New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041.

Geobacillus thermodenitrificans K1041 is an unusual thermophile that is highly transformable via electroporation, making it a promising host for screening genetic libraries at elevated temperatures. In this study, we determined its biological properties, draft genome sequence, and effective vectors and also optimized the electroporation procedures in an effort to enhance its utilization. The organism exhibited swarming motility but not detectable endospore formation, and growth was rapid at 60°C under neutral and relatively low-salt conditions. Although the cells showed negligible acceptance of shuttle plasmids from general strains of Escherichia coli, methylation-controlled plasmids from dam mutant strains were efficiently accepted, suggesting circumvention of a restriction-modification system in G. thermodenitrificans K1041. We optimized the electroporation procedure to achieve efficiencies of 103 to 105 CFU/µg for five types of plasmids, which exhibited the different copy numbers and segregational stabilities in G. thermodenitrificans K1041. Some sets of plasmids were compatible. Moreover, we observed substantial plasmid-directed production of heterologous proteins in the intracellular or extracellular environments. Our successful construction of a library of promoter mutants using K1041 cells as hosts and subsequent screening at elevated temperatures to identify improved promoters revealed that G. thermodenitrificans K1041 was practical as a library host. The draft genomic sequence of the organism contained 3,384 coding genes, including resA and mcrB genes, which are involved in restriction-modification systems. Further examination revealed that in-frame deletions of resA increased transformation efficiencies, but mcrB deletion had no effect. The ΔresA mutant exhibited transformation efficiencies of >105 CFU/µg for some plasmids. IMPORTANCE Geobacillus thermodenitrificans K1041 has yet to be fully characterized. Although it is transformable via electroporation, it rarely accepts Escherichia coli-derived plasmids. This study clarified the biological and genomic properties of G. thermodenitrificans K1041. Additionally, we developed an electroporation procedure resulting in efficient acceptance of E. coli-derived plasmids. This procedure produced transformants using small amounts of plasmids immediately after the ligation reaction. Thus, G. thermodenitrificans K1041 was identified as a host for screening promoter mutants at elevated temperatures. Furthermore, because this strain efficiently produced heterologous proteins, it could serve as a host for screening thermostable proteins encoded in random mutant libraries or metagenomes. We also generated a ΔresA mutant that exhibited transformation efficiencies of >105 CFU/µg, which were highest in cases of electroporation-based transformation of Geobacillus spp. with E. coli-derived plasmids. Our findings provide a new platform for screening diverse genetic libraries at elevated temperatures.

Transcriptome and growth efficiency comparisons of recombinant thermophiles that produce thermolabile and thermostable proteins: implications for burden-based selection of thermostable proteins.

Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.