Identification, functional characterization, assembly and structure of ToxIN type III toxin–antitoxin complex from E. coli

Abstract

Toxin–antitoxin (TA) systems are proposed to play crucial roles in bacterial growth under stress conditions such as phage infection. The type III TA systems consist of a protein toxin whose activity is inhibited by a noncoding RNA antitoxin. The toxin is an endoribonuclease, while the antitoxin consists of multiple repeats of RNA. The toxin assembles with the individual antitoxin repeats into a cyclic complex in which the antitoxin forms a pseudoknot structure. While structure and functions of some type III TA systems are characterized, the complex assembly process is not well understood. Using bioinformatics analysis, we have identified type III TA systems belonging to the ToxIN family across different Escherichia coli strains and found them to be clustered into at least five distinct clusters. Furthermore, we report a 2.097 Å resolution crystal structure of the first E. coli ToxIN complex that revealed the overall assembly of the protein-RNA complex. Isothermal titration calorimetry experiments showed that toxin forms a high-affinity complex with antitoxin RNA resulting from two independent (5′ and 3′ sides of RNA) RNA binding sites on the protein. These results further our understanding of the assembly of type III TA complexes in bacteria.

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from <em>Escherichia coli</em> for Biophysical Studies

“…The fractions can be pooled together and concentrated appropriately depending on the experiment that needs to be performed, such as NMR spectroscopy, crystallization, or ITC. Using this protocol, we could also successfully purify two other ToxIN complexes and their components (from cluster 4 and 5 of E. coli ) ( Manikandan et al, 2022 ) (Supplementary Figure 1 and Supplementary Figure 2).…”

“…The type III TA systems are classified into three families: ToxIN, CptIN, and TenpIN, based on toxin sequence homology ( Blower et al, 2012 ). Among these classes, the ToxIN family (here, protein toxin and antitoxin RNA are called ToxN and ToxI, respectively) is the most studied in terms of its structure, assembly, and functions ( Blower et al, 2011; Short et al, 2012; Guegler and Laub, 2021 ; Manikandan et al, 2022 ). In a recent study, we classified E. coli ToxIN TA systems into five clusters based on the sequence analysis of the toxin proteins ( Manikandan et al, 2022 ).…”

“…Among these classes, the ToxIN family (here, protein toxin and antitoxin RNA are called ToxN and ToxI, respectively) is the most studied in terms of its structure, assembly, and functions ( Blower et al, 2011; Short et al, 2012; Guegler and Laub, 2021 ; Manikandan et al, 2022 ). In a recent study, we classified E. coli ToxIN TA systems into five clusters based on the sequence analysis of the toxin proteins ( Manikandan et al, 2022 ). The structural and functional analysis of a CptIN TA complex from Eubacterium rectale has also been reported ( Rao et al, 2015 ).…”

“…Here, by adapting the methods published earlier ( Blower et al, 2011 ; Short et al, 2012 ; Rao et al, 2015 ; Manikandan et al, 2022 ), we have optimized a protocol to co-express and co-purify the ToxIN complexes and their components from three clusters of ToxIN family from E. coli ( Manikandan et al, 2022 ) for their biophysical and structural investigation [using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy]. The method described here yields TA RNP complex, free protein toxin, and free antitoxin RNA of the ToxIN system with an approximate molar ratio of 3:2:2 (with an approximate yield of 7, 3, and 1.5 mg, respectively, from one litre of LB media).…”

Acinetobacter Baumannii Phages: Past, Present and Future

“…The toxin-antitoxin (TA) system is widely present in A. baumannii [ 195 , 196 , 197 ] and is composed of toxin proteins and antitoxin proteins or antitoxin noncoding RNA [ 198 ]. Among them, toxins can regulate many important cellular processes in cells, which limits the growth of bacteria [ 199 ], and antitoxins can inhibit the activity of toxins.…”