JOURNAL OF BACTERIOLOGY, Sept. 2004, p. 5799–5807
0021-9193/04/$08.00?0 DOI: 10.1128/JB.186.17.5799–5807.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 186, No. 17
Genetic Evidence Identifying the True Gluconeogenic
Fructose-1,6-Bisphosphatase in Thermococcus
kodakaraensis and Other Hyperthermophiles
Takaaki Sato, Hiroyuki Imanaka, Naeem Rashid, Toshiaki Fukui,
Haruyuki Atomi, and Tadayuki Imanaka*
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan
Received 18 March 2004/Accepted 7 June 2004
Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase
activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases
from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase)
from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel
FBPase (FbpTk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed
as the “missing” FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which
candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis, ImpTk,
was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We
therefore constructed ?fbp and ?imp strains by applying a gene disruption system recently developed for T.
kodakaraensis and investigated their phenotypes. The ?fbp strain could not grow under gluconeogenic condi-
tions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of
intracellular FBPase activity. Evidently, fbpTkis an indispensable gene for gluconeogenesis and is responsible
for almost all intracellular FBPase activity. In contrast, the endogenous impTkgene could not complement the
defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the
conditions examined. These facts indicated that impTkis irrelevant to gluconeogenesis, despite the high FBPase
activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence
that the true FBPase for gluconeogenesis in T. kodakaraensis is the FbpTkortholog, not the IMPase/FBPase
Fructose-1,6-bisphosphatase (FBPase; EC 126.96.36.199), which
catalyzes the hydrolysis of D-fructose-1,6-bisphosphate (FBP)
to D-fructose-6-phosphate (F6P) and inorganic phosphate (Pi),
is a well-known key enzyme of gluconeogenesis. Along with
phosphofructokinase (EC 188.8.131.52), which catalyzes the reverse
reaction, the phosphorylation of F6P during glycolysis, the
unidirectional FBPase regulates the flux of sugar metabolism.
Therefore, FBPases have been identified and characterized
from a wide variety of bacteria and eukaryotes. In most cases,
the activity of FBPase is tightly controlled through various
mechanisms in order to alleviate a futile cycle, which occurs by
the simultaneous functioning of both FBPase and phospho-
fructokinase. For the yeast Saccharomyces cerevisiae, regula-
tion at the transcriptional level (catabolite repression) (13, 39),
reversible, short-term inactivation by protein modification
(21), and proteolytic degradation (catabolite degradation) (13,
30, 37) have been reported. In addition, the majority of
FBPases are allosterically regulated by AMP (9, 12, 18, 31, 44)
and inhibited by fructose-2,6-bisphosphate (14, 18, 28).
Bacterial FBPases have been classified into three classes
based on their primary structures, and eucaryal FBPases are
homologous to bacterial class I enzymes. In Escherichia coli,
the class I FBPase encoded by fbp has been demonstrated to
fulfill the major role in gluconeogenesis (6, 7, 38), while GlpX,
a class II FBPase, is not essential for gluconeogenic growth (5).
In contrast, a class II FBPase in Corynebacterium glutamicum
which is the sole FBPase in this bacterium has been confirmed
to function in gluconeogenesis (31). Bacillus subtilis possesses
a highly distinct FBPase belonging to class III which is an
essential enzyme for growth on gluconeogenic carbon sources
when a bypass of the FBPase reaction (10) is blocked by a
mutation in the bfd gene (11). Regulation at the transcriptional
level which is dependent on carbon sources has been observed
for FBPase I of E. coli (27), whereas FBPase II of C. glutami-
cum (31) and FBPase III of B. subtilis (11) appear to be
Recent complete genome analyses and subsequent compar-
ative genomics have enabled us to discuss the distribution and
diversity of particular genes in a variety of organisms. With
respect to archaeal and bacterial (hyper)thermophiles, one
intriguing finding is the absence of obvious orthologs for
known FBPases in these genomes, despite the presence of the
other genes involved in gluconeogenesis. Nevertheless, various
lineages of (hyper)thermophiles can grow on gluconeogenic
substrates, and FBPase activities have actually been detected
in several strains (8, 34). These facts suggested the presence of
an unknown class of FBPases in (hyper)thermophiles. This had
* Corresponding author. Mailing address: Department of Synthetic
Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Phone: 81 75-383-2777. Fax: 81 75-383-2778. E-mail: imanaka@sbchem
seemed to be resolved by an unexpected finding based on
protein structure. Stec et al. found that the three-dimensional
architecture of inositol monophosphatase (IMPase; EC
184.108.40.206) from a hyperthermophilic archaeon, Methanococcus
jannaschii (the MJ0109 product), was very similar to that of
FBPase from higher eukaryotes, and indeed the MJ0109 prod-
uct exhibited the dual activities of an IMPase and an FBPase
(41). Moreover, the orthologs of MJ0109 from other hyper-
thermophiles, specifically Archaeoglobus fulgidus, Thermotoga
maritima (41), and Pyrococcus furiosus (42), have also been
demonstrated to possess both activities. From these catalytic
properties, the MJ0109 orthologs have been considered to act
as the gluconeogenic FBPase in (hyper)thermophiles and have
been classified as class IV FBPases (42).
On the other hand, by purification of a protein responsible
for the intracellular FBPase activity, members of our labora-
tory recently identified a novel candidate for the true FBPase
in the sulfur-reducing hyperthermophilic euryarchaeon “Ther-
mococcus kodakaraensis” KOD1 (1, 26, 29). The gene of the
identified FBPase was designated fbpTk, and the recombinant
protein actually displayed FBPase activity with a strict sub-
strate specificity for FBP, unlike IMPase/FBPase IV orthologs.
Furthermore, transcription of the gene in T. kodakaraensis
cells was strongly repressed under glycolytic growth conditions
with starch. These catalytic and transcriptional properties
agreed well with a gluconeogenic function, although AMP did
not show inhibitory effects on the activity. The primary struc-
ture of FbpTkis quite different from those of previously re-
ported FBPases, including IMPase/FBPase IV, but shares sig-
nificant homologies with hypothetical proteins that are highly
conserved in (hyper)thermophiles. These facts have raised the
possibility that FbpTkorthologs may be the bona fide FBPases
in organisms grown under high-temperature conditions. How-
ever, to date, it still remains to be clarified which candidate,
IMPase/FBPase IV, the FbpTkortholog, or both, plays the
major gluconeogenic role in (hyper)thermophiles.
For this study, we aimed to solve this question by using T.
kodakaraensis, as the recently determined whole-genome se-
quence of strain KOD1 contains no ortholog for the classical
FBPases but harbors a gene for IMPase/FBPase IV (desig-
nated impTk) along with fbpTk, as is the case for many other
(hyper)thermophile genomes. There is a potent advantage in
the use of T. kodakaraensis for analyses of gene function in
vivo, as we have recently constructed a targeted gene disrup-
tion system for this organism (33) which is the first to be
described for hyperthermophiles. In this study, we applied the
gene disruption system to clarify the respective participation of
impTkand fbpTkin gluconeogenesis in T. kodakaraensis. The
results obtained here provide direct evidence that enables us to
conclude that FbpTk, not ImpTk, is the true missing FBPase in
the hyperthermophilic archaeon.
MATERIALS AND METHODS
Strains and growth conditions. The strains and plasmids used for this study are
listed in Table 1. T. kodakaraensis KOD1 and its derivatives were cultivated
under strictly anaerobic conditions at 85°C in a rich growth medium (ASW-YT)
or a synthetic medium (ASW-AA) (33). ASW-YT medium (pH 6.6) was com-
posed of 0.8? artificial seawater (ASW), 5.0 g of yeast extract/liter, 5.0 g of
tryptone/liter, and 2.0 g of elemental sulfur/liter. ASW-AA medium consisted of
0.8? ASW, 20 amino acids (total, 2,125 mg/liter) as carbon sources, modified
Wolfe’s trace minerals, a vitamin mixture (total, 0.44 mg/liter), and 2.0 g of
elemental sulfur/liter (the pH was adjusted to 6.9 with NaOH). The growth
properties of ?fbp and ?imp strains of T. kodakaraensis were investigated in
ASW-AA medium containing 5.0 g of soluble starch (Nacalai Tesque, Kyoto,
Japan)/liter or 5.0 g of sodium pyruvate (Nacalai Tesque)/liter after preculture in
ASW-YT medium. The preparation of plate medium and cultivation of the cells
on it were performed as described previously (33).
E. coli strains DH5? and BL21-CodonPlus(DE3)-RIL, used for general DNA
manipulation and heterologous gene expression, respectively, were routinely
cultivated at 37°C in Luria-Bertani (LB) medium (32).
Overexpression of impTkand fbpTkgenes in E. coli and purification of recom-
binant enzymes. The expression vector for impTkwas constructed as follows. A
DNA fragment containing the imp-coding region (771 bp) was amplified from
the genomic DNA of T. kodakaraensis KOD1 by a PCR using primers EIMP-R
and EIMP-F (5?-GGGGTGATCATATGGAGTTTAACTGGAGTGAG-3? and
quences indicate NdeI and EcoRI sites, respectively]). The amplified DNA
fragment (885 bp) was inserted into pUC118 at the HincII site. After confirma-
TABLE 1. Strains and plasmids used for this study
Strain or plasmidRelevant characteristicsSource or reference
supE44 ?lacU169 (?80 lacZ ?M15) hsdR17 recA1
endA1 gyrA96 thi-1 relA1
E. coli B F?ompT hsdS(rB
?(DE3) endA Hte (argU ileY leuW Camr)
Stratagene (La Jolla, Calif.)
Amprgeneral cloning vector
Amprgeneral expression vector
pET-21a(?) derivative; fbp
pET-21a(?) derivative; imp
pUC118 derivative; pyrF marker cassette (PpyrF::pyrF)
pUC118 derivative; trpE marker cassette (PpyrF::trpE)
pUC118 derivative; ?fbp::trpE
pUC118 derivative; ?imp::trpE
Takara (Kyoto, Japan)
Novagen (Madison, Wis.)
5800SATO ET AL.J. BACTERIOL.
tion of the absence of unintended mutations in the sequence, the NdeI-EcoRI
restriction fragment was inserted into the pET-21a(?) expression vector (Nova-
gen) at the corresponding sites. E. coli strain BL21-CodonPlus(DE3)-RIL was
transformed with the resulting plasmid, pET-imp (6,262 bp). The transformant
was cultivated at 37°C in LB medium containing 100 ?g of ampicillin/ml until the
cell density (optical density at 660 nm [OD660]) reached about 0.5. The culture
was then supplemented with 0.1 mM (final concentration) isopropyl-1-thio-?-D-
galactopyranoside to induce overexpression and was incubated for a further 14 h
The cells were harvested by centrifugation, resuspended in 100 mM Tris-HCl
buffer (pH 8.0), and then sonicated. After centrifugation (8,000 ? g, 30 min), the
soluble cell extract was heat treated for 30 min at 80°C to remove thermolabile
proteins derived from the host, followed by centrifugation (8,000 ? g, 30 min).
The supernatant was applied to a Resource Q anion exchange column (6 ml)
(Amersham Biosciences, Buckinghamshire, United Kingdom), and the recom-
binant protein was eluted with a linear gradient of NaCl (0 to 1.0 M) in 50 mM
Tris-HCl (pH 8.0) with a flow rate of 2.0 ml/min. The resulting fractions were
combined and concentrated by use of Centricon YM-30 columns (Millipore,
Bedford, Mass.) and then were further purified through a Superdex 200 HR
10/30 gel filtration column (Amersham Biosciences) with a mobile phase of 50
mM Tris-HCl (pH 8.0) containing 0.15 M NaCl at a flow rate of 0.6 ml/min. The
molecular mass of native ImpTkwas determined from a calibration curve con-
structed with the standard proteins ferritin (440 kDa), catalase (232 kDa),
ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa).
The protein concentration was determined with the Bio-Rad (Hercules, Calif.)
protein assay system, with bovine serum albumin as a standard.
Overexpression of fbpTkin E. coli and purification of the recombinant protein
were performed as described previously (29).
IMPase and FBPase assay. The IMPase activities of the recombinant proteins
were determined by measuring the release of free Pifrom D-myo-inositol-1-
monophosphate (IMP) (Sigma, St. Louis, Mo.) by the Malachite Green proce-
dure (17). The reaction was performed in a 100-?l mixture containing 50 mM
Tris-HCl (pH 8.0), 20 mM MgCl2, 2 mM IMP, and protein solution. The assay
mixture without the substrate was preincubated at 85°C for 3 min, and the
reaction was started by the addition of IMP. After 1 min at the same tempera-
ture, the reaction was stopped by rapid cooling on ice for 5 min. Next, 20 ?l of
0.2% Tween 20 and 500 ?l of Malachite Green-ammonium molybdate solution
(5 ml of 35% HCl, 562 mg of (NH4)6Mo7· 4H2O [Wako Pure Chemicals, Osaka,
Japan], and 75 mg of Malachite Green [Sigma] per 50 ml) were added to a
portion of the reaction mixture (95 ?l). The increase in liberated Piwas deter-
mined by measuring the increase in absorbance at 620 nm derived from the
formation of a complex between molybdophosphoric acid and Malachite Green.
The spontaneous increase in released Pidue to the thermal decomposition of
IMP was subtracted from the datum of each experiment. To determine the
activities in cell extracts from T. kodakaraensis strains, we cultivated cells in
ASW-YT medium supplemented with 5.0 g of soluble starch/liter or 5.0 g of
sodium pyruvate/liter at 85°C for 15 h and prepared cell extracts from cells in
early stationary phase as described previously (33). IMPase activity in the ex-
tracts was measured by the Malachite Green procedure as described above, with
modifications in the concentration of IMP (4 mM) and the reaction time (15
min). Alternatively, NAD-dependent inositol dehydrogenase was applied as a
specific coupling enzyme (19). In this discontinuous assay, the first reaction with
the extracts was performed at 85°C as described above. The second reaction,
containing the coupling enzyme, was carried out in the presence of 200 mM
NaCl, 0.2 U of inositol dehydrogenase (Sigma), and 4 mM NAD?in the reaction
mixture. After 5 min at 25°C, the amount of NADH was determined by mea-
suring the increase in the absorbance at 340 nm.
FBPase activities of the recombinant proteins and those of cell extracts were
measured by a spectrophotometric assay coupled with glucose-6-phosphate
isomerase and NADP-dependent glucose-6-phosphate dehydrogenase (29).
NADPH was quantified by measuring the increase in the absorbance at 340 nm.
An experiment without FBP was used as a blank value, and the thermal decom-
position of FBP was also considered in each measurement. One unit of activity
was defined as 1 ?mol of product produced per min for both assays.
Construction of disruption vectors pUDImp and pUDFbp. Two disruption
vectors, pUDImp and pUDFbp, were constructed for the targeted disruption of
imp and fbp genes, respectively, in T. kodakaraensis by homologous double-
crossover recombination. First, we prepared a trpE marker cassette in which trpE
is oriented downstream of a putative promoter region for pyrF. The trpE coding
region was amplified from the genomic DNA of T. kodakaraensis KOD1 by the
use of primers MTRP-R and MTRP-F (5?-GGGCATATGCCTCTCAAAAAG
CTGAAGCCCGTTGAC-3? and 5?-GGGGGATCCTCATTCCCTCACCCCC
AGCGCCTTCAGA-3? [the underlined sequences indicate NdeI and BamHI
sites, respectively]). A fragment digested with NdeI and BamHI was inserted into
pUD harboring the pyrF marker cassette (33) at the corresponding sites to
replace the coding region of pyrF with trpE. The pyrF promoter-trpE fusion was
again amplified from the resulting plasmid to introduce PvuII sites at both ends
with primers PJPRO-R2 and PJTRP-F2 (5?-CAGCTGCCGCAACGCGCATT
A-3? [the underlined sequences indicate PvuII sites]), followed by insertion into
pUC118 at the HincII site. The resulting plasmid harboring the trpE marker
cassette was designated pUMT2 (4,591 bp).
For construction of the two disruption vectors, pUDImp and pUDFbp, two
DNA fragments of imp and fbp along with their flanking regions (about 1,000 bp)
for homologous recombination were amplified from the genomic DNA of T.
kodakaraensis KOD1 by the use of primers PIMP-R and PIMP-F for pUDImp
(5?-TAACGAGTGCCTTTCCAGTAAG-3? and 5?-AGTGCTCCCTTTCTTTT
GACTTTC-3?) and PFBP-R and PFBP-F for pUDFbp (5?-CTCTTGATAGAC
GGGCAGAGAAGTGTGG-3? and 5?-GCATCTGCCAGTTGAGAATCGGG
ACGAAGTCGCCC-3?). Each fragment was subcloned into pUC118 at the
HincII site. DNA fragments including the homologous regions and the entire
pUC118 plasmid, but not the target gene coding regions, were amplified with
primers PDDIMP-R and PDDIMP-F for pUDImp (5?-CTGGAGGCGATGAA
GGATGAGCC-3? and 5?-AATATCACCCCAGCAAGGCTAT-3?) and PD
DFBP-R and PDDFBP-F for pUDFbp (5?-TTTCCAGCCCTTTTCTGTTCAT
TTTACCC-3? and 5?-GGCAACCACCGGTATTTTTAACCTCT-3?). The trpE
marker cassette, excised from pUMT2 by PvuII digestion (1,423 bp), was ligated
with each resulting fragment to give pUDImp (6,630 bp) and pUDFbp (6,668 bp)
(Fig. 1). In these plasmids, the trpE marker cassette replaced the entire coding
region of fbp (1,128 bp) and most of imp (a 27-bp sequence at the 3? terminus of
imp was retained in order to not disturb a downstream open reading frame and
its putative ribosome-binding sequence overlapping with imp), with the same
orientation as the original genes.
Transformation of T. kodakaraensis. T. kodakaraensis strain KW128 (?pyrF
?trpE::pyrF), in which tryptophan auxotrophy can be complemented by the
selectable marker trpE (unpublished results), was used for the targeted disrup-
tion of fbp or imp. The transformation of KW128 was performed as reported
previously (33), with slight modifications. Approximately 4 ? 108cells at the late
exponential phase were harvested, resuspended in 200 ?l of 0.8? ASW, and kept
on ice for 30 min. After treatment with DNA and a successive heat shock, the
cells were incubated in 1.5 ml of ASW-YT medium at 85°C for 2 h. The cells that
were harvested and resuspended in 200 ?l of 0.8? ASW were then directly
spread on a selective synthetic plate medium that lacked tryptophan (ASW-
AAW?) (33), with a supplementation of 5.0 g of soluble starch/liter. After
cultivation for 5 to 8 days at 85°C, the transformants grown on the plate medium
were analyzed by colony PCR or PCR with the genomic DNA as a template and
with primers CHDIMP-R and CHDIMP-F for ?imp candidates (5?-TCTCTAC
CAGCTATTTCCTTCGTTTTTGGG-3? and 5?-AACGTCGCGCAGGAAACT
TTTGGAAAAAGC-3?) and CHDFBP-R and CHDFBP-F for ?fbp candidates
(5?-TTGAATGTCTTCTTGATGTTGGCCTGATGCGG-3? and 5?-TCTTGAT
CCTCTCTTCTTTCGGGATGTAGG-3?) as primer sets that anneal outside of
the homologous regions (Fig. 1A and B, respectively). Control experiments
without any exogenous DNA gave no tryptophan prototrophs. The gene dis-
ruptant candidates were purified by repeated selection on ASW-AAW?plate
medium with starch and then were further analyzed. In order to confirm the
complete deletion of the respective target regions, primer sets that annealed
within the target genes were applied (CHIMP-R and CHIMP-F for imp [5?-G
GACTAACGTGAGCGGAGACGTAACAAAGT-3? and 5?-ACTCCCTTCCC
CTTTCGTCCGTTACTATTC-3?] and CHFBP-R and CHFBP-F for fbp [5?-A
GGATGTTCTTTCAAAAGCAGTCGAAGATG-3? and 5?-CGCATGTATTC
GGTTATCTCAAGGGCCTTCTGGCGGG-3?]) (Fig. 1).
Hybridization analyses. Southern blot analysis was performed with 5.0 ?g of
genomic DNA digested with HindIII for ?imp-2A and ApaI for ?fbp-8J. Total
RNAs were isolated from cells of T. kodakaraensis at the early exponential phase
by use of an RNeasy Midi kit (Qiagen, Hilden, Germany). For Northern blot
analysis, 30 ?g of RNA was applied. DNAs or RNAs were separated by agarose
gel electrophoresis according to standard procedures (32) and were transferred
onto positively charged nylon membranes (Roche Diagnostics, Mannheim, Ger-
many) by vacuum blotting. The preparation of specific probes, hybridization, and
signal detection were performed with a DIG-DNA labeling and detection kit
(Roche Diagnostics) according to the instructions from the manufacturer. The
primer sets used for the preparation of trpE, imp, and fbp probes (Fig. 1) were
PROTRP-R plus PROTRP-F (5?-TCCATCATCGGGGGGAAGATCGAAGA
GC-3? and 5?-CGAACGCGTTTTTCCCCTCATCGAGTT-3?), CHIMP-R plus
CHIMP-F, and CHFBP-R plus CHFBP-F, respectively. The Northern hybrid-
VOL. 186, 2004TRUE GLUCONEOGENIC FBPase IN T. KODAKARAENSIS 5801
FIG. 1. Schematic drawing of pUDImp (A) and pUDFbp (B) for disruption of imp and fbp in T. kodakaraensis KW128. The homologous regions between the circular DNAs and the
chromosome of T. kodakaraensis are shaded. Restriction site abbreviations: A, ApaI; H, HindIII. The bold gray bar below the trpE gene indicates the region spanned by the trpE probe for
Southern blot analyses. The bold black bars below the imp and fbp genes indicate the regions spanned by the imp and fbp probes used for Northern blot analyses.
5802SATO ET AL. J. BACTERIOL.
ization experiments were performed with a common RNA-blotted membrane to
which the fbp probe was applied after stripping of the initial imp probe.
FBPase and IMPase activities of recombinant Fbp and Imp
from T. kodakaraensis. Along with fbpTk, previously identified
as a gene for a novel candidate for the true FBPase in hyper-
thermophiles (29), T. kodakaraensis possesses a gene (impTk)
corresponding to the IMPase/FBPase IV genes PF2014 from P.
furiosus (53.8% identity at the protein level) (42), MJ0109 from
M. jannaschii (48.8% identity), AF2372 from A. fulgidus
(36.4% identity), and TM1415 from Thermotoga maritima
(31.9% identity) (41). To compare the respective activities of
FbpTkand ImpTk, we overexpressed each gene in E. coli and
individually purified the recombinant proteins to apparent ho-
mogeneity, as judged by sodium dodecyl sulfate-polyacrylam-
ide gel electrophoresis (data not shown). The native molecular
mass of ImpTkwas determined to be 54 kDa by gel filtration
column chromatography. According to the subunit molecular
mass determined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (33 kDa) and that deduced from the primary
structure (28.0 kDa), a homodimeric subunit assembly was
indicated for ImpTk, as in the cases of PF2014, MJ0109, and
AF2372. FbpTkhas been determined to be a homooctamer
We then measured the phosphatase activities of the recom-
binant enzymes toward FBP and IMP at 85°C (Table 2). As
reported for orthologs from other hyperthermophiles, ImpTk
was a bifunctional enzyme exhibiting a high FBPase activity as
well as IMPase activity. In fact, the FBPase activity (52.6
U/mg) was much higher than the IMPase activity (9.5 U/mg),
as was observed for the ortholog from the closely related ar-
chaeon P. furiosus (42). In contrast, FbpTkhas been reported to
display strict substrate specificity for FBP (29), and it actually
exhibited only negligible activity on IMP. Using an enzyme-
coupled assay, we confirmed that both enzymes released the
1-phosphate group of FBP regioselectively to generate F6P.
The FBPase activity of ImpTkfollowed Michaelis-Menten ki-
netics at 85°C, without homotropic allosteric properties or
substrate inhibition. The specific activity of ImpTktowards FBP
was 2.5-fold higher than that of FbpTk(18.9 U/mg). A kinetic
analysis of the FBPase reaction of ImpTkindicated that the
enzyme exhibited a higher affinity for the substrate and a larger
turnover number than FbpTkpreviously examined at 95°C (29)
(Table 2). Even at the lower assay temperature, ImpTkexhib-
ited a higher catalytic efficiency in the FBPase reaction, with a
kcat/Kmvalue of 1,170 s?1mM?1, which was ?6.5-fold higher
than that of FbpTk.
Construction of ?imp and ?fbp strains of T. kodakaraensis.
A direct method for examining the in vivo function of a par-
ticular gene is to analyze the phenotypic changes displayed in
corresponding knockout strains. We have previously reported
targeted gene disruption in T. kodakaraensis, which is expected
to be a powerful tool for research on hyperthermophilic ar-
chaea. The gene targeting system was applied here to disrupt
imp or fbp in order to clarify which gene is mainly responsible
for gluconeogenesis in T. kodakaraensis. For this purpose, we
adopted T. kodakaraensis strain KW128 (?pyrF ?trpE::pyrF)
and trpE as a host strain and a selectable marker, respectively.
The host strain, KW128, shows tryptophan auxotrophy due to
the replacement of trpE with pyrF on the chromosome, and the
exogenous trpE gene can be used as a marker that can com-
plement the auxotrophy (unpublished results). Two disruption
plasmids, pUDImp and pUDFbp, were constructed for the
targeted disruption of impTkand fbpTk, respectively (Fig. 1).
These vectors harbored the trpE marker cassette between the
upstream and downstream flanking regions (about 1,000 bp) of
the respective gene of interest. The host strain was individually
transformed with each plasmid as described in Materials and
Methods. The resulting transformants were isolated on a se-
lective plate medium without tryptophan and with supplemen-
tation of starch. The transformation efficiencies were deter-
mined to be 120 and 213 transformants/?g of DNA/4 ? 108
cells for the disruption of impTkand fbpTk, respectively. After
a second round of selection, we isolated a candidate strain for
each disruption and designated them the ?imp-2A and ?fbp-8J
The genotypes of ?imp-2A and ?fbp-8J were confirmed by
PCR, sequencing, and Southern blot analyses. PCR analyses of
the ?imp-2A and ?fbp-8J strains with primer sets that an-
nealed outside of the homologous regions (Fig. 1) resulted in
the amplification of fragments corresponding to the loci of
?imp::trpE (3,499 bp) and ?fbp::trpE (3,575 bp), respectively,
which were formed by double-crossover recombination (Fig.
2A and B). In both cases, no amplification was observed by
PCRs using primer sets that annealed within the respective
target genes (data not shown), indicating a complete deletion
of the target genes and the absence of contaminant strains
harboring the target genes. The replacement of imp and fbp
with the trpE marker in the respective transformants was also
confirmed by sequencing analysis of the targeted regions. Fur-
thermore, we performed a Southern blot analysis with a trpE
probe (Fig. 1). As shown in Fig. 2C and D, the wild-type KOD1
strain showed single signals deriving from the endogenous trpE
gene in the trp operon, while no positive signal could be de-
tected in the host strain KW128 with a ?trpE::pyrF genotype.
For the ?imp-2A and ?fbp-8J strains, the signals could be
detected with expected mobilities corresponding to a trpE in-
sertion within the targeted regions (2.8 kbp for ?imp-2A [Fig.
2C] and 11.4 kbp for ?fbp-8J [Fig. 2D]). The absence of other
signals confirmed the unique occurrence of the desired gene
replacement without unintended nonhomologous recombina-
tion in both disruptants.
Growth properties of the disruptants. The host strain and
the constructed disruptants were preincubated in an ASW-YT
TABLE 2. IMPase and FBPase activities and kinetic parameters of
recombinant ImpTkand FbpTk
9.5 ? 0.3
0.05 ? 0.01
52.6 ? 0.3
18.9 ? 0.7
0.02 ? 0.00
23.4 ? 0.4
aIMPase activity was determined by measuring released Piby the Malachite
Green method with 2 mM IMP at 85°C.
bFBPase activity was determined by a coupling assay with 2 mM FBP at 85°C.
cMeans and standard deviations were obtained from three independent ex-
dPreviously determined at 95°C (29).
VOL. 186, 2004 TRUE GLUCONEOGENIC FBPase IN T. KODAKARAENSIS5803
medium followed by cultivation in synthetic ASW-AA medium
containing 20 amino acids supplemented with soluble starch
(glycolytic conditions) or pyruvate (gluconeogenic conditions)
to investigate the contribution of the impTkand/or fbpTkgene
to gluconeogenesis. As shown in Fig. 3, all of the strains
showed comparable growth on starch as a glycolytic substrate.
On the other hand, under gluconeogenic conditions with pyru-
vate and 20 amino acids, the ?fbp-8J strain was not able to
grow, in contrast to the unimpaired growth of KW128 and
?imp-2A. The same results were also obtained for cultivation
in ASW-AA medium without the addition of pyruvate, in
which the amino acids can be utilized as gluconeogenic sub-
strates. PCR analyses confirmed that the gluconeogenic growth
of ?imp-2A was not due to contamination by any imp?strain,
such as the host strain (data not shown). These results clearly
demonstrate that fbp is an essential gene for the growth of T.
kodakaraensis under gluconeogenic conditions, whereas imp is
not only irrelevant to gluconeogenesis, but is also incapable of
complementing the defect of fbp.
Enzyme assay in cell extracts from disruptants. Cell extracts
of the host strain and the two disruptants were prepared from
cells grown in a nutrient-rich ASW-YT medium with starch or
pyruvate. FBPase activity in the extracts was determined by a
coupled assay. The host strain KW128 showed an FBPase
activity of 0.37 ? 0.02 U/mg under gluconeogenic conditions,
while the activity could not be observed under glycolytic con-
ditions (?0.01 U/mg), probably due to the strict glucose re-
pression of the gene, as reported previously (29). Likewise,
there was no detectable FBPase activity in the two disruptants
in the presence of starch. With respect to the cells grown on
pyruvate, ?imp-2A cells exhibited FBPase activity (0.34 ? 0.01
U/mg) comparable to that in the host strain, whereas activity
could not be detected in ?fbp-8J cells under the same condi-
tions. These results indicate that most of the FBPase activity
within the cells derives from the fbpTkproduct and also imply
that no other candidate for FBPase is present in this organism.
Furthermore, the results were also in agreement with the
growth properties of each strain described above. Note that the
?fbp-8J strain, which did not show gluconeogenic growth in the
synthetic medium, could grow in the rich ASW-YT medium
without supplementation of starch. This is presumed to be due
to the presence of some glycolytic substrates in yeast extract, a
component of the medium, to an extent that they sustain the
growth of strains that are deficient in gluconeogenesis. We
further examined IMPase activity in the cell extracts by mea-
suring the release of Pifrom IMP by the Malachite Green
method. We found that the levels of IMPase activity were
below the detection limit (?0.01 U/mg) in all extracts, even
those from strains KW128 and ?fbp-8J, which harbored the
FIG. 2. (A) Amplification of imp locus from T. kodakaraensis KOD1, KW128, and ?imp-2A, with CHDIMP-R and CHDIMP-F as primers.
(B) Amplification of fbp locus from T. kodakaraensis KOD1, KW128, and ?fbp-8J, with CHDFBP-R and CHDFBP-F as primers. (C) Southern
blot analysis using the trpE probe with genomic DNAs of KOD1, KW128, and ?imp-2A digested with HindIII. (D) Southern blot analysis using
the trpE probe with genomic DNAs of KOD1, KW128, and ?fbp-8J digested with ApaI. The region corresponding to the trpE probe is indicated
in Fig. 1. M, DNA size marker (HindIII-digested ? DNA).
FIG. 3. Growth properties of T. kodakaraensis KW128, ?imp-2A,
and ?fbp-8J under glycolytic (open symbols) or gluconeogenic (closed
symbols) conditions. The cells were cultured in ASW-AA medium
supplemented with soluble starch or pyruvate at 85°C. Symbols: open
circles, KW128 with starch; open squares, ?imp-2A with starch; open
triangles, ?fbp-8J with starch; closed circles, KW128 with pyruvate;
closed squares, ?imp-2A with pyruvate; closed triangles, ?fbp-8J with
pyruvate. Error bars represent standard deviations for repeated inde-
5804SATO ET AL.J. BACTERIOL.
intact imp gene. With cell extracts, this method was hampered
to some extent by the high levels of IMP-independent Pithat
were present and produced during incubation at a high tem-
perature. We therefore examined an alternative assay using
NAD-dependent inositol dehydrogenase as a specific coupling
enzyme. Even with this second method, the same results were
obtained. The low levels of IMPase activity indicate that the
contribution of ImpTkto the total amount of intracellular FB-
Pase activity is negligible, or at most very limited, regardless of
the high catalytic efficiency for the FBPase reaction observed
for recombinant ImpTk.
Transcriptional analysis of impTkand fbpTk. The transcrip-
tional profiles of impTkand fbpTkwere investigated by North-
ern blot analysis. The regions spanned by specific probes for
impTkand fbpTkare displayed in Fig. 1. Total RNAs were
isolated from cells grown in rich ASW-YT medium with starch
or pyruvate. A positive signal was not detected for the imp
probe with RNA from ?imp-2A (Fig. 4A) or for the fbp probe
with ?fbp-8J RNA (Fig. 4B), consistent with the complete
deletion of the respective target genes by homologous recom-
Compared with the clear signals for fbp with KW128 and
?imp-2A cells grown under gluconeogenic conditions, the sig-
nals with cells grown on starch were highly reduced, as shown
in Fig. 4B. This observation confirmed that the previously
described transcriptional repression of fbp found in the wild-
type cells (29) also occurred in these strains and coincided with
the results of the enzyme assay described above. Although
IMPase activity could hardly be detected in the cell extracts,
the use of the imp probe enabled us to identify transcripts of
the gene in KW128 and ?fbp-8J cells (Fig. 4A). However, the
signals were extremely weak and were detectable only after a
prolonged chromogenic reaction (?10 times longer than that
applied for the fbp probe), indicating much lower levels of
transcription of impTkthan of fbpTk. This weak transcription
was estimated to be a primary reason for the low intracellular
IMPase activity and the inability of the gene to complement
the defect of fbp. Unlike fbpTk, the transcription of impTkwas
constitutive, without regulation dependent on the carbon
The signal length for fbpTk(1.3 kb) corresponded to a mono-
cistronic transcript from the 1,128 bp fbpTkgene (Fig. 4B),
while the 2.6-kb signal length for impTksuggested a tricistronic
transcription of impTk(771 bp) together with an upstream (810
bp) and a downstream (621 bp) gene encoding uncharacterized
membrane proteins, both of which are probably unrelated to
sugar metabolism (deduced from T. kodakaraensis genome
analysis). The monocistronic transcription of fbpTkimplies that
the phenotype of the ?fbp-8J strain can be attributed to the
disruption of the fbpTkgene per se, most likely ruling out polar
effects accompanied by the gene disruption. We also observed
that in the ?imp-2A and ?fbp-8J strains, no remarkable en-
hancement of transcription occurred for one gene in the ab-
sence of the other. This fact and the different transcriptional
profiles of these genes demonstrate that the transcriptional
regulation of fbpTkand impTkare independent from one an-
The results obtained in this study indicate the following
points. (i) fbpTkis no doubt an indispensable gene for glucone-
ogenesis, and almost all FBPase activity within the cells derives
from fbpTk. (ii) impTkwas unable to complement the defect of
fbpTkin ?fbp-8J cells, demonstrating that the gene does not
participate in gluconeogenesis, in spite of the fact that the
recombinant protein of impTkexhibited a higher kcat/Kmvalue
for FBP than that of fbpTk. These results clearly provide evi-
FIG. 4. Northern blot analysis with imp probe (A) and fbp probe (B). Total RNAs were isolated from cells of strains KW128, ?imp-2A, and
?fbp-8J grown in ASW-YT medium supplemented with pyruvate (P) or starch (S). The regions corresponding to the respective probes are
indicated in Fig. 1. Each lane contained 30 ?g of total RNA. The signal intensities between the panels cannot be directly compared due to the
prolonged chromogenic reaction time (?10 times longer) for panel A compared to that for panel B. Numbers on the left are lengths of RNA size
markers (in bases).
VOL. 186, 2004 TRUE GLUCONEOGENIC FBPase IN T. KODAKARAENSIS 5805
dence that the true FBPase for gluconeogenesis in the hyper-
thermophilic archaeon T. kodakaraensis is the structurally di-
vergent FBPase encoded by fbpTk, not IMPase/FBPase IV. The
transcriptional profile of fbpTkwas also in good agreement with
the physiological function elucidated here. Although the pro-
tein product is a nonallosteric enzyme (29), the observed tran-
scriptional regulation of the gene allows fbpTkto play an im-
portant role in controlling the flux of gluconeogenesis. As
described previously, the divergent FBPase is not unique to T.
kodakaraensis; its orthologs (COG1980) are highly conserved
in hyperthermophiles (29). In contrast, the IMPase/FBPase IV
orthologs (COG0483) are widely distributed in organisms that
are unrelated in terms of domain classification and growth
temperature. As mentioned above, none of the 17 (hyper)ther-
mophiles whose genomes have been sequenced (including T.
kodakaraensis) harbor a classical FBPase. Among them, the
FbpTkortholog is present in 16 strains, including Aquifex ae-
olicus and Thermoanaerobacter tengcongensis, belonging to the
domain Bacteria, and 13 of the 16 strains also possess IMPase/
FBPase IV. The results obtained in this study imply that the
FbpTkorthologs in these (hyper)thermophiles most likely fulfill
the gluconeogenic role in vivo. Therefore, we propose that
orthologs of FbpTkshould be classified as class V FBPases,
representing the true gluconeogenic FBPases of (hyper)ther-
mophiles. At present, the bacterium Thermotoga maritima is
the only exception that exhibits hyperthermophily without an
obvious FBPase V ortholog. Since an IMPase/FBPase IV or-
tholog is present in its genome, that protein may function as
the gluconeogenic FBPase in Thermotoga maritima, or alter-
natively, a further divergent class of FBPase may exist in the
organism. The FBPase V ortholog is also absent from the three
mesophilic archaea Halobacterium sp. strain NRC-1, Methano-
sarcina acetivorans, and Methanosarcina mazei. However, these
archaea possess orthologs for classical FBPases (FBPase I in
Halobacterium sp. and FBPase II in Methanosarcina species)
that can fulfill this step in gluconeogenesis. These facts imply
that FBPase V is a (hyper)thermophile-specific enzyme rather
than an archaeal enzyme. It has been reported that COG1980
of FBPase V is one of the most striking COGs (clusters of
orthologous groups of proteins) whose presence is biased to-
ward hyperthermophiles, after reverse gyrase (22). It can be
speculated that FBPase V has structural features that limit
efficient functioning of the protein to high temperatures, and
thereby the enzyme is replaced by the structurally distinct
FBPases in mesophiles, or vice versa.
Despite the high catalytic efficiency of the ImpTkprotein for
the FBPase reaction in vitro, the enzyme cannot fulfill a glu-
coneogenic function in vivo. Northern blot analysis revealed
that impTkwas transcribed in T. kodakaraensis, as in the case of
IMPase/FBPase IV in P. furiosus (42). However, the constitu-
tive transcription of impTkwas estimated to be very weak.
IMPase activity was also trivial in all strains examined. At least
under the conditions examined in this study, the short supply of
ImpTkprotein is the main reason why the protein is unable to
function as a gluconeogenic enzyme in vivo. However, we can-
not exclude the possibility that the FBPase activity of the
weakly expressed protein is specifically suppressed by an un-
known mechanism in the cell.
IMPase in mammalian cells supplies myo-inositol from IMP
to synthesize phosphatidylinositols together with CDP-diacyl-
glycerol. In E. coli, the SuhB protein, which is orthologous to
eukaryotic IMPase, actually exhibits IMPase activity (25), but
the major role of this protein in vivo is suggested to be the
posttranscriptional control of gene expression (15, 16, 40, 43).
On the other hand, IMPase in some hyperthermophiles has
been thought to be related to the biosynthesis of di-myo-ino-
sitol-1,1?-phosphate (DIP), which is quite different from its
role in mammalian counterparts. DIP has been found in vari-
ous kinds of hyperthermophiles, e.g., Pyrococcus (23, 35), Ther-
mococcus (20), Methanococcus (4), and Thermotoga (24). This
unique compatible solute was presumed to serve as an os-
molyte against extracellular stresses such as high salinity or a
high growth temperature. Two DIP biosynthesis pathways have
been proposed, both of which share a common first step in
IMP formation from glucose-6-phosphate by IMP synthase
(EC 220.127.116.11). DIP was generated from myo-inositol and CDP-
inositol in Methanococcus igneus (3), while the coupling of two
IMP molecules in an NTP-dependent manner generated DIP
and Piin Pyrococcus woesei (36). IMPase activity is required for
the production of myo-inositol from IMP in the former path-
way. Although such DIP accumulation has not yet been exam-
ined in T. kodakaraensis, there is a possibility that imp may
function in DIP biosynthesis. The independent transcriptional
regulation of impTkfrom that of fbpTkdoes not contradict this
supposition. If this were the case, the transcription of imp in T.
kodakaraensis might be up-regulated in response to extracel-
lular stresses. Alternatively, ImpTk, with its broad substrate
specificity, might dephosphorylate other compounds in differ-
ent pathways, or as in the case of E. coli SuhB (2), it may
display a distinct function that is unrelated to its apparent
phosphatase activity. Further detailed analyses of the ?imp-2A
strain will help to clarify the function of the archaeal IMPase.
This study was supported by a grant-in-aid for scientific research to
T. I. (no. 14103011) and was partly supported by a grant-in-aid for
JSPS fellows to T.S. (no. 15005649) from the Ministry of Education,
Science, Sports, Culture, and Technology.
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