Glycolytic Control Mechanisms2310 Glycolytic Control Mechanisms. I Vol. 240, No. 6
Morales, J., Hashimoto, M. , Williams, T., Hirawake-Mogi, H., · Relocation of the first 7 of 10...
Transcript of Morales, J., Hashimoto, M. , Williams, T., Hirawake-Mogi, H., · Relocation of the first 7 of 10...
Morales, J., Hashimoto, M., Williams, T., Hirawake-Mogi, H.,Makiuchi, T., Tsubouchi, A., Kaga, N., Taka, H., Fujimura, T., Koike,M., Mita, T., Bringaud, F., Concepción, J. L., Hashimoto, T., Embley,T. M., & Nara, T. (2016). Differential remodelling of peroxisomefunction underpins the environmental and metabolic adaptability ofdiplonemids and kinetoplastids. Proceedings of the Royal Society B:Biological Sciences, 283(1830).https://doi.org/10.1098/rspb.2016.0520
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Differential remodelling of peroxisome function underpins the environmental
and metabolic adaptability of diplonemids and kinetoplastids
Short title: Gluconeogenic compartment in diplonemids
Jorge Morales1,9, Muneaki Hashimoto1,11, Tom A. Williams2,3,11, Hiroko Hirawake-Mogi1,11,
Takashi Makiuchi1,10, Akiko Tsubouchi1, Naoko Kaga4, Hikari Taka4, Tsutomu Fujimura4, Masato
Koike5, Toshihiro Mita1, Frédéric Bringaud6, Juan L. Concepción7, Tetsuo Hashimoto8, T. Martin
Embley2 and Takeshi Nara1*
1Department of Molecular and Cellular Parasitology, 4Division of Proteomics and Biomolecular
Science, and 5Department of Cellular and Molecular Neuropathology, Juntendo University
Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
2Institute for Cell and Molecular Biosciences, Catherine Cookson Building, Framlington Place,
Newcastle University, Newcastle upon Tyne NE2 4HH, UK
3School of Earth Sciences, University of Bristol, Bristol BS8 1TG, UK.
6Centre de Résonance Magnétique des Systèmes Biologiques (RMSB) UMR 5536 CNRS
Université de Bordeaux, France
7Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes,
Mérida 5101, Venezuela
8Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai,
Tsukuba, Ibaraki 305-8572, Japan
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*Correspondence: [email protected]
Footnote
9Present address: Emmy Noether-Gruppe “Microbial Symbiosis and Organelle Evolution”,
Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
10Present address: Department of Infectious Diseases, Tokai University School of Medicine,
Isehara, Kanagawa 259-1193, Japan
11These authors contributed equally to this work.
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Abstract
The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic
biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved
specialised peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway
as well as other enzymes, which underpin the physiological flexibility of these major human
pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most
abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalisation of
gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine
diplonemid, Diplonema papillatum. Our results suggest that peroxisome modification was
already underway in the common ancestor of kinetoplastids and diplonemids, and raise the
possibility that the central importance of gluconeogenesis to carbon metabolism in the
heterotrophic free-living ancestor may have been an important selective driver. Our data indicate
that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a
factor in the success of their free-living euglenozoan relatives.
Keywords
organelle evolution; metabolic compartmentalisation; peroxisomes; glycolysis; diplonemids;
kinetoplastids
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1. Introduction
Glycolysis (the Embden–Meyerhof-Parnas pathway) and gluconeogenesis are common to all
domains of life and play fundamental roles in essential cellular processes. Glycolysis consists of
10 enzymatic steps and generates ATP, NADH, and intermediate metabolites for a variety of
metabolic processes, while gluconeogenesis produces glucose 6-phosphate (G6P) from other
carbon sources including lipids and amino acids [1]. Glycolysis takes place in the cytosol but
glycolytic enzymes have also been associated with the plasma membrane of erythrocytes [2] and
with mitochondria [3, 4]. In addition, enzyme isoforms with non-glycolytic roles are found to
locate to other compartments [5].
Relocation of the first 7 of 10 glycolytic enzymes to microbody-like organelles, called
glycosomes, was first demonstrated in the kinetoplastid parasite, Trypanosoma brucei [6], and
subsequently in other kinetoplastids [7]. So far the kinetoplastids, which comprise bodonids and
trypanosomatids, are the only organism known to sequester a successive glycolytic cascade into
organelles. These enzymes are targeted to glycosomes by a peroxisomal targeting signal (PTS),
implying that glycosomes originated as modified peroxisomes. Glycosomes also contain enzymes
of other pathways including gluconeogenesis, the pentose phosphate pathway, energy/redox
metabolism, and nucleotide synthesis [7]. Glycosomes are thus a classic example of how complex
and physiologically important metabolic pathways can be relocated during evolution.
The relocation of entire metabolic pathways in eukaryotes is rarely observed, because
isolation of the individual pathway enzymes by transfer from one compartment to another can
abolish otherwise tightly coordinated enzymatic cascades. To overcome these difficulties, a
“minor-mistargeting” mechanism has been proposed to allow for the dual location of pathways
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[8]. According to this hypothesis, the imperfect targeting of PTS-tagged enzymes to peroxisomes
would allow for the retention of functional glycolysis in the cytosol until the newly routed
peroxisomal enzymes could reconstitute the pathway. This hypothesis provides a plausible
mechanism for how functional retargeting could occur, but does not identify the physiological
conditions or selective pressures that might drive the initial evolution of compartmentalisation.
The phylum Euglenozoa currently contains the euglenoids, diplonemids and kinetoplastids,
with diplonemids the sister group of kinetoplastids and euglenoids the outgroup to both [9]. In
euglenoids, glycolysis occurs in the cytosol but some enzymes are also located to its secondary
plastid to participate in the Calvin cycle, as in plants and green algae [10, 11]. In the free-living
marine diplonemid Diplonema papillatum, we have previously reported that fructose-1,6-
biphosphate aldolase (FBPA), the 4th enzyme of glycolysis, has a type-2 PTS (PTS2) and is
compartmentalized [12], suggesting that the transition to glycosomes may have already occurred
in the common ancestor of diplonemids and kinetoplastids.
2. Materials and methods
(a) Organisms
Diplonema papillatum (strain ATCC® 50162) and the kinetoplastid Trypanosoma cruzi
(Tulahuen stock), were grown axenically in ATCC® 1532 and 1029 media, respectively. ATCC
1532® medium contains 0.1% (w/v) tryptone and 1% horse serum, corresponding to 10 mM
amino acid ingredients and 40-60 µM glucose, respectively. ATCC 1029® LIT medium is a rich
medium that contains 6 mM glucose and 0.9% (w/v) liver infusion broth/0.5% tryptone,
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equivalent to > 100 mM amino acid ingredients. All experiments were carried out using the
standard medium unless otherwise stated.
(b) Draft genome sequencing of D. papillatum
D. papillatum genomic DNA was extracted using standard protocols, purified on agarose gel to
remove mitochondrial DNA, and used for genome sequencing. The D. papillatum genomic DNA
was sequenced using a HiSeq 2000 apparatus (Illumina K.K., Tokyo, Japan) by Takara Bio Inc.,
Shiga, Japan and Eurofins Genomics K.K., Tokyo, Japan. Total reads were 21.5 Gb and the
estimated genome size was 176.5 Mb. The D. papillatum open reading frames for genes of
interest were identified using TBLASTN. We predicted PTS1 by the Prosite pattern PS00342 for
the C-terminal tripeptides, [-(STAGCN)-(RKH)-(LIVMAFY)], and the PTS 1 Predictor
(http://mendel.imp.ac.at/pts1/) [13]; we required a positive identification from both methods to
diagnose the presence of a bona fide PTS. The presence of a PTS2 was manually inspected based
on the N-terminal nonameric consensus sequence, (RK)-(LIV)-X5-(QH)-(LA) [14]. The T.
brucei glycosomal proteins obtained by detailed proteomic analysis were used as references [15].
(c) Phylogenetics
We used the D. papillatum sequences as queries in BLASTP searches against the other
euglenozoan genomes and a broad selection of outgroups. The Euglena gracilis PFK sequence
was obtained from http://euglenadb.org/. Sequences were aligned with Meta-Coffee [16]. Poorly-
aligning regions were masked using the “automated1” option in trimAl [17]. Phylogenies were
built using the LG [18], C20, and C60 [19] in PhyloBayes. The trees inferred under the three
methods were very similar, with no differences impacting on our inferences of the relationships
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among the euglenozoans, and the consensus trees in figures 1, S1 to S5 and S8 to S10 were
inferred using the C60 model.
(d) Indirect immunofluorescence analysis
Antisera to glycolytic enzymes were obtained as described in supplementary materials and
methods. Indirect immunofluorescence analysis was performed as described [12]. Briefly, cells
were fixed with 2% paraformaldehyde, probed with antisera to each glycolytic enzyme, and then
treated with anti-rabbit Alexa-fluor 488-conjugate (Life Technologies), followed by
counterstaining of the nucleus with Hoechst 33342 dye. Fluorescence was observed using
fluorescence microscopy (Axio Imager M2, Carl Zeiss Co. Ltd., Tokyo, Japan).
(e) Glucose and amino acid consumption
D. papillatum cells of the mid-log phase were cultured in ATCC® 1532 medium supplemented
with 6 mM D-glucose, while T. cruzi cells were cultured in LIT medium. The glucose
concentration of the culture supernatant was measured using a Glucose-Oxidase assay kit
(Sigma-Aldrich). Amino acid consumption by cells was evaluated by the increase of NH4+ in the
culture supernatant using the Ammonia assay kit (Sigma-Aldrich).
(f) Metabolic labeling
Cells in the mid-log phase of growth were starved in artificial seawater (ATCC® 1405 HESNW
medium) for 2 hrs and then incubated with 6 mM 13C6 D-glucose or 6 mM 13C5 L-glutamine
(Sigma-Aldrich) in artificial seawater for 0, 4, and 8 hrs or with 30 mM 13C6 D-glucose in
ATCC® 1532 medium for 72 hrs without starvation. The detailed experimental procedures for
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extraction of metabolites and analysis by CE-Q-TOFMS and LC-MS were described in the
supplementary materials and methods.
3. Results
(a) Identification of peroxisome targeting signals in the glycolytic enzymes of D. papillatum
To investigate glycolytic compartmentalisation in diplonemids, we sequenced the genome of D.
papillatum and identified all of the genes for glycolysis (table 1). We identified putative PTS for
2 types of hexose kinase, namely a high-affinity type hexokinase (HK) and a low-affinity type
glucokinase (GLK), and for phosphoglucose isomerase (PGI) and phosphoglycerate kinase
(PGK), similar to kinetoplastid homologues. We identified two homologues of
phosphofructokinase (PFK), PFK1 and PFK2, in the genome assembly. PFK1 possesses a
predicted PTS1 but is likely a pseudogene or a bacterial contaminant (see below). We also found
a PTS for triose-phosphate isomerase (TIM), despite of the absence of an apparent PTS in the
kinetoplastid TIM. By contrast, putative PTS were absent from the predicted coding sequences of
PFK2, phosphoglycerate mutase (PGAM, 2,3-bisphosphoglycerate-independent type), enolase
(ENO), pyruvate kinase (PK) and two isoforms of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
Phylogenetic analysis demonstrated that most of these enzymes (HK, GLK, PGI, PGK,
ENO and PK) were present in the common ancestor of D. papillatum and kinetoplastids (figures
S1, S2, and S3). The phylogeny for TIM was weakly supported and hence cannot reject the
hypothesis of a single common origin for TIM (figure S1d). In contrast, three glycolytic enzymes
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– PFK1 and PFK2, the two isoforms of GAPDH, and PGAM – appear to have separate
evolutionary origins for D. papillatum and the kinetoplastids (figures 1, S1e, and S4).
The protein phylogeny for PFK revealed that E. gracilis and kinetoplastids are
monophyletic with high posterior support (PP = 0.99), suggesting their shared evolutionary origin
(clade A, figure 1). By contrast, D. papillatum PFK2 clusters with a PFK homologue from the
haptophyte Emiliania huxleyi (PP = 1) and is nested within a different eukaryotic clade (Clade B,
PP = 0.99), suggesting replacement of the ancestral euglenozoan PFK by lateral gene transfer. D.
papillatum PFK1 detected in our genome assembly is likely a pseudogene or a bacterial
contaminant: it does not group with other eukaryotic PFKs in the tree; the putative pfk1 gene
lacks a splice acceptor site at its 5’-untranslated region, which is essential for trans-splicing in
euglenozoans; and we were unable to detect any PFK activity in the D. papillatum lysate (table
1). In summary, our data suggest that PTS-driven relocation of most, but not all, glycolytic
enzymes had already occurred in the common ancestor of diplonemids and kinetoplastids.
We also investigated the presence of a PTS in the non-glycolytic enzymes of D. papillatum.
Among 18 PTS-harboring enzymes specific to kinetoplastid glycosomes, only three of these
enzymes were also predicted to have PTS in D. papillatum, which participate in gluconeogenesis
and energy metabolism (tables 1 and S1). These include the gluconeogenic fructose-1,6-
biphosphatase (FBPase) that catalyzes the reverse reaction to PFK, ATP-producing glycerol
kinase (GK) and pyruvate phosphate dikinase (PPDK). None of the enzymes identified for the
Diplonema pentose phosphate pathway (PPP), pyrimidine biosynthesis, or purine salvage were
predicted to locate to peroxisomes. Notably, despite of the presence of PTS1, the protein
phylogeny of FBPase revealed different evolutionary origins between D. papillatum and
kinetoplastids, implying independent LGT events and convergent PTS-driven relocation of
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FBPase in D. papillatum and kinetoplastids (table 1 and figure S5). Our data suggest that
metabolic compartmentalisation originated in association with glycolysis, gluconeogenesis and
energy metabolism in the common ancestor of D. papillatum and kinetoplastids.
(b) Localisation of PTS-harboring glycolytic enzymes in the peroxisomes of D. papillatum
To investigate the expression and confirm the cellular location of the D. papillatum glycolytic
enzymes, we raised antisera to 11 glycolytic enzymes including GLK. Western blot analysis of
the D. papillatum extracts showed the expected band for all enzymes tested, with the exception of
the two homologues of PFK and HK (figure S6a). The absence of the signals for both PFK1 and
PFK2 suggests that neither of the two PFK homologues is expressed or functional in the culture
condition of D. papillatum, consistent with the lack of PFK activity. To confirm the absence of
HK, we measured hexose kinase activity in D. papillatum cell extracts and found that the Km
value for glucose of the native enzyme harboring hexose kinase activity was 0.33 mM, whereas
the Km values of the recombinant HK and GLK were 0.064 and 0.66 mM, respectively. These
results suggest that the native hexose kinase activity is likely attributable to GLK in D.
papillatum under the experimental conditions.
Indirect immunofluorescence analysis (IFA) was performed to investigate the cellular
location of the glycolytic enzymes expressed in D. papillatum. Our previous study showed that
our mouse antiserum to D. papillatum FBPA detected a single band on western blots of the
membrane-rich fraction (10,500 x g sediment) from the D. papillatum extract and also
demonstrated the punctate pattern in the cell by IFA [12]. Immuno-electron microscopy using the
same antiserum also detected labeling of FBPA inside single-membrane compartments with the
characteristic morphology of peroxisomes (figure S6b). Therefore, we used FBPA as a
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peroxisomal marker. The signals for GLK, PGI, TIM, and PGK co-localised with that of FBPA,
indicating their shared peroxisomal location (figure 2). The signal for the anti-GAPDH1 antibody
gave a patchy distribution in the cytosol but did not co-localise with FBPA, consistent with the
absence of a PTS. PTS-lacking PGAM, ENO, and PK were also exclusively detected in the
cytosol of D. papillatum, similar to their kinetoplastid PTS-lacking homologues.
(c) Preference of amino acids as carbon source in D. papillatum
The failure to detect PFK activity in D. papillatum suggests that the glycolytic enzymes together
with FBPase play a replenishing role for gluconeogenesis in this organism. Notably, D.
papillatum can grow with a trace level of glucose. The modified ATCC 1532 medium
supplemented with 1% dialyzed fetal bovine serum (Thermo Fisher Scientific Inc.), which
corresponds to < 2.7 µM glucose, can also support the normal growth of D. papillatum (data not
shown). However, the growth of D. papillatum even in the absence of glucose does not
necessarily indicate the loss of glucose uptake and glycolytic activity. Therefore, we investigated
firstly the relative preference of carbon sources in D. papillatum. Consistent with our hypothesis,
D. papillatum does not significantly consume glucose (6 mM), but preferentially uses amino
acids (figure 3a, left). This is demonstrated by the significant production of ammonium in the
presence of glucose during early exponential growth. By contrast, epimastigotes (an insect form)
of the parasitic kinetoplastid Trypanosoma cruzi prefer glucose to amino acids, as previously
reported [20]. In experiments with T. cruzi (figure 3a, right), the levels of glucose decreased
significantly at day 3 to 6 along with exponential growth and became undetectable (< 10 µM) at
day 7, while the levels of ammonium remained constantly by day 5 and then increased
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significantly. Because amino acids are the major nutritional components in the diet of phagocytic
heterotrophs like D. papillatum, this preference likely reflects nutritional adaptation.
(d) Gluconeogenesis is the central carbon metabolism in D. papillatum
To investigate the metabolic fate of amino acids, we performed tracer experiments using 6 mM
13C-uniformly labeled (13C5, +5 carbons) L-glutamine, and D. papillatum cells which had been
starved in artificial seawater for 2 hours prior to labeling. We detected specific enrichment with
+5 carbon in the α-ketoglutarate fraction, indicating that glutamine is readily catabolized and
incorporated into metabolites of the TCA cycle of D. papillatum (figures 3b and S7a). The
presence of +5 and +6 carbons in the citrate fraction also indicated an active TCA cycle and the
recycling of labeled oxaloacetate. The time-dependent increase of 13C-labeling with +1 to +6
carbons for hexose phosphates provided evidence of active gluconeogenesis. In addition, the
extensive metabolic dilution of 13C-isotopologues in the hexose 6-phosphate fractions indicated
the presence of an internal carbon source. Quantitative metabolomics demonstrated that D.
papillatum contains higher amounts of free amino acids when compared to T. cruzi, suggesting
that they may serve as a carbon reservoir (table S2). We conclude that amino acids are the
preferred carbon/energy source for D. papillatum and that they are catabolized via the TCA cycle
and can subsequently be anabolized via gluconeogenesis.
(e) The pentose phosphate pathway complements PFK-deficiency in the glycolytic process
Metabolic labeling using 6 mM 13C6-D-glucose as a sole external carbon source showed
enrichment with +1 to +5 carbons in the hexose phosphate fractions, indicating the occurrence of
glucose catabolism in D. papillatum at a very low rate (figures 3c and S7b). Importantly,
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sedoheptulose 7-phosphate (S7P), an intermediate metabolite of PPP, was significantly labeled
with +1 to +6 carbons (figure 3c). Glucose uptake by D. papillatum was further investigated by
cells incubated for 72 hrs in normal medium containing 30 mM 13C6-glucose. Under these
conditions, the levels of +6 carbon were significantly lower than that of +3 carbon in the hexose
phosphate fractions (figure S7c). These data suggest that G6P is catabolized via the PPP to
produce triose phosphates and their subsequent catabolites, which can then be recycled to form
hexose phosphate species. Quantitative determination of the glycolytic metabolites also showed a
relative accumulation of hexose phosphates in D. papillatum compared to T. cruzi, regardless of
the nutritional conditions (table S2), suggesting the predominance of anabolism towards the
production of hexose phosphates. Taken together, these data demonstrate that glycolysis does not
significantly contribute to central carbon metabolism in D. papillatum and that gluconeogenesis
from amino acids is the dominant process.
(f) Retargeting of non-glycolytic pathway enzymes to peroxisomes occurred in the common
ancestor of kinetoplastids
To investigate the evolution of kinetoplastid glycosomes after the split from the diplonemid
lineage, we inferred additional phylogenies for PTS-harboring enzymes specific to kinetoplastids.
Both the phylogeny of adenylate kinase – which catalyzes the reversible interconversion of 2
ADP into ATP plus AMP – and of inosine 5-monophosphate dehydrogenase – a purine salvage
enzyme – revealed that the glycosomal forms of these enzymes have been acquired laterally from
bacteria in kinetoplastids (figures S8 and S9). Thus, kinetoplastids encode two copies of these
genes: an ancestral copy shared with D. papillatum, and a glycosomal copy obtained by LGT.
The phylogeny of a PPP enzyme, transketolase, showed monophyly of D. papillatum and
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kinetoplastids within the eukaryotic clade, indicating that the ancestral forms of this enzyme were
retargeted to the glycosomes in the lineage leading to kinetoplastids (figure S10). The phylogeny
of other PTS-targeted enzymes was ambiguous, largely due to a lack of resolution commonly
encountered in single-gene trees. We conclude that the transfer to the glycosomes of non-
glycolytic enzymes, including those for pyrimidine biosynthesis [21], occurred on the branch
leading to the kinetoplastids after their split from the diplonemid lineage (figure 4).
4. Discussion
Recent advances in our sampling of microbial eukaryotic diversity have begun to shed light on
the unique biological features of diplonemids, not only as a relative of important human
pathogens but also as a major protistan group in ocean plankton [22, 23]. The ecological success
of diplonemids as marine plankton [23] suggests that independence from glucose accompanied
by peroxisome remodelling is a viable strategy for heterotrophic life. Our draft genome
sequencing of Diplonema provides an important source of genomic data for this group.
The phylogeny of PFK revealed that kinetoplastid and E. gracilis PFKs share the same
evolutionary origin. Therefore, it is likely that, as well as euglenoids, a common ancestor of
diplonemids and kinetoplastids utilized PFK to perform glycolysis. Whether this ancestral PFK
had already relocated to peroxisomes in their common ancestor is, as yet, unclear. By contrast, D.
papillatum PFK2 shares the same origin with a PFK homologue of the haptophyte, E. huxleyi. E.
huxleyi is the most prominent marine cosmopolitan phytoplankton and often causes
coccolithophore blooms [24]. It is possible that marine heterotrophs like Diplonema feed on
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haptophytes, which may have been an evolutionary source of LGT; alternatively, both E. huxleyi
and D. papillatum may have obtained these PFK genes by LGT from other members of clade B.
Our data demonstrate that the remodelling of peroxisome function already occurred in the
common ancestor of diplonemids and kinetoplastids, and involved the retargeting of at least a
subset of glycolytic enzymes and GK and PPDK. It is unclear whether FBPase also relocated to
peroxisomes in their common ancestor, because both FBPases have different origins. Retargeting
to peroxisomes of ATP-producing GK and PPDK may be related to the requirement of ATP
hydrolysis in the first seven enzymatic steps of glycolysis, in terms of the maintenance of ATP
homeostasis inside the peroxisomes. That is, the maintenance of the ATP levels by the action of
GK, acting in the reverse direction, and PPDK in the peroxisomes might have played an
important role in maintaining the efficiency of glycolysis and gluconeogenesis in these
organisms.
As with enzymatic composition of the ancestral glycosomes, the primary metabolic status
in the common ancestor of diplonemids and kinetoplastids is still unclear. Our metabolome data
indicate that D. papillatum lacks a fully functional glycolytic pathway. By contrast, our
phylogenetic analyses suggest that the ancestor of diplonemids and kinetoplastids likely retained
glycolysis. A possible explanation is that the gluconeogenic state of Diplonema may not be an
ancestral, but is instead a derived feature, which was accompanied by nutritional adaptations to
heterotrophic life followed by the loss of expression and activity of PFK.
From a metabolic viewpoint, it seems rational that ancestral glycosomes harbored the first
seven enzymes of glycolysis and the corresponding enzymes of gluconeogenesis for completion
of a contiguous enzymatic cascade (figure 4). D. papillatum may represent an interesting case
whereby nutritional adaptations leading to glucose-independent metabolism have evolved,
15
resulting in peroxisomes housing gluconeogenesis (“gluconeosomes”). At the same time,
preservation of glycolytic activity in kinetoplastids might have potentiated adaptation to a
different mode of life, parasitism. Parasitism has developed repeatedly among kinetoplastids, and
this adaptability is made possible by the compartmentalisation into glycosomes of metabolic
processes that have to undergo drastic and rapid changes during some transitions in the complex
life cycles [25]. Hence, the sequestering of core metabolic pathways into peroxisomes, begun in
the common ancestor of diplonemids and kinetoplastids, may have laid the foundations for the
success of a major clade of medically and economically important parasites. Indeed, high levels
of glucose (in the millimolar range) are present in the blood and body fluids of vertebrates, which
can be readily utilized by hemoflagellates as carbon source.
Data accessibility. The D. papillatum genome was deposited in DDBJ/EMBL/GenBank under
the accession LMZG00000000. D. papillatum sequences for relevant enzymes reported here have
been deposited individually at DDBJ (accession numbers AB970479 - AB970481, AB970483 -
AB970501, LC127115, LC127507). The datasets supporting this article have been uploaded as
part of the supplementary material.
Competing interest. We have no competing interests.
Author contributions. T.N. designed research; J.M., M.H., T.A.W., H.H.-M., A.T., N.K., H.T.,
T.F., M.K., and T.N. performed research; J.M., T.A.W. and T.N. analyzed data; F.B. and J.L.C.
provided materials; T.A.W., T.Ma., T.Mi., F.B., J.L.C, and T.H. helped to draft manuscript;
T.A.W. and T.M.E edited the paper; and J.M. and T.N. wrote the paper.
16
Acknowledgements. We thank Paul Michels for critical reading and the Euglena gracilis
nucleotide sequencing consortium (http://euglenadb.org/) for E. gracilis PFK sequence.
Funding. T.N. acknowledges support from grants-in-aid for scientific research (24390102 and
25670205) and from the Foundation of Strategic Research Projects in Private Universities
(S1201013) from the Ministry of Education, Culture, Sport, Science, and Technology, Japan
(MEXT). T.M.E. acknowledges support from the Wellcome Trust and the European Advanced
Investigator Programme. T.A.W. is supported by a Royal Society University Research
Fellowship.
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20
Table 1. Characteristics of glycolytic enzymes of Diplonema papillatum (Dp).
Figure Legends
Figure 1. Diplonema papillatum lacks an orthologue of the glycosome-targeted
phosphofructokinase (PFK) gene found in other euglenozoans. A Bayesian consensus phylogeny
of PFK was inferred under the C60 model in PhyloBayes. E. gracilis and kinetoplastid PFKs
form a monophyletic group (clade A) with high posterior support (PP = 0.99). By contrast, D.
papillatum PFK2 clusters with a PFK homologue from the haptophyte Emiliania huxleyi (PP = 1)
and is nested within the clade “B” (PP = 0.99). Branch lengths are proportional to the expected
number of substitutions.
Figure 2. Cellular localisation of glycolytic enzymes in Diplonema papillatum using
immunofluorescence microscopy. Cells were labeled with polyclonal antisera to GLK, PGI, TIM,
GAPDH, PGK, PGAM, ENO, or PK (Target, red) and with antisera to D. papillatum FBPA as a
peroxisomal marker (FBPA, green). Images of the same cells were merged together with the
labels of Hoechst 33342 (blue) to visualize the nucleus (Merge). DIC, differential interference
contrast image; Merge, merged image. Scale bar = 10 µm.
Figure 3. Carbon metabolism in Diplonema papillatum. (a) Consumption of glucose and amino
acids in the culture media of D. papillatum (left) and Trypanosoma cruzi epimastigotes (right).
Production of ammonium, a catabolite of amino acids, denotes the consumption of amino acids.
(b and c) Metabolome analysis of D. papillatum. D. papillatum cells were incubated in
21
conditioned marine water for 2 hrs and then supplemented with 13C5-L-glutamine (b) or 13C6-D-
glucose (c) for 0, 4, and 8 hrs. The Y-axis indicates the relative abundance (%) of each
isotopomer of the relevant metabolite. The X-axis represents the number of 13C in the
isotopomers. Data for all metabolites in these experiments are shown in figures S7a and S7b,
respectively. Data points and error bars represent the mean ± s.d. of three independent
experiments.
Figure 4. A proposed metabolic model and peroxisome remodelling in Euglenozoa. (a)
Peroxisomes (Px) and mitochondria (Mt) have been present in the last eukaryotic common
ancestor (LECA), with cytosolic location of glycolysis (blue arrows), gluconeogenesis (red
arrows), pentose phosphate pathway (PPP), pyrimidine biosynthesis (Pyr), and purine salvage
(Pur). PFK and FBPase (FBP) are critical allosteric enzymes in glycolysis and gluconeogenesis,
respectively. (b) After the divergence of the lineage leading to Euglenoids, the peroxisomes of the
common ancestor of diplonemids and kinetoplastids were remodelled through the sequestration
of a subset of glycolytic enzymes, possibly FBPase, and others (boxed in yellow). In these
remodelled compartments, both glycolysis and gluconeogenesis were functional. (c) The
ancestral diplonemids or Diplonema lost glycolysis accompanied by the loss of PFK activity as
suggested in our analysis. (d) The ancestral kinetoplastid further sequestered into peroxisomes a
part of enzymes for PPP, Pyr, Pur, and others such as PEPCK and NADH-fumarate reductase
(boxed in blue).
22
Table 1. Characteristics of glycolytic enzymes of Diplonema papillatum.
EnzymePTS
Monophyly* Activity†D. papillatum T. cruzi
1. HK PTS2 PTS2 Yes (0.85)0.92‡
1. GLK PTS1 PTS1 Yes (0.96)2. PGI PTS1 PTS1 Yes (0.93) 39.13. PFK PTS1/ND PTS1 No (0.99) ND3. FBPase (gluconeogenic) PTS1 PTS1 No (1.00) 4.74. FBPA PTS2 PTS2 Yes (1.00)§ 14.25. TIM PTS1 ND Unresolved 123.26. GAPDH ND PTS1/ND No (0.90) 48.57. PGK PTS1 PTS1/ND Yes (0.81) 103.78. PGAM ND ND No (1.00) 2,1009. ENO ND ND Yes (0.99) 3.710. PK ND ND Yes (0.98) 15.1
*Monophyly of D. papillatum and kinetoplastids with posterior probability in parenthesis based on the relevant protein phylogeny (figures 1 and supplementary figures).
†nmol/min/mg protein of the D. papillatum cell extract (see also supplemental material). ‡The activities of HK (high-affinity type) and GLK (low-affinity type) are indistinguishable. §Ref. 12. ND; not detected.
Euglenozoa
Euglenozoa
Euglenozoa(Excavata)
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Euglenozoa (Excavata)
Archaea
Archaea
Methanocella paludicola SANAERheinheimera nanhaiensis E407-8
Alteromonas macleodii str. Deep ecotypeAlteromonas macleodii
Methanospirillum hungatei JF-1Methanoculleus marisnigri JR1
Magnetococcus marinus MC-1Trichomonas vaginalis G3 (Excavata)
Saccharomyces cerevisiae S288cSaccharomyces cerevisiae AWRI1631Neurospora crassa
Haemonchus contortusLactococcus lactis
Succinivibrionaceae bacterium WG-1Oceanimonas sp. GK1
Haemophilus sputorum CCUG 13788Haemophilus ducreyi 35000HP
Actinobacillus ureaeBuchnera aphidicola (Cinara tujafilina)
Escherichia coliThalassiosira pseudonana CCMP1335
Phaeodactylum tricornutumAureococcus anophagefferens
Guillardia theta CCMP2712Emiliania huxleyiCoraliomargarita akajimensis
Agrobacterium tumefaciens F2Sinorhizobium fredii
Bodo saltans PFK1Trichomonas vaginalis (Excavata)
Trichomonas vaginalis G3Trichomonas vaginalis G3
Trichomonas vaginalisPelosinus fermentans DSM 17108
Naegleria gruberi strain NEG-MNaegleria fowleri (Excavata)
Syntrophobacter fumaroxidansAccumulibacter phosphatis
Desulfobacca acetoxidansDesulfobacter postgatei 2ac9
Desulfobacterium autotrophicumUncultured Desulfobacterium sp.Desulfococcus oleovorans
Diplonema papillatum PFK1Waddlia chondrophila 2032/99
Paramecium tetraurelia strain d4-2Toxoplasma gondii ME49
Plasmodium falciparum 3D7Cryptosporidium hominisToxoplasma gondii GT1
Plasmodium falciparum 3D7Cryptosporidium hominis
Ectocarpus siliculosusOryza sativa Japonica Group
Arabidopsis thalianaArabidopsis thaliana
Toxoplasma gondii ME49Toxoplasma gondii
Oryza sativa Japonica GroupOryza sativa
Oryza sativa Japonica GroupOryza sativaArabidopsis thalianaArabidopsis thaliana
Giardia intestinalis (Excavata)Treponema phagedenis F0421
Treponema pallidumTreponema caldaria
Solobacterium moorei F0204Holdemanella biformis
Spirochaeta thermophila DSM 6578
Entamoeba histolyticaBorellia burgdoferi
Entamoeba histolytica (Amoebozoa)Monocercomonoides sp. (Oxymonadida, Excavata)
Euglena gracilisTrypanosoma cruziTrypanosoma brucei
Leishmania major strain FriedlinCrithidia fasciculata
Bodo saltans PFK2Nakamurella multipartita
Emiliania huxleyi CCMP1516 (Haptophyta)Diplonema papillatum PFK2
Phaeodactylum tricornutum CCAP 1055/1 (Stramenopiles)Phaeodactylum tricornutum (Stramenopiles)
Ectocarpus siliculosus (Stramenopiles)Ectocarpus siliculosus (Stramenopiles)Chlamydomonas reinhardtii (Plantae)
Tetrahymena thermophila SB210 (Alveolata)Paramecium tetraurelia (Alveolata)
Arabidopsis thaliana (Plantae)Arabidopsis thaliana (Plantae)
Nicotiana tomentosiformis (Plantae)
1
0.78
0.991
0.99
0.52
0.99
0.991
1
0.55
0.57 1
0.930.99
1
0.53
0.99
0.93
10.92
0.971 1 1
0.99
0.99
0.731
0.990.98
1
1
0.730.98
11
1
0.99
0.78
0.5
0.94
0.92
0.99
0.920.91
0.87
1
0.71
0.9511
0.98
0.93
10.81
10.99
1
0.96
0.991
0.7
1
0.970.92
0.99
0.91
0.970.99
11
1
1
0.64
0.99
1
0.59
1
0.99
1
0.98
1
0.99 10.2
Clade A
Clade B
Bacteria
DIC FBPA Target Merge
PGI
TIM
PGK
GAPDH
GLK
PGAM
ENO
PK
Time (days) Time (days)
0
15
10
5
2 84 106
Con
cent
ratio
n (m
M)
104
105
106
107
Cel
l cou
nt (/
ml,
----
)D. papillatum T. cruzi
10
10
10
Cel
l cou
nt (/
ml,
----
)
Glucose 6-phosphate
0 hr 4 hr 8 hr
20
10
15
0
5
20
10
30
0
40
20
50
30
0
10
(c)R
elat
ive
abun
danc
e (%
)R
elat
ive
abun
danc
e (%
)20
10
15
0
5
(b)
+1
20
10
15
0
5
0 hr 4 hr 8 hr
20
10
15
0
5
Sedoheptulose7-phosphate
Glucose 6-phosphate
Fructose 6-phosphate
Citrateα-Ketoglutarate
(a)
Rel
ativ
e ab
unda
nce
(%)
Rel
ativ
e ab
unda
nce
(%)
– – Glucose – – Ammonium 0
5
10
15
Con
cent
ratio
n (m
M)
+1 +2 +3 +4 +5 +6 +2 +3 +4 +5
+1 +2 +3 +4 +5 +6 +1 +2 +3 +4 +5 +640
+1 +2 +3 +4 +5 +6
+1 +2 +3 +4 +5 +6
2 84 106
+7
6
7
8
– – Glucose – – Ammonium
Diplonemids(D. papillatum) Kinetoplastids
Euglenoids
LECA
Mt
Glucose
Glucose-P
PEP
Amino acids
PPP
Pyr
Pur
PFKFBP
Px
(a)
Px
Glucose
PFK
PPP
Pyr
Mt
Glucose-P
PEP
PPDKGK
FBP
Amino acids
Pur
(c)
Px
PPP
Pyr
Pur
Mt
Glucose-P
PEP
Amino acids
PFKFBP
PPDKADK
GK
Glucose
PEPCK
(d)
Px
PPP
Pyr
Mt
Glucose-P
PEP
PPDKGK
Amino acids
Pur
(b)
PFKFBP
Glucose
Differential remodelling of peroxisome function underpins the environmental
and metabolic adaptability of diplonemids and kinetoplastids
Jorge Morales, Muneaki Hashimoto, Tom A. Williams, Hiroko Hirawake-Mogi, Takashi
Makiuchi, Akiko Tsubouchi, Naoko Kaga, Hikari Taka, Tsutomu Fujimura, Masato Koike,
Toshihiro Mita, Frédéric Bringaud, Juan L. Concepción, Tetsuo Hashimoto, T. Martin Embley
and Takeshi Nara
Supplementary material
Supplementary materials and methods
(a) Enzyme activity
Cells of the mid-log phase were collected, washed twice with 10% (v/v) mannitol and
resuspended in 50 mM Tris-HCl/2 mM EDTA pH 7 with protease inhibitor cocktail (Complete,
Mini, Roche Diagnostics K.K., Tokyo, Japan). Cells were disrupted by sonication, centrifuged at
26,000 x g for 20 minutes, and the supernatant was used to measure the enzymatic activity
described as follows; hexokinase and glucokinase [1], phosphoglucose isomerase [2],
phosphofructokinase [3], fructose-1,6-biphosphate aldolase [4], GAPDH [5], triose-phosphate
isomerase [6], phosphoglycerate kinase [7], phosphoglycerate mutase [8], enolase [9], pyruvate
kinase [10], and fructose-1,6-biphosphatase [11].
(b) Antibodies
The mouse antiserum to D. papillatum FBPA was produced as described [12]. To obtain antisera
to glycolytic enzymes, the cDNA clones of interest were obtained by PCR, cloned in pET151/D-
TOPO vector, and expressed in BL21(DE3)Star cells (Life Technologies Japan Inc., Tokyo,
Japan). The recombinant proteins were purified using His•Bind® Resin (Merck KGaA,
Darmstadt, Germany) and used for raising antisera in rabbits or mice. Immunization of animals
and collection of antisera were in part carried out by Medical & Biological Laboratories Co., Ltd.,
Nagoya, Japan. All animal experiments were carried out in accordance with the fundamental
guidelines for animal experiments under the jurisdiction of the Ministry of Education, Culture,
Sports, Science and Technology (Notice No. 71, 2006), and approved by the Committee for
Animal Experimentation of Juntendo University with the Approval No. 270081.
(c) Western blots
20 µg of protein from the cell supernatant were separated onto a SuperSep® Ace 10-20 % gel
(Wako Pure Chemical Industries, Ltd., Osaka, Japan) and transferred to a polyvinylidene fluoride
membrane under semi-dry conditions at 20 V for 30 minutes. After blocking with 5% fetal
bovine serum (FBS) in phosphate-buffered saline containing 0.25 % tween20 (PBST), the
membranes were incubated overnight at 4°C with the antisera to each glycolytic enzyme in 1%
FBS/PBST, washed with PBST, and incubated with HRP-conjugated secondary antibody
(Jackson ImmunoResearch Laboratories Inc., PA), followed by chemiluminescent development.
The reaction was visualized using ImageQuant LAS-4000 min (GE Healthcare Japan Inc., Tokyo,
Japan).
(d) Electron microscopy
Cells of the late-log phase were harvested, washed twice in artificial marine water, and fixed as
described [13]. Ultrathin sections were stained with lead citrate and uranyl acetate and examined
with a transmission electron microscope at 75 kV (Hitachi H-7100, Hitachi High-Technologies
Co. Ltd., Tokyo, Japan). For immunoelectron microscopy, cells were fixed using 4%
paraformaldehyde and 1% glutaraldehyde in PBS at 4ºC for 2 hrs, dehydrated with ethanol, and
embedded in LRWhite at -20ºC for 4 days under UV-light. Ultrathin sections were incubated
with 50 mM NH4Cl in PBS for 2 hours, blocked with 3% BSA in PBS/0.025 % tween20 for 1 hr,
and incubated with antisera to D. papillatum FBPA [12] for 1 hr. Cells were then incubated with
gold-labeled anti-mouse antibody (10 nm particle, Sigma-Aldrich Co. LLC., MO), counterstained
with uranyl acetate, and observed using Hitachi H-7100.
(e) Metabolic labeling
The mid-log phase of cells were starved in artificial seawater for 2 hrs and then incubated with 6
mM 13C6 D-glucose or 6 mM 13C5 L-glutamine (Sigma-Aldrich) in artificial seawater for 0, 4,
and 8 hrs or with 30 mM 13C6 D-glucose in normal culture medium for 72 hrs without starvation.
Cells in the mid-log phase of growth were starved in artificial seawater for 2 hrs and then
incubated with 6 mM 13C6 D-glucose or 6 mM 13C5 L-glutamine (Sigma-Aldrich) in artificial
seawater for 0, 4, and 8 hrs or with 30 mM 13C6 D-glucose in normal culture medium for 72 hrs
without starvation. Cells were then washed twice with 10% w/v mannitol and treated with
methanol by vortexing for 1 min. After removing the debris by centrifugation, the supernatant
was extracted with 1:0.4 of chloroform and milli-Q water by vortexing for 1 min. The aqueous
phase was filtered through an Ultrafree MC PHLCC HMT filter (Merck KGaA, Darmstadt,
Germany) having a cut-off of 5 kDa at 9,400 x g for 2 hrs to remove proteins. The eluate
containing the metabolites was evaporated and dissolved in 0.05 ml of Milli-Q water. CE-Q-
TOFMS analysis was performed using an Agilent CE system combined with a Q-TOFMS
(Agilent Technologies, Palo Alto, CA, USA) as described [14-16]. The data obtained by CE-Q-
TOFMS analysis were preprocessed using MassHunter workstation software Qualitative Analysis
Version B.06.00. Each metabolite was identified and quantified based on the peak information
including m/z, migration time, and peak area. The quantified data were then evaluated for
statistical significance by a Wilcoxon signed-rank test. Alternatively, liquid chromatography-
coupled mass spectrometry (LC-MS) was performed on a TSQ Quantum Ultra AM with the
analysis software Xcalibur 2.0.6 software (Thermo Fisher Scientific, San Jose, CA) coupled to a
Gilson-HPLC system and a Gilson 234 autosampler (Gilson Inc., WI) as described [17].
(f) Metabolome analysis
Metabolome measurements were carried by Human Metabolome Technology Inc., Tsuruoka,
Japan. Briefly, cells were collected in mid-log phase, washed twice with 10% mannitol, and
resuspended in methanol containing internal standards (H3304-1002, Human Metabolome
Technologies, Inc., Tsuruoka, Japan) followed by ultrasonication, to inactivate enzymes. The
metabolites were further extracted and finally dissolved in 50 µL of Milli-Q water for CE-MS
analysis.
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Table S1. Predicted PTS in non-glycolytic enzymes of Diplonema papillatum.
Enzyme PTS*
D. papillatum T. brucei
Gluconeogenesis
Malate dehydrogenase (MDH), glycosomal ND PTS1 Phosphoenolpyruvate carboxykinase (PEPCK) ND PTS1
Pentose phosphate pathway 6-phosphogluconolactonase ND PTS1 Ribokinase ND PTS1 Transketolase ND PTS1
Energy/redox metabolism
Adenylate kinase ND PTS1 Glycerol 3-phosphate dehydrogenase ND PTS1 Glycerol kinase (GK) PTS1 PTS1 Isocitrate dehydrogenase ND PTS1 NADH-dependent fumarate reductase ND PTS1 Pyruvate phosphate dikinase (PPDK) PTS1 PTS1
Pyrimidine biosynthesis/purine salvage
Adenine phosphoribosyltransferase ND PTS1 Guanylate kinase ND PTS1 Hypoxanthine-guanine phosphoribosyltransferase ND PTS1 Inosine-5'-monophosphate dehydrogenase ND PTS1 Orotate phosphoribosyltransferase (OPRT) ND PTS1† Orotidine monophosphate decarboxylase (OMPDC) ND PTS1†
*PTS1 represents being double positive for both the C-terminal consensus tripeptides, (STAGCN)-(RKH)-(LIVMAFY), and a category of “targeted” or “twilight zone” using the PTS1 predictor (see materials and methods). †Fused in OMPDC-OPRT. ND; No PTS detected.
Table S2. Metabolites of carbon metabolism in Diplonema papillatum. Metabolites (pmole/108 cells) T. cruzi D. papillatuma D. papillatumb D. papillatumc Glycolysis Hexose phosphate group
Glucose 6-phosphate 161 21,166 37,182 6,168 Glucose 1-phosphate 77 4,393 5,773 1,435 Fructose 6-phosphate 348 7,895 13,324 2,739 Fructose 1,6-bisphosphate 158 3,490 2,203 769
Triose phosphate group
Dihydroxyacetone phosphate 129 11,779 5,944 2,600 Glyceraldehyde 3-phosphate ND 186 ND ND 3-Phosphoglyceric acid 1,707 4,080 4,442 1,003 2-Phosphoglyceric acid 267 638 777 189 Phosphoenolpyruvic acid 1,036 564 903 ND
Ratio of total hexose-P:triose-P 744:3149
(1:4.2) 36944:17247
(2.1:1) 58482:12066
(4.8:1) 11111:3792
(2.9:1) TCA cycle
Citric acid 330 1,705 894 401 cis-aconitic acid ND 924 532 237 Isocitric acid ND 70,382 53,107 21,083 α-ketoglutaric acid ND 4,397 4,412 3,180 Succinic acid 35,203 108,395 113,315 22,092 Fumaric acid 126 6,454 2,204 1,492 Malic acid 535 22,612 15,545 9,739
Pentose phosphate pathway
6-Phosphogluconic acid 21 73 ND ND Ribulose 5-phosphate 144 1,859 1,906 511 Ribose 5-phosphate 38 585 638 128 Sedoheptulose 7-phosphate 282 1,729 5,199 443 Erythrose 4-phosphate ND 572 353 ND
Nucleotides
ATP 8,578 6,944 9,481 1,247 ADP 8,010 19,800 19,395 6,637 AMP 8,028 50,665 23,739 14,455 cAMP 15 ND ND ND GTP 2,264 1,228 2,116 607 GDP 2,196 4,572 4,143 2,223 GMP 1,208 6,643 3,884 3,248 UTP 4,974 1,984 2,259 290 UDP 5,083 4,847 4,626 1,502 UMP 5,571 11,936 5,445 2,657 CTP 583 139 400 ND CDP 360 302 528 280 CMP 457 2,560 2,407 1,424
a ATCC® 1405 artificial seawater supplemented with 10 % v/v horse serum. b ATCC® 1532 medium. c ATCC® 1532 medium supplemented with 6 mM D-glucose. ND, not detected.
Table S2. Continued. Metabolites (pmole/108 cells) T. cruzi D. papillatuma D. papillatumb D. papillatumc Others
Pyruvic acid 126 1,372 2,351 1,820 Lactic acid 11,800 82,962 84,245 20,330
Amino acids β-Ala 147 1,191,232 Glu 14,748 842,686 Lys 20,288 453,660 Pro 17,256 423,095 Ala 212,561 367,248 Gln 86 317,801 Arg 11,335 217,878 Gly 99,373 137,363 Val 36,898 134,090 His 200 109,362 Ser 507 98,449 Thr 338 85,611 Leu 3,968 78,799 Tyr 929 73,437 Asn 1,700 53,480 Asp 1,578 51,883 Ile 15,529 35,984 Phe 808 35,481 Trp 290 15,796 Met 244 12,534 Cys ND 569
a ATCC® 1405 artificial seawater supplemented with 10 % v/v horse serum. b ATCC® 1532 medium. c ATCC® 1532 medium supplemented with 6 mM D-glucose. ND, not detected.
Euglenozoa
Bacteria
Ruegeria sp.Micromonas pusilla
Geobacter sp.Synechococcus sp.Microcoleus chthonoplastes
Sideroxydans lithotrophicusXanthomonas albilineans
Metaseiulus occidentalisAcanthamoeba castellanii
Trichomonas vaginalis ATrichomonas vaginalis B
Naegleria gruberiTrypanosoma cruzi
Leishmania majorDiplonema papillatum
Dictyostelium discoideum
0.96
0.66
0.97
0.82 0.91
1
0.62
0.99
0.96 1
0.2
(a) Hexokinase (b) Glucokinase
Euglenozoa
Euglenozoa
BacteriaArchaea
Bacteria
Mus musculusHomo sapiens
Drosophila melanogasterDictyostelium discoideumSaccharomyces cerevisiae
Neurospora crassaDiplonema papillatum
Phaeodactylum tricornutumTrichomonas vaginalis
Naegleria gruberiTrimastix pyriformis
Giardia lambliaEctocarpus siliculosus
Cyanidioschyzon merolaeTrypanosoma cruzi
Trypanosoma bruceiLeishmania major
Tetrahymena thermophilaParamecium tetraurelia
Toxoplasma gondiiPlasmodium falciparum
Cryptosporidium parvumOryza sativa
Chlamydomonas reinhardtiiArabidopsis thaliana
Marinomonas sp. MED121Lautropia mirabilis
group III euryarchaeote KM3-28-E8Magnetococcus marinusGeobacter daltonii
Lactobacillus helveticusTrichodesmium erythraeum
Thermodesulfatator indicusPatulibacter sp. I11
0.58
0.81
0.5
0.96
0.54
0.71
0.521 0.98
0.78
1
0.92 0.640.94
10.7
0.97
0.99 0.5 0.91
0.2
(d) Triosephosphate isomerase
Euglenozoa
Bacteria
Bacteria
BacteriaBacteria
Bacteria
Streptomyces sp.Gloeobacter violaceus
Trypanosoma cruziTrypanosoma brucei
Leishmania majorDiplonema papillatum
Saccharomyces cerevisiaeNeurospora crassa
Aspergillus nidulansSyntrophobacter fumaroxidans
Dictyostelium discoideumDesulfovibrio africanusDrosophila melanogaster
Mus musculusHomo sapiens
Methylotenera mobilisTrimastix pyriformis
Magnetococcus marinusPhaeodactylum tricornutumEntamoeba histolytica
Fibrisoma limiEscherichia coli
Photobacterium damselaeVibrio metschnikoviiVibrio mimicusVibrio cholerae
Chloroherpeton thalassiumChloroherpeton thalassium
Ectocarpus siliculosusChlamydomonas reinhardtii
Oryza sativaArabidopsis thaliana
Toxoplasma gondiiToxoplasma gondii
Tetrahymena thermophilaParamecium tetraureliaParamecium tetraurelia
Plasmodium falciparumCryptosporidium hominisCryptosporidium parvum
Thalassiosira pseudonanaCyanidioschyzon merolae
1
0.63
0.58
0.93 1
0.98
0.7
11
0.991
0.7
0.81
0.96
1 11
1
1
0.591
0.99
1
1
0.67
11
0.67 1
0.980.2
(c) Phosphoglucose isomerase
Euglenozoa
Bacteria
Bacteria
Trimastix pyriformisToxoplasma gondii
Plasmodium falciparumCryptosporidium hominis
Trypanosoma bruceiTrypanosoma cruzi
Leishmania majorDiplonema papillatum
Chlamydomonas reinhardtiiMus musculus
Drosophila melanogasterSaccharomyces cerevisiae
Neurospora crassaTreponema primitiaDysgonomonas gadeiBacteroides sp.
Arabidopsis thalianaOryza sativa
Pelosinus fermentansDesulfotomaculum gibsoniae
Clostridium cellulolyticum
0.54
0.88
10.74
0.76
0.850.99
0.94
0.99
0.7
0.64
0.98
0.930.99
11
0.99
0.950.2
Bacteria
Euglenozoa
Euglenozoa
BacteriaArchaea
Bacteria
Sorangium cellulosum So ce 56Methylococcus capsulatuss tr.Bath
Dechloromonas aromatica RCBHoldemania filiformis DSM12042
Giardia lamblia ATCC50803Entameoba histolytica
Trimastix pyriformisDiplonema papillatum
Chlamydomonas reinhardtiiTrypanosoma cruzi Trypanosoma brucei
Leishmania majorArabidopsis thaliana
Oryza sativa Japonica GroupSlackia piriformis YIT12062
Silicibacterlacus caerulensis ITI-1157Methanosaeta harundinacea 6Ac
Trichomonas vaginalis G3Encephalitozoon cuniculi
Aspergillus nidulansNeurospora crassa OR74A
Fischerella sp. JSC-11
0.67 0.97
0.98
0.99
1
0.67
0.98
11
0.99
0.98
0.540.63
0.580.9
0.2
(e) Phosphoglycerate mutase
Euglenozoa
Bacteria
BacteriaArchaea
Paramecium tetraureliaEntamoeba histolytica
Encephalitozoon cuniculiDictyostelium discoideumCyanidioschyzon merolae
Arabidopsis thalianaToxoplasma gondii
Toxoplasma gondii ME49Plasmodium falciparum
Ustilago maydisSaccharomyces cerevisiae S288c
Aspergillus nidulansNeurospora crassa OR74A
Mus musculusMus musculusHomo sapiens
Drosophila melanogasterGiardia lamblia
Trimastix pyriformisTrypanosoma cruzi Trypanosoma brucei bruceiLeishmania major
Diplonema papillatumDictyostelium discoideum AX4
Cryptosporidium parvumCryptosporidium hominis TU502Chlamydomonas reinhardtii
Thalassiosira pseduonanaPhaeodactylum tricornutum
Ectocarpus siliculosusArabidopsis thaliana
Oryza sativaNaegleria gruberi
Trichomonas vaginalisTrichomonas vaginalis
Methanococcus voltaeA3Thermofilum pendens Hrk5
Korarchaeum cryptofilumScardovia wiggsiaePelobacter carbinolicus
Lactobacillus plantarumMagnetococcus marinus MC-1
Euglena gracilisEuglena gracilis
Glaciecola sp. 4H-3-7+YE-5Dechloromonas aromatica
Microcystis aeruginosaCandidatus Caldiarchaeum subterraneum
0.91
0.690.99
0.5
0.971
1
0.73
0.75
0.92
0.80.75
0.9910.99
1
0.99
0.910.97 0.99
0.99
11
0.570.97
0.88
0.75
0.90.53
0.860.76
1
0.610.2
Archaea
Euglenozoa
(f) Enolase
Figure S1. Consensus phylogenetic trees for glycolytic enzymes. (a) Hexokinase; (b) glucokinase; (c) phophoglucose isomerase; (d) triose phosphate isomerase; (e) phophoglycerate mutase; and (f) enolase. Euglenozoa, bacteria and archaea are labeled in green, brown, and gray, respectively. The detailed methods to reconstruct the phylogenetic trees are described in the materials and methods.
Archaea
Archaea
Bacteria
Bacteria
Bacteria
Euglenozoa
Euglenozoa
Bacteria
Euglenozoa
Bacteria
Bacteria
Bacteria
Methanosarcina barkeri str.FusaroMethanosaeta harundinacea 6Ac
Methanocella conradii HZ254Methanocella conradii HZ254
uncultured marine crenarchaeote HF4000 ANIW93H17Cenarchaeum symbiosum A
Candidatus Nitrosoarchaeum limnia SFB1Nitrosopumilus maritimus SCM1Candidatus Nitrosopumilus salaria BD31
Candidatus Nitrosoarchaeum koreensis MY1Candidatus Caldiarchaeum subterraneum
Vulcanisaeta distributa DSM14429Candidatus Korarchaeum cryptofilumOPF8
Metallosphaera cuprina Ar4Sulfolobus solfataricusSulfolobus islandicus REY15A
Caldivirga maquilingensis IC167Ignicoccus hospitalis KIN4I
Aeropyrum pernix K1Acidilobuss accharovorans 34515
Thermofilum pendensHrk5Encephalitozoon cuniculi
Phaeodactylum tricornutumCCAP10551Pelobacter carbinolicus DSM2380
Methanoculleus marisnigri JR1Methanoculleus bourgensis MS2
Methanosphaerula palustris E19cMethanoregula boonei 6A8
Methanocella paludicola SANAEMethanocella arvoryzae MRE50
Magnetococcus marinus MC1Trypanosoma cruzi strain CL BrenerEctocarpus siliculosus
Gloeobacter violaceus PCC7421Euglena gracilis
Trypanosoma cruzi strain CL BrenerLeishmania major strain Friedlin
Trypanosoma cruzi strain CL BrenerTrypanosoma cruzi strain CL Brener
Trypanosoma bruceiTrypanosoma brucei brucei
Trypanosoma bruceiLeishmania major strain FriedlinDiplonema papillatum
Clostridium novyi NTClostridium botulinum C str.StockholmClostridium perfringens SM101
Clostridium botulinum B str.Eklund17BClostridium sp.7243FAAClostridium beijerinckii NCIMB8052
Chlamydomonas reinhardtiiTrimastix pyriformis
Cryptosporidium parvumPlasmodium falciparum
Naegleria gruberiToxoplasma gondii VEG
Tetrahymena thermophilaParamecium tetraurelia strain d42
Paramecium tetraureliaTrichomonas vaginalis
Saccharomyces cerevisiae S288cUstilago maydis
Aspergillus nidulansNeurospora crassa OR74A
Homo sapiensMus musculus
Mus musculusHomo sapiens
Drosophila melanogasterDrosophila melanogaster
Dictyostelium discoideumAX4Thalassiosira pseduonana
Entamoeba histolyticaEuglena gracilis
Giardia lamblia ATCC50803Candidatus Accumulibacter phosphatis clad .e IIA str.UW1
Coriobacter iaceaebacterium JC110Collinsella stercoris DSM13279
Collinsella tanakaei YIT12063Collinsella intestinalis DSM13280
Coriobacterium glomerans PW2Olsenella uli DSM7084
Atopobium vaginae PB189T14Atopobium vaginae DSM15829
Atopobium rimae ATCC49626Atopobium parvulum DSM20469
Ectocarpus siliculosusEctocarpus siliculosus
Phaeodactylum tricornutumEctocarpus siliculosus
Chlamydomonas reinhardtiiCyanidioschyzon merolae
Toxoplas magondii ME49Ectocarpus siliculosus
Phaeodactylum tricornutum CCAP10551Ectocarpus siliculosus
Prochlorococcus marinus str. MIT9515Prochlorococcus marinus str. MIT9202
Fischerella sp. JSC11Raphidiopsis brookii D9Cylindrospermopsis raciborskii CS505
Cyanothece sp. PCC7822Oryza sativa Japonica GroupOryza sativa Indica GroupArabidopsis thalianaOryza sativa Japonica GroupOryza sativa Indica Group
Arabidopsis thalianaArabidopsis thalianaStigmatella aurantiaca DW431
Corallococcus coralloides DSM2259Chondromyces apiculatusDSM436
Anaeromyxobacter sp. KAnaeromyxobacter sp. Fw1095
gammaproteobacteriaFrancisella philomiragia subsp. philomiragia ATCC25015
Methylococcus capsulatus str.BathLautropia mirabilis ATCC51599
Oxalobacter formigenes OXCC13Herminiimonas arsenicoxydans
Cupriavidus basilensis OR16Burkholderia ambifaria AMMD
Rhodomicrobium vannielii ATCC17100Hyphomicrobium sp. MC1Methylocystis sp. SC2
Methylocystis sp. ATCC49242Methylobacterium populi BJ001
Rhodospirillum centenum SWCommensalibacter intestini A911
Acetobacter aceti NBRC14818Acetobactert ropicalis NBRC101654
0.99
0.990.84
1
1
10.52
0.98
0.59
0.99
0.51
0.73
0.860.92
0.991
1
0.99
0.93
0.980.99
0.890.99
0.99
0.55
0.78
0.81
0.99
0.990.83
0.990.99
0.86
0.990.89
0.94
0.950.98
0.56
0.78
0.7
0.50.53 0.99
0.99
0.71
0.59
0.850.99
0.99
0.75
0.990.99
0.89
0.99
0.99
0.810.9
0.96
0.93
0.89
0.99
0.99
0.99
0.71
0.750.99 0.99
0.99
0.52
0.980.99
0.96
0.53
0.510.83
0.7
0.87
0.98
0.95
0.95
0.99
0.820.85
0.94
0.87
0.99
0.94
0.990.99
0.96
0.99
0.99
0.991
0.611
0.99
0.76 0.99
0.97
0.92
0.990.89
0.99
0.990.99
0.2
Figure S2. Consensus phylogenetic tree for phosphoglycerate kinase. The methods and labeling are as in figure S1.
Archaea
Bacteria
Bacteria
Euglenozoa
Archaea
Bacteria
Archaea
Bacteria
Bacteria
Entamoeba histolyticaTrichomonas vaginalis
Trichomonas vaginalisG3Synechococcus sp. RS9916
Prochlorococcus marinus str.MIT9515Naegleria gruberi
Methanocella paludicola SANAEMethanocella conradii HZ254
Methanocella arvoryzae MRE50Giardia lamblia ATCC50803
Giardia lamblia ATCC50803Trimastix pyriformis
Cyanothece sp.PCC 7822Haliangium ochraceum DSM14365
Stigmatella aurantiaca DW4/3-1Corallococcus coralloides DSM2259
Candidatus KorarchaeumcryptofilumOPF8ThermofilumpendensHrk5
Candidatus Caldiarchaeum subterraneumToxoplasma gondii
Plasmodium falciparum 3D7Ectocarpus siliculosus
Pseudogulbenkiania sp. NH8BHerbaspirillum sp. GW103
Herbaspirillum sp. CF444Granulibacter bethesdensis CGDNIH1
Azospirillum sp.B510Azospirillum lipoferum 4B
Acidianus hospitalis W1Metallosphaera yellowstonensis MK1
Drosophila melanogasterCyanidioschyzon merolae
Arabidopsis thalianaParamecium tetraurelia
Tetrahymena thermophilaNaegleria gruberi
Vibrio metschnikovii CIP69.14Moritella sp. PE36Moritellamarina ATCC15381
Ectocarpus siliculosusUstilago maydis
Saccharomyce scerevisiae S288cSaccharomyces cerevisiae S288c
Aspergillus nidulansNeurospora crassa OR74A
Encephalitozoon cuniculiMus musculusHomo sapiensMus musculusDrosophila melanogaster
Trypanosoma cruzi strain CL BrenerTrypanosoma brucei brucei strain 927/4 GUTat10.1
Leishmania major strain FriedlinDiplonema papillatum
Dictyostelium discoideumDictyostelium discoideum AX4
Olsenellasp.oraltaxon 809 str.F0356Coriobacterium glomerans PW2
Collinsella tanakaei YIT12063Paenibacillus sp. oraltaxon 786 str.D14Paenibacillus alvei DSM29
Brevibacillus laterosporus LMG15441Cryptosporidium parvum
Toxoplasma gondii ME49Plasmodium falciparum 3D7
Tetrahymena thermophilaTetrahymena thermophila
Paramecium tetraurelia strain d4-2Thalassiosira pseudonana
Phaeodactylum tricornutum CCAP1055/1Phaeodactylum tricornutum CCAP1055/1
Ectocarpus siliculosusChlamydomonas reinhardtii
Chlamydomonas reinhardtiiChlaymdomonas reinhardtii
Oryza sativa Japonica GroupArabidopsis thaliana
Oryza sativa Indica GroupArabidopsis thaliana
0.74
0.99
0.98
0.950.97
0.51
0.78
0.51 0.99
0.79
0.6
0.970.92
0.96
0.990.99
0.64 0.99
0.55
0.77
0.87
0.99
0.99
0.94
0.91
0.98
0.99
0.99
0.99
0.87
0.99
0.8
0.98 0.99
0.91
0.99
0.990.99
0.980.99
0.95
0.94
0.97
0.99
0.70.99
0.93
0.99
0.99
0.96
0.98
0.850.86
0.94
0.890.2
Figure S3. Consensus phylogenetic tree for pyruvate kinase. The methods and labeling are as in figure S1.
Euglenozoa
Euglenozoa
Euglenozoa
Euglenozoa
Euglenozoa
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Archaea
Archaea
Archaea
Bacteria
Acetivibrio cellulolyticus CD2Clostridium thermocellum ATCC 27405
Clostridium clariflavum DSM 19732Paludibacter propionicigenes WB4
Clostridium papyrosolvens DSM 2782Clostridium sp. MSTE9
Clostridium leptum DSM 753Clostridium methylpentosum DSM 5476
Anaerotruncus colihominis DSM 17241Giardia lamblia
Entamoeba histolyticaPhaeodactylum tricornutum
Tetrahymena thermophilaTetrahymena thermophila
Paramecium tetraureliaParamecium tetraurelia strain d4 2
Trypanosoma bruceiTrypanosoma cruzi strain CL Brener
Leishmania major strain FriedlinEncephalitozoon cuniculi
Giardia lamblia ATCC 50803Toxoplasma gondii VEGToxoplasma gondiiToxoplasma gondii
Phaeodactylum tricornutum CCAP 1055 1Ectocarpus siliculosusThalassiosira pseudonana
Phaeodactylum tricornutum CCAP 1055 1Phaeodactylum tricornutum CCAP 1055 1
Ectocarpus siliculosusEctocarpus siliculosus
Naegleria gruberiDiplonema papillatum GAPDH2
Diplonema papillatum GAPDH1Diplonema sp. ATCC 50225
Syntrophus aciditrophicus SBTrypanosoma cruzi strain CL Brener
Trypanosoma cruzi strain CL BrenerTrypanosoma cruzi strain CL Brener
Trypanosoma cruzi strain CL BrenerTrypanosoma cruzi strain CL Brener
Trypanosoma bruceiXenorhabdus bovienii SS 2004
Morganella morganii subsp. morganii KTYersinia enterocolitica subsp. enterocolitica 8081Hafnia alvei ATCC 51873Erwinia tasmaniensis Et1 99
Synechococcus sp. WH 5701Synechococcus sp. CB0101Cyanobium sp. PCC 7001
Polaribacter irgensii 23 PNitrosospira sp. TCH716
Magnetospirillum magnetotacticum MS 1Magnetospirillum gryphiswaldense MSR 1
Hydra magnipapillatablood disease bacterium R229
Hylemonella gracilis ATCC 19624Cupriavidus metallidurans CH34
Toxoplasma gondii VEGPlasmodium falciparum
Ectocarpus siliculosusCryptosporidium parvum Iowa II
Saccharomyces cerevisiae S288cSaccharomyces cerevisiae S288c
Chlamydomonas reinhardtiiChlamydomonas reinhardtiiChlamydomonas reinhardtii
Arabidopsis thalianaOryza sativa Indica Group
Arabidopsis thalianaCyanidioschyzon merolae
Ustilago maydisNeurospora crassa OR74A
Mus musculusHomo sapiensHomo sapiens
Homo sapiensHomo sapiens
Mus musculusMus musculus
Drosophila melanogasterDrosophila melanogaster
Dictyostelium discoideum AX4Oryza sativa Japonica Group
Oryza sativa Japonica GroupOryza sativa Japonica Group
Arabidopsis thalianaArabidopsis thaliana
Microcystis aeruginosa NIES 843Cyanothece sp. PCC 7425
SAR324 cluster bacterium SCGC AAA001 C10SAR324 cluster bacterium JCVI SC AAA005
Leishmania majorTrypanosoma cruzi strain CL BrenerTrypanosoma brucei brucei strain 927 4 GUTat10.1Trypanosoma brucei brucei
Euglena gracilisPelobacter carbinolicus DSM 2380
Geobacter uraniireducens Rf4Aspergillus nidulans
Methanoculleus bourgensis MS2Halopiger xanaduensis SH 6
Natronobacterium gregoryi SP2Halobiforma lacisalsi AJ5
Natronobacterium gregoryi SP2Halobiforma lacisalsi AJ5
Halogranum salarium B 1Halogranum salarium B 1
Halalkalicoccus jeotgali B3Phaeodactylum tricornutum CCAP 1055 1
Ectocarpus siliculosusDiplonema sp. ATCC 50225
Rhynchopus sp. ATCC 50231Diplonema sp. ATCC 50225
Chlamydomonas reinhardtiiuncultured archaeon
Thermofilum pendens Hrk 5Candidatus Korarchaeum cryptofilum OPF8
delta proteobacterium NaphS2Geobacter uraniireducens Rf4
Geobacter sp. M21Geobacter sp. M18
Geobacter daltonii FRC 32alpha proteobacterium IMCC14465
Tistrella mobilis KA081020 065Parvularcula bermudensis HTCC2503
Azospirillum lipoferum 4BAzospirillum brasilense Sp245
Chlamydomonas reinhardtiiActinomyces urogenitalis DSM 15434
Candidatus Aquiluna sp. IMCC13023Renibacterium salmoninarum ATCC 33209
Arthrobacter sp. Rue61aArthrobacter globiformis NBRC 12137
Arthrobacter chlorophenolicus A6Actinomyces sp. oral taxon 448 str. F0400
Trimastix pyriformisTrichomonas vaginalis G3
Trichomonas vaginalis G3Trichomonas vaginalis G3
uncultured archaeon GZfos1D1Aciduliprofundum boonei T469
0.97
0.65
0.98
0.9
0.960.99
0.86
0.82
0.67
0.94
0.980.99
0.99
1
0.78
0.951
0.97
0.950.79 0.97
0.84
0.840.96
0.870.9
0.94
0.95
0.990.99
0.97
0.69
0.95
0.62
0.99
0.99
0.64 0.99 0.99
0.990.99
0.99
0.89
0.9
0.530.99
0.99
10.88
0.54
0.96
0.56
0.991
0.85
0.950.99
0.75
0.77
0.94
0.980.99
0.99
0.87
0.95
0.61
0.99
0.8
0.99
0.990.98
0.99
0.890.96
0.91
0.81
0.91
1
0.99
0.97
1
0.990.97
1
0.66
0.99
0.99
0.69 10.93
0.72
0.62
0.550.99 1
0.990.51
0.51 1
0.510.99
0.78
0.990.96
0.990.69
0.64
10.99
0.99
0.75
0.2
Figure S4. Consensus phylogenetic tree for glyceraldehyde-3-phosphate dehydrogenase. The methods and labeling are as in figure S1.
Euglenozoa (FBPase)
Bacteria
Euglenozoa (FBPase)
Euglenozoa (SBPase)
Bacteria
Archaea
Diplonema papillatumDesulfobulbus propionicus
delta proteobacterium MLMS-1Desulfurivibrio alkaliphilus
delta proteobacterium NaphS2Desulfovibrio aespoeensis
Desulfovibrio salexigensDesulfovibrio vulgaris
Toxoplasma gondiiToxoplasma gondii
Synechococcus sp. PCC7335Synechococcus sp. JA-3-3Ab
Synechococcus elongatusCylindrospermopsis raciborskii
Raphidiopsis brookiiFischerella sp. JSC-11Nostoc punctiformeAnabaena variabilisNostoc sp. PCC7120
Synechocystis sp. PCC6803Hippea maritima
Calditerrivibrio nitroreducensDeferribacter desulfuricans
Sorangium cellulosumDesulfurobacterium thermolithotrophum
Thermovibrio ammonificansThermodesulfobium narugense
Thermodesulfobium narugenseSulfurihydrogenibium yellowstonense
Sulfurihydrogenibium azorenseHydrogenivirga sp.
Tetrahymena thermophilaThalassiosira pseudonana
Phaeodactylum tricornutumEctocarpus siliculosus
Mus musculusMus musculus
Mus musculusHomo sapiens
Mus musculusHomo sapiensHomo sapiens
Drosophila melanogasterDictyostelium discoideum
Tetrahymena thermophilaTetrahymena thermophila
Cyanidioschyzon merolaePhaeodactylum tricornutum
Ectocarpus siliculosusPhaeodactylum tricornutum
Phaeodactylum tricornutumEctocarpus siliculosus
Phaeodactylum tricornutumEctocarpus siliculosus
Ectocarpus siliculosusOryza sativaArabidopsis thaliana
Chlamydomonas reinhardtiiOryza sativaOryza sativaArabidopsis thaliana
Oryza sativaOryza sativa
Oryza sativaOryza sativa
Arabidopsis thalianaUstilago maydis
Trypanosoma cruziTrypanosoma brucei
Leishmania majorSaccharomyces cerevisiaeSaccharomyces cerevisiae
Neurospora crassaAspergillus nidulans
Methylotenera versatilisMagnetospirillum magneticum
Sulfitobacter sp.Roseobacter sp. GAI101
Octadecabacter arcticusAgrobacterium rhizogenes
Agrobacterium rhizogenesPseudogulbenkiania sp. NH8BMethyloversatilis universalis
Oxalobacter formigenesSutterella parvirubra
Neisseria mucosaNeisseria weaveri
Neisseria meningitidisNeisseria meningitidis 93004Neisseria meningitidis MC58
Saccharophagus degradansPseudoalteromonas haloplanktis
Idiomarina sp. A28LShewanella baltica OS223Shewanella baltica OS195
Marinomonas mediterraneaMarinomonas sp. MWYL1
Oceanobacter sp. RED65Rhodovulum sp. PH10
Caulobacter sp. AP07Caulobacter sp. K31
Streptomyces coelicoflavusMycobacterium tusciae
Nocardia cyriacigeorgicaMicrolunatus phosphovorus
Pseudonocardia dioxanivoransMethanoplanus petrolearius
Halopiger xanaduensisNatrinema pellirubrum
Natronomonas pharaonisHaloarcula marismortuiHaloarcula hispanica
Haloferax mediterraneiHaloferax volcanii
Natrialba magadiiHaloterrigena turkmenica
Chlamydomonas reinhardtiiTrypanosoma cruzi
Trypanosoma bruceiTetrahymena thermophila
Paramecium tetraureliaParamecium tetraurelia
Paramecium tetraureliaParamecium tetraurelia
Toxoplasma gondiiPhaeodactylum tricornutum
Neurospora crassaEctocarpus siliculosus
Ectocarpus siliculosusChlamydomonas reinhardtii
Chlamydomonasr einhardtiiChlamydomonas reinhardtii
Arabidopsis thaliana
0.62
0.52
0.77
0.79
0.75
0.99
11
0.67 10.98
0.790.99
0.870.99
1
0.83
0.991
0.830.85 1
0.990.99
0.96
1
0.990.99
1
0.84
0.990.99
0.99
11
10.9
0.940.99
0.781
0.98
0.87
0.70.68
0.61
1
0.66
0.99
11
11
1
0.970.98
0.99
0.99
0.830.96
10.99
11
1
0.95
0.990.51
0.99
1
0.82
0.93
0.95
0.68
0.99
0.86 1
0.74
0.82
0.66
0.56
1
1
0.760.99
0.97
0.93
0.87
0.99
0.71
1
0.550.66 0.93
0.83
1
1
1
0.92
0.821
0.991
0.99
0.98
0.60.98
0.991
1
0.88
0.2
Figure S5. Consensus phylogenetic tree for fructose-1,6-bisphosphatase (FBPase). Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme for pentose phosphate pathway. The methods and labeling are as in figure S1.
(a)
C
E
F
D
D
N
M
A
M
B
B
Peroxisome
HK PGI GAPDH1 TIM PGK PGM PK ENO GLK
kDa
75-
50-
37-
25- 21-
15-
100-
150-
E F
Figure S6. Expression of glycolytic enzymes in D. papillatum. (a) Western blot analysis of the D. papillatum extracts using antisera to the relevant enzymes. The expected size of each protein is shown by a triangle in each panel. (b) Ultrastructure of D. papillatum. Peroxisomes (arrow) are present as dense bodies surrounded by a single-membrane (B, a magnified image of the inset of A). C-F, Immunoelectron microscopic observations of FBPA within peroxisomes. Note that gold particles are specifically detected inside peroxisomes (arrow). (D, E, and F are magnified images of the insets of C). N; nucleus. M; mitochondrion, P; peroxisome. Scale bar = 0.5 μm.
(b)
PFK2 PFK1
Rel
ativ
e ab
unda
nce
(%)
(a) 6 mM 13C5-L-glutamine
Rel
ativ
e ab
unda
nce
(%)
Rel
ativ
e ab
unda
nce
(%)
(b) 6 mM 13C6-D-glucose
(c) 30 mM 13C6-D-glucose
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Glucose 6-phosphate
0 hr 4 hr 8 hr
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Fructose 6-phosphate
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Fructose 1,6-isphosphate
0
5
10
15
20
+1 +2 +3
3-phospho-glycerate
0
5
10
15
20
+1 +2 +3
Phosphoenol-pyruvate
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Glucose 6-phosphate
0 hr 4 hr 8 hr
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Fructose 6-phosphate
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Fructose 1,6-bisphosphate
0
5
10
15
20
+1 +2 +3
3-phospho-glycerate
0
10
20
30
40
+1 +2 +3
Phosphoenol-pyruvate
0
5
10
15
20
+1 +2 +3
Lactate
0 hr 4 hr 8 hr
0
10
20
30
40
50
+1 +2 +3 +4 +5
α-ketoglutarate
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Citrate
0
10
20
30
+1 +2 +3 +4
Malate
0
10
20
30
40
+1 +2 +3 +4 +5 +6 +7
Sedoheptulose 7-phosphate
0
5
10
15
20
+1 +2 +3
Lactate
0 hr 4 hr 8 hr
0
5
10
15
20
+1 +2 +3 +4
Succinate
0
5
10
15
20
+1 +2 +3 +4
Fumarate
0
5
10
15
20
+1 +2 +3 +4
Malate
0
10
20
30
40
+1 +2 +3 +4 +5 +6 +7
Sedoheptulose 7-phosphate
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Citrate
0
10
20
30
+1 +2 +3 +4 +5 +6
Glucose 6-phosphate
12C 13C
0
10
20
30
+1 +2 +3 +4 +5 +6
Fructose 6-phosphate
0
10
20
30
+1 +2 +3 +4 +5 +6
Fructose 1,6-bisphosphate
0
10
20
30
40
+1 +2 +3
DHAP
0
10
20
30
+1 +2 +3
3-phospho-glycerate
0
5
10
15
20
+1 +2 +3
Lactate
0
5
10
15
20
+1 +2 +3 +4 +5 +6
Citrate
12C 13C
0
5
10
15
20
+1 +2 +3 +4
Succinate
0
5
10
15
20
+1 +2 +3 +4
Malate
0
10
20
30
40
+1 +2 +3 +4 +5
Ribose 5-phosphate
0
10
20
30
+1 +2 +3 +4 +5
Ribulose 5-phosphate
0
10
20
30
40
+1 +2 +3 +4 +5 +6 +7
Sedoheptulose 7-phosphate
Figure S7. Metabolomic analysis of 13C-labeled metabolites in D. papillatum. D. papillatum cells were incubated with 6 mM 13C5-glutamine for 0, 4, and 8 hrs (a), 6 mM 13C6-D-glucose for 0, 4, and 8 hrs (b), or 30 mM 13C6-D-glucose for 72 hrs (c). The Y-axis indicates the relative abundance (%) of each isotopomer of the relevant metabolite. The X-axis represents the number of 13C in the isotopomers. Data points and error bars represent the mean ± s.d. of three independent experiments.
Euglenozoa (glycosomal)
Euglenozoa
Euglenozoa
Euglenozoa
Bacteria
Bacteria
Bacteria
Leptotrichia hofstadiiStreptomyces sp. NRRL S 87
Desulfotalea psychrophilaEuglena gracilis
Desulfonatronum thiodismutansClostridiales bacterium VE202 09
Francisella philomiragiaTrypanosoma brucei brucei TREU927
Trypanosoma grayiTrypanosoma cruzi CL Brener
Trypanosoma brucei brucei TREU927Phytomonas sp. isolate Hart1
Leishmania major FriedlinStrigomonas culicis
Angomonas deaneiNecropsobacter rosorum
Pasteurella sp. FF6Haemophilus sp. FF7
Pseudomonas aeruginosaMethylococcaceae bacterium Sn10 6
Burkholderiales bacterium 1 1 47Acinetobacter soli
Toxoplasma gondiiNeospora caninum Liverpool
Diplonema papillatumBlastocystis hominis
Phytophthora infestans T30 4Saprolegnia parasitica
Aphanomyces invadansTrypanosoma grayiTrypanosoma cruzi CL Brener
Trypanosoma brucei brucei TREU927Strigomonas culicis
Phytomonas sp. isolate Hart1Leptomonas pyrrhocoris
Leishmania major FriedlinAngomonas deanei
Volvox carteri f. nagariensisHelicosporidium sp. ATCC 50920
Ectocarpus siliculosusOstreococcus tauri
Micromonas sp. RCC299Bathycoccus prasinos
Arabidopsis thalianaRicinus communisOryza sativa Japonica Group
Glycine sojaAegilops tauschii
Oxytricha trifallaxGaldieria sulphuraria
Rhizopus delemar RA 99 880Coprinopsis cinerea cytosolic
Wallemia sebi CBS 633.66Cryptococcus neoformans var. grubii H99
Trichophyton soudanense CBS 452.61Podospora anserina S mat+
Saccharomyces cerevisiae S288cCandida albicans 12C
Salpingoeca rosettaCapsaspora owczarzaki ATCC 30864
Solenopsis invictaDrosophila melanogaster
Danaus plexippusPlutella xylostella
Bombyx moriXenopus laevis mitochondrial 2
Homo sapiens mitochondrialAscaris suum
Acanthamoeba castellanii str. NeffDictyostelium discoideum
0.99
0.99
0.991
0.94
0.771
1
0.98
0.9
0.630.94
0.56
1
1
0.98
0.560.99
1
0.99
0.99
1
0.76
0.68
0.67
0.910.99
0.53
0.95
0.96
1
0.990.950.99
0.5
0.95
0.59
0.76
0.991
0.74
0.92
0.990.9 1
0.59
0.931
10.64
0.950.64
0.76
1
0.530.2
Figure S8. Consensus phylogenetic tree for adenylate kinase. Note that E. gracilis enzyme and the glycosomal enzymes of kinetoplastids are nested independently within the bacterial clade (PP = 0.99), while D. papillatum and cytosolic kinetoplastid homologues cluster in the eukaryotic subtree (PP = 0.99). The methods and labeling are as in figure S1.
Euglenozoa(glycosomal)
Euglenozoa
Bacteria
Bacteria
Archaea
Clostridium botulinum A ATCC 3502Thermoanaerobacter tengcongensis
Lactococcus lactisBacillus subtilis
Helicobacter pylori 26695Thermus thermophilus HB27
Spirochaeta thermophila DSM 6192Pyrococcus furiosus DSM 3638
Tetrahymena thermophilaParamecium tetraurelia
Trypanosoma bruceiTrypanosoma cruziLeishmania major
Fibrobacter succinogenesChlorobium tepidum
Fusobacterium nucleatum ATCC 25586Bacteroides thetaiotaomicron
Thermotoga maritima MSB8Aquifex aeolicus
Neisseria gonorrhoeaeEscherichia coli
Acidobacterium capsulatumGeobacter sulfurreducens
Phytophthora infestansToxoplasma gondii
Plasmodium falciparum 3D7Thalassiosira pseudonana
Phaeodactylum tricornutumGuillardia theta
Dictyostelium discoideumEmiliania huxleyi
Cyanidioschyzon merolaeEuglena gracilis
Trypanosoma cruzi BrenerTrypanosoma brucei
Leishmania major
Diplonema papillatum
Saccharomyces cerevisiaeMus musculusHomo sapiens
Drosophila melanogasterMonosiga brevicollis
Caenorhabditis elegansChlamydomonas reinhardtii
Zea maysArabidopsis thaliana
0.56
0.91
1
0.94
0.5
0.99
0.75 11
1
0.59
0.7
0.93
0.99
1
0.85
1
0.54
0.68
0.99
1
0.7
0.63
0.99
0.97
0.81 1
0.93
0.710.59
1
0.660.99
10.2
Figure S9. Consensus phylogenetic tree for inosine-5-monophosphate dehydrogenase. Note that glycosomal isoforms of kinetoplastids are nested within the bacterial clade, while E. gracilis, D. papillatum and cytosolic kinetoplastid homologues are monophyletic and cluster in the eukaryotic clade, consistent with the organismal tree (PP = 1). The methods and labeling are as in figure S1.
Figure S10. Consensus phylogenetic tree for transketolase. Note that the euglenozoan enzymes are monophyletic (PP = 1). The methods and labeling are as in figure S1.
Euglenozoa
Bacteria
Bacteria
Mycobacterium tuberculosisTrichomonas vaginalis
Giardia lambliaGeobacter metallireducens
Deinococcus radioduransChlorobium tepidum
Synechocystis sp. PCC 6803Chloroflexus aurantiacus
Chlamydia trachomatisBorrelia burgdorferi B31
Bacillus subtilisThermotoga maritima MSB8
Bacteroides thetaiotaomicronAquifex aeolicus
Agrobacterium sp. H13 3Neisseria gonorrhoeae FA 1090
Naegleria gruberiZea mays
Cyanidioschyzon merolaeToxoplasma gondii
Phytophthora sojaeSaccharomyces cerevisiae
Capsaspora owczarzaki ATCC 30864Caenorhabditis elegans
Solenopsis invictaHomo sapiensBos taurus
Arabidopsis thalianaDiplonema papillatum
Trypanosoma cruzi strain CL BrenerTrypanosoma brucei brucei TREU927Phytomonas sp. isolate EM1
Leishmania major strain FriedlinStrigomonas culicisAngomonas deanei
0.87
0.780.69
0.67
0.97
1
0.99
0.61
0.62
0.78
1
0.99 1
1
1
0.651
0.79
0.770.2