Elucidation of gene regulation systems based on genome analysis

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We have identified a novel regulator, Tgr (Thermococcales glycolytic regulator), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD 1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ABC transporter genes, but is more closely related (67%) to a TrmB paralog in P.furiosus (PF0124). Growth of a tgr disruption strain (Δtgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared to the wild-type strain, while comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Δtgr mutant even under gluconeogenic conditions. The Δtgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, TGM (Thermococcales glycolytic motif). The TGM was positioned upstream of the BRE/TATA sequence in gluconeogenic promoters, and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T.kodakaraensis via its direct binding to the TGM.