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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 and protocols, 2021-10, Vol.4 (4), p.71 [Periódico revisado por pares]

Basel: MDPI AG

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Título:
Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems
Autor: Maggi, Stefano ; Ferrari, Alberto ; Yabre, Korotoum ; Bonini, Aleksandra Anna ; Rivetti, Claudio ; Folli, Claudia
Assuntos: Atomic force microscopy ; Bacteria ; Chromosomes ; Cloning ; E coli ; Ethidium bromide ; Fluorescence microscopy ; inhibition growth assays ; Membrane permeability ; membrane-associated toxins ; Microscopy ; Phenotypes ; Proteins ; Ribonuclease ; ribonuclease toxins ; RNA polymerase ; single-cell fluorescence microscopy ; Study Protocol ; Toxicity ; Toxins ; toxin–antitoxin systems
É parte de: Methods and protocols, 2021-10, Vol.4 (4), p.71
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Descrição: 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.
Editor: Basel: MDPI AG
Idioma: Inglês

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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

Basel: MDPI AG

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