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Sabine Brantl - One of the best experts on this subject based on the ideXlab platform.
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in vitro characterization of the type i toxin antitoxin System bsre sr5 from bacillus subtilis
Journal of Biological Chemistry, 2016Co-Authors: Christin Meisner, Natalie Jahn, Sabine BrantlAbstract:BsrE/SR5 is a new type I toxin/antitoxin System located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3' ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (kapp) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA System from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences.
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In Vitro Characterization of the Type I Toxin-Antitoxin System bsrE/SR5 from Bacillus subtilis.
The Journal of biological chemistry, 2015Co-Authors: Christin Meißner, Natalie Jahn, Sabine BrantlAbstract:BsrE/SR5 is a new type I toxin/antitoxin System located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3' ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (kapp) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA System from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences.
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BsrG/SR4 from Bacillus subtilis--the first temperature-dependent type I Toxin-Antitoxin System.
Molecular microbiology, 2012Co-Authors: Natalie Jahn, Heike Preis, Christoph Wiedemann, Sabine BrantlAbstract:Summary Here, we describe bsrG/SR4, a novel type I toxin–antitoxin System from the SPβ prophage region of the Bacillus subtilis chromosome. The 294-nucleotide bsrG RNA encodes a 38-amino-acid toxin, whereas SR4 is a 180-nucleotide antisense RNA that acts as the antitoxin. Both genes overlap by 123 nucleotides. BsrG expression increases at the onset of stationary phase. The sr4 promoter is 6- to 10-fold stronger than the bsrG promoter. Deletion of sr4 stabilizes bsrG mRNA and causes cell lysis on agar plates, which is due to the BsrG peptide and not the bsrG mRNA. SR4 overexpression could compensate cell lysis caused by overexpression of bsrG. SR4 interacts with the 3′ UTR of bsrG RNA, thereby promoting its degradation. RNase III cleaves the bsrG RNA/SR4 duplex at position 185 of bsrG RNA, but is not essential for the function of the toxin–antitoxin System. Endoribonuclease Y and 3′-5′ exoribonuclease R participate in the degradation of both bsrG RNA and SR4, whereas PnpA processes three SR4 precursors to the mature RNA. A heat shock at 48°C results in faster degradation and, therefore, significantly decreased amounts of bsrG RNA.
Laurence Van Melderen - One of the best experts on this subject based on the ideXlab platform.
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Reassessing the Role of the Type II MqsRA Toxin-Antitoxin System in Stress Response and Biofilm Formation: mqsA Is Transcriptionally Uncoupled from mqsR.
mBio, 2019Co-Authors: Nathan Fraikin, Clothilde J. Rousseau, Nathalie Goeders, Laurence Van MelderenAbstract:ABSTRACT Toxin-Antitoxin (TA) Systems are broadly distributed modules whose biological roles remain mostly unknown. The mqsRA System is a noncanonical TA System in which the toxin and antitoxins genes are organized in operon but with the particularity that the toxin gene precedes that of the antitoxin. This System was shown to regulate global processes such as resistance to bile salts, motility, and biofilm formation. In addition, the MqsA antitoxin was shown to be a master regulator that represses the transcription of the csgD, cspD, and rpoS global regulator genes, thereby displaying a pleiotropic regulatory role. Here, we identified two promoters located in the toxin sequence driving the constitutive expression of mqsA, allowing thereby excess production of the MqsA antitoxin compared to the MqsR toxin. Our results show that both antitoxin-specific and operon promoters are not regulated by stresses such as amino acid starvation, oxidative shock, or bile salts. Moreover, we show that the MqsA antitoxin is not a global regulator as suggested, since the expression of csgD, cspD and rpoS is similar in wild-type and ΔmqsRA mutant strains. Moreover, these two strains behave similarly in terms of biofilm formation and sensitivity to oxidative stress or bile salts. IMPORTANCE There is growing controversy regarding the role of chromosomal Toxin-Antitoxin Systems in bacterial physiology. mqsRA is a peculiar Toxin-Antitoxin System, as the gene encoding the toxin precedes that of the antitoxin. This System was previously shown to play a role in stress response and biofilm formation. In this work, we identified two promoters specifically driving the constitutive expression of the antitoxin, thereby decoupling the expression of antitoxin from the toxin. We also showed that mqsRA contributes neither to the regulation of biofilm formation nor to the sensitivity to oxidative stress and bile salts. Finally, we were unable to confirm that the MqsA antitoxin is a global regulator. Altogether, our data are ruling out the involvement of the mqsRA System in Escherichia coli regulatory networks.
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overproduction of the lon protease triggers inhibition of translation in escherichia coli involvement of the yefm yoeb toxin antitoxin System
Molecular Microbiology, 2004Co-Authors: Susanne K Christensen, Genevieve Maenhautmichel, Natacha Mine, Susan Gottesman, Kenn Gerdes, Laurence Van MelderenAbstract:In Escherichia coli, the Lon ATP-dependent protease is responsible for degradation of several regulatory proteins and for the elimination of abnormal proteins. Previous studies have shown that the overproduction of Lon is lethal. Here, we showed that Lon overproduction specifically inhibits translation through at least two different pathways. We have identified one of the pathways as being the chromosomal yefM-yoeB Toxin-Antitoxin System. The existence of a second pathway is demonstrated by the observation that the deletion of the yefM-yoeB System did not completely suppress lethality and translation inhibition. We also showed that the YoeB toxin induces cleavage of translated mRNAs and that Lon overproduction specifically activates YoeB-dependent mRNAs cleavage. Indeed, none of the other identified chromosomal Toxin-Antitoxin Systems (relBE, mazEF, chpB and dinJ-yafQ) was involved in Lon-dependent lethality, translation inhibition and mRNA cleavage even though the RelB and MazE antitoxins are known to be Lon substrates. Based on our results and other studies, translation inhibition appears to be the key element that triggers chromosomal Toxin-Antitoxin Systems. We propose that under Lon overproduction conditions, translation inhibition is mediated by Lon degradation of a component of the YoeB-independent pathway, in turn activating the YoeB toxin by preventing synthesis of its unstable YefM antidote.
Natalie Jahn - One of the best experts on this subject based on the ideXlab platform.
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in vitro characterization of the type i toxin antitoxin System bsre sr5 from bacillus subtilis
Journal of Biological Chemistry, 2016Co-Authors: Christin Meisner, Natalie Jahn, Sabine BrantlAbstract:BsrE/SR5 is a new type I toxin/antitoxin System located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3' ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (kapp) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA System from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences.
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In Vitro Characterization of the Type I Toxin-Antitoxin System bsrE/SR5 from Bacillus subtilis.
The Journal of biological chemistry, 2015Co-Authors: Christin Meißner, Natalie Jahn, Sabine BrantlAbstract:BsrE/SR5 is a new type I toxin/antitoxin System located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3' ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (kapp) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA System from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences.
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BsrG/SR4 from Bacillus subtilis--the first temperature-dependent type I Toxin-Antitoxin System.
Molecular microbiology, 2012Co-Authors: Natalie Jahn, Heike Preis, Christoph Wiedemann, Sabine BrantlAbstract:Summary Here, we describe bsrG/SR4, a novel type I toxin–antitoxin System from the SPβ prophage region of the Bacillus subtilis chromosome. The 294-nucleotide bsrG RNA encodes a 38-amino-acid toxin, whereas SR4 is a 180-nucleotide antisense RNA that acts as the antitoxin. Both genes overlap by 123 nucleotides. BsrG expression increases at the onset of stationary phase. The sr4 promoter is 6- to 10-fold stronger than the bsrG promoter. Deletion of sr4 stabilizes bsrG mRNA and causes cell lysis on agar plates, which is due to the BsrG peptide and not the bsrG mRNA. SR4 overexpression could compensate cell lysis caused by overexpression of bsrG. SR4 interacts with the 3′ UTR of bsrG RNA, thereby promoting its degradation. RNase III cleaves the bsrG RNA/SR4 duplex at position 185 of bsrG RNA, but is not essential for the function of the toxin–antitoxin System. Endoribonuclease Y and 3′-5′ exoribonuclease R participate in the degradation of both bsrG RNA and SR4, whereas PnpA processes three SR4 precursors to the mature RNA. A heat shock at 48°C results in faster degradation and, therefore, significantly decreased amounts of bsrG RNA.
Sylvain Moineau - One of the best experts on this subject based on the ideXlab platform.
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Mutational Analysis of the Antitoxin in the Lactococcal Type III Toxin-Antitoxin System AbiQ
Applied and environmental microbiology, 2015Co-Authors: Maxime Bélanger, Sylvain MoineauAbstract:ABSTRACT The lactococcal abortive phage infection mechanism AbiQ recently was classified as a type III Toxin-Antitoxin System in which the toxic protein (ABIQ) is regulated following cleavage of its repeated noncoding RNA antitoxin ( antiQ ). In this study, we investigated the role of the antitoxin in antiphage activity. The cleavage of antiQ by ABIQ was characterized using 5′ rapid amplification of cDNA ends PCR and was located in an adenine-rich region of antiQ . We next generated a series of derivatives with point mutations within antiQ or with various numbers of antiQ repetitions. These modifications were analyzed for their effect on the antiphage activity (efficiency of plaquing) and on the endoribonuclease activity (Northern hybridization). We observed that increasing or reducing the number of antiQ repeats significantly decreased the antiphage activity of the System. Several point mutations had a similar effect on the antiphage activity and were associated with changes in the digestion profile of antiQ . Interestingly, a point mutation in the putative pseudoknot structure of antiQ mutants led to an increased AbiQ antiphage activity, thereby offering a novel way to increase the activity of an abortive infection mechanism.
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Structure and activity of AbiQ, a lactococcal endoribonuclease belonging to the type III Toxin-Antitoxin System.
Molecular microbiology, 2013Co-Authors: Julie E. Samson, Silvia Spinelli, Christian Cambillau, Sylvain MoineauAbstract:Summary AbiQ is a phage resistance mechanism found on a native plasmid of Lactococcus lactis that abort virulent phage infections. In this study, we experimentally demonstrate that AbiQ belongs to the recently described type III toxin–antitoxin Systems. When overexpressed, the AbiQ protein (ABIQ) is toxic and causes bacterial death in a bacteriostatic manner. Northern and Western blot experiments revealed that the abiQ gene is transcribed and translated constitutively, and its expression is not activated by a phage product. ABIQ is an endoribonuclease that specifically cleaves its cognate antitoxin RNA molecule in vivo. The crystal structure of ABIQ was solved and site-directed mutagenesis identified key amino acids for its anti-phage and/or its RNase function. The AbiQ System is the first lactococcal abortive infection System characterized to date at a structural level.
Baolin Sun - One of the best experts on this subject based on the ideXlab platform.
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A novel type I Toxin-Antitoxin System modulates persister cell formation in Staphylococcus aureus
International journal of medical microbiology : IJMM, 2020Co-Authors: Gul Habib, Jiade Zhu, Baolin SunAbstract:A plethora of Toxin-Antitoxin Systems exist in bacteria and has multilateral roles in bacterial pathogenesis and virulence. Toxin-Antitoxin Systems have been involved in persister cell formation in Escherichia coli and Mycobacterium but have not been reported to be associated with Staphylococcus aureus persistence. Persistence is the ability of bacterial cells to tolerate unfavorable conditions and multiple stresses. There are less known and more unknown factors that either alleviate or aggravate bacterial persistence phenomenon. For the first time, we reported a new chromosomally encoded tripartite Toxin-Antitoxin System and its role in S. aureus persister cell formation. The toxin gene is bacteriostatic in action and counterbalanced by antitoxin RNA that could basepair with the toxin mRNA and formed a duplex. The transcriptional regulator positively regulates the toxin expression under certain stress conditions. The toxin ectopic induction increased S. aureus susceptibility to norfloxacin, ciprofloxacin, and ofloxacin. Whole-genome RNA sequencing revealed that MDR efflux pump norA is significantly down-regulated by toxin ectopic induction. The deletion of norA from S. aureus genome reduced resistance toward ciprofloxacin, norfloxacin, and ofloxacin, as well as resulted in a decrease in minimal inhibitory concentration while complementation of norA successfully restored the phenotypes. The persistence assay of the norA mutant revealed that deletion of norA increased persister cell survival in S. aureus. Altogether, we have provided insight into the first tripartite type-I TA System and revealed the role of MDR NorA in the persister cell formation of S. aureus.
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Autoregulation and Virulence Control by the Toxin-Antitoxin System SavRS in Staphylococcus aureus.
Infection and immunity, 2018Co-Authors: Wen Wen, Banghui Liu, Lu Xue, Zhongliang Zhu, Liwen Niu, Baolin SunAbstract:ABSTRACT Toxin-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 in Staphylococcus 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 the savRS System led to the increased hemolytic activity and pathogenicity in a mouse subcutaneous abscess model. We further identified two virulence genes, hla and efb, 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.