Abstract
Hydrogen peroxide (H2O2) produces hydroxyl radicals that directly attack a variety of biomolecules and cause severe cellular dysfunction. An extremely thermophilic bacterium, Thermus thermophilus HB8, possesses at least three enzymes that can scavenge H2O2: manganese-containing catalase (TTHA0122, MnCAT), a possible peroxiredoxin homologue (TTHA1300), and a possible heme peroxidase (HPX) homologue (TTHA1714). To investigate the roles of these proteins, we attempted to disrupt each of these genes in T. thermophilus HB8. Although we were able to completely disrupt ttha1300, we were unable to completely delete ttha0122 and ttha1714 because of polyploidy. Quantitative real-time PCR showed that, compared to the wild type, 31 % of ttha0122 and 11 % of ttha1714 remained in the ∆ttha0122 and ∆ttha1714 disruption mutants, respectively. Mutants with reduced levels of ttha0122 or ttha1714 exhibited a significant increase in spontaneous mutation frequency. ∆ttha1714 grew slower than the wild type under normal conditions. ∆ttha0122 grew very poorly after exposure to H2O2. Moreover, ∆ttha0122 did not show H2O2-scavenging activity, whereas ∆ttha1300 and ∆ttha1714 scavenged H2O2, a property similar to that exhibited by the wild type. MnCAT purified from T. thermophilus HB8 cells scavenged H2O2 in vitro. The recombinant form of the possible HPX homologue, reconstituted with hemin, showed peroxidase activity with H2O2 as an oxidant substrate. Based on these results, we propose that not only MnCAT but also the possible HPX homologue is involved in protecting the cell from oxidative stress in T. thermophilus.
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Abbreviations
- ROS:
-
Reactive oxygen species
- MnCAT:
-
Manganese-containing catalase
- HPX:
-
Heme peroxidase
- HTK :
-
Thermostable kanamycin-resistance gene
- HRP:
-
Horseradish peroxidase
- ABTS:
-
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
- MES:
-
2-morpholinoethanesulfonic acid
- MOPS:
-
3-(N-morpholino)propanesulfonic acid
- SDS-PAGE:
-
SDS-polyacrylamide gel electrophoresis
- MALDI-TOF MS:
-
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
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We thank all the members of SR System Biology Research Group, RIKEN SPring-8, Harima Institute.
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Communicated by S. Albers.
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Ebihara, A., Manzoku, M., Fukui, K. et al. Roles of Mn-catalase and a possible heme peroxidase homologue in protection from oxidative stress in Thermus thermophilus . Extremophiles 19, 775–785 (2015). https://doi.org/10.1007/s00792-015-0753-2
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DOI: https://doi.org/10.1007/s00792-015-0753-2