Abstract
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.
Highlights
Numerous efforts have been addressed to understand the mechanism of action of different toxin–antitoxin (TA) systems identified in various bacterial strains
In this article we collectively describe these strategies by reporting simple protocols for culture growth, protein expression, and toxicity assessment in the bacterial host Escherichia coli
The in vivo activity of bacterial toxins is generally evaluated by inhibition growth assays conducted in liquid or on solid media, and E. coli host cells are usually chosen for their ease and rapid growth and for the plethora of expression systems commercially available [17,21]
Summary
Numerous efforts have been addressed to understand the mechanism of action of different toxin–antitoxin (TA) systems identified in various bacterial strains. TA systems consist of two genetic elements, the toxin capable of interfering with essential cellular processes and the counterpart antitoxin which inhibits toxin activity. TA systems are widespread in bacteria, both in chromosomes where they can be present in multiple copies, and in mobile elements [1]. TA systems are grouped into 8 classes (type I-VIII) based on the molecular nature of the antitoxin and its action mechanism [6,7]. In type I TA systems, the antitoxin is a sRNA that binds the mRNA coding for the toxin peptide resulting in translation inhibition. Type I toxins are peptides or small proteins (less than 60 aa) containing a potential trans-membrane domain and they have been associated with membrane depolarization, ATP leakage, and with nucleoid condensation, with the exception of SymE and RalR which act as RNase and DNase, respectively [8,9,10]
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