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

Abstract

We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (⌬tgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas 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 ⌬tgr mutant even under gluconeogenic conditions. The ⌬tgr 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, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (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.Proper control of glycolytic and gluconeogenic activities in the cell is vital for the efficient assimilation of carbon and generation of energy and has been considered a paradigm for metabolic regulation. Stringent regulation is generally observed to avoid futile cycles that potentially lead to the depletion of energy; hence one pathway is suppressed while the other is active.Mechanisms underlying the regulation of glycolysis/gluconeogenesis have been extensively studied in different bacterial and eukaryotic species. In Escherichia coli, genes involved in these pathways are generally expressed in a constitutive manner (1), and control is brought about predominantly by allosteric regulation of the enzymes themselves. The major sites of allosteric regulation are the two glycolytic enzymes, phosphofructokinase (PFK) 3 (2) and pyruvate kinase (3), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) (4). The reactions catalyzed by the three enzymes are irreversible under physiological conditions and are therefore considered key steps in the respective pathways. Besides the allosteric control, recent analyses have also indicated the p...

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

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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.…”

Structural Insights into Nonspecific Binding of DNA by TrmBL2, an Archaeal Chromatin Protein

“…The ortholog of TrmBL1 in Thermococcus kodakarensis, named Tgr (Thermococcales glycolytic regulator), was found to prominently recognize the palindromic TGM (Thermococcales glycolytic motif) sequence [6,7]. Depending on its position within the respective promoter, Tgr binds to it, acting as an activator when present upstream of BRE/TATA box of genes encoding gluconeogenic proteins but as a repressor when positioned downstream of the BRE/ TATA box of genes encoding glycolytic genes.…”