Expression, purification, and functional analysis of novel RelE operon from X. nematophila.

Jitendra Singh Rathore,Lalit Kumar Gautam

doi:10.1155/2014/428159

Jitendra Singh Rathore, Lalit Kumar Gautam

Open Access

https://doi.org/10.1155/2014/428159

Copy DOI

Abstract

Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

Highlights

In bacterial and archaeal genome toxin-antitoxin (TA) complex is evident [1]
When bacterium comes under stress toxin gene is overexpressed and the produced toxin protein gets activated to interfere with cellular targets, while its cognate antitoxin protein is truncated by various means like Lon and Clp protease mediated degradation [3, 4]
Restriction endonucleases, and Taq polymerase were purchased from New England Biolabs (NEB), glutathione S-transferase (GST) agarose resin was from Gold bioscience, USA, and Ni-NTA agarose resin and QIAquick spin columns were from Qiagen, Germany

Summary

Introduction

In bacterial and archaeal genome toxin-antitoxin (TA) complex is evident [1]. Toxin-antitoxin modules are known to play role in plasmid maintenance and confer stability to them [2]. Either toxicity of the toxin molecules in a cell is normally neutralized with the cognate antitoxin by transcriptional repression of TA operon through binding to its palindromic sequences within its promoter region or it forms TA complex and resists toxin from binding to its target; all this happens in a manner defined as condition cooperativity [13,14,15].

Results

Conclusion