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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 tsrH258Y 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 tsrH258Y was more thermostable than that from tsr. In fact, the tsrH258Y 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 thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426