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 peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids. Proceedings of the Royal Society B: Biological Sciences, 283(1830). https://doi.org/10.1098/rspb.2016.0520
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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
1 *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.
2 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
3 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
4 [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,
5 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
6 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
13 13 medium) for 2 hrs and then incubated with 6 mM C6 D-glucose or 6 mM C5 L-glutamine
13 (Sigma-Aldrich) in artificial seawater for 0, 4, and 8 hrs or with 30 mM C6 D-glucose in
ATCC® 1532 medium for 72 hrs without starvation. The detailed experimental procedures for
7 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
8 – 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
9 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
10 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
11 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
13 13 C-uniformly labeled ( C5, +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
13 Metabolic labeling using 6 mM C6-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,
12 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
13 cells incubated for 72 hrs in normal medium containing 30 mM C6-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
13 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
14 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 13 13 conditioned marine water for 2 hrs and then supplemented with C5-L-glutamine (b) or C6-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. PTS Enzyme 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) 39.1 3. PFK PTS1/ND PTS1 No (0.99) ND 3. FBPase (gluconeogenic) PTS1 PTS1 No (1.00) 4.7 4. FBPA PTS2 PTS2 Yes (1.00)§ 14.2 5. TIM PTS1 ND Unresolved 123.2 6. GAPDH ND PTS1/ND No (0.90) 48.5 7. PGK PTS1 PTS1/ND Yes (0.81) 103.7 8. PGAM ND ND No (1.00) 2,100 9. ENO ND ND Yes (0.99) 3.7 10. 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. Methanocella paludicola SANAE Archaea 0.99 Rheinheimera nanhaiensis E407-8 1 1 Alteromonas macleodii str. Deep ecotype Bacteria Alteromonas macleodii 0.99 Methanospirillum hungatei JF-1 Methanoculleus marisnigri JR1 Archaea 0.78 Magnetococcus marinus MC-1 Bacteria Trichomonas vaginalis G3 (Excavata) 0.99 1 Saccharomyces cerevisiae S288c 0.52 1 Saccharomyces cerevisiae AWRI1631 0.55 Neurospora crassa Haemonchus contortus 0.99 Lactococcus lactis Succinivibrionaceae bacterium WG-1 0.57 1 Oceanimonas sp. GK1 1 Haemophilus sputorum CCUG 13788 Bacteria 0.93 Haemophilus ducreyi 35000HP 0.53 Actinobacillus ureae 0.99 Buchnera aphidicola (Cinara tujafilina) 0.99 Escherichia coli 0.92 Thalassiosira pseudonana CCMP1335 1 Phaeodactylum tricornutum Aureococcus anophagefferens Guillardia theta CCMP2712 0.97 Emiliania huxleyi 1 Coraliomargarita akajimensis 1 1 Agrobacterium tumefaciens F2 Bacteria Sinorhizobium fredii Bodo saltans PFK1 Euglenozoa 0.73 Trichomonas vaginalis (Excavata) 0.93 1 Trichomonas vaginalis G3 0.99 Trichomonas vaginalis G3 0.99 Trichomonas vaginalis 0.98 Pelosinus fermentans DSM 17108 Bacteria 1 Naegleria fowleri (Excavata) Naegleria gruberi strain NEG-M 0.98 Syntrophobacter fumaroxidans Accumulibacter phosphatis 0.73 Desulfobacca acetoxidans 1 0.99 1 Desulfobacter postgatei 2ac9 Bacteria 1 Desulfobacterium autotrophicum Uncultured Desulfobacterium sp. 0.99 Desulfococcus oleovorans Diplonema papillatum PFK1 Euglenozoa Waddlia chondrophila 2032/99 Bacteria Paramecium tetraurelia strain d4-2 0.99 Toxoplasma gondii ME49 0.87 Plasmodium falciparum 3D7 1 0.5 0.92 Cryptosporidium hominis 0.911 Toxoplasma gondii GT1 0.92 Plasmodium falciparum 3D7 0.71 Cryptosporidium hominis Ectocarpus siliculosus 0.95 Oryza sativa Japonica Group Arabidopsis thaliana 0.94 1 1 Arabidopsis thaliana 0.93 Toxoplasma gondii ME49 Toxoplasma gondii 0.81 Oryza sativa Japonica Group 0.98 0.78 1 Oryza sativa 0.99 Oryza sativa Japonica Group 1 Oryza sativa 1 Arabidopsis thaliana Arabidopsis thaliana Giardia intestinalis (Excavata) 1 Treponema phagedenis F0421 0.99 Treponema pallidum Treponema caldaria 1 Solobacterium moorei F0204 Bacteria 0.96 Holdemanella biformis 0.7 Spirochaeta thermophila DSM 6578 0.97 Borellia burgdoferi 0.92 Entamoeba histolytica Entamoeba histolytica (Amoebozoa) Monocercomonoides sp. (Oxymonadida, Excavata) 0.97 Euglena gracilis 0.99 0.99 1 Trypanosoma cruzi 1 Trypanosoma brucei Euglenozoa Clade A 1 Leishmania major strain Friedlin 1 (Excavata) 0.91 Crithidia fasciculata Bodo saltans PFK2 Nakamurella multipartita Bacteria 1 Emiliania huxleyi CCMP1516 (Haptophyta) Diplonema papillatum PFK2 Euglenozoa (Excavata) 0.64 0.99 Phaeodactylum tricornutum (Stramenopiles) Phaeodactylum tricornutum CCAP 1055/1 (Stramenopiles) 0.99 1 1 Ectocarpus siliculosus (Stramenopiles) Ectocarpus siliculosus (Stramenopiles) Clade B Chlamydomonas reinhardtii (Plantae) 0.59 1 Tetrahymena thermophila SB210 (Alveolata) Paramecium tetraurelia (Alveolata) 0.98 Arabidopsis thaliana (Plantae) 0.2 1 Arabidopsis thaliana (Plantae) 0.99 Nicotiana tomentosiformis (Plantae) DIC Target FBPA Merge GLK
PGI
TIM
GAPDH
PGK
PGAM
ENO
PK (a) D. papillatum T. cruzi 7
10 --) 8 --) 10 15 15
106 10 10 107
5 Cell count (/ml, -- Concentration (mM) Cell count (/ml, -- 10 Concentration (mM) 5 5 – – Glucose – – Glucose – – Ammonium – – Ammonium 6 0 104 0 10 2 4 6 8 10 2 4 6 8 10 Time (days) Time (days) (b) (c) 20 20 20 Glucose Fructose Glucose 15 6-phosphate 15 6-phosphate 15 6-phosphate
10 0 hr 10 10 0 hr 4 hr 4 hr 5 8 hr 5 5 8 hr
0 0 0 Relative abundance (%) +1 +2 +3 +4 +5 +6 +1 +2 +3 +4 +5 +6 Relative abundance (%) +1 +2 +3 +4 +5 +6 50 20 40 α-Ketoglutarate Citrate Sedoheptulose 40 15 30 7-phosphate 30 10 20 20 10 10 5 0 0 0 Relative abundance (%) +1 +2 +3 +4 +5 +1 +2 +3 +4 +5 +6 Relative abundance (%) +1 +2 +3 +4 +5 +6 +7 (a) (b) (c) (d) Glucose Glucose Glucose Glucose
Glucose-P PPP Glucose-P PPP Glucose-P PPP Glucose-P PPP FBP PFK FBP PFK FBP FBP GK Pyr Pyr Pyr PFK PPDK Pyr GK GK ADK Pur Pur Pur Pur PEP Px PPDK Px PPDK Px PEPCK PEP PEP PEP Px PFK
Mt Amino acids Amino acids Amino acids Mt Mt Mt Amino acids
Diplonemids (D. papillatum) LECA Kinetoplastids Euglenoids 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
13 13 mM C6 D-glucose or 6 mM C5 L-glutamine (Sigma-Aldrich) in artificial seawater for 0, 4,
13 and 8 hrs or with 30 mM C6 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
13 13 incubated with 6 mM C6 D-glucose or 6 mM C5 L-glutamine (Sigma-Aldrich) in artificial
13 seawater for 0, 4, and 8 hrs or with 30 mM C6 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. PTS* Enzyme 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 36944:17247 58482:12066 11111:3792 (1:4.2) (2.1:1) (4.8:1) (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.
(a) Hexokinase (b) Glucokinase
Trimastix pyriformis Ruegeria sp. 0.74 Toxoplasma gondii Micromonas pusilla 1 0.96 0.97 Geobacter sp. 0.54 Plasmodium falciparum Cryptosporidium hominis 1 Synechococcus sp. 0.9 Bacteria 0.99 Trypanosoma brucei 0.66 0.82 Microcoleus chthonoplastes Sideroxydans lithotrophicus 0.85 Trypanosoma cruzi 0.94 Leishmania major Euglenozoa Xanthomonas albilineans 0.88 Diplonema papillatum Metaseiulus occidentalis Chlamydomonas reinhardtii Acanthamoeba castellanii 0.99 Trichomonas vaginalis A 0.7 0.98 Mus musculus 1 Drosophila melanogaster Trichomonas vaginalis B 0.76 Naegleria gruberi 0.99 Saccharomyces cerevisiae 0.62 0.64 0.93 Neurospora crassa Bacteria 1 Trypanosoma cruzi 0.96 Leishmania major 1 Treponema primitia Euglenozoa 0.99 1 Dysgonomonas gadei 0.2 Diplonema papillatum Bacteroides sp. Dictyostelium discoideum Arabidopsis thaliana Oryza sativa 0.99 Pelosinus fermentans Desulfotomaculum gibsoniae Bacteria (d) Triosephosphate isomerase 0.2 0.95 Clostridium cellulolyticum 1 Mus musculus 0.8 Homo sapiens Drosophila melanogaster Dictyostelium discoideum 0.96 Saccharomyces cerevisiae (c) Phosphoglucose isomerase 0.58 Neurospora crassa Diplonema papillatum Euglenozoa Phaeodactylum tricornutum 0.63 Streptomyces sp. Bacteria 0.54 Trichomonas vaginalis Gloeobacter violaceus Naegleria gruberi 0.98 Trypanosoma cruzi 0.5 Trimastix pyriformis Trypanosoma brucei 0.71 Giardia lamblia 1 0.93 1 Leishmania major Euglenozoa Ectocarpus siliculosus Diplonema papillatum Cyanidioschyzon merolae 0.98 Trypanosoma cruzi 1 Saccharomyces cerevisiae 1 1 Neurospora crassa Trypanosoma brucei Euglenozoa 0.58 Aspergillus nidulans 0.52 Leishmania major 1 Tetrahymena thermophila Syntrophobacter fumaroxidans Bacteria Paramecium tetraurelia Dictyostelium discoideum 0.78 0.92 Toxoplasma gondii Desulfovibrio africanus 0.64 Plasmodium falciparum 0.99 Drosophila melanogaster Cryptosporidium parvum 0.7 1 Mus musculus 0.94 Oryza sativa Homo sapiens Chlamydomonas reinhardtii 0.7 Methylotenera mobilis Bacteria Arabidopsis thaliana Trimastix pyriformis 0.7 Marinomonas sp. MED121 Bacteria Magnetococcus marinus Bacteria Lautropia mirabilis 0.81 Phaeodactylum tricornutum 1 group III euryarchaeote KM3-28-E8 Archaea Entamoeba histolytica 0.97 Magnetococcus marinus Fibrisoma limi Geobacter daltonii Escherichia coli 0.91 Lactobacillus helveticus 0.96 0.99 0.5 Trichodesmium erythraeum Bacteria Photobacterium damselae Thermodesulfatator indicus 1 1 Vibrio metschnikovii Bacteria 0.2 Patulibacter sp. I11 1 Vibrio mimicus Vibrio cholerae 1 Chloroherpeton thalassium Chloroherpeton thalassium Ectocarpus siliculosus 1 Chlamydomonas reinhardtii (f) Enolase Oryza sativa 0.59 0.99 Arabidopsis thaliana 1 Toxoplasma gondii Paramecium tetraurelia Toxoplasma gondii Entamoeba histolytica 1 Tetrahymena thermophila Encephalitozoon cuniculi 1 Dictyostelium discoideum 1 1 Paramecium tetraurelia Paramecium tetraurelia Cyanidioschyzon merolae 0.67 Plasmodium falciparum Arabidopsis thaliana 0.99 Toxoplasma gondii 0.67 1 Cryptosporidium hominis Cryptosporidium parvum 0.69 Toxoplasma gondii ME49 0.2 Plasmodium falciparum 0.98 Thalassiosira pseudonana Ustilago maydis Cyanidioschyzon merolae 0.97 1 Saccharomyces cerevisiae S288c 1 Aspergillus nidulans 0.5 Neurospora crassa OR74A 0.75 Mus musculus 0.91 Mus musculus 0.92 Homo sapiens (e) Phosphoglycerate mutase 0.73 Drosophila melanogaster 0.75 Giardia lamblia Trimastix pyriformis 0.8 Sorangium cellulosum So ce 56 0.99 Trypanosoma cruzi 1 Trypanosoma brucei brucei Methylococcus capsulatuss tr.Bath Bacteria 0.99 Euglenozoa 0.97 Dechloromonas aromatica RCB Leishmania major 0.67 Diplonema papillatum Holdemania filiformis DSM12042 Dictyostelium discoideum AX4 Giardia lamblia ATCC50803 1 Cryptosporidium parvum 1 Entameoba histolytica Cryptosporidium hominis TU502 0.98 Trimastix pyriformis Chlamydomonas reinhardtii 0.91 0.98 Diplonema papillatum 0.99 Thalassiosira pseduonana Euglenozoa 0.97 Phaeodactylum tricornutum 0.99 Chlamydomonas reinhardtii Trypanosoma cruzi Ectocarpus siliculosus 0.99 0.99 Arabidopsis thaliana 0.67 Trypanosoma brucei Euglenozoa Oryza sativa Leishmania major 1 1 Naegleria gruberi 1 Arabidopsis thaliana 1 Trichomonas vaginalis Oryza sativa Japonica Group Trichomonas vaginalis Slackia piriformis YIT12062 0.57 Methanococcus voltaeA3 Silicibacterlacus caerulensis ITI-1157 Bacteria 0.97 Thermofilum pendens Hrk5 Archaea Korarchaeum cryptofilum 0.98 Methanosaeta harundinacea 6Ac Archaea 0.99 Scardovia wiggsiae Trichomonas vaginalis G3 0.63 0.53 Pelobacter carbinolicus Encephalitozoon cuniculi Lactobacillus plantarum Bacteria 0.58 0.88 0.9 0.54 0.9 Aspergillus nidulans Magnetococcus marinus MC-1 0.2 Neurospora crassa OR74A 1 Euglena gracilis Euglenozoa Fischerella sp. JSC-11 Bacteria 0.75 0.86 Euglena gracilis Glaciecola sp. 4H-3-7+YE-5 0.76 Dechloromonas aromatica Bacteria 0.2 0.61 Microcystis aeruginosa Candidatus Caldiarchaeum subterraneum Archaea
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. 0.84 Methanosarcina barkeri str.Fusaro 0.99 Methanosaeta harundinacea 6Ac 1 Methanocella conradii HZ254 Methanocella conradii HZ254 uncultured marine crenarchaeote HF4000 ANIW93H17 1 Cenarchaeum symbiosum A 0.52 Candidatus Nitrosoarchaeum limnia SFB1 1 0.98 0.99 Nitrosopumilus maritimus SCM1 Candidatus Nitrosopumilus salaria BD31 0.59 Candidatus Nitrosoarchaeum koreensis MY1 0.99 Candidatus Caldiarchaeum subterraneum 0.73 Vulcanisaeta distributa DSM14429 Candidatus Korarchaeum cryptofilumOPF8 Metallosphaera cuprina Ar4 0.51 0.99 1 Sulfolobus solfataricus 1 0.92 Sulfolobus islandicus REY15A Caldivirga maquilingensis IC167 0.86 0.99 Ignicoccus hospitalis KIN4I Aeropyrum pernix K1 Acidilobuss accharovorans 34515 Thermofilum pendensHrk5 Encephalitozoon cuniculi Phaeodactylum tricornutumCCAP10551 Archaea Pelobacter carbinolicus DSM2380 0.99 Methanoculleus marisnigri JR1 Bacteria Methanoculleus bourgensis MS2 0.98 0.99 Methanosphaerula palustris E19c 0.89 Methanoregula boonei 6A8 Archaea 0.99 Methanocella paludicola SANAE Methanocella arvoryzae MRE50 Magnetococcus marinus MC1 Bacteria 0.55 Trypanosoma cruzi strain CL Brener Ectocarpus siliculosus Euglenozoa Gloeobacter violaceus PCC7421 Bacteria Euglena gracilis 0.78 0.99 Trypanosoma cruzi strain CL Brener Leishmania major strain Friedlin 0.99 Trypanosoma cruzi strain CL Brener 0.81 0.99 Trypanosoma cruzi strain CL Brener 0.83 Trypanosoma brucei 0.99 Trypanosoma brucei brucei 0.86 Trypanosoma brucei Leishmania major strain Friedlin Diplonema papillatum 0.89 Clostridium novyi NT 0.99 Clostridium botulinum C str.Stockholm Euglenozoa 0.94 Clostridium perfringens SM101 Clostridium botulinum B str.Eklund17B Bacteria Clostridium sp.7243FAA 0.95 0.98 Clostridium beijerinckii NCIMB8052 Chlamydomonas reinhardtii Trimastix pyriformis Cryptosporidium parvum 0.7 Plasmodium falciparum 0.56 Naegleria gruberi 0.5 Toxoplasma gondii VEG 0.99 Tetrahymena thermophila 0.78 0.53 0.99 Paramecium tetraurelia strain d42 Paramecium tetraurelia Trichomonas vaginalis 0.85 Saccharomyces cerevisiae S288c 0.99 Ustilago maydis 0.99 Aspergillus nidulans 0.71 Neurospora crassa OR74A 0.89 Homo sapiens 0.99 Mus musculus 0.59 0.99 Mus musculus 0.99 Homo sapiens 0.99 Drosophila melanogaster Drosophila melanogaster 0.93 0.75 Dictyostelium discoideumAX4 0.96 Thalassiosira pseduonana Entamoeba histolytica 0.81 0.93 Euglena gracilis Euglenozoa 0.9 Giardia lamblia ATCC50803 Candidatus Accumulibacter phosphatis clad.e IIA str.UW1 0.2 Coriobacter iaceaebacterium JC110 0.99 Collinsella stercoris DSM13279 0.89 Collinsella tanakaei YIT12063 Collinsella intestinalis DSM13280 0.99 0.99 0.75 Coriobacterium glomerans PW2 Bacteria 0.99 Olsenella uli DSM7084 0.99 Atopobium vaginae PB189T14 Atopobium vaginae DSM15829 0.71 0.99 Atopobium rimae ATCC49626 Atopobium parvulum DSM20469 0.99 Ectocarpus siliculosus 0.98 Ectocarpus siliculosus 0.96 Phaeodactylum tricornutum Ectocarpus siliculosus Chlamydomonas reinhardtii Cyanidioschyzon merolae 0.83 Toxoplas magondii ME49 0.52 Ectocarpus siliculosus 0.51 0.7 Phaeodactylum tricornutum CCAP10551 Ectocarpus siliculosus 0.98 Prochlorococcus marinus str. MIT9515 Prochlorococcus marinus str. MIT9202 0.87 0.95 Fischerella sp. JSC11 Bacteria 0.53 Raphidiopsis brookii D9 0.99 Cylindrospermopsis raciborskii CS505 0.95 Cyanothece sp. PCC7822 0.94 Oryza sativa Japonica Group 0.85 Oryza sativa Indica Group 0.82 Arabidopsis thaliana 0.99 Oryza sativa Japonica Group Oryza sativa Indica Group 0.87 Arabidopsis thaliana 0.94 Arabidopsis thaliana Bacteria 0.99 Stigmatella aurantiaca DW431 Corallococcus coralloides DSM2259 0.99 0.96 Chondromyces apiculatusDSM436 Anaeromyxobacter sp. K 0.99 Anaeromyxobacter sp. Fw1095 1 gammaproteobacteria 0.99 Francisella philomiragia subsp. philomiragia ATCC25015 Methylococcus capsulatus str.Bath 0.61 1 Lautropia mirabilis ATCC51599 0.99 Oxalobacter formigenes OXCC13 Herminiimonas arsenicoxydans 0.99 0.99 Cupriavidus basilensis OR16 0.76 Burkholderia ambifaria AMMD 0.92 Rhodomicrobium vannielii ATCC17100 Hyphomicrobium sp. MC1 0.97 0.99 Methylocystis sp. SC2 0.89 Methylocystis sp. ATCC49242 0.99 Methylobacterium populi BJ001 0.99 Rhodospirillum centenum SW 0.99 Commensalibacter intestini A911 Acetobacter aceti NBRC14818 Acetobactert ropicalis NBRC101654
Figure S2. Consensus phylogenetic tree for phosphoglycerate kinase. The methods and labeling are as in figure S1. Entamoeba histolytica 0.99 Trichomonas vaginalis Trichomonas vaginalisG3 0.98 Synechococcus sp. RS9916 Prochlorococcus marinus str.MIT9515 Bacteria Naegleria gruberi 0.97 Methanocella paludicola SANAE 0.95 Methanocella conradii HZ254 Archaea Methanocella arvoryzae MRE50 0.51 Giardia lamblia ATCC50803 Giardia lamblia ATCC50803 0.78 Trimastix pyriformis Cyanothece sp.PCC 7822 Haliangium ochraceum DSM14365 Bacteria 0.74 0.51 0.99 Stigmatella aurantiaca DW4/3-1 Corallococcus coralloides DSM2259 Candidatus KorarchaeumcryptofilumOPF8 0.79 ThermofilumpendensHrk5 Archaea Candidatus Caldiarchaeum subterraneum 0.92 Toxoplasma gondii 0.97 Plasmodium falciparum 3D7 Ectocarpus siliculosus 0.6 0.99 Pseudogulbenkiania sp. NH8B 0.99 Herbaspirillum sp. GW103 Herbaspirillum sp. CF444 0.96 Granulibacter bethesdensis CGDNIH1 Bacteria 0.99 Azospirillum sp.B510 0.64 Azospirillum lipoferum 4B Acidianus hospitalis W1 Metallosphaera yellowstonensis MK1 Archaea Drosophila melanogaster 0.77 Cyanidioschyzon merolae Arabidopsis thaliana 0.87 Paramecium tetraurelia Tetrahymena thermophila Naegleria gruberi 0.99 Vibrio metschnikovii CIP69.14 Moritella sp. PE36 Bacteria 0.99 Moritellamarina ATCC15381 Ectocarpus siliculosus Ustilago maydis 0.98 0.99 Saccharomyce scerevisiae S288c Saccharomyces cerevisiae S288c Aspergillus nidulans 0.91 0.99 0.99 Neurospora crassa OR74A Encephalitozoon cuniculi 0.99 Mus musculus Homo sapiens 0.94 0.87 0.8 Mus musculus Drosophila melanogaster 0.91 Trypanosoma cruzi strain CL Brener 0.55 Trypanosoma brucei brucei strain 927/4 GUTat10.1 Euglenozoa 0.98 0.99 Leishmania major strain Friedlin Diplonema papillatum 0.99 Dictyostelium discoideum Dictyostelium discoideum AX4 0.99 Olsenellasp.oraltaxon 809 str.F0356 0.99 Coriobacterium glomerans PW2 Collinsella tanakaei YIT12063 0.99 Paenibacillus sp. oraltaxon 786 str.D14 Bacteria 0.98 Paenibacillus alvei DSM29 Brevibacillus laterosporus LMG15441 0.97 Cryptosporidium parvum Toxoplasma gondii ME49 0.99 Plasmodium falciparum 3D7 0.93 Tetrahymena thermophila Tetrahymena thermophila 0.94 0.99 Paramecium tetraurelia strain d4-2 0.7 0.99 Thalassiosira pseudonana Phaeodactylum tricornutum CCAP1055/1 0.95 0.96 Phaeodactylum tricornutum CCAP1055/1 0.99 Ectocarpus siliculosus Chlamydomonas reinhardtii Chlamydomonas reinhardtii 0.98 Chlaymdomonas reinhardtii 0.94 Oryza sativa Japonica Group 0.85 Arabidopsis thaliana 0.86 Oryza sativa Indica Group 0.2 0.89 Arabidopsis thaliana
Figure S3. Consensus phylogenetic tree for pyruvate kinase. The methods and labeling are as in figure S1. Acetivibrio cellulolyticus CD2 0.97 Clostridium thermocellum ATCC 27405 Clostridium clariflavum DSM 19732 0.98 Paludibacter propionicigenes WB4 Clostridium papyrosolvens DSM 2782 Bacteria 0.99 Clostridium sp. MSTE9 0.96 Clostridium leptum DSM 753 Clostridium methylpentosum DSM 5476 0.86 Anaerotruncus colihominis DSM 17241 Giardia lamblia 0.94 Entamoeba histolytica Phaeodactylum tricornutum 0.65 0.99 Tetrahymena thermophila 0.98 Tetrahymena thermophila 0.99 Paramecium tetraurelia Paramecium tetraurelia strain d4 2 Trypanosoma brucei 1 Trypanosoma cruzi strain CL Brener Euglenozoa Leishmania major strain Friedlin 0.9 0.78 Encephalitozoon cuniculi Giardia lamblia ATCC 50803 Toxoplasma gondii VEG 1 Toxoplasma gondii 0.95 Toxoplasma gondii 0.97 Phaeodactylum tricornutum CCAP 1055 1 Ectocarpus siliculosus 0.84 Thalassiosira pseudonana 0.97 Phaeodactylum tricornutum CCAP 1055 1 0.79 Phaeodactylum tricornutum CCAP 1055 1 0.95 Ectocarpus siliculosus Ectocarpus siliculosus 0.82 Naegleria gruberi 0.84 Diplonema papillatum GAPDH2 0.96 Diplonema papillatum GAPDH1 Euglenozoa Diplonema sp. ATCC 50225 Syntrophus aciditrophicus SB Bacteria Trypanosoma cruzi strain CL Brener 0.94 0.99 Trypanosoma cruzi strain CL Brener 0.99 Trypanosoma cruzi strain CL Brener Trypanosoma cruzi strain CL Brener Euglenozoa 0.9 0.95 Trypanosoma cruzi strain CL Brener Trypanosoma brucei 0.87 0.69 Xenorhabdus bovienii SS 2004 Morganella morganii subsp. morganii KT Yersinia enterocolitica subsp. enterocolitica 8081 0.97 Hafnia alvei ATCC 51873 0.95 Erwinia tasmaniensis Et1 99 0.99 Synechococcus sp. WH 5701 Synechococcus sp. CB0101 0.99 Cyanobium sp. PCC 7001 Polaribacter irgensii 23 P Bacteria 0.62 Nitrosospira sp. TCH716 0.67 0.64 0.99 Magnetospirillum magnetotacticum MS 1 0.99 Magnetospirillum gryphiswaldense MSR 1 Hydra magnipapillata blood disease bacterium R229 0.99 0.99 Hylemonella gracilis ATCC 19624 0.99 Cupriavidus metallidurans CH34 Toxoplasma gondii VEG 0.89 Plasmodium falciparum Ectocarpus siliculosus Cryptosporidium parvum Iowa II 0.99 Saccharomyces cerevisiae S288c 0.53 Saccharomyces cerevisiae S288c Chlamydomonas reinhardtii 0.99 Chlamydomonas reinhardtii 0.9 Chlamydomonas reinhardtii 0.88 Arabidopsis thaliana 1 Oryza sativa Indica Group Arabidopsis thaliana Cyanidioschyzon merolae 0.96 Ustilago maydis Neurospora crassa OR74A 0.99 Mus musculus 1 Homo sapiens Homo sapiens 0.56 0.75 Homo sapiens 0.99 Homo sapiens Mus musculus 0.54 0.95 0.77 Mus musculus Drosophila melanogaster 0.85 0.94 Drosophila melanogaster Dictyostelium discoideum AX4 0.99 Oryza sativa Japonica Group Oryza sativa Japonica Group 0.98 Oryza sativa Japonica Group 0.99 Arabidopsis thaliana Arabidopsis thaliana 0.95 Microcystis aeruginosa NIES 843 Cyanothece sp. PCC 7425 0.99 SAR324 cluster bacterium SCGC AAA001 C10 Bacteria 0.87 SAR324 cluster bacterium JCVI SC AAA005 0.99 Leishmania major 0.98 Trypanosoma cruzi strain CL Brener 0.99 Trypanosoma brucei brucei strain 9274GUTat10.1 Euglenozoa 0.99 Trypanosoma brucei brucei 0.61 Euglena gracilis Pelobacter carbinolicus DSM 2380 0.89 0.96 Geobacter uraniireducens Rf4 Bacteria Aspergillus nidulans Methanoculleus bourgensis MS2 0.8 Halopiger xanaduensis SH 6 0.91 0.99 1 Natronobacterium gregoryi SP2 Halobiforma lacisalsi AJ5 1 0.97 Natronobacterium gregoryi SP2 Archaea 0.99 Halobiforma lacisalsi AJ5 0.91 1 Halogranum salariumB1 0.97 Halogranum salariumB1 Halalkalicoccus jeotgali B3 Phaeodactylum tricornutum CCAP 1055 1 0.99 Ectocarpus siliculosus Diplonema sp. ATCC 50225 0.99 1 0.81 0.69 0.93 Rhynchopus sp. ATCC 50231 Diplonema sp. ATCC 50225 Chlamydomonas reinhardtii uncultured archaeon Euglenozoa Thermofilum pendens Hrk 5 Archaea Candidatus Korarchaeum cryptofilum OPF8 delta proteobacterium NaphS2 0.55 Geobacter uraniireducens Rf4 0.66 0.99 1 Geobacter sp. M21 0.62 Geobacter sp. M18 Geobacter daltonii FRC 32 0.2 0.99 alpha proteobacterium IMCC14465 0.51 Tistrella mobilis KA081020 065 Parvularcula bermudensis HTCC2503 1 Azospirillum lipoferum 4B 0.51 Azospirillum brasilense Sp245 Bacteria 0.72 Chlamydomonas reinhardtii 0.51 Actinomyces urogenitalis DSM 15434 Candidatus Aquiluna sp. IMCC13023 0.99 0.99 Renibacterium salmoninarum ATCC 33209 0.96 0.69 Arthrobacter sp. Rue61a 0.78 0.99 Arthrobacter globiformis NBRC 12137 Arthrobacter chlorophenolicus A6 Actinomyces sp. oral taxon 448 str. F0400 1 Trimastix pyriformis 0.99 Trichomonas vaginalis G3 0.64 0.99 Trichomonas vaginalis G3 Trichomonas vaginalis G3 0.75 uncultured archaeon GZfos1D1 Aciduliprofundum boonei T469 Archaea
Figure S4. Consensus phylogenetic tree for glyceraldehyde-3-phosphate dehydrogenase. The methods and labeling are as in figure S1. Diplonema papillatum Euglenozoa (FBPase) 1 Desulfobulbus propionicus 1 delta proteobacterium MLMS-1 0.99 Desulfurivibrio alkaliphilus delta proteobacterium NaphS2 0.98 Desulfovibrio aespoeensis 0.67 0.75 1 Desulfovibrio salexigens Desulfovibrio vulgaris 0.99 Toxoplasma gondii Toxoplasma gondii 0.79 Synechococcus sp. PCC7335 0.87 Synechococcus sp. JA-3-3Ab 0.99 Synechococcus elongatus 0.79 1 Cylindrospermopsis raciborskii 1 0.99 Raphidiopsis brookii Bacteria Fischerella sp. JSC-11 Nostoc punctiforme 0.83 1 Anabaena variabilis 0.83 0.85 Nostoc sp. PCC7120 0.77 Synechocystis sp. PCC6803 Hippea maritima Calditerrivibrio nitroreducens 0.99 0.99 Deferribacter desulfuricans Sorangium cellulosum 1 Desulfurobacterium thermolithotrophum Thermovibrio ammonificans 0.96 Thermodesulfobium narugense 0.99 Thermodesulfobium narugense 1 Sulfurihydrogenibium yellowstonense 0.99 Sulfurihydrogenibium azorense Hydrogenivirga sp. Tetrahymena thermophila 0.99 Thalassiosira pseudonana 0.99 Phaeodactylum tricornutum 0.52 Ectocarpus siliculosus 0.9 Mus musculus 1 Mus musculus 1 Mus musculus 1 Homo sapiens Mus musculus 0.99 0.94 Homo sapiens 0.99 Homo sapiens Drosophila melanogaster Dictyostelium discoideum 1 Tetrahymena thermophila 0.78 Tetrahymena thermophila Cyanidioschyzon merolae 0.68 Phaeodactylum tricornutum Ectocarpus siliculosus 0.7 0.84 1 Phaeodactylum tricornutum Phaeodactylum tricornutum 0.87 0.61 Ectocarpus siliculosus 0.99 Phaeodactylum tricornutum Ectocarpus siliculosus 0.98 0.66 1 Ectocarpus siliculosus 1 Oryza sativa Arabidopsis thaliana Chlamydomonas reinhardtii 1 1 Oryza sativa 1 Oryza sativa Arabidopsis thaliana 0.62 0.99 Oryza sativa 0.98 Oryza sativa 0.97 Oryza sativa 0.99 Oryza sativa Arabidopsis thaliana Ustilago maydis 0.99 Trypanosoma cruzi 1 Trypanosoma brucei 0.83 Leishmania major Euglenozoa (FBPase) 0.96 1 Saccharomyces cerevisiae 1 Saccharomyces cerevisiae 1 Neurospora crassa Aspergillus nidulans Methylotenera versatilis Magnetospirillum magneticum 0.99 Sulfitobacter sp. 0.51 Roseobacter sp. GAI101 0.99 Octadecabacter arcticus 1 Agrobacterium rhizogenes Agrobacterium rhizogenes 0.93 Pseudogulbenkiania sp. NH8B Methyloversatilis universalis 0.95 Oxalobacter formigenes Sutterella parvirubra Neisseria mucosa 0.99 Neisseria weaveri 0.82 Neisseria meningitidis 0.86 1 0.95 Neisseria meningitidis 93004 0.74 Neisseria meningitidis MC58 0.56 Saccharophagus degradans Bacteria Pseudoalteromonas haloplanktis Idiomarina sp. A28L 0.68 0.66 1 Shewanella baltica OS223 Shewanella baltica OS195 0.82 1 Marinomonas mediterranea Marinomonas sp. MWYL1 Oceanobacter sp. RED65 Rhodovulum sp. PH10 0.99 Caulobacter sp. AP07 0.76 Caulobacter sp. K31 Streptomyces coelicoflavus Mycobacterium tusciae 0.97 0.87 Nocardia cyriacigeorgica Microlunatus phosphovorus 0.93 Pseudonocardia dioxanivorans Methanoplanus petrolearius Halopiger xanaduensis Natrinema pellirubrum 0.71 0.66 Natronomonas pharaonis 0.55 0.93 1 Haloarcula marismortui Haloarcula hispanica Archaea 1 0.83 1 Haloferax mediterranei Haloferax volcanii Natrialba magadii Haloterrigena turkmenica 0.99 Chlamydomonas reinhardtii 1 Trypanosoma cruzi 0.82 Trypanosoma brucei Euglenozoa (SBPase) Tetrahymena thermophila 1 0.99 Paramecium tetraurelia 1 0.98 Paramecium tetraurelia Paramecium tetraurelia 0.99 Paramecium tetraurelia 0.92 0.98 Toxoplasma gondii 0.6 Phaeodactylum tricornutum Neurospora crassa Ectocarpus siliculosus 0.2 Ectocarpus siliculosus 0.99 1 Chlamydomonas reinhardtii Chlamydomonasr einhardtii 1 0.88 Chlamydomonas reinhardtii Arabidopsis thaliana
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) HK GLK PGI PFK1 PFK2 TIM GAPDH1 PGK PGM ENO PK kDa
150- 100- 75-
50-
37-
25- 21-
15-
(b)
A B C
D
M
M E F N
B Peroxisome
D 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. X-axis representsthenumber of for 72hrs(c).TheY-axisindicatesthe relative abundance(%)ofeachisotopomertherelevant metabolite.The Figure S7. three independentexperiments. 6 mM (b) 6mM (a) 6mM (c) 30mM Relative abundance (%) Relative abundance (%) Relative abundance (%) 10 15 20 10 15 20 10 20 30 10 15 20 10 15 20 10 15 20 13 0 0 5 0 5 0 5 0 5 0 5 C 1 2 3 4 5 +6 +5 +4 +3 +2 +1 1 2 3 4 5 +6 +5 +4 +3 +2 +1 1 2 3 4 5 +6 +5 +4 +3 +2 +1 5 1 2 3 4 5 +6 +5 +4 +3 +2 +1 1 2 +3 +2 +1 1 2 +3 +2 +1 Glucose 6-phosphate Glucose 6-phosphate -glutamine for0,4,and8hrs (a),6mM 6-phosphate Lactate Lactate Metabolomic analysisof 13 13 Glucose Citrate C C 13 C 6 5 -L-glutamine -D-glucose 6 8 hr 4 hr 0 hr -D-glucose 8 hr 4 hr 0 hr 13C 12C
13C 12C 10 20 30 40 50 10 15 20 0 0 5 10 15 20 0 5 10 20 30 8 hr 4 hr 0 hr 8 hr 4 hr 0 hr 1 2 3 4 5 +6 +5 +4 +3 +2 +1 1 2 3 4 +5 +4 +3 +2 +1 0 1 2 3 +4 +3 +2 +1 Succinate α-ketoglutarate 1 2 3 4 5 +6 +5 +4 +3 +2 +1 6-phosphate 10 15 20 0 5 10 15 20 Fructose 0 5 Citrate 1 2 3 4 5 +6 +5 +4 +3 +2 +1 Fructose 6-phosphate 13 1 2 3 4 5 +6 +5 +4 +3 +2 +1 Fructose 6-phosphate C intheisotopomers.Data pointsanderrorbarsrepresentthemean±s.d.of 10 15 20 13 0 5
C-labeled metabolitesin 1 2 3 +4 +3 +2 +1 Malate 10 20 30 10 20 30 0 0 10 15 20 0 5 1 2 3 4 5 +6 +5 +4 +3 +2 +1 1, 1 2 3 +4 +3 +2 +1 6-bis 1 2 3 +4 +3 +2 +1 10 20 30 40 Fructose 0 Succinate Malate 13 phos 1 2 3 4 +5 +4 +3 +2 +1 C 10 15 20 5-phosphate 0 5 6 10 15 20 -D-glucose for0,4,and8hrs (b),or30mM Ribose phate 0 5 Fructose 1,6-isphosphate 1 2 3 4 5 +6 +5 +4 +3 +2 +1
1 2 3 4 5 +6 +5 +4 +3 +2 +1 10 15 20 10 15 20 bisphosphate Fructose 1,6- 0 5 0 5 10 20 30 40 0 D. papillatum 1 2 3 +4 +3 +2 +1 10 20 30 1 2 3 4 5 +6 +5 +4 +3 +2 +1 0 1 2 +3 +2 +1 Fumarate DHAP 1 2 3 4 +5 +4 +3 +2 +1 5-phosphate Ribulose Citrate 10 20 30 10 15 20 0 10 15 20 0 5 . D.papillatum 0 5 3-phospho- 1 2 +3 +2 +1 glycerate 10 15 20 1 2 +3 +2 +1 0 5 1 2 3 +4 +3 +2 +1 3-phospho- 10 20 30 40 glycerate 0 Malate 1 2 +3 +2 +1 3-phospho- 1 2 3 4 5 6 +7 +6 +5 +4 +3 +2 +1 glycerate Sedoheptulose 10 20 30 40 7-phosphate 0 10 15 20 1 2 3 4 5 6 +7 +6 +5 +4 +3 +2 +1 0 5 1 2 +3 +2 +1 10 15 20 Lactate cellswereincubatedwith 0 5 10 20 30 40 Sedoheptulose 0 7-phosphate Phosphoenol- 1 2 +3 +2 +1 1 2 3 4 5 6 +7 +6 +5 +4 +3 +2 +1 10 20 30 40 pyruvate 0 Phosphoenol- 1 2 +3 +2 +1 Sedoheptulose 13 7-phosphate pyruvate C 6 -D-glucose Leptotrichia hofstadii 0.99 Streptomyces sp. NRRL S 87 Bacteria 1 Desulfotalea psychrophila 0.99 0.94 Euglena gracilis Euglenozoa Desulfonatronum thiodismutans Clostridiales bacterium VE202 09 Bacteria Francisella philomiragia 0.77 Trypanosoma brucei brucei TREU927 Trypanosoma grayi 0.99 1 0.98 Trypanosoma cruzi CL Brener 1 Trypanosoma brucei brucei TREU927 Phytomonas sp. isolate Hart1 0.9 Leishmania major Friedlin Strigomonas culicis 0.63 0.94 Angomonas deanei 1 Necropsobacter rosorum Euglenozoa (glycosomal) Pasteurella sp. FF6 1 Haemophilus sp. FF7 0.99 Pseudomonas aeruginosa Bacteria 0.56 0.56 Methylococcaceae bacterium Sn10 6 Burkholderiales bacterium 1147 0.98 Acinetobacter soli Toxoplasma gondii 1 Neospora caninum Liverpool Diplonema papillatum Euglenozoa Blastocystis hominis 0.99 Phytophthora infestans T30 4 Saprolegnia parasitica 0.99 Aphanomyces invadans Trypanosoma grayi 0.76 Trypanosoma cruzi CL Brener Trypanosoma brucei brucei TREU927 1 Strigomonas culicis 0.67 Euglenozoa Phytomonas sp. isolate Hart1 0.91 Leptomonas pyrrhocoris 0.68 0.99 Leishmania major Friedlin Angomonas deanei 0.95 Volvox carteri f. nagariensis Helicosporidium sp. ATCC 50920 Ectocarpus siliculosus Ostreococcus tauri 0.96 Micromonas sp. RCC299 0.53 Bathycoccus prasinos Arabidopsis thaliana 1 Ricinus communis Oryza sativa Japonica Group 0.99 Glycine soja 0.95 0.99 Aegilops tauschii Oxytricha trifallax Galdieria sulphuraria Rhizopus delemar RA 99 880 0.99 0.5 Coprinopsis cinerea cytosolic 1 0.92 Wallemia sebi CBS 633.66 Cryptococcus neoformans var. grubii H99 0.74 Trichophyton soudanense CBS 452.61 0.76 Podospora anserina S mat+ 0.95 0.99 0.9 1 Saccharomyces cerevisiae S288c Candida albicans 12C Salpingoeca rosetta Capsaspora owczarzaki ATCC 30864 0.59 0.64 Solenopsis invicta 1 Drosophila melanogaster 0.93 Danaus plexippus 0.59 Plutella xylostella 1 0.95 0.64 Bombyx mori 1 Xenopus laevis mitochondrial 2 Homo sapiens mitochondrial 0.76 Ascaris suum Acanthamoeba castellanii str. Neff 0.2 0.53 Dictyostelium discoideum
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. Clostridium botulinum A ATCC 3502 Thermoanaerobacter tengcongensis 1 Lactococcus lactis Bacillus subtilis Bacteria Helicobacter pylori 26695 0.56 Thermus thermophilus HB27 Spirochaeta thermophila DSM 6192 0.99 Pyrococcus furiosus DSM 3638 Archaea 0.91 1 Tetrahymena thermophila 0.75 1 Paramecium tetraurelia 0.5 1 Trypanosoma brucei Trypanosoma cruzi Euglenozoa 0.59 Leishmania major (glycosomal) 0.94 Fibrobacter succinogenes Chlorobium tepidum Fusobacterium nucleatum ATCC 25586 0.7 Bacteroides thetaiotaomicron Thermotoga maritima MSB8 0.93 Aquifex aeolicus Bacteria 1 Neisseria gonorrhoeae Escherichia coli 0.99 Acidobacterium capsulatum 0.85 Geobacter sulfurreducens Phytophthora infestans 0.99 Toxoplasma gondii Plasmodium falciparum 3D7 0.68 1 Thalassiosira pseudonana 0.54 Phaeodactylum tricornutum Guillardia theta Dictyostelium discoideum Emiliania huxleyi 0.63 Cyanidioschyzon merolae Euglena gracilis 0.7 0.97 Diplonema papillatum Trypanosoma cruzi Brener Euglenozoa 0.81 1 1 Trypanosoma brucei 0.93 Leishmania major 0.99 Saccharomyces cerevisiae 1 Mus musculus 0.59 Homo sapiens 0.71 Drosophila melanogaster Monosiga brevicollis 0.66 Caenorhabditis elegans 0.99 Chlamydomonas reinhardtii 1 Zea mays 0.2 Arabidopsis thaliana
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. Mycobacterium tuberculosis Bacteria 0.87 Trichomonas vaginalis Giardia lamblia Geobacter metallireducens Deinococcus radiodurans Chlorobium tepidum Synechocystis sp. PCC 6803 0.69Chloroflexus aurantiacus 0.78 Chlamydia trachomatis Borrelia burgdorferi B31 Bacteria Bacillus subtilis Thermotoga maritima MSB8 0.67 Bacteroides thetaiotaomicron Aquifex aeolicus 0.97 Agrobacterium sp. H13 3 Neisseria gonorrhoeae FA 1090 Naegleria gruberi Zea mays 0.99 Cyanidioschyzon merolae 0.62 Toxoplasma gondii 1 Phytophthora sojae Saccharomyces cerevisiae 0.78 Capsaspora owczarzaki ATCC 30864 Caenorhabditis elegans 1 Solenopsis invicta 0.61 0.99 1 Homo sapiens Bos taurus Arabidopsis thaliana Diplonema papillatum 1 Trypanosoma cruzi strain CL Brener 1 0.65 Trypanosoma brucei brucei TREU927 Phytomonas sp. isolate EM1 Euglenozoa 1 0.79 Leishmania major strain Friedlin Strigomonas culicis 0.2 0.77 Angomonas deanei
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.