Extremophiles

, Volume 21, Issue 1, pp 153–161

Identification of a VapBC toxin–antitoxin system in a thermophilic bacterium Thermus thermophilus HB27

Original Paper

DOI: 10.1007/s00792-016-0891-1

Cite this article as:
Fan, Y., Hoshino, T. & Nakamura, A. Extremophiles (2017) 21: 153. doi:10.1007/s00792-016-0891-1
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Abstract

There are 12 putative toxin–antitoxin (TA) loci in the Thermus thermophilus HB27 genome, including four VapBC and three HicBA families. Expression of these seven putative toxin genes in Escherichia coli demonstrated that one putative VapC toxin TTC0125 and two putative HicA toxins, TTC1395 and TTC1705, inhibited cell growth, and co-expression with cognate antitoxin genes rescued growth, indicating that these genes function as TA loci. In vitro analysis with the purified TTC0125 and total RNA/mRNA from E. coli and T. thermophilus showed that TTC0125 has RNase activity to rRNA and mRNA; this activity was inhibited by the addition of the purified TTC0126. Translation inhibition assays showed that TTC0125 inhibited protein synthesis by degrading mRNA but not by inactivating ribosomes. Amino acid substitutions of 14 predicted catalytic and conserved residues in VapC toxins to Ala or Asp in TTC0125 indicated that nine residues are important for its in vivo toxin activity and in vitro RNase activity. These data demonstrate that TTC0125-TTC0126 functions as a VapBC TA module and causes growth inhibition by degrading free RNA. This is the first study to identify the function of TA systems in T. thermophilus.

Keywords

Thermus thermophilus Toxin–antitoxin system VapBC Ribonuclease 

Abbreviations

Dox

Doxycycline

DTT

Dithiothreitol

IPTG

Isopropyl-β-d-thiogalactopyranoside

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate-buffered saline

PMSF

Phenylmethanesulfonylfluoride

RNase

Ribonuclease

TA

Toxin–antitoxin

Supplementary material

792_2016_891_MOESM1_ESM.pdf (425 kb)
Supplementary material 1 (PDF 425 kb)

Copyright information

© Springer Japan 2016

Authors and Affiliations

  1. 1.Faculty of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

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