A GLOBAL TRANSCRIPTIONAL REGULATOR IN THERMOCOCCUS ... Keywords: Glycolysis, Gluconeogenesis, Archaea,

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    Tamotsu Kanai 1#

    , Jasper Akerboom 2#

    , Shogo Takedomi 1#

    , Harmen J.G. van de Werken 2 ,

    Fabian Blombach 2 , John van der Oost

    2 , Taira Murakami

    1 , Haruyuki Atomi

    1 and

    Tadayuki Imanaka 1 *

    1 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of

    Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan 2 Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen

    University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands # Authors contributed equally

    *Address correspondence to: Tadayuki Imanaka, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.

    Tel: +81-75-383-2777; Fax: +81-75-383-2778; E-mail: imanaka@sbchem.kyoto-u.ac.jp

    Running Title: Transcriptional regulator of archaeal sugar metabolism

    Keywords: Glycolysis, Gluconeogenesis, Archaea, Transcriptional regulation

    We have identified a novel regulator, Tgr

    (Thermococcales glycolytic regulator),

    functioning as both an activator and a

    repressor of transcription in the

    hyperthermophilic archaeon

    Thermococcus kodakaraensis KOD1. Tgr

    (TK1769) displays similarity (28%

    identical) to Pyrococcus furiosus TrmB

    (PF1743), a transcriptional repressor

    regulating the trehalose/maltose ABC

    transporter genes, but is more closely

    related (67%) to a TrmB paralog in P.

    furiosus (PF0124). Growth of a tgr

    disruption strain ( tgr) displayed a

    significant decrease in growth rate under

    gluconeogenic conditions compared to the

    wild-type strain, while comparable growth

    rates were observed under glycolytic

    conditions. A whole genome microarray

    analysis revealed that transcript levels of

    almost all genes related to glycolysis and

    maltodextrin metabolism were at

    relatively high levels in the 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, TGM (Thermococcales glycolytic

    motif). The TGM was positioned upstream

    of the BRE/TATA sequence in

    gluconeogenic promoters, and downstream

    of it in glycolytic promoters.

    Electrophoretic mobility shift assay

    indicated that recombinant Tgr protein

    specifically binds to promoter regions

    containing a TGM. Tgr was released from

    the DNA when maltotriose was added,

    suggesting that this sugar is most likely the

    physiological effector. Our results strongly

    suggest that Tgr is a global transcriptional

    regulator that simultaneously controls, in

    response to sugar availability, both

    glycolytic and gluconeogenic metabolism

    in T. kodakaraensis via its direct binding to

    the TGM.


    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 in order 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) (2) and pyruvate

    kinase (3), and the gluconeogenic enzyme,

    http://www.jbc.org/cgi/doi/10.1074/jbc.M703424200The latest version is at JBC Papers in Press. Published on September 17, 2007 as Manuscript M703424200

    Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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    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 (reviewed in (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


    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

    (reviewed in (8,9)). 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-Dourdoroff 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


    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 (GLK) (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 to

    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 transcriptional level (24). We

    have recently identified a potential

    cis-regulatory element

    (TATCAC-N5-GTGATA) 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