Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus

Abstract

Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70–75 °C, pH 7.5. In extremely high temperature environment, DNA damages in cells occur at a much higher frequency in thermophiles than mesophiles such as E. coli. When temperature rises, the deamination of cytosine residues in double-strand DNA is expected to increase greatly. T. thermophilus HB27 has two putative uracil-DNA glycosylase genes ( udgA and udgB). Expression level of udgA gene was 2–3 times higher than that of udgB at 70, 74, and 78 °C when it was monitored by β-glucosidase reporter assay. We developed hisD 3110 , hisD 3113 , hisD 3115 , and hisD 174 marker allele that can specifically detect G:C → A:T, C:G → A:T, T:A → A:T, and A:T → G:C base-substitutions, respectively, by His + reverse mutations. We then disrupted udgA and udgB by thermostable kanamycin-resistant gene ( htk) or pyrE gene insertion in each hisD background, and their spontaneous His + reversion frequencies were compared. A udgA,B double mutant showed a pronounced increase in G:C → A:T reversion frequency compared with each single udg mutant, udgA or udgB. Estimated mutation rates of the udgA,B mutant cultured at 60, 70, and 78 °C were about 2, 12, and 117 His +/10 8/generation, respectively. At 70 °C culture, increased ratio of the mutation rate compared with the udg + strain was 12-fold in udgA, 3-fold in udgB, and 56-fold in udgA,B mutant. On the other hand, no difference was observed in other mutations of C:G → A:T, T:A → A:T, and A:T → G:C between udgA,B double mutant and the parent udg + strain. The present results indicated that gene products of udgB as well as udgA functioned in vivo to remove uracil in DNA and prevent G:C → A:T transition mutations.