Identification and Characterization of HEPN-MNT Type II TA System from Methanothermobacter thermautotrophicus ?H

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea plasmids and genomes to regulate DNA replication, gene transcription, or protein translation. Higher eukaryotic and prokaryotic nucleotide-binding (HEPN) and minimal nucleotidyltransferase (MNT) domains are prevalent in prokaryotic genomes and constitute TA pairs. However, three gene pairs (MTH304/305, 408/409, and 463/464) of Methanothermobacter thermautotropicus ?HH HEPN-MNT family have not been studied as TA systems. Among these candidates, our study characterizes the MTH463/MTH464 TA system. MTH463 expression inhibited Escherichia coli growth, whereas MTH464 did not and blocked MTH463 instead. Using site-directed MTH463 mutagenesis, we determined that amino acids R99G, H104A, and Y106A from the R[?X]4-6H motif are involved with MTH463 cell toxicity. Furthermore, we established that purified MTH463 could degrade MS2 phage RNA, whereas purified MTH464 neutralized MTH463 activity in vitro. Our results indicate that the endonuclease toxin MTH463 (encoding a HEPN domain) and its cognate antitoxin MTH464 (encoding the MNT domain) may act as a type II TA system in M. thermautotropicus ?H. This study provides initial and essential information studying TA system functions, primarily archaea HEPN-MNT family.

Automated summary

Toxin-antitoxin (TA) systems, which regulate various cellular processes, are prevalent in bacteria and archaea. In this study, the MTH463/MTH464 TA system from Methanothermobacter thermautotropicus ?H was characterized. MTH463 was shown to inhibit the growth of Escherichia coli, while MTH464 blocked the activity of MTH463. Mutagenesis experiments revealed specific amino acids involved in the toxicity of MTH463. Furthermore, purified MTH463 was able to degrade phage RNA, while purified MTH464 neutralized the activity of MTH463 in vitro. These findings suggest that MTH463 and MTH464 function as a type II TA system in M. thermautotropicus ?H.