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• W2029063714 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 (Δtgr) 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 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, 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. 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 (Δtgr) 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 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, 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 (1Fraenkel D.G. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C1987: 142-150Google Scholar), 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) 3The 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. (2Blangy D. Buc H. Monod J. J. Mol. Biol. 1968; 31: 13-35Crossref PubMed Scopus (305) Google Scholar) and pyruvate kinase (3Waygood E.B. Mort J.S. Sanwal B.D. Biochemistry. 1976; 15: 277-282Crossref PubMed Scopus (40) Google Scholar), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) (4Fraenkel D.G. Pontremoli S. Horecker B.L. Arch. Biochem. Biophys. 1966; 114: 4-12Crossref PubMed Scopus (36) Google Scholar). 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 (5Oh M.-K. Rohlin L. Kao K.C. Liao J.C. J. Biol. Chem. 2002; 277: 13175-13183Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) and post-transcriptional levels (6Romeo T. Mol. Microbiol. 1998; 29: 1321-1330Crossref PubMed Scopus (375) Google Scholar). In the eukaryote Saccharomyces cerevisiae, a number of mechanisms involved in glycolysis/gluconeogenesis regulation have been identified (for a review, see Ref. 7Gonçalves P. Planta R.J. Trends Microbiol. 1998; 6: 314-319Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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. 8Verhees C.H. Kengen S.W.M. Tuininga J.E. Schut G.J. Adams M.W.W. de Vos W.M. van der Oost J. Biochem. J. 2003; 375: 231-246Crossref PubMed Scopus (191) Google Scholar and 9Siebers B. Schönheit P. Curr. Opin. Microbiol. 2005; 8: 695-705Crossref PubMed Scopus (161) Google Scholar). 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 (10Siebers B. Tjaden B. Michalke K. Dörr C. Ahmed H. Zaparty M. Gordon P. Sensen C.W. Zibat A. Klenk H.-P. Schuster S.C. Hensel R. J. Bacteriol. 2004; 186: 2179-2194Crossref PubMed Scopus (61) Google Scholar). 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 (11Lorentzen E. Hensel R. Knura T. Ahmed H. Pohl E. J. Mol. Biol. 2004; 341: 815-828Crossref PubMed Scopus (46) Google Scholar, 12Brunner N.A. Brinkmann H. Siebers B. Hensel R. J. Biol. Chem. 1998; 273: 6149-6156Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Brunner N.A. Siebers B. Hensel R. Extremophiles. 2001; 5: 101-109Crossref PubMed Scopus (40) Google Scholar) as well as for phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase (14Tjaden B. Plagens A. Dörr C. Siebers B. Hensel R. Mol. Microbiol. 2006; 60: 287-298Crossref PubMed Scopus (57) Google Scholar). 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 (15Itoh T. J. Biosci. Bioeng. 2003; 96: 203-212Crossref PubMed Scopus (31) Google Scholar). Some members of this order can also grow on carbohydrates using a modified EM pathway (16Kengen S.W.M. de Bok F.A.M. van Loo N.-D. Dijkema C. Stams A.J.M. de Vos W.M. J. Biol. Chem. 1994; 269: 17537-17541Abstract Full Text PDF PubMed Google Scholar), which differs from the classical EM pathway by the presence of several unique enzymes, such as ADP-dependent glucokinase (17Kengen S.W.M. Tuininga J.E. de Bok F.A.M. Stams A.J.M. de Vos W.M. J. Biol. Chem. 1995; 270: 30453-30457Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), ADP-dependent PFK (18Tuininga J.E. Verhees C.H. van der Oost J. Kengen S.W.M. Stams A.J.M. de Vos W.M. J. Biol. Chem. 1999; 274: 21023-21028Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and GAP:ferredoxin oxidoreductase (19Mukund S. Adams M.W.W. J. Biol. Chem. 1995; 270: 8389-8392Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 20van der Oost J. Schut G. Kengen S.W.M. Hagen W.R. Thomm M. de Vos W.M. J. Biol. Chem. 1998; 273: 28149-28154Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). 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) (20van der Oost J. Schut G. Kengen S.W.M. Hagen W.R. Thomm M. de Vos W.M. J. Biol. Chem. 1998; 273: 28149-28154Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 21Verhees C.H. Huynen M.A. Ward D.E. Schiltz E. de Vos W.M. van der Oost J. J. Biol. Chem. 2001; 276: 40926-40932Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 22Siebers B. Brinkmann H. Dörr C. Tjaden B. Lilie H. van der Oost J. Verhees C.H. J. Biol. Chem. 2001; 276: 28710-28718Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 23Schäfer T. Schönheit P. Arch. Microbiol. 1993; 159: 354-363Crossref Scopus (68) Google Scholar), and the activity of gluconeogenic enzymes significantly increases under gluconeogenic conditions (23Schäfer T. Schönheit P. Arch. Microbiol. 1993; 159: 354-363Crossref Scopus (68) Google Scholar), 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 (24Schut G.J. Brehm S.D. Datta S. Adams M.W.W. J. Bacteriol. 2003; 185: 3935-3947Crossref PubMed Scopus (115) Google Scholar). We have recently identified a potential cis-regulatory element (TATCACN5GTGATA) in the glycolytic promoters of P. furiosus (25van de Werken H.J.G. Verhees C.H. Akerboom J. de Vos W.M. van der Oost J. FEMS Microbiol. Lett. 2006; 260: 69-76Crossref PubMed Scopus (28) Google Scholar). 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 (26Atomi H. Fukui T. Kanai T. Morikawa M. Imanaka T. Archaea. 2004; 1: 263-267Crossref PubMed Scopus (252) Google Scholar, 27Morikawa M. Izawa Y. Rashid N. Hoaki T. Imanaka T. Appl. Environ. Microbiol. 1994; 60: 4559-4566Crossref PubMed Google Scholar). 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 (25van de Werken H.J.G. Verhees C.H. Akerboom J. de Vos W.M. van der Oost J. FEMS Microbiol. Lett. 2006; 260: 69-76Crossref PubMed Scopus (28) Google Scholar). The development of a gene disruption system for T. kodakaraensis (28Sato T. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2003; 185: 210-220Crossref PubMed Scopus (229) Google Scholar, 29Sato T. Fukui T. Atomi H. Imanaka T. Appl. Environ. Microbiol. 2005; 71: 3889-3899Crossref PubMed Scopus (176) Google Scholar) as well as the availability of genome information (30Fukui T. Atomi H. Kanai T. Matsumi R. Fujiwara S. Imanaka T. Genome Res. 2005; 15: 352-363Crossref PubMed Scopus (358) Google Scholar) makes this archaeon an attractive model organism for the elucidation of the physiological role of unknown gene function in Thermococcales (31Imanaka H. Yamatsu A. Fukui T. Atomi H. Imanaka T. Mol. Microbiol. 2006; 61: 898-909Crossref PubMed Scopus (59) Google Scholar, 32Sato T. Imanaka H. Rashid N. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2004; 186: 5799-5807Crossref PubMed Scopus (81) Google Scholar, 33Orita I. Sato T. Yurimoto H. Kato N. Atomi H. Imanaka T. Sakai Y. J. Bacteriol. 2006; 188: 4698-4704Crossref PubMed Scopus (94) Google Scholar). 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. Microorganisms and Culture Conditions—For general DNA manipulation and sequencing, E. coli DH5α (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 (34Kanai T. Imanaka H. Nakajima A. Uwamori K. Omori Y. Fukui T. Atomi H. Imanaka T. J. Biotechnol. 2005; 116: 271-282Crossref PubMed Scopus (134) Google Scholar). The ASW-AA-based medium contained artificial seawater (ASW), vitamin mixture, modified Wolfeʼns trace minerals, and 20 amino acids as described previously (26Atomi H. Fukui T. Kanai T. Morikawa M. Imanaka T. Archaea. 2004; 1: 263-267Crossref PubMed Scopus (252) Google Scholar, 35Robb F.T. Place A.R. Archaea: A Laboratory Manual—Thermophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1995: 167-168Google Scholar). As members in the Thermococcales are reported to have several tungstoenzymes (36Kletzin A. Adams M.W.W. FEMS Microbiol. Rev. 1996; 18: 5-63Crossref PubMed Google Scholar), NaWO4·2H2O was also added to ASW-AA medium at a final concentration of 10 μm. Construction of the T. kodakaraensis Δtgr Strain—Disruption of the Tgr gene (TK1769, tgr) by double crossover homologous recombination was performed using the gene disruption system developed for T. kodakaraensis (28Sato T. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2003; 185: 210-220Crossref PubMed Scopus (229) Google Scholar, 29Sato T. Fukui T. Atomi H. Imanaka T. Appl. Environ. Microbiol. 2005; 71: 3889-3899Crossref PubMed Scopus (176) Google Scholar). 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 (29Sato T. Fukui T. Atomi H. Imanaka T. Appl. Environ. Microbiol. 2005; 71: 3889-3899Crossref PubMed Scopus (176) Google Scholar), resulting in the plasmid for tgr disruption (pUTGR). A T. kodakaraensis uracil auxotroph strain, KU216 (29Sato T. Fukui T. Atomi H. Imanaka T. Appl. Environ. Microbiol. 2005; 71: 3889-3899Crossref PubMed Scopus (176) Google Scholar), 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 Δtgr were digested with PstI, separated by 1% agarose gel electrophoresis, and transferred to a nylon membrane Hybond™-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 Δtgr strain was designated KGR1. Growth Measurements—Growth characteristics of wild-type cells (strain KOD1) and Δtgr 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 (26Atomi H. Fukui T. Kanai T. Morikawa M. Imanaka T. Archaea. 2004; 1: 263-267Crossref PubMed Scopus (252) Google Scholar). 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 ≈ 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 manufacturerʼns instructions. The labeled cDNA was dissolved in hybridization buffer (30 μl) containing 6× SSC (1× SSC is 0.15 m NaCl, 0.015 m sodium citrate), 0.2% SDS, 5× Denhardtʼns 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 2× SSC and 0.2% SDS at 55 °C for 5 min, rinsed in 0.05× 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 ≈ 0.5, and protein expression was induced by addition of 0.5 mm isopropyl β-d-thiogalactopyranoside. After 15 h of incubation at 37 °C, cells were harvested and centrifuged (20 min at 5000 × 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 × 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 × 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 (25van de Werken H.J.G. Verhees C.H. Akerboom J. de Vos W.M. van der Oost J. FEMS Microbiol. Lett. 2006; 260: 69-76Crossref PubMed Scopus (28) Google Scholar). DNA was purified with the QIAquick PCR Purification kit (Qiagen) and radioactively end-labeled with 32P with phosphonucleotide kinase (Invitrogen) according to the manufacturerʼns 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 1× TBE (89 mm Tris borate, 2 mm EDTA, pH 8.3) at 15 mA, 200 V at room temperature until satisfactory migration. TK1769 Encodes a Protein Similar to TrmB—TrmB is an archaeal transcriptional regulator involved in sugar metabolism originally identified in Thermococcus litoralis (37Lee S.-J. Engelmann A. Horlacher R. Qu Q. Vierke G. Hebbeln C. Thomm M. Boos W. J. Biol. Chem. 2003; 278: 983-990Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 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 (38DiRuggiero J. Dunn D. Maeder D.L. Holley-Shanks R. Chatard J. Horlacher R. Robb F.T. Boos W. Weiss R.B. Mol. Microbiol. 2000; 38: 684-693Crossref PubMed Scopus (80) Google Scholar). 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) (39Lee S.-J. Moulakakis C. Koning S.M. Hausner W. Thomm M. Boos W. Mol. Microbiol. 2005; 57: 1797-1807Crossref PubMed Scopus (47) Google Scholar). The latter transporter functions in the uptake of maltooligosaccharides with three or more glucose units (40Koning S.M. Konings W.N. Driessen A.J.M. Archaea. 2002; 1: 19-25Crossref PubMed Scopus (34) Google Scholar). 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 (25van de Werken H.J.G. Verhees C.H. Akerboom J. de Vos W.M. van der Oost J. FEMS Microbiol. Lett. 2006; 260: 69-76Crossref PubMed Scopus (28) Google Scholar) 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. kodaka" @default.
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• W2029063714 title "A Global Transcriptional Regulator in Thermococcus kodakaraensis Controls the Expression Levels of Both Glycolytic and Gluconeogenic Enzyme-encoding Genes" @default.
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