ABSTRACT: Examination of E. faecalis toxin-antitoxin (TA) toxin Fst function utilizing a pheromone-inducible expression vector with tight repression and large dynamic range
Project description:Tools for regulated gene expression in E. faecalis are extremely limited. In this study we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin loci present in E. faecalis, parpAD1 of the pAD1 plasmid and parEF0409 located on the E. faecalis chromosome. Results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalis.
Project description:Toxin-antitoxin (TA) systems are ubiquitous throughout bacterial and archaeal genomes. TA systems consist of a stable toxin that inhibits growth and a labile antitoxin that prevents toxicity of the toxin. Here we made an artificial TA system (arT/arA) and performed a DNA microarray study for overproduction of the toxin. arT was overexpressed in Escherichia coli BW25113 and compared to the empty vector.
Project description:Toxin-antitoxin (TA) systems are ubiquitous throughout bacterial and archaeal genomes. TA systems consist of a stable toxin that inhibits growth and a labile antitoxin that prevents toxicity of the toxin. Here we made an artificial TA system (arT/arA) and performed a DNA microarray study for overproduction of the toxin.
Project description:As the number of bacterial genomes and transcriptomes increases, so does the number of newly identified toxin–antitoxin (TA) systems. However, their functional characterization remains challenging, often requiring the use of overexpression vectors that can lead to misinterpretations. To fill this gap, we developed a systematic approach called FASTBAC-Seq (Functional AnalysiS of Toxin–Antitoxin Systems in BACteria by Deep Sequencing). Combining life/death phenotypic selection with next-generation sequencing, FASTBAC-Seq allows the rapid identification of loss-of- function (toxicity) mutations in toxin-encoding genes belonging to TA loci with nucleotide resolution. Here, we apply this new tool to study aapA3/IsoA3, a member of a new family of type I TA systems hosted on the chromosome of the major human gastric pathogen Helicobacter pylori.
Project description:The previously uncharacterized proteins HigB (unannotated) and HigA (PA4674) of Pseudomonas aeruginosa PA14 were found to form a type II TA system in which antitoxin HigA masks the RNase activity of toxin HigB through direct binding. To determine the physiological role of HigB/HigA in P. aeruginosa, a whole-transcriptome experiment was performed for the higA antitoxin deletion mutant of the PA14 strain compared to the wild-type PA14 strain. The rationale was that for the strain that lacks the antitoxin, the effect of the toxin could be discerned due to enhanced activity of the toxin. Furthermore, toxin HigB reduces production of the virulence factors pyochelin, pyocyanin, swarming, and biofilm formation.
Project description:The role of six toxin-antitoxin (TA) systems on biofilm development was investigated (MazEF, RelBEF, ChpB, YefM-YoeB, DinJ-YafQ, and TomB-Hha). Although these TA systems were reported previously to not impact bacterial fitness, we found that biofilm formation is decreased by toxins and increased by anti-toxins, in part, through YjgK. Hence, one role of TA systems is to regulate biofilm formation.
Project description:The role of six toxin-antitoxin (TA) systems on biofilm development was investigated (MazEF, RelBEF, ChpB, YefM-YoeB, DinJ-YafQ, and TomB-Hha). Although these TA systems were reported previously to not impact bacterial fitness, we found that biofilm formation is decreased by toxins and increased by anti-toxins, in part, through YjgK. Hence, one role of TA systems is to regulate biofilm formation.
Project description: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 a type I TA system, the SprF1/SprG1 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 between the strains points on SprF1 antitoxin as an agent 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 toxin-driven release of the cytoplasmic proteins may modulate the host inflammatory response that, in turn, may amplify the S. aureus infection spread.
Project description:The role of six toxin-antitoxin (TA) systems on biofilm development was investigated (MazEF, RelBEF, ChpB, YefM-YoeB, DinJ-YafQ, and TomB-Hha). Although these TA systems were reported previously to not impact bacterial fitness, we found that biofilm formation is decreased by toxins and increased by anti-toxins, in part, through YjgK. Hence, one role of TA systems is to regulate biofilm formation. Experiment Overall Design: Strains: E. coli K-12 MG1655 and E. coli K-12 LVM100 delta 5 TA mutant Experiment Overall Design: Medium: LB Experiment Overall Design: Cells: biofilm cells on glass wool Experiment Overall Design: Time: 15 h Experiment Overall Design: Temperature: 37oC
