Our structure-function studies therefore elucidated the transcriptional regulation mechanism of the ParESO-CopASO pair, and may help to understand the regulation of CP4So maintenance in S. oneidensis.
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Kang, Sung-Min. "Mycobacterium tuberculosis Rv0229c Shows Ribonuclease Activity and Reveals Its Corresponding Role as Toxin VapC51." Antibiotics 12, no. 5 (May 1, 2023): 840. http://dx.doi.org/10.3390/antibiotics12050840.
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The VapBC system, which belongs to the type II toxin–antitoxin (TA) system, is the most abundant and widely studied system in Mycobacterium tuberculosis. The VapB antitoxin suppresses the activity of the VapC toxin through a stable protein–protein complex. However, under environmental stress, the balance between toxin and antitoxin is disrupted, leading to the release of free toxin and bacteriostatic state. This study introduces the Rv0229c, a putative VapC51 toxin, and aims to provide a better understanding of its discovered function. The structure of the Rv0229c shows a typical PIN-domain protein, exhibiting an β1-α1-α2-β2-α3-α4-β3-α5-α6-β4-α7-β5 topology. The structure-based sequence alignment showed four electronegative residues in the active site of Rv0229c, which is composed of Asp8, Glu42, Asp95, and Asp113. By comparing the active site with existing VapC proteins, we have demonstrated the justification for naming it VapC51 at the molecular level. In an in vitro ribonuclease activity assay, Rv0229c showed ribonuclease activity dependent on the concentration of metal ions such as Mg2+ and Mn2+. In addition, magnesium was found to have a greater effect on VapC51 activity than manganese. Through these structural and experimental studies, we provide evidence for the functional role of Rv0229c as a VapC51 toxin. Overall, this study aims to enhance our understanding of the VapBC system in M. tuberculosis.
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Kang, Sung-Min, Ji Sung Koo, Chang-Min Kim, Do-Hee Kim, and Bong-Jin Lee. "mRNA Interferase Bacillus cereus BC0266 Shows MazF-Like Characteristics Through Structural and Functional Study." Toxins 12, no. 6 (June 8, 2020): 380. http://dx.doi.org/10.3390/toxins12060380.
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Toxin–antitoxin (TA) systems are prevalent in bacteria and are known to regulate cellular growth in response to stress. As various functions related to TA systems have been revealed, the importance of TA systems are rapidly emerging. Here, we present the crystal structure of putative mRNA interferase BC0266 and report it as a type II toxin MazF. The MazF toxin is a ribonuclease activated upon and during stressful conditions, in which it cleaves mRNA in a sequence-specific, ribosome-independent manner. Its prolonged activity causes toxic consequences to the bacteria which, in turn, may lead to bacterial death. In this study, we conducted structural and functional investigations of Bacillus cereus MazF and present the first toxin structure in the TA system of B. cereus. Specifically, B. cereus MazF adopts a PemK-like fold and also has an RNA substrate-recognizing loop, which is clearly observed in the high-resolution structure. Key residues of B. cereus MazF involved in the catalytic activity are also proposed, and in vitro assay together with mutational studies affirm the ribonucleic activity and the active sites essential for its cellular toxicity.
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Nonin-Lecomte, Sylvie, Laurence Fermon, Brice Felden, and Marie-Laure Pinel-Marie. "Bacterial Type I Toxins: Folding and Membrane Interactions." Toxins 13, no. 7 (July 14, 2021): 490. http://dx.doi.org/10.3390/toxins13070490.
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Bacterial type I toxin-antitoxin systems are two-component genetic modules that encode a stable toxic protein whose ectopic overexpression can lead to growth arrest or cell death, and an unstable RNA antitoxin that inhibits toxin translation during growth. These systems are widely spread among bacterial species. Type I antitoxins are cis- or trans-encoded antisense small RNAs that interact with toxin-encoding mRNAs by pairing, thereby inhibiting toxin mRNA translation and/or inducing its degradation. Under environmental stress conditions, the up-regulation of the toxin and/or the antitoxin degradation by specific RNases promote toxin translation. Most type I toxins are small hydrophobic peptides with a predicted α-helical transmembrane domain that induces membrane depolarization and/or permeabilization followed by a decrease of intracellular ATP, leading to plasmid maintenance, growth adaptation to environmental stresses, or persister cell formation. In this review, we describe the current state of the art on the folding and the membrane interactions of these membrane-associated type I toxins from either Gram-negative or Gram-positive bacteria and establish a chronology of their toxic effects on the bacterial cell. This review also includes novel structural results obtained by NMR concerning the sprG1-encoded membrane peptides that belong to the sprG1/SprF1 type I TA system expressed in Staphylococcus aureus and discusses the putative membrane interactions allowing the lysis of competing bacteria and host cells.
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Kim, Do-Hee, Sung-Min Kang, Sung-Min Baek, Hye-Jin Yoon, Dong Man Jang, Hyoun Sook Kim, Sang Jae Lee, and Bong-Jin Lee. "Role of PemI in the Staphylococcus aureus PemIK toxin–antitoxin complex: PemI controls PemK by acting as a PemK loop mimic." Nucleic Acids Research 50, no. 4 (February 10, 2022): 2319–33. http://dx.doi.org/10.1093/nar/gkab1288.
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Abstract Staphylococcus aureus is a notorious and globally distributed pathogenic bacterium. New strategies to develop novel antibiotics based on intrinsic bacterial toxin–antitoxin (TA) systems have been recently reported. Because TA systems are present only in bacteria and not in humans, these distinctive systems are attractive targets for developing antibiotics with new modes of action. S. aureus PemIK is a type II TA system, comprising the toxin protein PemK and the labile antitoxin protein PemI. Here, we determined the crystal structures of both PemK and the PemIK complex, in which PemK is neutralized by PemI. Our biochemical approaches, including fluorescence quenching and polarization assays, identified Glu20, Arg25, Thr48, Thr49, and Arg84 of PemK as being important for RNase function. Our study indicates that the active site and RNA-binding residues of PemK are covered by PemI, leading to unique conformational changes in PemK accompanied by repositioning of the loop between β1 and β2. These changes can interfere with RNA binding by PemK. Overall, PemK adopts particular open and closed forms for precise neutralization by PemI. This structural and functional information on PemIK will contribute to the discovery and development of novel antibiotics in the form of peptides or small molecules inhibiting direct binding between PemI and PemK.
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Jurėnas, Dukas, Laurence Van Melderen, and Abel Garcia-Pino. "Crystallization and X-ray analysis of all of the players in the autoregulation of theataRTtoxin–antitoxin system." Acta Crystallographica Section F Structural Biology Communications 74, no. 7 (June 26, 2018): 391–401. http://dx.doi.org/10.1107/s2053230x18007914.
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TheataRToperon from enteropathogenicEscherichia coliencodes a toxin–antitoxin (TA) module with a recently discovered novel toxin activity. This new type II TA module targets translation initiation for cell-growth arrest. Virtually nothing is known regarding the molecular mechanisms of neutralization, toxin catalytic action or translation autoregulation. Here, the production, biochemical analysis and crystallization of the intrinsically disordered antitoxin AtaR, the toxin AtaT, the AtaR–AtaT complex and the complex of AtaR–AtaT with a double-stranded DNA fragment of the operator region of the promoter are reported. Because they contain large regions that are intrinsically disordered, TA antitoxins are notoriously difficult to crystallize. AtaR forms a homodimer in solution and crystallizes in space groupP6122, with unit-cell parametersa = b = 56.3,c= 160.8 Å. The crystals are likely to contain an AtaR monomer in the asymmetric unit and diffracted to 3.8 Å resolution. The Y144F catalytic mutant of AtaT (AtaTY144F) bound to the cofactor acetyl coenzyme A (AcCoA) and the C-terminal neutralization domain of AtaR (AtaR44–86) were also crystallized. The crystals of the AtaTY144F–AcCoA complex diffracted to 2.5 Å resolution and the crystals of AtaR44–86diffracted to 2.2 Å resolution. Analysis of these structures should reveal the full scope of the neutralization of the toxin AtaT by AtaR. The crystals belonged to space groupsP6522 andP3121, with unit-cell parametersa=b= 58.1,c= 216.7 Å anda=b= 87.6,c = 125.5 Å, respectively. The AtaR–AtaT–DNA complex contains a 22 bp DNA duplex that was optimized to obtain high-resolution data based on the sequence of two inverted repeats detected in the operator region. It crystallizes in space groupC2221, with unit-cell parametersa= 75.6,b= 87.9,c= 190.5 Å. These crystals diffracted to 3.5 Å resolution.
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Bajaj, R. Alexandra, Mark A. Arbing, Annie Shin, Duilio Cascio, and Linda Miallau. "Crystal structure of the toxin Msmeg_6760, the structural homolog ofMycobacterium tuberculosisRv2035, a novel type II toxin involved in the hypoxic response." Acta Crystallographica Section F Structural Biology Communications 72, no. 12 (November 19, 2016): 863–69. http://dx.doi.org/10.1107/s2053230x16017957.
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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 inMycobacterium tuberculosislatency 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.
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Ariyachaokun, Kanchiyaphat, Anna D. Grabowska, Claude Gutierrez, and Olivier Neyrolles. "Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System." Toxins 12, no. 5 (May 16, 2020): 329. http://dx.doi.org/10.3390/toxins12050329.
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MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.
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Wen, Wen, Banghui Liu, Lu Xue, Zhongliang Zhu, Liwen Niu, and Baolin Sun. "Autoregulation and Virulence Control by the Toxin-Antitoxin System SavRS inStaphylococcus aureus." Infection and Immunity 86, no. 5 (February 12, 2018): e00032-18. http://dx.doi.org/10.1128/iai.00032-18.
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ABSTRACTToxin-antitoxin (TA) systems play diverse physiological roles, such as plasmid maintenance, growth control, and persister cell formation, but their involvement in bacterial pathogenicity remains largely unknown. Here, we have identified a novel type II toxin-antitoxin system, SavRS, and revealed the molecular mechanisms of its autoregulation and virulence control inStaphylococcus aureus. Electrophoretic mobility shift assay and isothermal titration calorimetry data indicated that the antitoxin SavR acted as the primary repressor bound to its own promoter, while the toxin SavS formed a complex with SavR to enhance the ability to bind to the operator site. DNase I footprinting assay identified the SavRS-binding site containing a short and long palindrome in the promoter region. Further, mutation and DNase I footprinting assay demonstrated that the two palindromes were crucial for DNA binding and transcriptional repression. More interestingly, genetic deletion of thesavRSsystem led to the increased hemolytic activity and pathogenicity in a mouse subcutaneous abscess model. We further identified two virulence genes,hlaandefb, by real-time quantitative reverse transcription-PCR and demonstrated that SavR and SavRS could directly bind to their promoter regions to repress virulence gene expression.
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Levante, Alessia, Camilla Lazzi, Giannis Vatsellas, Dimitris Chatzopoulos, Vasilis S. Dionellis, Periklis Makrythanasis, Erasmo Neviani, and Claudia Folli. "Genome Sequencing of five Lacticaseibacillus Strains and Analysis of Type I and II Toxin-Antitoxin System Distribution." Microorganisms 9, no. 3 (March 21, 2021): 648. http://dx.doi.org/10.3390/microorganisms9030648.
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The analysis of bacterial genomes is a potent tool to investigate the distribution of specific traits related to the ability of surviving in particular environments. Among the traits associated with the adaptation to hostile conditions, toxin–antitoxin (TA) systems have recently gained attention in lactic acid bacteria. In this work, genome sequences of Lacticaseibacillus strains of dairy origin were compared, focusing on the distribution of type I TA systems homologous to Lpt/RNAII and of the most common type II TA systems. A high number of TA systems have been identified spread in all the analyzed strains, with type I TA systems mainly located on plasmid DNA. The type II TA systems identified in these strains highlight the diversity of encoded toxins and antitoxins and their organization. This study opens future perspectives on the use of genomic data as a resource for the study of TA systems distribution and prevalence in microorganisms of industrial relevance.