RESEARCH ARTICLES
Mosaic Origin of the Heme Biosynthesis Pathway in Photosynthetic Eukaryotes
Miroslav Obornı́k* à and Beverley R. Green*
*Department of Botany, University of British Columbia, Vancouver BC, Canada; Institute of Parasitology, Academy of
Sciences of Czech Republic, Branišovská, České Budějovice, Czech Republic; and àFaculty of Biological Sciences,
University of South Bohemia, Branišovská, České Budějovice, Czech Republic
Heme biosynthesis represents one of the most essential metabolic pathways in living organisms, providing the precursors
for cytochrome prosthetic groups, photosynthetic pigments, and vitamin B12. Using genomic data, we have compared the
heme pathway in the diatom Thalassiosira pseudonana and the red alga Cyanidioschyzon merolae to those of green algae
and higher plants, as well as to those of heterotrophic eukaryotes (fungi, apicomplexans, and animals). Phylogenetic
analyses showed the mosaic character of this pathway in photosynthetic eukaryotes. Although most of the algal and plant
enzymes showed the expected plastid (cyanobacterial) origin, at least one of them (porphobilinogen deaminase) appears to
have a mitochondrial (a-proteobacterial) origin. Another enzyme, glutamyl-tRNA synthase, obviously originated in the
eukaryotic nucleus. Because all the plastid-targeted sequences consistently form a well-supported cluster, this suggests that
genes were either transferred from the primary endosymbiont (cyanobacteria) to the primary host nucleus shortly after the
primary endosymbiotic event or replaced with genes from other sources at an equally early time, i.e., before the formation
of three primary plastid lineages. The one striking exception to this pattern is ferrochelatase, the enzyme catalyzing the first
committed step to heme and bilin pigments. In this case, two red algal sequences do not cluster either with the other plastid
sequences or with cyanobacterial sequences and appear to have a proteobacterial origin like that of the apicomplexan
parasites Plasmodium and Toxoplasma. Although the heterokonts also acquired their plastid via secondary endosymbiosis
from a red alga, the diatom has a typical plastid-cyanobacterial ferrochelatase. We have not found any remnants of the
plastidlike heme pathway in the nonphotosynthetic heterokonts Phytophthora ramorum and Phytophthora sojae.
Introduction
The heme biosynthesis pathway is common to prokaryotes and eukaryotes. It is a fundamental metabolic pathway
needed for biosynthesis of cytochromes, chlorophylls, phycobilins, and the corrin nucleus of vitamin B12. However, the
first part of the pathway, the synthesis of 5-aminolevulinate
(ALA), differs in various organisms. In photosynthetic
eukaryotes and all prokaryotes that are not members of
the a-proteobacterial group, ALA is synthesized by the
C5 pathway starting with the five-carbon precursor glutamate, which is ligated to tRNAGlu by glutamyl-tRNA
synthase (GluRS) (fig. 1A), reduced to form glutamate-1semialdehyde by glutamyl-tRNA reductase, and then transaminated by glutamate-1-semialdehyde 2,1-aminomutase
(GSA) to give ALA (Beale 1999). On the other hand, all
eukaryotes that do not possess a photosynthetic plastid (animals, fungi, and apicomplexans), as well as a-proteobacteria,
form ALA by condensation of succinyl-CoA with glycine
in a reaction catalyzed by ALA synthase (fig.1B).
From ALA to protoporphyrin IX, the pathway is generally the same in all organisms. In photoautotrophs, the
pathway branches at protoporphyrin IX (fig. 1), with the
insertion of Fe (II) to give heme or Mg (II) to give Mgprotoporphyrin IX, the first committed step of chlorophyll
synthesis. In photosynthetic eukaryotes, the story is further
complicated by the fact that heme is required for cytochrome
biosynthesis in at least three cellular compartments: the
cytoplasm, the mitochondrion, and the chloroplast. In the
chloroplast, heme is needed not only for the assembly of
Key words: heme biosynthetic pathway, algae, chloroplasts,
evogenomics.
E-mail: [email protected].
Mol. Biol. Evol. 22(12):2343–2353. 2005
doi:10.1093/molbev/msi230
Advance Access publication August 10, 2005
Ó The Author 2005. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
the cytochrome b6f complex of the photosynthetic electron
transport chain but also as the precursor of phytochromobilin (the chromophore of the photoregulatory molecule phytochrome) and the phycobilin pigments in those algae that
use phycobilisomes for light harvesting. In plants, the steps
from Mg-protoporphyrin IX to chlorophyll are localized in
the chloroplast, but the subcellular localization of the reactions common to heme and chlorophyll biosynthesis have
been the subject of much discussion. However, it now
appears that all these enzymes are localized only in the
chloroplast (Papenbrock and Grimm 2001; Cornah, Terry,
and Smith 2003), with the exception of protoporphyrinogen
oxidase, which is dually targeted to chloroplasts and mitochondria (Watanabe et al. 2001), and a small amount of ferrochelatase activity found in mitochondria (Cornah, Terry,
and Smith 2003).
Many lines of evidence support the idea that the first
chloroplast was the result of an endosymbiotic relationship
between a cyanobacterium and a nonphotosynthetic eukaryote (Delwiche and Palmer 1997; McFadden 2001).
This eukaryote almost certainly had a mitochondrion and
was able to synthesize heme, although we have no idea
where the heme biosynthesis pathway was located. Most
of the cyanobacterial genes were lost from the plastid genome, but as many as 2,000 were transferred to the host
nucleus (Martin et al. 2002). In order for the products of
transferred genes to function in the plastid, they had to acquire presequences that would target them to the plastid. At
the same time, some of the host’s nuclear genes could also
have acquired such presequences. An example of the latter
case is the plastid-targeted fructose bisphosphate aldolase
of plants and green algae, which appears to have resulted
from duplication of the gene encoding the cytosolic enzyme
(Gross et al. 1999).
2344 Obornı́k and Green
A
1
Glutamyl-tRNA
reductase
2
B
Succinyl-CoA
L-glutamate
Glutamyl-tRNA
synthase
Glycine
Glutamyl-tRNA
5-aminolevulinate
(ALA) synthase
(S)Glutamate-1-semialdehyde
Glutamate-1-semialdehyde
2,3 aminomutase (GSA)
3
5-aminolevulinate (ALA)
Porphobilinogen
synthase
4
Porphobilinogen
deaminase
5
Porphobilinogen
synthesis of cytochromes
Preuroporphyrinogen
Uroporphyrinogen
synthase
6
Uroporphyrinogen
decarboxylase
7
Uroporphyrinogen III
Protoheme (heme)
Coproporphyrinogen III
Coproporphyrinogen
oxidase
10
8
Ferrochelatase
Protoporphyrin IX
Protoporphyrinogen IX
Protoporphyrinogen
oxidase
9
Mg-chelatase
Mg-protoporphyrin IX
synthesis of chlorophyll
FIG. 1.—General scheme of the heme biosynthesis pathway. The first part of the pathway, synthesis of ALA, differs in various organisms. In
photosynthetic eukaryotes, ALA is synthesized in the plastid via the ‘‘glutamate’’ or C5 pathway (A), while in animals, fungi, and apicomplexans,
ALA is produced in the mitochondrion using the Shemin or ‘‘succinyl-CoA’’ pathway (B).
All the algae with chlorophyll c are believed to have
acquired their plastids by a process called secondary endosymbiogenesis, in which a red algal endosymbiont was
engulfed and domesticated by a nonphotosynthetic eukaryotic host (Delwiche and Palmer 1997; McFadden 2001).
These algae include the photosynthetic heterokonts such
as diatoms, brown algae, and chrysophytes, as well as haptophytes, cryptophytes, and dinoflagellates. It is not clear if
they all originated in a single endosymbiotic event, but comparison of heterokont, haptophyte, and cryptophyte plastid
genes suggests that these three groups, collectively referred
to as Kingdom Chromista (Cavalier-Smith 2002), had a common ancestor (Yoon et al. 2002). However, the dinoflagellates belong to the Kingdom Alveolata along with ciliates
and apicomplexans. Although the apicomplexan parasites
such as Plasmodium and Toxoplasma are not photosynthetic,
they do have a relict plastid with a reduced plastid genome,
and the plastid is the site of a number of metabolic pathways
(Ralph et al. 2004). In fact, several pieces of evidence support
the suggestion that there was only one secondary endosymbiotic event involving a red algal endosymbiont, giving rise
to the chromalveolates (Cavalier-Smith 2002), a group including both the chromists and the alveolates (Fast et al.
2001; Patron, Rogers, and Keeling 2004).
In any case, in order for the secondary endosymbiotic
relationship(s) to become established, there must have been
a substantial amount of gene transfer from the red algal nucleus to the host nucleus, and these transferred genes would
have included many that encoded chloroplast-targeted proteins such as the enzymes of the heme biosynthesis pathway. Now that the draft genome sequences of the diatom
Thalassiosira pseudonana (Armbrust et al. 2004) (http://
genome.jgi-psf.org/thaps1/thaps1.home.html) and the red
alga Cyanidioschyzon merolae (Matsuzaki et al. 2004)
(http://merolae.biol.s.u-tokyo.ac.jp/) are available, it is possible to investigate the evolutionary history of the heme
biosynthesis enzymes in photosynthetic eukaryotes with
primary and secondary plastids. We also looked for these
genes in the draft genomes of two nonphotosynthetic heterokonts, the plant pathogens Phytophthora ramorum and
Phytophthora sojae (http://genome.jgi-psf.org/ramorum/
and http://genome.jgi-psf.org/sojae/). If the common ancestor of all the heterokonts had a plastid, the oomycetes
might have retained red algal–like genes, even though they
now have no trace of a plastid. We also analyzed sequences
from Plasmodium falciparum, Plasmodium yoelii (http://
www.ncbi.nlm.nih.gov/), and Toxoplasma gondii (http://
www.toxodb.org/ToxoDB.shtml).
Materials and Methods
Sequences
The genome of the diatom T. pseudonana (http://
genome.jgi-psf.org/) was searched for putative genes of
the heme pathway (Armbrust et al. 2004). The diatom
gene models were compared to the entire National Center
Mosaic Origin of Heme Biosynthesis 2345
for Biotechnology Information protein database to make
sure a well-identified gene was one of the best matches.
Each gene model was examined and, if necessary, extended
to make sure it had the N-terminal endoplasmic reticulum
(ER) signal sequence required for the first step of chloroplast
targeting in heterokont algae (Lang, Apt, and Kroth 1998;
Kroth and Strotmann 1999; Kroth 2002). The identity of the
T. pseudonana ferrochelatase was confirmed by sequencing.
Sequences of the red alga C. merolae were obtained from the
genomic Web site (http://merolae.biol.s.u-tokyo.ac.jp/):
they were well annotated and did not need any further editing. A partial sequence of ferrochelatase from the red alga
Porphyra yezoensis was assembled from two largely overlapping expressed sequence tags (ESTs) (http://www.kazusa.
or.jp/en/plant/porphyra/EST/) giving a total length of 185
amino acids. The Galdieria sulfuraria EST database
(http://genomics.msu.edu/galdieria) had a single EST
(A438G06) encoding a 285-residue sequence clearly related to red algal ferrochelatases. Ferrochelatase from
the apicomplexan parasite T. gondii was extracted from
the ToxoDB Web site (http://www.toxodb.org/ToxoDB.
shtml): from 30 contigs, the 2 longest were selected for
phylogenetic analysis. Because they display some variability in their amino acid sequences, we used both
sequences for tree reconstructions. Annotated gene models
from P. sojae and P. ramorum with good EST support
were obtained at the Joint Genome Initiative genome
Web site (http://genome.jgi-psf.org/).
Phylogenetic Analysis
Phylogenetic trees of all enzymes of the heme biosynthesis pathway were constructed using amino acid sequences from photosynthetic eukaryotes, fungi, animals,
proteobacteria, cyanobacteria, Firmicutes, and Archaea
downloaded from GenBankTM (http://www.ncbi.nlm.nih.
gov/); accession numbers are given in Supplementary
Table 1 (Supplementary Material online). The origins of sequences not in Genbank are given in Supplementary Table 2
(Supplementary Material online). Sequences were aligned
using the ClustalX program (Thompson et al. 1997), and
the alignment was manually corrected to exclude gaps
and ambiguously aligned regions. Phylogenetic trees were
constructed using maximum likelihood (ML) and neighborjoining (NJ) methods. PhyML program (Guindon and
Gascuel 2003) was used to construct ML trees with the
JTT substitutional matrix (Jones, Taylor, and Thornton
1992) and discrete gamma distribution in eight categories 1
invariant sites. Gamma shape parameter a and the fraction
of invariant sites were estimated from the data set. ML bootstrap support was computed using SEQBOOT in PHYLIP
version 3.6a3 (Felsenstein 2001) and PhyML. NJ trees were
constructed with the JTT substitutional matrix, using the
AsaturA program which is designed to deal with mutational
saturation of amino acids (Van de Peer et al. 2002). The
AsaturA program defines amino acid substitutions with high
and low probabilities of occurrence as ‘‘frequent’’ or ‘‘rare,’’
respectively; for each sequence pair, the number of frequent
and rare amino acid replacements is plotted against the calculated pairwise evolutionary distance. By testing several
cutoff values for each tree, it is possible to determine what
fraction of positions is mutationally saturated, and those positions are omitted from further phylogenetic analysis. We
used the cutoff value that gave the highest number of rare
(i.e., unsaturated) substitutions without change of the tree
topology. NJ bootstrap support was computed in 1,000 replicates. Most trees were rooted with a representative Firmicute because (1) Firmicutes are considered a monophyletic
group (e.g., Battistuzzi, Feijao, and Hedges, 2004), (2) they
are not related to organelle donors (proteobacteria, cyanobacteria), and (3) their use produced trees where the major
taxa formed the expected monophyletic groups. It must be
pointed out that the choice of an out-group is almost always
somewhat arbitrary; our trees could just as well have been
presented as unrooted, with no change to our conclusions. In
the case of GluRS, the tree is rooted by Thermotoga
maritima, representing the most basal taxon in the data
set (Woese 1987). The tree based on oxygen-independent
coproporphyrinogen oxidase is presented as unrooted
because there are no available Firmicutes sequences.
Targeting Prediction
Putative diatom signal peptides were predicted by
SignalP (Nielsen et al. 1997; Nielsen, Brunak, and von
Heijne 1999; http://www.cbs.dtu.dk/services/SignalP) and
green algal and plant chloroplast transit peptides (cTPs)
by TargetP (plant option) (Nielsen et al. 1997; Emanuelsson
et al. 2000; http://www.cbs.dtu.dk/services/TargetP) and
Predotar (http://www.inra.fr/predotar/). TargetP (plant and
nonplant options) and Predotar were also used to predict
putative mitochondrial-targeting presequences. The targeting of nuclear-encoded proteins from apicomplexans was
predicted using the Prediction of Apicoplast Targeting
Sequences program (http://gecco.org.chemie.uni-frankfurt.
de/pats/pats-index.php) (Zuegge et al. 2001).
Results
Overview
In the T. pseudonana (diatom) and C. merolae (red algal)
genomes, we found gene models for all the enzymes of the
plant heme pathway (fig. 1A). Most of them were single copy,
but as in other organisms, there were two unrelated coproporphyrinogen oxidases, an oxygen-dependent and an oxygenindependent form (Dailey 1997), and both a ‘‘cytosolic’’
and an ‘‘organellar’’ GluRS. We did not find genes encoding
the alternative ‘‘a-proteobacterial–type’’ (succinyl-CoA)
pathway for ALA synthesis (fig.1B) in either alga.
In the diatom, almost all the genes encoded an ERtargeting signal sequence at the 5#-end, as would be expected for a nuclear-encoded protein that is transported to
the plastid via the endomembrane system (Apt et al. 2002,
Kroth 2002). We used the ER signal sequence as our
criterion for plastid targeting because signal sequences
are very conserved across taxonomic groups and are predicted with 80%–85% reliability by the program SignalP
(Nielsen et al. 1997; Nielsen, Brunak, and von Heijne
1999). We did not rely upon computer-based predictions
for the diatom cTPs (which target the protein across the
inner two membranes, i.e., those derived from the primary
plastid) because both TargetP and Predotar are trained on
2346 Obornı́k and Green
A
Maximum likelihood
100
100
77
100
81
100
52
Arabidopsis thaliana 2 PT
Oryza sativa 2 PT
Plasmodium falciparum
Caenorhabditis elegans
Homo sapiens
100 Phytophthora ramorum 1 MT
Phytophthora sojae
Cyanidioschyzon merolae 2
Thalassiosira pseudonana 2
Encephalitozoon cuniculi
Cyanidioschyzon merolae 3
Phytophthora ramorum 2
Schizosaccharomyces pombe
Oryza sativa 3
Archea
68
Gamma
proteobacteria
99/99
82/98
Alpha
proteobacteria
100/100
Neighbour-joining
B
63/65
100/100
97/94
95/67
Arabidopsis thaliana 1 MT
Hordeum vulgare PT
Nicotiana tabacum MT
Cyanidioschyzon merolae 1 MT
Thalassiosira pseudonana 1 MT
100/100
100/100
100
Actino
bacteria
Chlamydiae
93/88
Spirochaetes
100/99
Archea
100
Firmicutes
70/97/99
100/100
Cyanobacteria
100
Oryza sativa 2
Arabidopsis thaliana 2
Plasmodium falciparum
Homo sapiens
56
Caenorhabditis elegans
85 97
Encephalitozoon cuniculi
68
Thalassiosira pseudonana 2
100 Phytophthora ramorum 1
99
Phytophthora sojae 1
Cyanidioschyzon merolae 2
100
99
Oryza sativa 3
61
Schizosaccharomyces pombe
99
Phytophthora ramorum 2
Cyanidioschyzon merolae 3
60
0.1
100/100
Thermotoga maritima
0.1
FIG. 2.—Phylogenetic trees of GluRS (glutamate tRNA ligase) amino acid sequences. (A) ML tree (loglk 5 ÿ37,096.71883) constructed using
PhyML with JTT substitutional matrix and discrete gamma distribution in eight 1 one categories. All parameters were estimated from data set (gamma
shape 5 1.473; Pinv 5 0.017). Tree was rooted using Thermotoga maritima (Thermotogales) as out-group. Numbers above branches indicate ML
bootstrap support (JTT, one category of sites, 100 replicates)/NJ bootstrap support (JTT, AsaturA cutoff 5 446, 1,000 replicates); ML support only
in eukaryotic-archaeal cluster. –(B) Alternative topology (NJ tree) of the eukaryotic-archaeal cluster obtained using AsaturA program (cutoff 5
446). Numbers above branches indicate NJ bootstrap support. PT (black box), sequences with putative plastid targeting leaders; MT (white box),
sequences possessing putative mitochondrial leaders.
sets derived from green plants and did not give consistent
results with diatom homologs of proteins experimentally
demonstrated to be plastid located in other photosynthetic
eukaryotes (Armbrust et al. 2004; B. K. Chaal and B. R.
Green, unpublished data).
Step 1: GluRSs
Genes encoding two types of GluRS were found in the
diatom, in the red alga, and in plants. The sequences form
two distinct clusters in all trees constructed (fig. 2). One
diatom gene, T. pseudonana 2, and two C. merolae genes
cluster with sequences from oomycetes, fungi, microsporidia, animals, and plants. This highly supported cluster is
strongly affiliated with GluRSs from Archaea and is quite
different from eubacterial homologues, suggesting that
these gene sequences were derived from the original host
cytosolic genes. There appears to have been an early gene
duplication in the eukaryote lineage. Neither ML (fig. 2A)
nor NJ (fig. 2B) analysis was able to resolve the branching
order within the eukaryotes. Two of the plant sequences (O.
sativa 2 and A. thaliana 2) possess putative cTPs at their
Mosaic Origin of Heme Biosynthesis 2347
N-termini according to the TargetP program (Emanuelsson
et al. 2000). No ER-targeting domain or potential transit
peptide was found for the diatom gene model, even after
searching the nucleotide sequence for 1,000 nt upstream.
The N-terminus of the translated gene model did not give
a strong prediction with any program, so we do not know in
what cell compartment its product functions.
The second diatom sequence, T. pseudonana 1, clusters with several plant and red algal sequences with good
support but lies within a large badly resolved eubacterial
clade. Even if the eukaryotic-archaeal cluster is excluded
from the analysis, the phylogenetic position of the second
plant-algal cluster cannot be resolved (data not shown). For
all the proteins in this cluster, TargetP and Predotar gave
inconsistent or ambiguous (mitochondrion and plastid) predictions for an N-terminal–targeting presequence. The diatom sequence has a potential mitochondrial leader but
no ER signal sequence, so this protein may be imported
by the mitochondrion.
Steps 2–4 and 7–9: Cyanobacterial-Plastid Clade
Glutamyl-tRNA reductase, the next enzyme of the
pathway, is clearly of cyanobacterial origin (fig. 3A).
The green algal and plant sequences form a well-supported
clade and with the red algal and diatom sequences are sister
group to the cyanobacterial sequences. Cyanobacterial and
plastid-targeted GSA sequences also form a well-supported
clade (fig. 3B), but the branching order within the clade is
not well resolved. No sequences for either of these enzymes
have been found in the genomes of Phytophthora or the
apicomplexans.
Porphobilinogen synthase (Step 4, fig. 3C) is the only
protein for which there is a good selection of secondary
endosymbiont sequences: T. pseudonana forms a wellsupported cluster with the diatom Odontella sinensis and
other heterokonts (Fucus vesiculosis and Laminaria
digitata). This cluster is sister group to a cluster consisting
of two rhodophyte sequences (Gracilaria gracilis and
C. merolae) and the sequence from the chlorarachniophyte
Bigelowiela natans. A recently published analysis of
nuclear-encoded plastid-targeted proteins in B. natans
showed that a significant fraction of these proteins (including GSA, fig. 3B) was not evolutionarily related to a green
plastid ancestor (Archibald et al. 2003). The Plasmodium
sequences form very long branches but are part of the
cyanobacterial-plastid cluster. The two Phytophthora sequences branch with the fungal sequences, rather than with
the other heterokont sequences.
Plastid and cyanobacterial sequences are on the
same branch for uroporphyrinogen decarboxylase (Step 7,
fig. 4A) and one form of protoporphyrinogen oxidase
(Step 9, fig. 4D). In both cases, the red algal and diatom
sequences are on the same branch but with poor statistical
support, as was the case for glutamyl-tRNA reductase and
porphobilinogen synthase. The two forms of red algal and
diatom uroporphyrinogen decarboxylase appear to be the
result of a gene duplication prior to the secondary
endosymbiotic event, and both are predicted to be plastid
targeted. The second form of protoporphyrinogen oxidase
is found only in the green plants and appears to be of
proteobacterial origin. Neither the Plasmodium nor the
Phytophthora sequence is part of the cyanobacterialplastid clade.
There are two unrelated coproporphyrinogen oxidases,
the O2-independent (fig. 4B) and O2-dependent (fig. 4C). The
cyanobacterial-plastid clade is well supported for the O2independent enzyme. This gene was not found in apicomplexan or Phytophthora genomes. For the O2-dependent
form, there is a plastid clade next to the Phytophthora branch
but it is obviously not related to cyanobacteria. The tree as
a whole does not support any particular origin of eukaryotic
O2-dependent coproporphyrinogen oxidases.
Step 5: Porphobilinogen Deaminase: Proteobacterial
Origin
In the case of porphobilinogen deaminase (fig. 3D),
the plastid sequences form one cluster, which is invariably
placed within an a-proteobacterial clade with high bootstrap support, suggesting a possible mitochondrial origin
of this particular enzyme. The Plasmodium (apicomplexan)
sequences appeared with low support at the root of the
a-proteobacteria–plastid cluster, suggesting that they also
might have an a-proteobacterial origin. The cyanobacterial
sequences are quite separate, forming a sister group to the
cluster containing opisthokonts and the two sequences from
Phytophthora.
Step 10: Ferrochelatase
The red algal ferrochelatase sequences did not fit into
the general plastid pattern (fig. 5). All three of them (from
C. merolae, P. yezoensis, and G. sulfuraria) formed a wellsupported branch which clustered with the apicomplexan
and some proteobacterial sequences. The proteobacterialapicomplexan-red algal branch was well supported in both
ML and NJ trees. All the other plastid ferrochelatase sequences, including those of the diatoms T. pseudonana
and Phaeodactylum tricornutum, formed a separate wellsupported cluster with cyanobacterial sequences.
The Plasmodium ferrochelatase was previously proposed to be of proteobacterial origin on the basis of NJ
and parsimony analysis, before the diatom and red algal sequences were available (Sato and Wilson 2003). Figure 5
supports this interpretation and suggests that the three
red algae have also replaced their cyanobacterial-type
ferrochelatase with a proteobacterial one. This was somewhat unexpected because the diatoms (with plastids of red
algal origin) have the cyanobacterial form. The Phytophthora sequences again branch with the fungal sequences
rather than with those of the photosynthetic heterokonts.
Discussion
Mosaic Origin
The evolution of metabolic pathways is a subject of
much current interest, with a number of different hypotheses
to explain the origin of new enzymes (Nara, Hshimoto, and
Aoki 2000; Illingworth et al. 2003; Schmidt et al. 2003). In
this work, we address the next level of evolution: gene acquisition/replacement related to the endosymbiotic origins
of organelles. Because all known prokaryotes, eubacteria
2348 Obornı́k and Green
PT
Phytophthora sojae
Phytophthora ramorum
FIG. 3.—ML trees based on amino acid sequences of enzymes from Steps 2–5. (A) Glutamyl-tRNA reductase (loglk 5 ÿ17,399.23256, gamma shape 5
1.507; Pinv 5 0.025). NJ AsaturA cutoff 5 693. Tree rooted using Heliobacillus mobilis as out-group. (B) GSA (loglk 5 ÿ13,748.91073, gamma shape 5
1.032;Pinv5 0.000).NJAsaturAcutoffvalue5 514.TreerootedusingBacillusanthracisasout-group.(C)Porphobilinogensynthase(loglk5 ÿ15,061.76323,
gamma 5 1.164; Pinv 5 0.043). AsaturA cutoff 5 526. Tree rooted using H. mobilis as out-group. (D) Porphobilinogen deaminase (loglk 5 ÿ15,466.38941;
gamma 5 1.340; Pinv 5 0.081). AsaturA cutoff value 5 614. Tree rooted using Staphylococcus epidermidis as out-group. Numbers above branches indicate ML
bootstrap support (JTT, one category of sites, 300 replicates)/NJ bootstrap support (JTT, AsaturA cutoff value as specified for each tree, 1,000 replicates).
Sequences putatively targeted to plastid are marked by PT (black box); sequences possessing putative mitochondrial leaders are indicated by MT (white box).
Mosaic Origin of Heme Biosynthesis 2349
FIG. 4.—ML trees based on amino acid sequences of enzymes from Steps 7–9. (A) Uroporphyrinogen decarboxylase (loglk 5 ÿ18,487.67565; gamma
shape 5 1.453; Pinv 5 0.040). AsaturA cutoff 5 559. Tree rooted using Staphylococcus epidermidis as out-group. (B) Oxygen-independent coproporphyrinogen oxidase (loglk 5 ÿ17,056.01128; gamma shape 5 1.661; Pinv 5 0.106). AsaturA cutoff 5 446. Tree rooted using Thermoanaerobacter tengcongens
sequenceas out-group.(C) Oxygen-dependent coproporphyrinogenoxidase(loglk 5 ÿ8305.35663;gamma shape5 1.369;Pinv5 0.100).AsaturAcutoff5
578. Treerooted using Caulobactercrescentus (Proteobacteria) sequence as an out-group.(D) Protoporphyrinogen oxidase (loglk 5 ÿ20,058.38689; gamma
shape 5 2.530; Pinv 5 0.005). AsaturA cutoff 5 343. Tree rooted using Listeria innocua sequence as out-group. Numbers above branches indicate ML
bootstrap support (JTT, one category of sites, 300 replicates)/NJ bootstrap support (JTT, AsaturA cutoff value as specified, 1,000 replicates). Sequences
putatively targeted to plastid are marked by PT (black box); sequences possessing putative mitochondrial leaders are indicated by MT (white box).
2350 Obornı́k and Green
A
100/100
74/-
-/100
Arabidopsis thaliana 4 PT
Arabidopsis thaliana 1 PT
Arabidopsis thaliana 2 PT
53/59
Solanum tuberosum PT
80/94 Nicotiana tabacum PT
Plants and
Oryza sativa PT
92/94
green algae
Cucumis sativum 1 PT
99/78
PT
Arabidopsis thaliana 3
Hordeum vulgare PT
Cucumis sativum 2 PT
70/Chlamydomonas reinhardtii PT
48/60
Polytomella sp. PT
Synechocystis sp.
Prochlorococcus marinus
Thermosynechococcus elongatus
88/96
Cyanobacteria
Nostoc punctiforme
Nostoc sp.
Trichodesmium
erythraeum
88/69
Phaeodactylum tricornutum (EST)
100/98
Diatoms
Thalassiosira pseudonana PT
100/99
76/60 Bos taurus MT
98/86 Mus musculus MT
Homo sapiens MT
82/43/54
Xenopus laevis MT
99/100
Animals
Gallus gallus MT
74/Danio rerio MT
Chironomus sp. MT
Drosophila melanogaster MT
100/91
100/99
100/100 Phytophthora sojae MT
Stramenopiles
Phytophthora ramorum MT
54/Schizosaccharomyces pombe MT
Saccharomyces cerevisiae MT
Fungi
94/91
Neurospora crassa MT
100/91 Rickettsia connorii
100/100 Rickettsia sibirica
Rickettsia prowazekii
Alpha
99/90
Caulobacter crescentus
proteobacteria
Rhodospirillum rubrum
96/100
Magnetospirillum magnetotacticum
100 Yersinia pestis
Maximum likelihood
98
Yersinia enterocolitica
85
Salmonella typhimurium
Proteobacteria
Vibrio vulnificus
74
Vibrio cholerae
95
69
Haemophillus influenzae
Brucella melitensis
65
100
Plasmodium yoelii
60
Plasmodium falciparum
Apicomplexa
100
Toxoplasma gondii 1
94
Toxoplasma gondii 2
46
Shewanella oneidensis
100
Coxiella burnetii
94
Porphyra yezoensis (EST)
100
Cyanidioschyzon merolae MT ? Red algae
Galdieria sulfuraria (EST)
Bacillus anthracis
Bacillus halodurans
Bacillus cereus
Listeria monocytogenes
Staphylococcus aureus
92/61
Firmicutes
0.1
B
94
97
100 Yersinia enterocolitica
Neighbour-joining
Yersinia pestis
89
Salmonella typhimurium
Vibrio cholerae
84
79
Vibrio vulnificus
Proteobacteria
Haemophillus influenzae
Brucella melitensis
Coxiella burnetii
Shewanella oneidensis
83
100
Toxoplasma gondii 1
Toxoplasma gondii 2 Apicomplexa
100
Plasmodium falciparum
46
Plasmodium yoelii
Cyanidioschyzon merolae MT ?
60
100
Porphyra yezoensis (EST)
Red algae
Galdieria sulfuraria (EST)
FIG. 5.—Ferrochelatase (Step 10). (A) ML tree based on amino acid sequences (loglk 5 ÿ15,844.57515; gamma shape 5 1.630; Pinv 5 0.023).
Numbers above branches indicate ML bootstrap support (JTT, one category of sites, 300 replicates). (B) Cluster containing red algal and apicomplexan
ferrochelatase sequences from NJ tree (JTT matrix, AsaturA cutoff 5 395). PT (black box), sequences putatively targeted to plastid; MT (white box),
sequences possessing putative mitochondrial leaders.
and archaea, require heme for numerous cellular functions, it
is safe to assume that both the original amitochondriate eukaryote (assumed to have arisen from the archaeal lineage)
and the a-proteobacterium that became the mitochondrion
were able to synthesize heme. This may underlie the fact
that in animal cells ALA is synthesized in the mitochondrion
via succinyl-CoA, while the following four steps in the
pathway (fig. 1) take place in the cytosol, ending with
coproporphyrinogen III. Coproporphyrinogen III is then
transported back to the mitochondrion where the last three
steps of the heme pathway are located (Ralph et al. 2004).
The primary chloroplast endosymbiosis led to a massive transfer of cyanobacterial genes to the nucleus: at least
1,700 are still recognizable as cyanobacterial homologues
in Arabidopsis (Martin et al. 2002). Some of them replaced
the preexisting nuclear genes, but in other cases, a duplicate
Mosaic Origin of Heme Biosynthesis 2351
of the nuclear gene acquired a targeting sequence and took
over part of the chloroplast pathway (Martin and Herrmann
1998). According to our phylogenetic analyses, most of the
genes for enzymes involved in heme biosynthesis in photosynthetic eukaryotes originated in the cyanobacterial
endosymbiont. For oxygen-dependent coproporphyrinogen
oxidase (Step 8, fig. 4C) and uroporphyrinogen synthase
(Step 6, data not shown), the plastid sequences form
a well-supported cluster that is unrelated to the cyanobacterial cluster but not clearly related to any other group. The
close relationship among sequences from all photosynthetic
eukaryotes suggests that these genes were among those
transferred to the host nucleus during the primary endosymbiogenesis, before the divergence of the two major primary
plastid-containing lineages.
However, several enzymes in the pathway do not appear to be of cyanobacterial origin. In the case of porphobilinogen deaminase, the plastid enzymes are clearly related
to those of a-proteobacteria (fig. 3D). One possibility is that
the cyanobacterial gene was transferred to the nucleus after
the primary endosymbiotic event but replaced by a gene of
proteobacterial origin before the divergence of the red,
green, and glaucophyte algae. Another possibility is that
the cyanobacterial gene was never transferred to the nucleus
and a copy of the preexisting mitochondrial nuclearencoded gene was retargeted to the chloroplast. The animal
and fungal enzymes are located in the cytosol and could
be of either cytosolic or proteobacterial (mitochondrial) origin, but on our phylogenetic trees, they appear closely
related to cyanobacterial sequences (fig. 3D). Such clustering of proteins from cyanobacteria and nonphotosynthetic
eukaryotes has also been found in phosphoadenosine
phosphosulfate reductase and heme oxygenase (M.O.,
unpublished data) and may be an artifact due to the limited
sampling of eukaryotes.
The evolution of the GluRSs is complicated by the fact
that they belong to the Glx-tRNA synthase family, which
contains both GluRSs and glutaminyl-tRNA synthases
(GlnRS) (Siatecka et al. 1998). Phylogenetic trees divide
this family into the a group, which contains only eubacterial
GluRS, and the b group, which has three well-separated subgroups: archaeal GluRS, eukaryotic GluRS, and eukaryotic
GlnRS (Siatecka et al. 1998). Plants, C. merolae, and T.
pseudonana have a eukaryotic GluRS that forms a sister
group to the archaeal GluRS (fig. 2A). This gene is clearly
not of cyanobacterial or a-proteobacterial origin: it most
likely originated in the nucleus of the first photosynthetic
eukaryote. Plants and the two algae have a second GluRS
sequence that appears within the a group of eubacterial
genes. However, we could not determine whether they originated from within the cyanobacteria (plastid) or the proteobacteria (mitochondrion), even with separate phylogenetic
analysis of a and b groups. Four of the a group sequences
(A. thaliana, Nicotiana tabacum, C. merolae, and T. pseudonana) possess putative mitochondrial leaders based on
TargetP predictions, but Hordeum vulgare has a putative
plastid leader.
It is clear that the heme biosynthesis pathway has a mosaic character in photosynthetic eukaryotes. This is not the
only example of a mosaic metabolic pathway. The polyamine biosynthesis pathway in Arabidopsis (Illingworth
et al. 2003), the eukaryotic pyrimidine biosynthesis pathway (Nara, Hshimoto, and Aoki 2000), and the glycolytic
pathway in plants (Martin and Herrmann 1998) are all composed of enzymes with different evolutionary origins. We
have also found that the shikimate pathway is an evolutionary mosaic (M.O. and B.R.G., unpublished data).
Secondary Endosymbiosis and the Red Lineage
Whether or not they are of cyanobacterial origin, the
enzymes of plants, red algae, and diatoms appear to have
a common origin, with the exception of red algal ferrochelatase (fig. 4). The simplest explanation is that the cyanobacterial ferrochelatase gene was replaced by one from
a proteobacterium in the lines leading to Porphyra and
Cyanidioschyzon but not in the line involved in secondary
endosymbiosis.
Sato and Wilson (2003) first pointed out the proteobacterial origin of apicomplexan ferrochelatase, confirmed
by our trees which have larger taxon sampling (fig. 5). It
must be noted that the heme biosynthesis pathway in apicomplexans complicates any scenario involving a common
ancestor of all groups with red algal plastids. Although heterokonts and apicomplexans may have had a common ancestor (Fast et al. 2001), the heme pathway in apicomplexan
parasites is very different from that in plants and diatoms
(Armbrust et al. 2004; Ralph et al. 2004). Part of the pathway appears to be localized in the apicomplexan complex
plastid (apicoplast) (Ralph et al. 2004; Sato et al. 2004), but
ALA is synthesized via the succinyl-CoA pathway in the
mitochondrion and the two penultimate steps may be localized in the cytosol (Ralph et al. 2004). Three apicoplast
enzymes, porphobilinogen deaminase (fig. 3D), uroporphyrinogen decarboxylase (fig. 4A), and ferrochelatase,
are of proteobacterial origin. It has recently been shown that
the ferrochelatase encoded by the P. falciparum genome is
located in the apicoplast (Varadharajan et al. 2004), in spite
of the fact that it does not have a typical apicoplast-targeting
sequence (Sato and Wilson 2003). However, a substantial
fraction of heme biosynthesis in the intraerythrocyte
stage is due to host enzymes imported into the cytosol
(Varadharajan et al. 2004). What effect this has had on
the evolution of the pathway is unknown.
Nonphotosynthetic Heterokonts
Phytophthora species are plant pathogens classified as
oomycetes, the sister group to the photosynthetic heterokonts (e.g., diatoms). It is still unclear whether the oomycetes once had a chloroplast and lost it or whether the
photosynthetic heterokonts acquired plastids after the
two branches of the family separated. If the chromalveolate
hypothesis is correct, the common ancestor of the oomycetes and the diatoms must have had a plastid. However,
the oomycete sequences invariably branch with fungal
and animal sequences (figs. 2A, 2B, 3, and 4) and not with
those of the diatom or the cyanobacteria. This suggests that
the red algal nuclear genes were not successful in replacing
the endogenous nuclear genes in the oomycete lineage. It
also suggests that the oomycetes diverged from the other
chromalveolate lineages before the secondary endosymbiont was fully integrated, which might imply a very rapid
2352 Obornı́k and Green
divergence of all the chromalveolate lineages after the
secondary endosymbiotic event.
A recently published hypothesis suggested that all
eukaryotes that currently have plastids had a primary plastid
in their evolutionary history (Nozaki et al. 2003). This
rather startling suggestion was based on phylogenetic
trees of four concatenated proteins (a- and b-tubulin, actin,
and elongation factor-1a) that divided eukaryotes into two
major groups: Group A, opisthokonts (metazoans and
fungi), and Group B, all photosynthetic eukaryotes and
their nonphotosynthetic relatives, with red algae at the base.
Group B therefore included all members of the Alveolata
(ciliates, dinoflagellates, and apicomplexans) and the Discicristata (euglenoids and trypanosomatids) as well as the
Heterokontophyta and green algae/plants. If this were true,
the secondary host nucleus would already have contained
genes for chloroplast-targeted proteins because it had once
had a primary plastid. However, Phytophthora clusters with
their Group A (opisthokonts) in most of our trees and shows
no trace of having had a primary or a secondary plastid.
Although the Nozaki et al. (2003) hypothesis cannot be rigorously tested until genomic sequences of a much wider
sampling of eukaryotes are available, it is important to point
out that it also would require that the mosaic character of the
heme pathway evolved during the primary endosymbiogenesis, before the primary plastids and their hosts diverged
into the red, green, and glaucophyte lineages.
Conclusions
In summary, we found that the evolutionary history of
the heme biosynthesis pathway is very much the same in
plants possessing primary green plastids as in the red algae
and in the diatom with a red secondary plastid. In all photosynthetic eukaryotes, the pathway has a mosaic character
and the enzymes involved display cyanobacterial (plastid),
a-proteobacterial (mitochondrial), and cytosolic (eukaryotic nucleus) origin.
Supplementary Material
Supplementary Tables 1 and 2 and Supplementary
alignments are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
Supplementary Table 1. Accession numbers of
sequences used in this analysis.
Supplementary Table 2. Origin of sequences not in
Genbank that were used in this analysis.
Acknowledgments
We thank Ross Waller for useful discussions and for
suggesting the inclusion of Phytophthora sequences and
Katerina Jiroutova for sequencing the T. pseudonana ferrochelatase. Financial support was provided by the Natural
Sciences and Engineering Council of Canada and the award
of a Canada Council Killam Fellowship to B.R.G., by the
Research Plan of the Institute of Parasitology ASCR no.
z60220518, and the Academy of Sciences of the Czech
Republic, project no. A500220502.
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Accepted July 18, 2005