# MazEF Homologs in Symbiobacterium thermophilum Exhibit Cross-Neutralization with Non-Cognate MazEFs

## TL;DR

This study shows that a toxin-antitoxin system in Symbiobacterium thermophilum interacts with similar systems from other bacteria, suggesting a shared evolutionary origin.

## Contribution

The study reveals cross-neutralization between MazF-sth and non-cognate MazE-bs, and identifies key residues for MazF-sth activity.

## Key findings

- MazF-sth cleaves RNA at the UACAUA motif, similar to MazF from Gram-positive bacteria.
- Arg29 and Thr52 are critical residues for MazF-sth ribonuclease activity.
- Cross-neutralization between MazF-sth and MazE-bs suggests horizontal gene transfer.

## Abstract

Toxin–antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer.