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Originally published In Press as doi:10.1074/jbc.M703424200 on September 17, 2007

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*Formula

Tamotsu Kanai{ddagger}1, Jasper Akerboom§1, Shogo Takedomi{ddagger}1, Harmen J. G. van de Werken§, Fabian Blombach§, John van der Oost§, Taira Murakami{ddagger}, Haruyuki Atomi{ddagger}, and Tadayuki Imanaka{ddagger}2

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ({Delta}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 {Delta}tgr mutant even under gluconeogenic conditions. The {Delta}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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 presence of regulatory mechanisms at the transcriptional (5) and post-transcriptional levels (6). In the eukaryote Saccharomyces cerevisiae, a number of mechanisms involved in glycolysis/gluconeogenesis regulation have been identified (for a review, see Ref. 7). Similar to the PFK, FBPase, and pyruvate kinase from E. coli, the S. cerevisiae enzymes also display allosteric properties. In eukaryotes, fructose 2,6-bisphosphate is a major allosteric effector of gluconeogenesis and glycolysis; it is an activator of PFK and an inhibitor of FBPase. Allosteric activation of PFK by fructose 2,6-bisphosphate leads to an increase in the concentration of fructose 1,6-bisphosphate, which in turn activates pyruvate kinase, thereby further increasing the glycolytic flux. In addition, genes encoding the three enzymes as well as the gluconeogenic phosphoenolpyruvate carboxykinase are controlled in response to the presence or absence of glucose at the transcription level. Furthermore a transition from a gluconeogenic to glycolytic environment triggers rapid inactivation of FBPase and phosphoenolpyruvate carboxykinase via protein phosphorylation and specific proteolysis.

Although studies on the metabolic regulation in Archaea are still in the initial phase, valuable insight has been obtained on the control of the glycolytic pathway in a number of archaeal strains including those of the Thermococcales and Thermoproteales (for reviews, see Refs. 8 and 9). Thermoproteus tenax, a member of the Thermoproteales that exhibits both autotrophic and heterotrophic modes of growth, utilizes a variant of the Embden-Meyerhof (EM) pathway as well as the semi- and non-phosphorylating Entner-Doudoroff pathways for glycolysis (10). The variant EM pathway is characterized by the absence of allosteric control in the reactions of the PPi-dependent PFK and pyruvate kinase. Instead transcriptional and allosteric regulation is observed for the enzymes involved in the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (11–13) as well as for phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase (14).

The Thermococcales order is composed of two major genera, Pyrococcus and Thermococcus, and its members are hyperthermophilic, anaerobic sulfur reducers that display growth on complex proteinaceous substrates (15). Some members of this order can also grow on carbohydrates using a modified EM pathway (16), which differs from the classical EM pathway by the presence of several unique enzymes, such as ADP-dependent glucokinase (17), ADP-dependent PFK (18), and GAP:ferredoxin oxidoreductase (19, 20). Pyrococcus furiosus is one of the most studied species of the Thermococcales and is capable of growth on a variety of sugars including maltose, starch, cellobiose, and laminarin. The level of activity of enzymes present in the modified EM pathway is generally higher in P. furiosus cells grown on sugars (glycolytic conditions) compared with cells grown on peptides or pyruvate (gluconeogenic conditions) (20–23), and the activity of gluconeogenic enzymes significantly increases under gluconeogenic conditions (23), both indicating tight control. Allosteric regulation has not been observed in the enzymes characterized so far in the modified EM and gluconeogenic pathways of the Thermococcales, and therefore, the regulation of enzyme activity is presumed to be primarily at the transcriptional level (24). We have recently identified a potential cis-regulatory element (TATCACN5GTGATA) in the glycolytic promoters of P. furiosus (25). This putative element was not found in Pyrococcus species that have a significantly lower capacity to metabolize sugars (Pyrococcus abyssi and Pyrococcus horikoshii); however, nearly identical motifs are present in the glycolytic and starch-utilizing promoters of the sugar-metabolizing Thermococcales. The sequence motif was thus designated Thermococcales glycolytic motif (TGM).

Thermococcus kodakaraensis is a member of the Thermococcales and grows between 60 and 100 °C with an optimum at 85 °C (26, 27). Both glycolytic (starch or maltodextrin) and gluconeogenic (peptides or pyruvate) modes of growth are observed for this archaeon, and the TGM sequences are found on most of the glycolytic and starch-utilizing gene promoters (25). The development of a gene disruption system for T. kodakaraensis (28, 29) as well as the availability of genome information (30) makes this archaeon an attractive model organism for the elucidation of the physiological role of unknown gene function in Thermococcales (31–33).

In this study, we characterized a gene (TK1769) disruption mutant of a putative transcriptional regulator in T. kodakaraensis. Whole genome microarray analysis and electrophoretic mobility shift assay strongly suggest that TK1769 encodes a transcriptional regulation factor controlling both the glycolytic (modified EM) and gluconeogenic pathways via binding to the TGM motif present in the glycolytic and gluconeogenic genes. The protein is therefore designated Tgr for Thermococcales glycolytic regulator.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Microorganisms and Culture Conditions—For general DNA manipulation and sequencing, E. coli DH5{alpha} (Invitrogen) was used. For recombinant production of Tgr, E. coli strain BL21(DE3) (Novagen, Madison, WI) containing the tRNA accessory plasmid pRIL (Stratagene, La Jolla, CA) was used. E. coli cells were cultivated at 37 °C in LB medium (10 g liter-1 tryptone, 5 g liter-1 yeast extract, and 10 g liter-1 NaCl) supplemented with either ampicillin (100 µg/ml) or kanamycin (100 µg/ml) and chloramphenicol (34 µg/ml) when necessary.

T. kodakaraensis strains were routinely cultivated under anaerobic conditions at 85 °C using a nutrient rich medium (MA-YT) or a synthetic medium (ASW-AA). The MA-YT-based medium contained synthetic sea salts (Marine Art SF; Tomita Pharmaceutical, Tokushima, Japan), yeast extract, and tryptone as described previously (34). The ASW-AA-based medium contained artificial seawater (ASW), vitamin mixture, modified Wolfe's trace minerals, and 20 amino acids as described previously (26, 35). As members in the Thermococcales are reported to have several tungstoenzymes (36), NaWO4·2H2O was also added to ASW-AA medium at a final concentration of 10 µM.

Construction of the T. kodakaraensis {Delta}tgr Strain—Disruption of the Tgr gene (TK1769, tgr) by double crossover homologous recombination was performed using the gene disruption system developed for T. kodakaraensis (28, 29). The plasmid used for disruption of tgr was constructed as follows. A DNA fragment containing the tgr-coding region together with its flanking regions (about 1,000 bp) was amplified with the primer set TGR-L1 (5'-CGGTTATCACTTTCACGTTC-3') and TGR-R2 (5'-GGTGGAAAACGCCGTCGAGT-3') using genomic DNA of T. kodakaraensis KOD1 as a template and inserted into the HincII site of pUC118. Using the constructed plasmid DNA as a template, the flanking regions of tgr along with the plasmid backbone were amplified using the primer set TGR-L2 (5'-CCCATCATTTTTAATTTCTA-3') and TGR-R1 (5'-CCAAAGACATTTAAGTTCAC-3'). The amplified fragment was ligated with a PvuII-PvuII restriction fragment (763 bp) containing the pyrF marker gene excised from pUD2 (29), resulting in the plasmid for tgr disruption (pUTGR). A T. kodakaraensis uracil auxotroph strain, KU216 (29), was used as the host strain for transformation. After transformation, a pyrF+ strain exhibiting uracil prototrophy was selected. The genotype of the tgr locus was confirmed by PCR amplification using the primer set tgr-US1 (5'-TACCGTTGAAGACGTGGG-3') and tgr-DS2 (5'-GCGTCAAGCCTGAATGGTGC-3'). Genotypes were also confirmed by Southern blot analyses. Two micrograms of genomic DNA from KU216 and {Delta}tgr were digested with PstI, separated by 1% agarose gel electrophoresis, and transferred to a nylon membrane HybondTM-N+ (GE Healthcare). The preparation of specific probes, hybridization, and signal detection were performed with the DIG DNA Labeling and Detection kit (Roche Diagnostics) according to the instructions from the manufacturer. The constructed {Delta}tgr strain was designated KGR1.

Growth Measurements—Growth characteristics of wild-type cells (strain KOD1) and {Delta}tgr mutant cells (strain KGR1) were determined as follows. Each strain was precultured in MA-YT medium supplemented with elemental sulfur (S0) (0.2%, w/v) at 85 °C for 10 h. S0 is required by T. kodakaraensis during growth on peptides or amino acids (26). After the preculture, cells were inoculated into MA-YT medium supplemented either with S0 (0.5%, w/v) (MA-YT-S0), sodium pyruvate (0.5%, w/v) (MA-YT-Pyr), or maltodextrin (0.5%, w/v) (MA-YT-Mdx). As a source of maltodextrin, Amycol number 3-L (Nippon Starch Chemical, Osaka, Japan), which consists of maltooligosaccharides of 1–12 glucose units, was used. Growth characteristics of KOD1 and KGR1 cells were also determined in synthetic ASW-AA medium containing S0 (0.2%, w/v) (ASW-AA-S0), S0 (0.2%, w/v) and sodium pyruvate (0.5%, w/v) (ASW-AA-S0-Pyr), or S0 (0.2%, w/v) and maltodextrin (0.5%, w/v) (ASW-AA-S0-Mdx). Cell densities (A660) were recorded at appropriate intervals with a UV spectrometer Ultraspec 3300 pro (GE Healthcare).

Microarray Analysis—The microarray plate used in this study (Array Tko1) was manufactured at Takara Bio (Otsu, Japan) and covers 2,226 genes among the total predicted 2,306 genes of T. kodakaraensis KOD1 (96.5% coverage). DNA fragments of about 300 bp, corresponding to the 3'-terminal regions of each coding region, were spotted on the glass plate. Two identical sets (left and right) were loaded on each plate. Therefore, two sets of data are obtained from each microarray plate. In the data files, individual signal intensity ratios obtained from each set as well as the average ratio value and the S.D. are shown.

T. kodakaraensis KOD1 and KGR1 were cultivated at 85 °C in MA-YT-S0, MA-YT-Pyr, or MA-YT-Mdx medium. Cells were harvested in the early log phase (A660 {approx} 0.2), and total RNA was extracted using the RNeasy Midi kit (Qiagen, Hilden, Germany). Fluorescently labeled cDNA used for hybridization was prepared using the RNA Fluorescence Labeling Core kit version 2.0 (Takara Bio). Total RNA (10 µg) was annealed with random hexamers, and reverse transcription was performed in solutions containing CyDye-labeled dUTP (Cy3-dUTP or Cy5-dUTP) (GE Healthcare). RNA was subsequently degraded with RNase H, and the labeled cDNA was purified using a column supplied in the kit according to the manufacturer's instructions. The labeled cDNA was dissolved in hybridization buffer (30 µl) containing 6x SSC (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.2% SDS, 5x Denhardt's solution (Sigma-Aldrich), and 0.1 mg/ml denatured salmon sperm DNA. Hybridization was performed under a coverslip (Spaced Cover Glass XL, Takara Bio) in a humidity chamber at 65 °C for 12–15 h. After hybridization, the microarray plates were washed four times with 2x SSC and 0.2% SDS at 55 °C for 5 min, rinsed in 0.05x SSC, and dried by centrifugation. The intensities of the Cy3 and Cy5 dyes were measured by using an Affymetrix 428 Array Scanner (Affymetrix, Santa Clara, CA). The microarray images were analyzed using ImaGene version 5.5 software (BioDiscovery, Marina Del Ray, CA).

Recombinant Production of Tgr in E. coli—The tgr gene of T. kodakaraensis KOD1 was amplified by PCR from genomic DNA using Pfu TURBO polymerase (Stratagene) by standard methods. Primers used were BG2072 (5'-GGGCGGCGCATATGAGGGAAGACGAGATAATTG-3') and BG2073 (5'-GCCGCCGGATCCTCACTCAAGGAGGATGAACTT-3') (NdeI and BamHI sites are underlined). BG2073 contained a stop codon to ensure overexpression of Tgr without a histidine tag to prevent interference during DNA binding assays. The PCR-amplified DNA fragment was digested with NdeI and BamHI and ligated in pET26b (Novagen), and the resulting plasmid was named pWUR278. The QIAprep Spin Miniprep kit (Qiagen) was used for plasmid purification. The correct sequence of the construct was verified (Baseclear, Leiden, The Netherlands).

Overexpression of Tgr was achieved by induction of E. coli BL21(DE3)/pRIL cells harboring pWUR278. A 1.5-liter culture was grown until A600 {approx} 0.5, and protein expression was induced by addition of 0.5 mM isopropyl beta-D-thiogalactopyranoside. After 15 h of incubation at 37 °C, cells were harvested and centrifuged (20 min at 5000 x g at 4 °C). Cells were resuspended in lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), 100 mM NaCl, pH 8.0) and disrupted by sonication at 0 °C. Insoluble material was removed by centrifugation (30 min at 26,000 x g at 2 °C). Ten millimolar MgCl2 and 0.1 mg/ml DNase I (Ambion, Austin, TX) were added, and the cell-free extract was incubated for 30 min at room temperature. DNase I and contaminating E. coli proteins were denatured by a subsequent heat treatment (20 min at 80 °C) and removed by centrifugation (30 min at 26,000 x g at 2 °C). Resulting heat-stable cell-free extract was slowly mixed with 5 ml of cross-linked agarose-heparin resin (Sigma-Aldrich) at 4 °C for 60 min. After mixing, the resin was allowed to settle in a 10-ml syringe. Contaminant proteins were eluted by washing the resin with 10 ml of wash buffer (20 mM Tris-HCl, 2 mM EDTA, 1 mM DTT, 200 mM NaCl, pH 8.0). Tgr was eluted in elution buffer (20 mM Tris-HCl, 2 mM EDTA, 1 mM DTT, 500 mM NaCl, pH 8.0). Partial desalting of the sample was accomplished by dialysis to lysis buffer at 4 °C for 16 h after which the sample was loaded on a Mono Q Column (pre-equilibrated with lysis buffer) (GE Healthcare). Tgr was eluted in a linear gradient to 1 M NaCl in lysis buffer. Samples containing Tgr were collected, pooled, and dialyzed to lysis buffer at 4 °C for 16 h.

Electrophoresis Mobility Shift Assay—Promoter sequences of 100 bp of the ADP-dependent PFK (pfk, TK0376), FBPase (fbp, TK2164), and archaeal histone A (hpkA, TK1413) were PCR-amplified using primers BG2113 (5'-GGCCGGCTGCAGTTTCACGGAGTACTGACTTTTC-3') and BG2114 (5'-CGGCCGGCATATGTATCACCCTCAGTGACTAA-3'), BG2117 (5'-GGCCGGCTGCAGCCGCTTCTATCACCTTCGAA-3') and BG2118 (5'-CCGGGCCCATATGAACCACCGGTATTTTTAACCTC-3'), and BG2115 (5'-GGCCGGCTGCAGTTCGTTGTTAGACCCTGAGAA-3') and BG2116 (5'-CGGCCGGCATATGCAACACCTCCTTAAAGGGCT-3'), respectively. The PCR-amplified pfk and fbp promoter fragments contained the TGM (25). DNA was purified with the QIAquick PCR Purification kit (Qiagen) and radioactively end-labeled with 32P with phosphonucleotide kinase (Invitrogen) according to the manufacturer's instructions. Unincorporated label was removed by the QIAquick Nucleotide Removal kit (Qiagen). For electrophoretic mobility shift assays (EMSAs), 43 nM Tgr was incubated with 0.2 pmol of labeled DNA in 15 µl of binding buffer (25 mM HEPES, 150 mM potassium glutamate, 10% glycerol, 1 mM DTT, 1 µg of poly(dI-dC)·poly(dI-dC), pH 7.5) at 70 °C for 30 min. Different concentrations of possible carbohydrate ligands (analytical grade) were added as indicated in the text. After incubation, samples were allowed to cool to room temperature for 3 min and loaded onto a prerun 4% native PAGE gel. Gels were run in 1x TBE (89 mM Tris borate, 2 mM EDTA, pH 8.3) at 15 mA, 200 V at room temperature until satisfactory migration.


Figure 1
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  		FIGURE 1.
Overall structure and amino acid sequence alignment of Tgr and TrmB proteins. The N-terminal helix-turn-helix motif is indicated as "HTH domain." Asterisks below the alignment indicate amino acid residues common in all four protein sequences. Amino acid residues of PF1743 (TrmB) involved in the interaction with the non-reducing glucosyl residue of maltose (50) are shown in bold. Among them, two residues, Gly324 and Glu330, that are particularly involved in the binding, are indicated with arrows.

 


Figure 2
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  		FIGURE 2.
A, a schematic diagram illustrating the expected homologous recombination that should occur using the disruption vector pUTGR. The homologous 5'- and 3'-flanking regions of tgr (1,000 bp each) are shown in white boxes. The positions and orientations of primers used for PCR analysis are indicated by arrowheads. The expected lengths of linear DNA fragments amplified by PCR or obtained by PstI digestion are indicated. B, confirmation of gene disruption by PCR. PCR analyses of the tgr locus with the primer set tgr-US1/tgr-DS2 using the genomic DNA of the KU216 strain (left lane) and the KGR1 strain (right lane) as a template. C, confirmation of gene disruption by Southern blot analyses. Southern blot analyses of PstI-digested genomic DNA of KU216 and KGR1 using probes corresponding to regions within the coding regions of tgr (left lane) and pyrF (right lane).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TK1769 Encodes a Protein Similar to TrmB—TrmB is an archaeal transcriptional regulator involved in sugar metabolism originally identified in Thermococcus litoralis (37). In vitro studies have indicated that in the absence of trehalose or maltose TrmB blocks transcription of the trehalose/maltose ATP-binding cassette (ABC) transporter operon, in which the TrmB gene itself is included, through the direct binding to its promoter region. In the presence of trehalose or maltose, TrmB is released from the promoter region, resulting in subsequent transcriptional initiation. A nearly identical trehalose/maltose ABC transporter operon (PF1739–PF1744, including a TrmB ortholog) is also present on the genome of P. furiosus that was proposed to be the result of lateral gene transfer (38). A recent in vitro analysis of P. furiosus TrmB (encoded by PF1743) indicated a dual function of this protein; it regulates not only the trehalose/maltose ABC transporter operon but also the maltodextrin ABC transporter operon (PF1938–PF1933) (39). The latter transporter functions in the uptake of maltooligosaccharides with three or more glucose units (40).

T. kodakaraensis contains neither an ortholog of TrmB nor that of a trehalose/maltose ABC transporter but contains an ortholog corresponding to the maltodextrin ABC transporter (TK1771–TK1775). This is consistent with the fact that maltotriose and longer oligomers, including starch, can support the growth of T. kodakaraensis, whereas maltose cannot. Despite the absence of a TrmB ortholog, two TrmB-like genes (paralogs TK0471 and TK1769) are present on the T. kodakaraensis genome. Orthologous genes corresponding to TK0471 are present on all of the four sequenced genomes of the Thermococcales, whereas TK1769 orthologs are found only in P. furiosus (PF0124) and T. kodakaraensis. We speculated previously (25) that the latter TrmB-like gene might encode the transcriptional regulator responsible for controlling the sugar metabolism in T. kodakaraensis. This was because (i) a TrmB ortholog is absent in T. kodakaraensis, (ii) the TK1769 orthologs are present only in the sugar-metabolizing Thermococcales species, and (iii) the TK1769 gene is located adjacent to the maltodextrin ABC transporter operon on the T. kodakaraensis genome.


Figure 3
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  		FIGURE 3.
Growth of T. kodakaraensis KOD1 (circles) and KGR1 (triangles) in MA-YT medium with S0 (A), sodium pyruvate (B), or maltodextrin (C) and in ASW-AA medium with S0 (D), S0 and sodium pyruvate (E), or S0 and maltodextrin (F). Batch cultivations of each strain were carried out at 85 °C. Error bars represent S.D. for three independent experiments. In D, E, and F, the specific growth rates of KOD1 (µWT) and those of KGR1 (µ{Delta}) calculated from multiple growth curves are indicated. OD660, optical density at 660 nm.

 
TK1769 encodes a protein (Tgr) of 341 amino acids with a calculated molecular mass of 39,386 Da (Fig. 1). Tgr is 67% identical to the PF0124 protein, but it is only 28% identical to TrmB of P. furiosus. It is annotated as a "predicted transcription regulator, DUF118 helix-turn-helix family" (30), and its helixturn-helix motif is positioned at the N terminus (~100 amino acids) as in the case of TrmB. TrmB-like regulators including TK1769 are assigned to the COG1378 group, whose members are mostly distributed in Archaea.

Construction of tgr-disrupted Mutant Strain—To clarify the physiological function of Tgr in T. kodakaraensis, its deletion mutant was constructed with a gene disruption system previously developed in this organism (28, 29). A tgr disruption vector that harbors the pyrF (pUTGR) was constructed and used to transform the host strain (KU216, {Delta}pyrF) by double crossover homologous recombination (Fig. 2A). A transformant exhibiting uracil prototrophy was confirmed to have the expected genotype; PCR amplification resulted in a DNA fragment with a length corresponding to that of the {Delta}tgr locus (Fig. 2B). The genetic recombination at the {Delta}tgr locus and the absence of the tgr gene on the chromosome was confirmed by Southern blot analysis (Fig. 2C), and the mutant was designated KGR1.

Growth Properties of KGR1—Growth characteristics of the KGR1 strain under several growth conditions were examined and compared with those of the wild-type strain, KOD1 (Fig. 3). First a nutrient rich MA-YT medium was used to examine the growth rate under gluconeogenic (MA-YT-S0 and MA-YT-Pyr) and glycolytic (MA-YT-Mdx) conditions. However, under all medium conditions tested, no significant difference in growth phenotype was observed between the two strains (Fig. 3, A–C). As yeast extract, which is a component of the MA-YT medium, is presumed to include (poly)saccharides that may affect the growth of the mutant (32), we next examined growth characteristics in a synthetic medium containing 20 amino acids (ASW-AA) under both gluconeogenic (ASW-AA-S0 and ASW-AA-S0-Pyr) and glycolytic (ASW-AA-S0-Mdx) conditions. It should be noted that stable growth of T. kodakaraensis cells in ASW-AA medium requires S0 even when sodium pyruvate or maltodextrin is present. In the ASW-AA medium, the growth rate under gluconeogenic conditions was severely affected by the disruption of tgr (Fig. 3, D and E). In contrast, although a prolonged lag time was observed, the KGR1 strain exhibited a growth rate comparable to that of the wild-type strain under glycolytic conditions (Fig. 3F). These results imply a significant function of the Tgr protein for growth under gluconeogenic conditions.

Transcriptome Analysis of the KOD1 and KGR1 Strains—To determine the entire set of genes that are controlled by Tgr, a whole genome DNA microarray analysis was performed on the wild-type strain KOD1 and the tgr disruptant strain KGR1. The media applied for gluconeogenic conditions were MA-YT-S0 and MA-YT-Pyr, and the medium representing glycolytic conditions was MA-YT-Mdx.


Figure 4
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  		FIGURE 4.
A, relative transcript levels of selected genes related to glycolysis and gluconeogenesis under glycolytic (MA-YT-Mdx) and gluconeogenic (MA-YT-S0 or MA-YT-Pyr) conditions in KOD1. The presence (O) or absence (X) of a TGM on each promoter is indicated. Error bars were calculated from two sets of microarrays. GLK, ADP-dependent glucokinase (TK1110); PGI, glucose-6-phosphate isomerase (TK1111); PFK, ADP-dependent PFK (TK0376); ALD, fructose-1,6-bisphosphate aldolase (TK0989); TPI, triose-phosphate isomerase (TK2129); GAPOR, GAP:ferredoxin oxidoreductase (TK2163); GAPN, GAP dehydrogenase (non-phosphorylating) (TK0705); PGM, phosphoglycerate mutase (TK0866); ENO, enolase (TK2106); PEPS, phosphoenolpyruvate synthase (TK1292); PYK, pyruvate kinase (TK0511); FBPase, TK2164; GAPDH, GAP dehydrogenase (phosphorylating) (TK0765); PGK, 3-phosphoglycerate kinase (TK1146). B, relative transcript levels of selected genes related to maltodextrin metabolism under glycolytic (MA-YT-Mdx) and gluconeogenic conditions (MA-YT-S0 or MA-YT-Pyr) in the wild-type strain. Genes constituting a putative operon are boxed with a dotted line. TK0977, pullulanase type II, GH13 family; TK1108, phosphohexomutase; TK1406, maltodextrin phosphorylase; TK1770, cyclomaltodextrinase; TK1771, maltodextrin-binding protein precursor; TK1772, maltodextrin transport system, permease component; TK1773, maltodextrin transport system, permease component; TK1774, amylopullulanase; TK1775, maltodextrin transport system, ATPase component; TK1809, 4-{alpha}-glucanotransferase; TK1884, {alpha}-amylase; TK2148, {alpha}-glucosidase; TK2172, cyclomaltodextrin glucanotransferase.

 
When the wild-type KOD1 strain was grown under glycolytic and gluconeogenic conditions, we observed a significant increase in the transcript levels of the genes encoding enzymes of the modified EM pathway (Fig. 4A, Table 1, and supplemental Figs. S1 and S2). The transcription of the key enzymes of the pathway, the ADP-dependent sugar kinases and GAP:ferredoxin oxidoreductase, increased 4–8-fold. We also observed up-regulation of genes involved in the degradation and uptake of maltodextrins (Fig. 4B, Table 1, and supplemental Figs. 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. The results obtained with the wild-type KOD1 strain were as expected and confirm that the three media although containing low levels of sugars deriving from yeast extract are applicable for transcriptome analysis between glycolytic and gluconeogenic modes of growth.


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  		TABLE 1
Transcript analysis of selected genes in T. kodakaraensis KOD1 grown under gluconeogenic and glycolytic conditions

 
As the effects on growth resulting from the tgr gene disruption were greater under gluconeogenic conditions, we compared the transcript levels of KOD1 and KGR1 cells grown in MA-YT-S0 or MA-YT-Pyr (see supplemental Tables S1–S3 for specific microarray data). The results of a biological replicate performed with the strains grown in MA-YT-S0 are shown in supplemental Tables S4–S6. In both media we observed a large number of genes whose transcript levels increased in the KGR1 strain compared with the KOD1 strain. Among the genes whose signal intensities increased over 4-fold in the KGR1 strain, 12 of 15 genes observed in MA-YT-S0 and 14 of 16 in MA-YT-Pyr were related to glycolysis or maltodextrin metabolism. When genes constituting these pathways were examined (Fig. 5, A and B; Table 2; supplemental Figs. S5–S8; and supplemental Tables S3 and S6), all genes displayed higher signal intensities in the KGR1 strain with the exception of the pyruvate kinase gene (see below). Among the glycolytic members, genes responsible for the unidirectional reaction steps, such as ADP-dependent glucokinase (TK1110), ADP-dependent PFK (TK0376), and non-phosphorylating GAP dehydrogenase (TK0705), showed high intensity ratios ({Delta}tgr/wild type). This may reflect a strong repression of these genes by Tgr under gluconeogenic conditions. Similarly high intensity ratios were observed for genes encoding the maltodextrin ABC transporter (TK1771–TK1773 and TK1775), amylopullulanase (TK1774), maltodextrin phosphorylase (TK1809), 4-{alpha}-glucanotransferase (TK1809), and phosphohexomutase (TK1108) in maltodextrin metabolism, suggesting an important role of these genes in the processing and uptake of maltodextrins, as has been reported in P. furiosus (42). Moreover the signal intensity ratios of the glycolytic and maltodextrin metabolism genes observed in the absence/presence of the tgr gene (Fig. 5, A and B) displayed the same tendencies as those observed in KOD1 grown under glycolytic/gluconeogenic conditions (Fig. 4, A and B). The results suggest that under gluconeogenic conditions Tgr functions as a global transcriptional regulator that represses the entire gene network involved in glycolysis and maltodextrin utilization.


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  		TABLE 2
Transcript analysis of selected genes in T. kodakaraensis KOD1 and KGR1 grown under gluconeogenic conditions

 
Cell-free extracts of KOD1 and KGR1 cells were tested for different enzyme activities: ADP-dependent glucokinase, ADP-dependent PFK, non-phosphorylating GAP dehydrogenase, phosphoglucomutase, and maltodextrin phosphorylase. The activity of ADP-dependent glucokinase and non-phosphorylating GAP dehydrogenase in KGR1 cells grown under gluconeogenic conditions was comparable to the activity observed in KOD1 cells grown under glycolytic conditions (supplemental Fig. S9), indicating that the regulation of these enzymes is predominantly brought about by Tgr. For the phosphoglucomutase and maltodextrin phosphorylase, the activity level in KGR1 cell extracts was 2–7-fold higher than the activity level measured in KOD1 cell extracts (both under gluconeogenic conditions; supplemental Fig. S9), indicating that Tgr plays a major role in regulating these enzyme activities. However, the activity levels were still 25–55% of the levels observed in KOD1 cells grown under glycolytic conditions, indicating possible additional post-transcriptional regulation. The strongest effect was observed with the ADP-dependent PFK; derepression after disruption of tgr in KGR1 cells was initially observed to only a low extent (supplemental Fig. S9). However, we found that activity levels increased severalfold when glucose 1-phosphate (0.4 mM) was added to the assay mixtures in KGR1 cell extracts, suggesting allosteric regulation (supplemental Fig. S10) in addition to the above mentioned Tgr-mediated transcriptional control (Fig. 5A). However, as appears to be the case of phosphoglucomutase and maltodextrin phosphorylase, activity levels were still 20–30% of those observed in KOD1 cells grown under glycolytic conditions, again suggesting additional post-transcriptional regulation in the case of PFK.

As described above, a common sequence motif (TGM) has been identified on almost all genes in P. furiosus and T. kodakaraensis that is predicted to be involved in glycolysis and maltodextrin metabolism. The pyruvate kinase gene (TK0511) was an exception, and a TGM sequence could not be found on its promoter. The gene also exhibited exceptional characteristics in our transcriptome analyses; no significant increase in transcript levels was observed when KOD1 cells were grown under glycolytic conditions (Fig. 4A and Table 1), and the effects of tgr gene disruption were minimal (Fig. 5A and Table 2). This is consistent with the recent finding that phosphoenolpyruvate synthase (encoded by TK1292) and not pyruvate kinase is the major glycolytic enzyme involved in the conversion of phosphoenolpyruvate to pyruvate in the modified EM pathway in T. kodakaraensis (31). Therefore, the correlation between the presence of a TGM and the occurrence of increased transcription brought about by tgr disruption suggests that TGM is involved in the Tgr-dependent gene repression mechanism, possibly acting as a direct binding site for Tgr.


Figure 5
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  		FIGURE 5.
A, relative transcript levels of selected genes related to glycolysis and gluconeogenesis in KOD1 and KGR1 cells grown under gluconeogenic (MA-YT-S0 or MA-YT-Pyr) conditions. B, relative transcript levels of selected genes related to maltodextrin metabolism in KOD1 and KGR1 cells grown under gluconeogenic conditions (MA-YT-S0 or MA-YT-Pyr). The asterisk indicates that the datum was not reliable because of low signal intensity. Error bars were calculated from two sets of microarrays. See Fig. 4 legend for abbreviations.

 
Although few in number, we observed the presence of genes whose transcript levels decreased in the {Delta}tgr strain under gluconeogenic conditions. It was of particular interest that genes encoding gluconeogenic enzymes (TK2164 encoding FBPase, TK0765 encoding phosphorylating GAP dehydrogenase, and TK1146 encoding phosphoglycerate kinase), which were up-regulated under gluconeogenic conditions in the KOD1 strain (Fig. 4A and Table 1), were included in this group (Fig. 5A and Table 2). The microarray data presented here suggest another aspect of the Tgr protein functioning as a transcriptional activator for genes involved in gluconeogenesis. Consistent with this role, we found that the increase in FBPase activity observed in KOD1 cells under gluconeogenic conditions could not be detected in KGR1 cells (supplemental Fig. S9).

The presence of a TGM in the FBPase gene promoter is described elsewhere (25). We have found additional TGM-like sequences in the promoter regions of the other gluconeogenic genes (TK0765: (-62)TTTCACN5GTGAAA; TK1146: (-65)AACCACN5GTGTTC (bases matching the consensus sequence are underlined; numbers in parentheses indicate the positions relative to the initiation codon of each gene)). These TGM sequences, including the TGM present in the FBPase promoter, are located upstream of the predicted Transcription factor B-responsive element (BRE)/TATA sequence. The TGM present in the promoters of glycolytic and maltodextrin-metabolizing genes is positioned down-stream of the predicted BRE/TATA sequence in the near vicinity of the transcriptional initiation site. The relative position of TGM with respect to the BRE/TATA sequence might determine the role of the Tgr protein, functioning as a repressor for the glycolytic genes and as an activator for the gluconeogenic genes (see "Discussion").

Besides the genes indicated in Table 2, there are a large number of genes whose transcription levels are affected by tgr gene disruption. Genes that had been reported previously to harbor TGM sequences in their promoters along with genes that exhibited particularly high increases in transcript levels are shown in Table 3. Among the former group, genes with increased transcript levels in KGR1 were TK1404 (phosphosugar mutase with unknown substrate specificity) (43), TK1436 (branching enzyme, glycoside hydrolase family 57) (44), and TK1743 (glycoside hydrolase family 57), all likely to be involved in {alpha}-glucan metabolism. The branching enzyme and the phosphohexomutase (TK1108; shown in Table 2) are key enzymes for glycogen biosynthesis from glucose 6-phosphate, suggesting that glycogen synthesis is repressed by Tgr under gluconeogenic conditions. Genes that displayed a decrease in transcript levels were those encoding a putative thiol protease (TK1295) and the subunits of a putative methylmalonyl-CoA decarboxylase (TK1622–TK1625). The genes in Table 3 that are repressed by Tgr harbored a TGM downstream of the BRE/TATA sequence, whereas the TGMs of activated genes were positioned upstream of the BRE/TATA sequence. Although TGM sequences had been identified in their promoter regions, a few genes apparently do not respond to the presence or absence of Tgr.


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  		TABLE 3
Transcript analysis of other genes with TGM and genes without a TGM but with increased transcription levels in KGR1

 
A number of genes that had not been identified as harboring a TGM were also found to be affected by tgr gene disruption. Five genes with the highest intensity ratios ({Delta}tgr/wild type) are shown in Table 3. Genes encoding putative sugar-phosphate nucleotidyltransferase (TK0955), bifunctional mannose-6-phosphate isomerase/mannose-1-phosphate guanylyltransferase (TK1109), and myo-inositol-1-phosphate synthase (TK2278) fit well to the presumed role of Tgr as they are expected to be involved in biosynthetic pathways activated in the presence of abundant sugar. The relationship of the ABC-type manganese/zinc transport system, ATPase component (TK0803) and conserved protein, radical S-adenosylmethionine superfamily (TK1135) with sugar metabolism cannot be understood at present. A detailed search in the upstream regions of these genes led to the identification of putative TGM sequences in four of the genes.


Figure 6
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  		FIGURE 6.
A, EMSAs of Tgr with DNA fragments corresponding to the 5'-flanking regions of the pfk (lanes 1 and 2), fbp (lanes 3 and 4), or hpkA (lane 5 and 6) genes. The concentration of DNA was 13 nM, and Tgr was added (lanes 2, 4, and 6) at a concentration of 43 nM. B, left panel, titration experiment. Tgr at concentrations of 0, 11, 32, or 43 nM (lanes 1, 2, 3, and 4) was incubated with the 5'-flanking region of the pfk gene (13 nM). Right panel, determination of the binding affinity of Tgr with the 5'-flanking region of the pfk gene. PhosphorImager software was used to quantify the amount of DNA bound to Tgr. C, Tgr (43 nM) was incubated with the 5'-flanking region of the pfk gene (13 nM) in the presence of maltotriose (left panel) and maltose (right panel). Lanes 1 and 7, DNA only; lanes 2 and 8, DNA with Tgr; lanes 3–6, DNA with Tgr and maltotriose added at concentrations of 100, 250, 500, and 1000 µM, respectively; lanes 9–12, DNA with Tgr and maltose added at concentrations of 100, 250, 500, and 1000 µM, respectively.

 
The Tgr gene (TK1769) itself contains the TGM sequence. When we compared the microarray signals of TK1769 using KOD1 grown on pyruvate (MA-YT-Pyr) and maltodextrin (MA-YT-Mdx), a higher signal was detected for cells grown on maltodextrin (log2(Mdx/Pyr) = 0.57 ± 0.03), suggesting that transcription of Tgr gene is autoregulated. It was also reported in P. furiosus that the Tgr gene ortholog (PF0124) is more actively transcribed in maltose-grown cells than in peptide-grown cells (log2(maltose/peptide) = 0.4) (24).

Electrophoretic Mobility Shift Assay—To test whether Tgr has the ability to bind to the TGM sequence, EMSAs were performed. T. kodakaraensis Tgr was overexpressed and purified to homogeneity as confirmed by SDS-PAGE analysis. Tgr had a tendency to partially break down in two parts of ~27 and 12 kDa in the absence of EDTA and DTT and when being concentrated (data not shown).

Using the purified Tgr, EMSAs were performed with three different DNA fragments, i.e. promoters of the ADP-dependent PFK (pfk, TK0376), FBPase (fbp, TK2164), and archaeal histone A (hpkA, TK1413). A clear band exhibiting the formation of Tgr-DNA complex was detected when TGM-containing promoters (pfk and fbp) were used (Fig. 6A). The hpkA promoter does not contain a TGM, and indeed no interaction could be detected. The motifs in the pfk promoter (AGTCACN5GTGATA) and in the fbp promoter (TATCATN5GTGATA) both slightly deviate from the TGM consensus in T. kodakaraensis. The EMSAs revealed that, at least under the conditions tested, Tgr has a higher affinity for the pfk promoter fragment (Fig. 6A). When the pfk promoter was titrated with increasing amounts of Tgr, the amount of retarded DNA clearly increased. Tgr protein bound to pfk promoter with an apparent dissociation constant (Kd) of 31 nM (Fig. 6B).

Several maltooligosaccharides (maltose, isomaltose, maltotriose, maltotetraose, and maltopentaose) were included in the Tgr-mediated EMSA, and their effects on the binding affinity of Tgr were analyzed. When maltose was added at various concentrations, no change in the amount of Tgr-DNA complex was observed (Fig. 6C). Similarly additions of isomaltose, maltotetraose, and maltopentaose gave no change in the complex formation (supplemental Fig. S11). However, when maltotriose was added, the complex formation between Tgr and pfk promoter was clearly inhibited in a concentration-dependent manner (Fig. 6C), suggesting that maltotriose functions as a physiological effector to regulate Tgr-mediated transcriptional activation/repression. Maltotriose inhibited the binding of Tgr with a Kd value of {approx}520 µM. The addition of glucose in the above described EMSA experiment did not affect the observed retardation patterns (supplemental Fig. S12), indicating that glucose is not a co-repressor in the case of Tgr, unlike the effect that was recently reported for P. furiosus TrmB (45).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the modified EM and gluconeogenic pathways of the Thermococcales, allosteric control has not been observed in the key enzymes characterized so far, including PFK (18, 46), FBPase (47), and pyruvate kinase.4 As the transcript levels of these genes have been found to respond to glycolytic/gluconeogenic conditions in P. furiosus, it has been suggested that regulation occurs primarily at the transcriptional level (24). Although our results have raised the possibilities of post-transcriptional regulation for several specific enzymes, the transcriptome analyses clearly indicated that all glycolytic/gluconeogenic genes in T. kodakaraensis are controlled at the transcription level via Tgr function. The presence of TGM sequences and a Tgr ortholog on the P. furiosus genome suggests that a similar regulon is also present in P. furiosus and possibly in other sugar-metabolizing Thermococcales species (25). The Thermococcales glycolytic regulon, or the Tgr regulon, of T. kodakaraensis comprises more than 30 genes and is the largest regulon that has presently been identified in the Archaea.


Figure 7
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  		FIGURE 7.
A model of the mechanisms by which Tgr regulates gene transcription in T. kodakaraensis under gluconeogenic (A) and glycolytic (B) growth conditions. RNAP, RNA polymerase.

 
A model illustrating the regulation mechanism of Tgr-mediated transcriptional repression/activation in T. kodakaraensis is shown in Fig. 7. Under gluconeogenic conditions, Tgr binds to the TGM of target genes. When TGM is downstream of the BRE/TATA sequence (in the case of the glycolytic and maltodextrin-metabolizing genes; Fig. 7A, left), Tgr most likely blocks RNA polymerase recruitment, leading to transcriptional inhibition. There is also the possibility that Tgr affects the binding of transcription factor B (TFB) and/or TATA-binding protein (TBP) to their respective binding sites. In T. kodakaraensis, two TFB paralogs (TK1280 and TK2287) whose functions cannot be distinguished (48) and one TBP ortholog (TK0132) are present. When TGM is upstream of the BRE/TATA sequence (in the case of the gluconeogenic genes; Fig. 7A, right), transcription is activated. Tgr may exhibit affinity toward RNA polymerase, TFB, and/or TBP, facilitating recruitment of these components to the promoter regions. When maltodextrins are available in the medium, the intermediate maltotriose binds to the Tgr protein, leading to displacement of Tgr from TGM. Dissociation relieves the inhibiting effects of Tgr on the glycolytic and maltodextrin-metabolizing genes (Fig. 7B, left) while diminishing the activating effect of Tgr as in the case of the gluconeogenic genes (Fig. 7B, right).

Tgr is the first archaeal transcription regulator that displays both activation and repression activities in a single protein. A transcription factor with similar dual activities has been identified in E. coli that also controls a series of genes involved in glycolysis and gluconeogenesis (49). The control mechanism of this protein, Cra (for catabolite repressor/activator; alternative name FruR), is very similar to that of Tgr. Cra binds to the operator regions upstream of glycolytic genes, and derepression takes place when Cra is released from its binding site by binding to specific inducers such as fructose 1-phosphate and fructose 1,6-bisphosphate. Furthermore transcriptional activation by Cra occurs in cases when the Cra-binding operator sequence precedes the RNA polymerase-binding site, whereas transcriptional repression occurs when it overlaps or follows the RNA polymerase-binding site (49). Despite the functional similarity between Tgr and Cra, comparison of their amino acid sequences indicates that these proteins are classified into distinct families of transcriptional factors; Cra is a member of the LacI-GalR family and assigned to the COG1609 group, whereas Tgr is assigned to the COG1378 group. Therefore, it can be presumed that Tgr and Cra originally evolved independently of one another but have come to adopt similar mechanisms of function, an example of convergent evolution.

Dissociation of TrmB from its operator sequence occurs upon binding of sugar ligands to TrmB (37, 39). The present results suggest that a similar binding/release mechanism also occurs in the case of Tgr. The sugar interaction domain of TrmB (TrmB{Delta}2–109) binds strongly with maltose and to a lesser degree with sucrose and maltotriose (50). Determination of the crystal structure of a maltose-TrmB{Delta}2–109 complex revealed that the C-terminal sugar-binding site of TrmB involved seven amino acid residues, six of them recognizing the non-reducing glucosyl residue (50). Two amino acid residues (Gly320 and Glu326) in TrmB that were necessary for the complex formation are also conserved in Tgr (corresponding to Gly324 and Glu330) (Fig. 1). As for the other residues involved in sugar binding, similar amino acids are conserved in Tgr, suggesting a possibility that the basic mechanisms for sugar recognition are shared by Tgr and TrmB. On the other hand, the other TrmB-like protein in T. kodakaraensis, TK0471, lacks a greater part of the corresponding sugar-binding site, indicating that this regulator may be triggered by a distinct effector in T. kodakaraensis.

Most archaeal transcriptional regulators characterized thus far act in a negative manner, and little is known about positive regulators. Ptr2 of Methanocaldococcus jannaschii, an Lrp family regulator, is one of the best studied positive regulators in the Archaea (51–53). Two genes activated by Ptr2 (rb2 and rbr) have a binding site just upstream of the BRE/TATA sequence, whereas binding site exists further upstream of the rb2 promoter. The presence of a binding site immediately upstream of the BRE/TATA sequence is reported in the lysWXJK operon of Sulfolobus solfataricus that is controlled by a proposed positive regulator, LysM (54), and in the haloarchaeal bop gene promoter, which is activated by Bat (55). The position of these binding sites, including that of TGM in the gluconeogenic genes, suggests a direct interaction of regulator proteins with TFB and/or TBP. Actually an in vitro reconstitution experiment indicated that binding of Ptr2 to the operator sequence enhances the recruitment of TBP to a weak TATA box (53). At present, the molecular mechanisms of the anticipated interaction between archaeal activators and TFB and/or TBP are still unclear and will be an important subject of research for the elucidation of the mechanisms governing archaeal transcriptional activation.

Addendum—During the reviewing process of this manuscript, Lee et al. (56) reported an in vitro study on the Tgr ortholog (TrmBL1) in P. furiosus. The results of the study are consistent with those obtained in the in vitro analysis of Tgr reported in this study and also display through footprint analysis that TrmBL1 recognizes TGM.


   FOOTNOTES
 
* 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. Back

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

1 These authors contributed equally to this work. Back

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.

3 The abbreviations used are: PFK, phosphofructokinase; FBPase, fructose-1,6-bisphosphatase; EM, Embden-Meyerhof; GAP, glyceraldehyde 3-phosphate; TGM, Thermococcales glycolytic motif; Tgr, Thermococcales glycolytic regulator; ASW, artificial seawater; EMSA, electrophoresis mobility shift assay; TFB, transcription factor B; TBP, TATA-binding protein; Cra, catabolite repressor/activator; ABC, ATP-binding cassette; DTT, dithiothreitol; Mdx, maltodextrin; Pyr, pyruvate; BRE, Transcription factor B-responsive element. Back

4 J. E. Tuininga and S. W. Kengen, personal communication. Back


   ACKNOWLEDGMENTS
 
Maltotetraose and maltopentaose were a kind gift from Dr. H. A. Schols (Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands).



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