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Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems
A large number of bacterial toxin–antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cel...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8544347/ https://www.ncbi.nlm.nih.gov/pubmed/34698227 http://dx.doi.org/10.3390/mps4040071 |
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author | Maggi, Stefano Ferrari, Alberto Yabre, Korotoum Bonini, Aleksandra Anna Rivetti, Claudio Folli, Claudia |
author_facet | Maggi, Stefano Ferrari, Alberto Yabre, Korotoum Bonini, Aleksandra Anna Rivetti, Claudio Folli, Claudia |
author_sort | Maggi, Stefano |
collection | PubMed |
description | A large number of bacterial toxin–antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cell transformation, culturing, and stability of the transformants, due to the expression of highly toxic proteins. Recently, in dealing with the type I Lpt/RNAII and the type II YafQ/DinJ TA systems, we encountered several of these problems that urged us to optimize methodological strategies to study the phenotype of recombinant Escherichia coli host cells. In particular, we have found conditions to tightly repress toxin expression by combining the pET expression system with the E. coli C41(DE3) pLysS strain. To monitor the RNase activity of the YafQ toxin, we developed a fluorescence approach based on Thioflavin-T which fluoresces brightly when complexed with bacterial RNA. Fluorescence microscopy was also applied to reveal loss of membrane integrity associated with the activity of the type I toxin Lpt, by using DAPI and ethidium bromide to selectively stain cells with impaired membrane permeability. We further found that atomic force microscopy can readily be employed to characterize toxin-induced membrane damages. |
format | Online Article Text |
id | pubmed-8544347 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-85443472021-10-26 Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems Maggi, Stefano Ferrari, Alberto Yabre, Korotoum Bonini, Aleksandra Anna Rivetti, Claudio Folli, Claudia Methods Protoc Study Protocol A large number of bacterial toxin–antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cell transformation, culturing, and stability of the transformants, due to the expression of highly toxic proteins. Recently, in dealing with the type I Lpt/RNAII and the type II YafQ/DinJ TA systems, we encountered several of these problems that urged us to optimize methodological strategies to study the phenotype of recombinant Escherichia coli host cells. In particular, we have found conditions to tightly repress toxin expression by combining the pET expression system with the E. coli C41(DE3) pLysS strain. To monitor the RNase activity of the YafQ toxin, we developed a fluorescence approach based on Thioflavin-T which fluoresces brightly when complexed with bacterial RNA. Fluorescence microscopy was also applied to reveal loss of membrane integrity associated with the activity of the type I toxin Lpt, by using DAPI and ethidium bromide to selectively stain cells with impaired membrane permeability. We further found that atomic force microscopy can readily be employed to characterize toxin-induced membrane damages. MDPI 2021-10-08 /pmc/articles/PMC8544347/ /pubmed/34698227 http://dx.doi.org/10.3390/mps4040071 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Study Protocol Maggi, Stefano Ferrari, Alberto Yabre, Korotoum Bonini, Aleksandra Anna Rivetti, Claudio Folli, Claudia Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems |
title | Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems |
title_full | Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems |
title_fullStr | Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems |
title_full_unstemmed | Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems |
title_short | Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems |
title_sort | strategies to investigate membrane damage, nucleoid condensation, and rnase activity of bacterial toxin–antitoxin systems |
topic | Study Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8544347/ https://www.ncbi.nlm.nih.gov/pubmed/34698227 http://dx.doi.org/10.3390/mps4040071 |
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