期刊
METHODS AND PROTOCOLS
卷 4, 期 4, 页码 -出版社
MDPI
DOI: 10.3390/mps4040071
关键词
toxin-antitoxin systems; ribonuclease toxins; membrane-associated toxins; inhibition growth assays; single-cell fluorescence microscopy; atomic force microscopy
资金
- Italian Ministero dell'Istruzione, dell'Universita e della Ricerca (MIUR) [262]
- Italian Ministry of Foreign Affairs and International Cooperation [MAE0057459]
Optimizing methodological strategies to study recombinant Escherichia coli host cells by tightly repressing toxin expression and using fluorescence approaches to monitor toxin activity; Fluorescence microscopy and stains like DAPI and ethidium bromide are effective tools to observe membrane integrity changes associated with toxin activity; Atomic force microscopy can be readily employed to characterize toxin-induced membrane damages.
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.
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