A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.
Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.
a Faculty of Agriculture, Applied Molecular Microbiology and Biomass Chemistry, Bioscience and Biotechnology , Kyushu University , Fukuoka , Japan.
Keisuke
K
Wada
b Faculty of Agriculture, Functional Genomics of Extremophiles , Graduate School, Kyushu University , Fukuoka , Japan.|c Department of Chemistry and Biotechnology , Graduate School of Engineering, Tottori University , Tottori , Japan.
Jyumpei
J
Kobayashi
b Faculty of Agriculture, Functional Genomics of Extremophiles , Graduate School, Kyushu University , Fukuoka , Japan.
Megumi
M
Furukawa
d Faculty of Agriculture, Microbial Genetics Division, Institute of Genetic Resources , Graduate School, Kyushu University , Fukuoka , Japan.
Katsumi
K
Doi
c Department of Chemistry and Biotechnology , Graduate School of Engineering, Tottori University , Tottori , Japan.
Takashi
T
Ohshiro
b Faculty of Agriculture, Functional Genomics of Extremophiles , Graduate School, Kyushu University , Fukuoka , Japan.|c Department of Chemistry and Biotechnology , Graduate School of Engineering, Tottori University , Tottori , Japan.
Hirokazu
H
Suzuki
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005819:Genetic Markers; D056506:Geobacillus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013883:Thiostrepton / Q000494:pharmacology*; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005819:Genetic Markers; D011994:Recombinant Proteins; D013883:Thiostrepton
Geobacillus kaustophilus; error-prone thermophile; selectable marker; thermoadaptation-directed evolution; thiostrepton resistance