A thiostrepton resistance gene and its mutants serve as selectable markers inGeobacillus kaustophilusHTA426

Abstract

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.

Draft Genome Sequence of Thiostrepton-Producing Streptomyces azureus ATCC 14921

“…Streptomyces azureus ATCC 14921 is categorized in the blue-spore Streptomyces cyaneus cluster and is known to be a thiostrepton producer ( 4 ). The thiostrepton resistance gene ( tsr ), which is a selective marker often used in the genetic engineering of actinomycetes, was isolated from the strain ( 5 , 6 ). S. azureus contains the pock-forming conjugative plasmid pSA1.1 ( 7 ), and spontaneous development of pocks in the strain is caused by the action of the plasmid and lysogenic phage SAt2 ( 8 ).…”

A Cassette Containing Thiostrepton, Gentamicin Resistance Genes, and dif sequences Is Effective in Construction of Recombinant Mycobacteria

“…In addition, Wada et al (2016) also demonstrated tsr as a viable selection marker for the thermophilic Geobacillus kaustophilus besides demonstrating accurate selection as a single copy in Streptomyces strains.…”

“…Mycobacteria are naturally resistant to many antibiotics and requires use of stable drugs with low frequency of spontaneous resistance for selection, hence limiting the alternative choices (Parish and Brown, 2008). The combined use of multiple markers enables more versatile genetic modifications, including the stable maintenance of multiple plasmids and inactivation of multiple genes (Wada et al, 2016). …”

Plasmid Curing is a Promising Approach to Improve Thermophiles for Biotechnological Applications: Perspectives in Archaea

“…Genetic tools are available for this strain [10][11][12][13][14][15][16]. The whole genome sequence has been determined [17], showing that G. kaustophilus HTA426 harbors the circular plasmid pHTA426 (Figure 1).…”