Identification of the Phr-dependent heat shock regulon in the hyperthermophilic archaeon, Thermococcus kodakaraensis

Abstract

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a putative transcriptional regulator (Tk-Phr) that is orthologous to the Pyrococcus furiosus Phr (Pf-Phr). Pf-Phr, a transcriptional regulator, represses genes encoding the small heat shock protein (sHSP), AAA(+) ATPase and Pf-Phr itself under normal growth temperatures. Here we constructed a gene disruption strain of Tk-Phr (strain KHR1). KHR1 cells showed similar specific growth rates with those of the wild-type strain under various temperatures. A whole genome microarray analysis was performed between KHR1 and wild-type cells grown at 80 degrees C. Transcript levels of more than 20 genes were significantly higher in KHR1 cells. Most genes contained a sequence motif virtually identical to that of Pf-Phr in their 5'-flanking regions. The Tk-Phr regulon included genes encoding sHSP, AAA(+) ATPase, prefoldin, RecA superfamily ATPase and Tip49. On the other hand, more than half of the members in the regulon encoded conserved/hypothetical proteins, raising the possibility that these proteins participate in unidentified processes of the heat shock response. In contrast, Tk-Phr deletion did not lead to dramatic increase in transcript and protein levels of a chaperonin (CpkB) previously shown to respond to heat shock, suggesting the presence of a second, Phr-independent heat shock response mechanism in T. kodakaraensis.

Mannosylglycerate and Di- myo -Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

“…An increase of 8°C above the optimal temperature resulted in a clear impairment of the final cell density (the OD 600 varied from 0.88 to 0.32), while it did not significantly affect the growth rate ( Table 1). The lack of an effect of temperature on the growth rate over the temperature range of 75 to 90°C was also reported for Thermococcus kodakarensis by Kanai et al (21). On the other hand, in medium containing 4.5% NaCl, both the growth rate and the final OD were reduced by about 40% compared with those found under optimal growth conditions.…”

“…(21). The central role of the transcriptional regulator Phr is well established in members of the order Thermococcales (21,26).…”

“…(21). The central role of the transcriptional regulator Phr is well established in members of the order Thermococcales (21,26). At the optimal temperature, Phr binds to the promoter region of several genes, such as the hsp20 and AAA ϩ ATPase genes and the gene encoding IPCT/DIPPS, the enzyme implied in DIP synthesis, thereby blocking their transcription.…”

An Archaeal Histone Is Required for Transformation of Thermococcus kodakarensis

“…RNA extractions and microarray hybridizations were performed as previously described (16). T. kodakarensis cultures were grown at 85°C in medium containing ASWyeast extract-tryptone plus sodium pyruvate (ASW-YT-Pyr medium), cells were harvested during exponential growth (OD 660 Ϸ 0.2), and RNA preparations were isolated by using RNeasy Midi kits (Qiagen).…”

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.…”