(A) MutS family proteins can be classified into MutSI and MutSII subfamilies. MutSI includes bacterial MutS, and eukaryotic MutSα and MutSβ, which are involved in MMR [69], [72]. MutSII includes eukaryotic MutSγ, and bacterial MutS2, which are involved in promotion and suppression of homologous recombination, respectively. Bacterial MutS and MutS2 are homodimeric protein, while eukaryotic MutSα, MutSβ, and MutSγ are heterodimeric proteins comprised of MSH2/MSH6, MSH2/MSH3, and MSH4/MSH5, respectively. (B) Divergence in functions of bacterial MutS and MutS2. In this study, it was suggested that MutS and MutS2 cooperatively participate in repair of oxidative DNA damages.

(A) Co-immunoprecipitation. “Cell” indicates strains used for immunoprecipitation, where W, ΔS, ΔL, and ΔS2 represent the wild-type, ΔmutS, ΔmutL, and ΔmutS2 strains, respectively. IP and WB indicate antibodies used for immunoprecipitation and Western blotting, respectively. S, L, S2, ATL, and UvrA indicate anti-MutS, -MutL, -MutS2, -ATL protein, and -UvrA antibodies, respectively. C indicates pre-immune antibody. (B) Pull-down assay. S2 indicates recombinant His₆-tagged MutS2 used as a bait protein. WB indicates antibodies used for Western blotting. L and S indicate anti-MutL and -MutS antibodies used for Western blotting.

(A) A Venn diagram shows the up-regulated genes in the three disruptants. (B) A schematic representation of vitamin B₁ biosynthesis operon in T. thermophilus HB8. (C) Vitamin B₁ biosynthesis genes were up-regulated in all three disruptants. Expression in the disruptants relative to that in the wild-type strain is indicated, where the P-values are less than 0.00076. The respective P-values are listed in Supplementary Tables S2, S3, S4. The values for ttha0675 were determined by using the definition in the platform GPL9209 (GEO accession number: GPL9209). (D) A predicted model of biosynthesis pathway of thiamine diphosphate in T. thermophilus HB8. Pyrimidine and thiazole moieties are synthesized separately and then combined to form thiamine phosphate. (E) RT-PCR confirmed the up-regulation of vitamin B₁ biosynthesis genes in each disruptant. DNA fragments were amplified using total RNAs as templates, and then subjected to agarose gel electrophoresis. M, W, ΔL, ΔS, and ΔS2 represent the 100-bp ladder DNA size marker, and the wild-type, ΔmutS, ΔmutL, and ΔmutS2 strains, respectively. Primers were designed to amplify 161-, 289-, 295-, 320-, 231-, 290-, and 365-bp DNA fragments from the cDNAs of ttha0675, ttha0674, ttha0676, ttha0677, ttha0678, ttha0679, and ttha0680, respectively.

(A) The effect of 0 (upper panel) or 50 (lower panel) mM vitamin B₁ on the tolerance of T. thermophilus HB8 wild-type strain to H₂O₂. Cells were incubated with various concentrations of H₂O₂ and then spotted onto plates as described under Methods. The concentrations of H₂O₂ are indicated at the top of the panel. (B) The vitamin B₁ dose dependence of the H₂O₂ sensitivity. T. thermophilus HB8 wild-type strain was incubated in broth containing 50 mM H₂O₂ and the indicated concentrations of vitamin B₁. (C) Sensitivity of Δttha0675 cells to H₂O₂. Wild-type and Δttha0675 strains were incubated in the broth containing 0, 10, 20, 30, 50, and 80 mM H₂O₂. After treatment with H₂O₂, cells were spread onto plates and incubated at 70°C for 24 h. The survival ratios of the wild-type (triangles) and Δttha0675 (circles) strains were estimated based on the numbers of colonies on the plates and plotted against H₂O₂ concentration. Bars indicate standard deviations.

(A) Sensitivity to H₂O₂. The wild-type, ΔmutS2, ΔmutS, ΔmutL, and ΔmutM strains of T. thermophilus HB8 were incubated in medium containing the indicated concentrations of H₂O₂. After incubation with H₂O₂, cells were spotted onto plates. (B) Effect of 10 mM H₂O₂ on growth curves of the wild-type (red), ΔmutS (pink), ΔmutL (purple), ΔmutS2 (blue), and ΔmutM (orange) strains of T. thermophilus HB8. Precultured cells were inoculated to 5 ml of medium to an OD₆₀₀ value of 0.10. After incubation at 70°C for 3 h, 50 µl of 0 (circles) or 1 M (triangles) H₂O₂ was added. (C) Rate of H₂O₂-induced mutagenesis. The wild-type, ΔmutS, ΔmutL, and ΔmutS2 strains of T. thermophilus HB8 were incubated in medium containing 0 or 10 mM H₂O₂ for 30 min. After incubation, cells were spread onto plates containing 0 or 50 µg/ml streptomycin. Frequency of streptomycin-resistant mutants per 10⁸ cells was calculated from the numbers of colonies formed on the streptomycin-containing and drug-free plates. Bars indicate standard deviations.

Extracellular oxidative stress and intracellular redox metabolism generate ROS, which can attack DNA to yield oxidatively damaged DNA. (A) In the wild-type strain, oxidatively damaged DNA is repaired by DNA repair enzymes including MutS, MutL, and MutS2. (B) In the ΔmutS, ΔmutL, and ΔmutS2 strains, the genes for vitamin B₁ biosynthesis are activated to prevent the accumulation of oxidative damage in DNA via an unknown mechanism.