Development of a supF-based mutation-detection system in the extreme thermophile Thermus thermophilus HB27
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Thermus thermophilus (T. thermophilus) HB27 is an extreme thermophile that grows optimally at 65–72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T. thermophilus than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a supF-based system to detect a broad spectrum of mutations in T. thermophilus. This system was validated by measuring spontaneous mutations in the wild type and a udgA, B double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the udgA, B strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in T. thermophilus. These results show that this system allowed for the rapid analysis of mutations in T. thermophilus, and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.
KeywordsThermus thermophilus supF Mutation Shuttle vector Uracil-DNA glycosylase DNA repair
We thank Prof. Isao Kuraoka (Fukuoka University) for helpful discussion. This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Duncan BK, Weiss B (1982) Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli. J Bacteriol 151:750–755Google Scholar
- Henne A, Brüggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H, Johann A, Lienard T, Gohl O, Martinez-Arias R, Jacobi C, Starkuviene V, Schlenczeck S, Dencker S, Huber R, Klenk HP, Kramer W, Merkl R, Gottschalk G, Fritz HJ (2004) The genome sequence of the extreme thermophile Thermus thermophilus. Nat Biotechnol 22:547–553CrossRefGoogle Scholar
- Nakamura A, Takakura Y, Kobayashi H, Hoshino T (2005) In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus. J Biosci Bioeng 100:158–163CrossRefGoogle Scholar
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar