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J. Biol. Chem., Vol. 282, Issue 46, 33659-33670, November 16, 2007

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

Tamotsu Kanai¹, Jasper Akerboom¹, Shogo Takedomi¹, Harmen J. G. van de Werken, Fabian Blombach, John van der Oost, Taira Murakami, Haruyuki Atomi, and Tadayuki Imanaka²

From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan and Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands

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.

Received for publication, April 24, 2007 , and in revised form, September 4, 2007.

^* This work was supported by Grants-in-aid for Scientific Research 14103011 (to T. I.) and 17350083 (to H. A.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Part of this research was supported by the Carbohydrate Research Centre Wageningen and by Biomolecular Informatics Project 050.50.206 that is subsidized by the Netherlands Organization for Scientific Research (NWO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S12 and Tables S1–S6.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 81-75-383-2777; Fax: 81-75-383-2778; E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

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