Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

Abstract

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg Tk ) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu Tk ) or a gene cluster which includes apu Tk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 M simvastatin were isolated. The transformants exhibited growth in the presence of 20 M simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg Tk locus when the endogenous hmg Tk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg Pf ) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ⌬apu Tk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu Tk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

A Global Transcriptional Regulator in Thermococcus kodakaraensis Controls the Expression Levels of Both Glycolytic and Gluconeogenic Enzyme-encoding Genes

“…S3 and S4). The maltodextrin ABC transporter system and amylopullulanase, both confirmed to play major roles in maltopolysaccharide assimilation (41), increased over 4-19-fold. In contrast, transcript levels of FBPase, which is essential for gluconeogenesis in T. kodakaraensis (32), were much higher under gluconeogenic conditions.…”

Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

“…One interesting example involved the in vivo disruption and analysis of a gene encoding reverse gyrase on the T. kodakaraensis genome, which revealed that the gene product functions in adaptation to high temperature, because reverse gyrase is highly conserved only in hyperthermophiles (Atomi et al, 2004). These efficient genetic tools have already been used to investigate several biological systems, such as the glycolysis pathway (Yoshida et al, 2006;Matsumi et al, 2007), natural product biosynthesis (Yokooji et al, 2009;Borges et al, 2010), transcription factors (Kanai et al, 2007(Kanai et al, , 2010, and operon transcription and translation machinery Hirata et al, 2008). However, these useful and powerful techniques have not yet been applied to the analysis of DNA repair.…”

A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection

“…In such a selection system, simvastatin, a competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was utilized to select for overexpression of the HMG-CoA reductase gene (hmgA) under the control of a strong constitutive promoter of glutamate dehydrogenase (Matsumi et al, 2007;Santangelo et al, 2008). This selection strategy has now been exploited in another hyperthermophilc euryarchaeon, Pyrococcus furious (optimal temperature 100 u C), with great success (Lipscomb et al, 2011;Waege et al, 2010).…”