Expression of  functionalBacillusSpoIISAB toxin–antitoxin modules inEscherichia coli

Abstract

SpoIISA and SpoIISB proteins from Bacillus subtilis belong to a recently described bacterial programmed-cell death system. The current work demonstrates that the toxin-antitoxin module is also functional in Escherichia coli cells, where the expression of SpoIISA toxin leads to transient growth arrest coupled with cell lysis, and SpoIISA-induced death can be prevented by coexpression of its cognate antitoxin, SpoIISB. Escherichia coli cells appear to be able to escape the SpoIISA killing by activation of a specific, as yet unidentified protease that cleaves out the cytosolic part of the protein. Analysis of the toxic effects of the transmembrane and cytosolic portions of SpoIISA showed that neither of them separately can function as a toxin; therefore, both parts of the protein have to act in concert to exert the killing. This work also identifies genes encoding putative homologues of SpoIISA and SpoIISB proteins on chromosomes of other Bacilli species. The SpoIISA-like proteins from Bacillus anthracis and Bacillus cereus were shown to manifest the same effect on the viability of E. coli as their homologue from B. subtilis. Moreover, expression of the proposed spoIISB-like gene rescues E. coli cells from death induced by the SpoIISA homologue.

The Chromosomal mazEF Locus of Streptococcus mutans Encodes a Functional Type II Toxin-Antitoxin Addiction System

“…E. coli cells have been successfully used previously as a host for verification of the function of various heterologically expressed TA loci (1,11,21,36,46). The S. mutans mazF gene was thus cloned into the pBAD expression system for induction of gene expression using araBAD promoter dose-dependent regulation.…”

Expression and localization of SpoIISA toxin during the life cycle of Bacillus subtilis

“…In B. subtilis , large plasmolysis zones and holes in the peptidoglycan layer were observed in cells with complete or incomplete septa, suggesting that the SpoIISA toxin could act as a holin (Adler et al., 2001). The disruption of the cytoplasmic membrane was also seen in E. coli cells (Florek et al., 2008). It was also shown that intact SpoIISA is integral to cell killing activity, since neither the cytosolic region nor the transmembrane domain alone can exert a toxic effect (Florek et al., 2008).…”

“…The disruption of the cytoplasmic membrane was also seen in E. coli cells (Florek et al., 2008). It was also shown that intact SpoIISA is integral to cell killing activity, since neither the cytosolic region nor the transmembrane domain alone can exert a toxic effect (Florek et al., 2008). …”

“…These results indicate that the SpoIIS system does not function as an addiction module through PSK in E. coli . However, it was observed previously that the SpoIISA protein is degraded in E. coli by the activity of some proteases (Florek et al., 2008), which can prevent functioning of the SpoIIS system as an addiction module in this microorganism.…”

“…Under these conditions, we were unable to detect SpoIISB using a standard western blot method, which was used for analysis of SpoIISA toxin. There exists the possibility that SpoIISB is rapidly turned over; however, it was shown previously that SpoIISB can be detected with a SpoIISB antibody when the protein is overexpressed in both B. subtilis (unpublished data) and E. coli (Florek et al., 2008). …”