EUKARYOTIC CELL, Aug. 2006, p.

11751183
1535-9778/06/$08.000 doi:10.1128/EC.00097-06
Copyright 2006, American Society for Microbiology. All Rights Reserved.

Vol. 5, No. 8

MINIREVIEWS
Algae Need Their Vitamins
Martin T. Croft,1* Martin J. Warren,2 and Alison G. Smith1
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom,1 and
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom2
in 1937 discovered that some members of the chlorophyta and
cryptophyta require thiamine as a growth factor in culture (39).
Over the following 40 years, many studies described algal species that required different combinations of three B vitamins:
vitamin B12 (cobalamin), vitamin B1 (thiamine), and vitamin
B7 (biotin) (50). A compilation of all of the available data (see
Table S1 in the supplemental material; summarized in Table 1)
reveals the widespread nature of vitamin auxotrophy within the
algal kingdom. Of 306 species surveyed, more than half required cobalamin, while 22% required thiamine and a smaller
proportion (5%) required biotin. Remarkably, for all three
vitamins, the algal species that have an obligate requirement
for the different cofactors do not appear to fall into any one
lineage, but rather auxotrophy is present in several unrelated
phyla, indicating that it must have arisen independently several
times through evolution. Even more surprisingly, this pattern is
also mirrored within individual genera. For example, Hematococcus in the chlorophyta, Peridinium in the dinophyta, Hymenomonas in the haptophyta, and Nitzschia in the heterokontophyta all have species that require cobalamin and others that
do not (see Table S1 in the supplemental material). Similarly,
the dinoflagellate Gymnodinium brevis requires all three vitamins whereas G. spendens requires only cobalamin. For the
auxotrophy to have evolved so frequently in algae, the simplest
explanation is that it is due to the loss of a single gene. A
plausible hypothesis would therefore be that species with a
requirement for a particular vitamin have lost a gene involved
in the biosynthesis of that cofactor. An intriguing parallel is
seen with vitamin C auxotrophy in mammals, where loss of the
terminal enzyme of the pathway has occurred in both primates
and guinea pigs (10).
Until now, however, it has not been possible to test this
hypothesis because nothing was known of the biosynthetic
routes for vitamins in the algal kingdom and only very little was
known about the roles of the cofactors in algal metabolism.
With the advent of genome sequencing, our ability to address
questions like this has been revolutionized. Currently, the sequences of four algal genomes are available. The first to become available was that of P. falciparum (25), a unicellular
nonphotosynthetic apicomplexan, which lives as a parasite in
insects and humans and is the causative agent of the devastating tropical disease malaria. The release of the P. falciparum
genome sequence was quickly followed by that of Chlamydomonas reinhardtii (www.jgi.doe.gov), a unicellular green alga,
which is commonly isolated from soils in North America. Two
further genome sequences were released in 2004; Thalassiosira
pseudonana is an ecologically important centric diatom found

Algae are simple photosynthetic eukaryotes whichowing

to their colonization of the oceansare responsible for up to
50% of the planets atmospheric carbon fixation (22). They
comprise a diverse group that can broadly be defined as unicellular (microalgae) or multicellular (macroalgae) photosynthetic organisms that lack roots, stems, leaves, conducting vessels, and complex sex organs (55). A single endosymbiotic
event between a cyanobacterium-like organism and a nonphotosynthetic eukaryote is thought to have given rise to the
three basal groups of algae: the chlorophyta (from which
higher plants arose), the glaucocystophyta, and the rhodophyta (Fig. 1).
The chlorophyta and the rhodophyta, via secondary and
tertiary endosymbiotic events with different nonphotosynthetic
eukaryotes, gave rise to algal groups with complex plastids
(Fig. 1). Some groups, such as the apicomplexans (e.g., Plasmodium falciparum) subsequently lost the ability to photosynthesize, although they still retain plastids. The number of symbioses that have occurred during the evolution of algae has
been heavily debated, and the details are discussed elsewhere
(46), but there are likely to have been several events. Consequently, it is not surprising that the physiology and metabolism
of algae are extremely varied. For example, while the majority
of green algae contain a highly structured cell wall comprising
glycoproteins (19), euglenophyta simply contain a protein layer
(known as the pellicle) beneath the cell membrane, and the cell
walls of diatoms are made from silica (24). Dinophyta, euglenophyta, and heterokontophyta contain members that are
phagotrophic on bacterial prey, but this characteristic is absent
from the groups with simple plastids. Furthermore, although
most algae are regarded as free-living organisms, many
dinoflagellates are closely associated with corals, and members
of several algal groups live with fungi as lichens (13), providing
photosynthate for their heterotrophic partner.
VITAMIN AUXOTROPHY IN ALGAE
The ability to photosynthesize has led to the perception of
algae as autotrophic organisms requiring light and a mixture of
inorganic nutrients only. However, studies by Lwoff and Dusi

* Corresponding author. Mailing address: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2
3EA, United Kingdom. Phone: 44 1223 330219. Fax: 44 1223 333953.
E-mail: mtc29@cam.ac.uk.
Supplemental material for this article may be found at http://ec
.asm.org/.
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FIG. 1. Summary of algal evolution. The three basal groups chlorophyta, rhodophyta, and glaucocystophyta are shown with green, red,
and blue plastids, respectively. Those groups derived from the chlorophyta and rhodophyta by secondary endosymbioses are shown with the
appropriate colored plastids. Tertiary endosymbiotic events are not
shown in this diagram. The boxed phyla contain at least one organism
with a sequenced genome.

in many of the worlds oceans (2), and Cyanidioschyzon merolae

is a unicellular thermophilic red alga isolated from sulfate-rich
hot springs (pH 1.5, 45C) (42). In this article, we use the
genome sequence data available for these four species to investigate the question of vitamin metabolism in algae, thus
providing the first clues as to why and how some algae have a
requirement for these cofactors. Each vitamin will be discussed
in turn before we focus on possible routes for their acquisition
by algae, which, given the extremely low free concentrations of
these nutrients in the natural environment, are likely to be
complex.
BIOTIN
Biotin (vitamin B7) was discovered in 1901 as a growthpromoting factor for yeast (69) and was finally isolated and

TABLE 1. Vitamin requirements of the individual species

detailed in the supplemental material compiled
under the different algal groupsa
Phylum

Chlorophyta
Rhodophyta
Cryptophyta
Dinophyta
Euglenophyta
Haptophyta
Heterokontophyta
Total
a

No. of species requiring:

No. of species
surveyed

Cobalamin

Thiamine

Biotin

148
13
6
27
15
17
80

44
12
5
24
13
10
47

19
0
5
7
11
14
11

0
0
1
7
1
0
5

306

155

67

14

Only those species that have had their cobalamin, thiamine, and biotin
requirements assessed have been included in this survey, and for this reason
those data do not include any glaucocystophytes, chloroarachnophytes, or apicomplexans. A requirement for biotin is found only in species that contain
complex plastids, i.e., those that have arisen as the result of secondary and
tertiary endosymbiosis with a eukaryotic alga. Furthermore, every species that
requires biotin also requires cobalamin, thiamine, or both.

FIG. 2. The biotin biosynthetic pathway as elucidated in eubacteria. There are several different pathways for the synthesis of pimeloylCoA. The gene names are followed by the symbol F indicating the
presence of the gene in C. merolae (Cm), the symbol E indicating the
presence of a gene in C. reinhardtii (Cr), the symbol indicating that
the given gene is present in the genome of T. pseudonana (Tp), or the
symbol indicating that the gene is in the genome of P. falciparum
(Pf). Genes with sequence similarity to bioF and bioA can also be
found in the genome sequence of D. discoideum, while only bioF can be
found in E. histolytica (see text for details).

purified in 1941 (64). Biotin is a cofactor for several essential

carboxylase enzymes (62), including acetyl coenzyme A (CoA)
carboxylase, which is involved in fatty acid synthesis, and so is
universally required. The molecule consists of an imidazole
ring fused to a sulfur-containing tetrahydrothiophene ring with
a fatty acid side chain (Fig. 2). In eubacteria, the first precursor
for biotin synthesis is pimeloyl-CoA but the source of this
differs among different species. Thereafter, the concerted action of four enzymes, BioF, BioA, BioD, and BioB, converts
pimeloyl-CoA to biotin (20) (Fig. 2). In the budding yeast
Saccharomyces cerevisiae, homologues of bioA, bioD, and bioB,
but not bioF, are present, so the source of 7-keto-8-aminopelargonic acid remains unknown. The higher plant Arabidopsis
thaliana contains genes for BioF, BioA (also called BIO1), and
BioB (also called BIO2) (3, 49), but the genome does not
appear to contain a gene with sequence similarity to known
bioD genes. Since A. thaliana can synthesize biotin de novo, the
absence of a bioD gene from the genome suggests that in
higher plants the conversion of 7,8-diaminopelargonic acid to

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TABLE 2. Genes involved in biotin thiamine and cobalamin metabolism in C. reinhardtii, T. pseudonana, C. merolae, and P. falciparuma
Model (length amino acids)

Gene
C. reinhardtii

Biotin biosynthesis
bioF
bioA
bioB
Thiamine biosynthesis
thiS
thiF
iscS
dxs
thiG
thiH/O
thiC
thiD
thiE
TPK
thiM
Cobalamin metabolism
metE
metH
cblE
cblA
cblB
mmcM

T. pseudonana

C. merolae

P. falciparum

chr12.glimmerm_1307 (630)

e_gwW.18.89.1
e_gwH.100.1.1 (675)
Chlre2_kg.scaffold_3000262 (PS)

2.889.1 (370)
128.16.1 (417)
10.577.1 (324)

CML225C (433)
CMG023C (802)
CML210C (379)

e_gwW.11.33.1 (270)
e_gwH.31.56.1 (396)
estExt_fgenesh2_pg.C_710047 (744)

54.113.1 (385)
20.15.1 (432)
6.114.1 (703)
169.11.1 (254)
7.273.1 (353)
12.129.1 (636)
54.158.1 (267)
54.123.1 (1,930)
30.73.1 (217)

CMV092C (67)
CMM289c (539)
CMT234C (453)
CMF089C (772)
CMV077C (258)
CMM298C (781)
CMG171C (673)
CMO125C (297)
CMP214C (308)
CMH016C (251)

MTC_estExt_fgenesh2_pg.C_120281 (567)
estExt_fgenesh2_pg.C_360009 (637)
mtc_168251 (711)
mtc_168251 (7110
gwW.1.690.1 (358)
estExt_gwp_1H.C_30409 (242)
Chlre2_kg.scaffold_82000007 (842)
estExt_GenewiseW_1.C_30026 (1,357)
e_gwH.14.9.1 (1,381)

chr13.genefinder 27r (584)

chr7.phat_291 (1,250)
chr13.phat_385 (1,177)

chr5.glimmerm_537 (310)
MAL6PI.285 (545)
PFL1920c (302)

CMJ234C (767)
65.17.1 (1,248)
80.8.1 (579)
2.1075.1 (301)
89.13.1 (190)
1.311.1 (726)

a
Model numbers for C. reinhardtii are the version 3.0 models, the T. pseudonana genes correspond to the filtered version II models, the C. merolae genes follow the
C. merolae genome annotation models, and the P. falciparum genes are either glimmerM, phat, or final models. Bacterial and yeast genes were used to search the algal
genomes by using tBLASTn, and hits with an expectation coefficient of less than E20 were considered significant. PS indicates that only part of the putative gene is
present in the genome sequence. The thiH/O gene products are isozymes that catalyze the same reaction.

D-desthiobiotin must be carried out by an as-yet-unidentified

enzyme.
Using the data from the literature (see Table S1 in the
supplemental material), we found that 14 out of 306 species
surveyed were biotin auxotrophs (Table 1). All of these are
confined to algal groups with complex plastids, such as Amphidinium carterae (dinophyte) and Ochromonas danica (heterokontophyte). Interestingly, all biotin auxotrophs also have a
requirement for either cobalamin, thiamine, or both (see Table
S1 in the supplemental material). C. reinhardtii, T. pseudonana,
and C. merolae are not biotin auxotrophs (see Table S1 in the
supplemental material), and as they contain several biotindependent carboxylases, they must have a functional biosynthetic pathway. Whether or not P. falciparum requires exogenous biotin is unknown. We used the BLAST algorithm (1) to
query the latest versions of their genome sequences with the
four bacterial biotin biosynthesis genes, bioF, bioA, bioD, and
bioB. In C. reinhardtii, T. pseudonana, and C. merolae, we could
identify genes with high sequence similarity to bioF, bioA, and
bioB, but not bioD (Table 2), a situation analogous to that in A.
thaliana. The coded boxes in Fig. 2 indicate the presence of
these genes. Intriguingly, P. falciparum contains bioF only and
none of the other three genes, so it will be of interest to
determine whether or not it requires the vitamin for growth. If
not, it suggests that there is a different biotin biosynthetic route
in P. falciparum. Two known biotin auxotrophs whose genomes
have been completely sequenced are the single-celled amoebae
Dictyostelium discoideum (17) and Entamoeba histolytica (38).
E. histolytica, like P. falciparum, is an obligate parasite that can

presumably obtain biotin from its host, while D. discoideum is

a slime mold that preys on soil microorganisms. The genomes
of these two amoebae contain a gene with sequence similarity
to bioF, while D. discoideum also contains a gene with sequence similarity to bioA. Given our current knowledge of
biotin biosynthesis in eukaryotes, it is not possible to conclude
how biotin auxotrophy arose initially in these lineages. Nevertheless, the simplest explanation is that it was caused by the
loss of a single biosynthetic gene, although this might not be
the same gene in every case.

THIAMINE
Like biotin, thiamine also plays a pivotal role in intermediary
carbon metabolism. The active form of the vitamin is thiamine
pyrophosphate (TPP), which is essential for all organisms. The
cofactor associates with a number of enzymes involved in primary carbohydrate and branched-chain amino acid metabolism, including pyruvate dehydrogenase, transketolase, -ketoacid decarboxylase, and -ketoacid oxidase (57). Recent work
on the biosynthesis of thiamine has mainly concentrated on
three prokaryotic organisms, Escherichia coli, Salmonella enterica serovar Typhimurium, and Bacillus subtilis (5). Thiamine
consists of a thiazole and a pyrimidine moiety, which are produced in separate branches of the biosynthetic pathway before
being coupled together to produce thiamine phosphate. This is
then further phosphorylated to produce the active cofactor
TPP (Fig. 3). Many of the genes encoding thiamine biosyn-

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FIG. 3. The TPP biosynthetic pathway. TPP is composed of a thiazole and a pyrimidine moiety, each synthesized through a separate pathway.
The thiazole branch is shaded in gray. The bacterial genes responsible for each step are shown, followed by the symbol F, E, , or indicating
the presence of the gene in different algae (see Fig. 2 for the key). Glyceraldehyde-3-phosphate (Ga3P) and pyruvate (Pyr) are combined to form
DXP in the thiazole branch, while 5-aminoimidazole ribonucleotide is converted to hydroxymethylpyrimidine diphosphate (HMP-PP) in the
pyrimidine branch. Prokaryotes use the thiL gene product to convert thiamine monophosphate to TPP, while eukaryotes appear to dephosphorylate thiamine monophosphate before pyrophosphorylating thiamine with TPK.

thetic enzymes from bacteria have been cloned, and in several

cases the structures of the enzymes have been solved (60).
We have a less complete understanding of the pathway in
eukaryotes, and what knowledge there is comes mainly from
the yeast S. cerevisiae. The overall pathway is similar to that in
bacteria, with thiamine monophosphate formed from thiazole
and pyrimidine moieties, but the enzymes involved appear to
be different. None of the bacterial genes have homologues in
the yeast genome. In contrast, one enzyme of the thiazole
branch, thi4, and one pyrimidine biosynthetic gene, thi5, have
been cloned from yeast, but neither shows any sequence similarity to the bacterial enzymes. Furthermore, thiL is absent
and the terminal enzyme of the pathway is thiamine pyrophosphokinase (TPK), which pyrophosphorylates thiamine to form
thiamine pyrophosphate (Fig. 3).
Thiamine was the first vitamin found to be an algal growth
factor (39). Early studies on the specificity of this requirement
showed that in some cases thiamine auxotrophy could be relieved by addition of the thiazole moiety to the growth medium, in others cases the pyrimidine moiety was sufficient,
while in the final group of auxotrophs the full thiamine molecule was essential for growth (50). These studies show that in
algae the thiamine biosynthetic pathway follows the same general pattern as in other organisms, with two separate branches
to make each of the moieties, which are then combined together to make thiamine (Fig. 3). Furthermore, the presence of
some parts of the pathway in thiamine auxotrophs suggests that
they require the vitamin because they have lost one or more of
the essential genes involved in its biosynthesis.
C. reinhardtii, C. merolae, and T. pseudonana do not require

thiamine or any of the intermediates in its biosynthesis for

growth, demonstrating that they can synthesize the vitamin de
novo. BLAST searches with the thiamine biosynthetic genes
from E. coli, S. enterica serovar Typhimurium, and B. subtilis
against the genome of the red alga C. merolae demonstrates
that it has all of the genes necessary to synthesize thiamine
monophosphate via the bacterial route (Fig. 3 and Table 2).
However, it does not contain a gene with similarity to bacterial
thiamine monophosphate kinase (ThiL) and instead has a homologue of the yeast TPK. The current versions of the C.
reinhardtii and T. pseudonana genomes suggest they contain
the genes for most of the enzymes in the pathway, but they do
not contain genes with sequence similarity to the short bacterial thiS gene, and C. reinhardtii also lacks a gene with sequence
similarity to thiG, which is involved in the synthesis of thiazole
phosphate. Many of the enzymes in the thiazole branch are
similar to those involved in molybdopterin biosynthesis, and so
one must be careful when assigning a role to these proteins
purely on the basis of sequence similarity. Unlike T. pseudonana and C. merolae, C. reinhardtii contains a gene with sequence similarity to thiM, which in eubacteria is involved in
scavenging the thiazole moiety from the environment (60).
However, in C. reinhardtii this gene appears to be essential for
thiamine biosynthesis, since mutations in thiM lead to thiamine
auxotrophy (21). This suggests that synthesis of the thiazole
moiety in C. reinhardtii follows a route different from that in
eubacteria. Another difference in C. reinhardtii is that the ThiD
and ThiE proteins are predicted to be part of the same large
polypeptide (mtc_168251), with the central region corresponding to thiD and the 3 end containing thiE. The N terminus of

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the protein has no bacterial homologue, and the significance of

the fusion protein thus remains unknown.
A further complication when extrapolating biochemical
pathways from genome sequences is that the thiazole branch of
the pathway initially involves the formation of deoxy-D-xylulose-5-phosphate (DXP) from glyceraldehyde-3-phosphate
and pyruvate. In many organisms, DXP is also the precursor to
isoprenoids. There are two known routes to isoprenoids, the
DXP pathway and the mevalonate (MEV) pathway (37). Animals use the MEV pathway, while eubacteria use either the
DXP or the MEV pathway (53). Higher plants have the ability
to synthesize isoprenoids via both routes; the MEV pathway is
in the cytosol and endoplasmic reticulum (as it is in animals),
whereas the DXP pathway is confined to the plastids. The
chlorophytes, such as Scenedesmus obliquus, C. reinhardtii, and
Chlorella fusca (14), and P. falciparum (53) use the DXP pathway exclusively, whereas the euglenophyte Euglena gracilis uses
only the MEV pathway (14) and has an obligate requirement
for thiamine, suggesting that DXP is not used in the biosynthesis of either thiamine or isoprenoids in this organism. The
rhodophyte Cyanidium caldarum and the heterokontophyte
Ochromonas danica use both the DXP and MEV pathways
(14), but while C. caldarum does not require thiamine, O.
danica has an obligate requirement for the vitamin. This demonstrates that it is not simply the ability to synthesize DXP that
determines whether or not an alga has a requirement for thiamine.
The thiamine requirement of P. falciparum has not been
categorically established, although previous reports have suggested that it possesses the enzymes that catalyze the final steps
in the pathway (6). The P. falciparum genome has a complement of thiamine biosynthesis genes similar to that of C. reinhardtii, with the exception of thiC, suggesting that it cannot
synthesize the pyrimidine moiety from 5-aminoimidazole ribonucleotide. It also lacks either a thiL or a TPK gene but does
have a gene for ThiM. Given the parasitic lifestyle of P. falciparum, it is quite possible that it is able to acquire either thiamine or its constituent parts from its host.
The two single-celled amoebae E. histolytica and D. discoideum require thiamine for growth, and none of the genes
specific for thiamine biosynthesis are found in their genomes.
Thus, although the currently available genome sequences do
not allow us to determine the initial process leading to thiamine auxotrophy, it appears that once this has arisen, there is
no selection pressure for the retention of any of the biosynthetic genes and these are lost from the genome.
COBALAMIN
Cobalamin is a cobalt-containing tetrapyrrole related to
chlorophyll and heme (Fig. 4A). Minot and Murphy first identified this cofactor in the 1920s, when they described how they
were able to cure the symptoms of pernicious anemia with liver
extracts (44). The active factor was isolated (61) and crystallized (51) in 1948; it was given the name vitamin B12 or, as it
contained cobalt, cobalamin. Cobalamin acts as a cofactor for
enzymes that catalyze either rearrangement-reduction reactions or methyl transfer reactions. In bacteria there are more
than 20 cobalamin-dependent enzymes (40), including those
important for methanogenesis, but in eukaryotes there are

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FIG. 4. Vitamin B12 metabolism in algal and human cells.

(A) Structure of vitamin B12 (cobalamin). The molecule consists of a
highly modified tetrapyrrole ring to which a lower nucleotide loop is
attached. R can be either a methyl (Me) or an adenosyl (Ado) group.
(B) Genes involved in vitamin B12 metabolism in eukaryotic cells.
Those genes responsible for each step are shown, followed by the
symbol E or indicating the presence of the gene in different algae
(see Fig. 2 for the key). Cobalamin derivatives (Cbl) and the oxidation
state of cobalt are shown. Cobalamin containing Co3 (CblIII) is
converted to methylcobalamin, while cobalamin containing Co (CblI)
is converted to adenosylcobalamin.

many fewer. In animals, there are two, methionine synthase

and methylmalonyl-CoA mutase, which is involved in the utilization of odd-chain fatty acids (40). Higher plants have no
cobalamin-dependent enzymes and so neither utilize nor synthesize cobalamin.
Cobalamin biosynthesis has been well characterized in bacteria. There are essentially two alternative routes, comprising
up to 20 enzymatic steps from the tetrapyrrole primogenitor
uroporphyrinogen III (66). The first to be characterized was
the so-called late-insertion pathway (4, 63), which has an absolute requirement for molecular oxygen (58) and in which the
cobalt ion is inserted into the tetrapyrrole macrocycle after
ring contraction. The second route is called the early-insertion
pathway (54), where the cobalt ion is chelated before ring
contraction and which can operate under anaerobic conditions.
All archaea and many eubacteria are able to synthesize cobalamin de novo, but several eubacteria lack the biosynthetic
pathway. An example of the latter is E. coli, which utilizes
cobalamin from the environment if it is available but is able to
alter its metabolism in the absence of the cofactor.
More than half of all microalgae surveyed (Table 1; see
Table S1 in the supplemental material) (11) have an obligate
requirement for exogenous vitamin B12, leading to the remarkable conclusion that auxotrophy is the norm rather than the
exception in the algal kingdom, despite the fact that these
organisms are photosynthetic. Of the algal species that did not
require an exogenous supply for growth, some were found to
take up cobalamin if it was available (11; see below). However,
when grown in its absence, the cells did not contain measurable
amounts of cobalamin. This demonstrates that, rather than
being able to synthesize it, these vitamin B12-independent algae
had no need for the cofactor in their metabolism, a situation
similar to that found in E. coli.
Inspection of the available algal genome sequences confirmed these observations. T. pseudonana has an obligate requirement for vitamin B12, but C. reinhardtii and C. merolae do

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not require the vitamin. BLAST searches of the C. reinhardtii,

C. merolae, and P. falciparum genomes did not identify any
genes with sequence similarity to known cobalamin biosynthetic genes, and while a gene with sequence similarity to cbiP,
encoding adenosyl-cobyric acid-a,c-diamide synthase, is
present in the genome of T. pseudonana (new V2.0 genewise
7.511.1), this organism does not possess any other genes required for cobalamin biosynthesis (11). Thus, algae do not
have the ability to synthesize cobalamin de novo, indicating
that cobalamin auxotrophy is likely to have arisen because of
an obligate requirement for the cofactor in algal metabolism
rather than from the inability to synthesize it. It is interesting
that this is different from the situation observed for thiamine
and biotin auxotrophy, which appears to have arisen because of
the loss of one or more genes involved in the biosynthesis of
the cofactors.
Soon after the isolation of vitamin B12 as the mammalian
anti-pernicious anemia factor (44), E. gracilis was shown to
require the vitamin for growth (52). Early studies showed that
the requirement of many auxotrophic algae for vitamin B12 was
reduced, but not completely removed, if methionine was added
to the culture medium (29). This observation can now be explained by the fact that cobalamin is a cofactor for methionine
synthase. More-recent studies (30) have shown that E. gracilis
contains a vitamin B12-dependent methionine synthase (also
called MetH), consistent with the idea that cobalamin plays a
role in algal methionine biosynthesis.
Higher plants do not require vitamin B12 for methionine
biosynthesis because they contain vitamin B12-independent
methionine synthase (MetE) and not MetH. By contrast, animals contain MetH and not MetE and thus require cobalamin.
The recent genome-sequencing projects have demonstrated
that both MetH and MetE can be found in different algae.
While T. pseudonana contains MetH only and C. merolae contains MetE only, C. reinhardtii contains both enzymes (Table
2). In the presence of vitamin B12, C. reinhardtii uses MetH but
in the absence of the vitamin it uses MetE (11). This phenomenon is analogous to the situation in eubacteria such as E. coli,
which also switch between MetE and MetH, depending upon
the availability of exogenous cobalamin (68). MetH has a much
higher turnover rate than MetE, and so it is a preferred route
for methionine synthesis when cobalamin is present (27). Interestingly, the two obligate parasites P. falciparum and E.
histolytica do not appear to contain either methionine synthase,
suggesting that they may acquire methionine from their hosts.
In contrast, the genome of D. discoideum, like that of C. reinhardtii, contains both metE and metH.
The fact that addition of methionine does not completely
remove vitamin B12 auxotrophy in algae prompted some investigators to look for other vitamin B12-dependent enzymes. A
vitamin B12-dependent ribonucleotide reductase has been partially purified from E. gracilis (28), suggesting that this organism may require cobalamin for DNA biosynthesis. This is consistent with the observation that DNA biosynthesis appears to
be inhibited during vitamin B12 deprivation. However, the vitamin B12-dependent type II ribonucleotide reductase, which is
generally thought to be present in prokaryotes only, is one of
three isoforms of ribonucleotide reductase (33). Other studies
have shown that ribonucleotide reductase activity increases in
E. gracilis during vitamin B12 deficiency (8), suggesting that, as

EUKARYOT. CELL

with many bacteria, there is more than one isoform of ribonucleotide reductase in this organism.
An alternative explanation for the reduction in DNA biosynthesis during vitamin B12 deprivation is that it is a result of
a perturbation of folate metabolism which results from reduced methionine synthase activity; this enzyme uses folate as
a cofactor. Such a metabolic abnormality, which is termed
folate trapping, is characteristic of vitamin B12 deficiency in
humans (59). Vitamin B12 auxotrophy in the green alga
Lobomonas rostrata can only be rescued when both folate and
methionine are added to the culture medium together (11),
demonstrating that folate trapping also occurs in algae and
providing an explaining as to why earlier studies (29) could
only partially rescue vitamin B12 auxotrophy in algae with the
addition of methionine alone.
Crude cell extracts of E. gracilis have been reported to contain methylmalonyl-CoA mutase activity (67), leading the authors to suggest that this organism contains a third vitamin
B12-dependent enzyme. This enzyme catalyzes the reversible
conversion of succinyl-CoA to methylmalonyl-CoA. In mammals, methylmalonyl-CoA mutase is essential for the degradation of odd-chain fatty acids (40), but in other organisms, the
enzyme has a role in anaerobic metabolism during propionate
fermentation, as well as in the biosynthesis of branched-chain
fatty acids. E. gracilis is able to grow on propionate (67), providing further evidence that an active methylmalonyl-CoA mutase may be present in the cell. A methylmalonyl-CoA mutase
gene is present in the genome of T. pseudonana, and there is
also an expressed sequence tag with sequence similarity to this
gene from the diatom Phaeodactylum tricornutum (PTMM04237).
Furthermore, the enzyme has recently been purified from the
vitamin B12-dependent haptophyte Pleurochrysis carterae (45).
Interestingly, all of the algae that have been found to contain
methylmalonyl-CoA mutase have complex plastids.
In mammalian cells, methylmalonyl-CoA mutase is located
in the mitochondrion. The proteins CblA and CblB are
thought to be responsible for the intracellular transport of
cobalamin into the mitochondria of mammalian cells (15, 16).
Proteins with sequence similarity to both CblA and CblB can
be found in the genome of T. pseudonana (Table 2), suggesting
that the methylmalonyl-CoA mutase in this alga is likely to be
located in the mitochondrion and that the machinery for the
intracellular transport of cobalamin is conserved between animals and algae. Not surprisingly for organisms that do not
contain methylmalonyl-CoA mutase, C. reinhardtii, C. merolae,
and P. falciparum do not possess genes with sequence similarity
to cblA and cblB (Table 2) but both C. reinhardtii and T.
pseudonana contain a gene with sequence similarity to cblE,
which encodes methionine synthase reductase, required in organisms containing MetH.
Why is it that so many algae have an absolute requirement
for vitamin B12? The exact role of methylmalonyl-CoA mutase
in algae is not known, but the fact that E. gracilis can use
propionate as a carbon source suggests that it allows these
organisms to grow heterotrophically when vitamin B12 is available. The isolation of an expressed sequence tag encoding this
enzyme from P. tricornutum, a vitamin B12-independent alga,
indicates that the presence of this enzyme in an algal cell does
not in itself result in cobalamin auxotrophy. Instead, vitamin
B12 auxotrophy appears to be determined by the enzymes in-

VOL. 5, 2006

MINIREVIEWS

volved in methionine biosynthesis. The red alga C. merolae

does not require vitamin B12 and, like higher plants, contains
metE only. In contrast T. pseudonana, which does require vitamin B12, contains metH only, whereas C. reinhardtii possesses
both enzymes. It seems likely that, as with many eubacteria,
early eukaryotes contained both metE and metH and later lost
one of the genes. In animals and some algae such as T. pseudonana, loss of metE in an environment that must have contained
a readily available source of the vitamin resulted in the creation of a vitamin B12-auxotrophic organism.
ACQUISITION OF VITAMINS
The requirement for biotin, thiamine, and cobalamin by so
many disparate algae indicates that the vitamins are available
in the environment and that mechanisms exist for their uptake
into algal cells. These three vitamins are all water soluble and
comparatively stable, suggesting that they can be rescued by
salvage. Indeed, thiamine-scavenging pathways are known in
animals, fungi, and eubacteria (60). These vitamins are cofactors for a limited number of enzymes and are thus required in
small quantities, reducing the pressure on biosynthetic flux and
making salvage a viable option.
However, the uptake of these compounds is not as simple as
it may at first seem because their concentration in the natural
environment is extremely low. Indeed, the minute amount of
these organic micronutrients has made them difficult to measure (50). The concentration of vitamin B12 in seawater is
thought to vary between 0 and 3 ng/liter (9), and while higher
levels have been reported in some freshwaters (12, 34), these
levels are generally too low to support algal growth. Several
studies have shown that different vitamin B12-dependent algae
require at least 10 ng/liter cobalamin in order to grow (50).
Similarly, the concentrations of both thiamine and biotin in the
natural environment are below that normally required in culture, with thiamine levels typically varying between 8 and 15
ng/liter at different points in the Pacific Ocean and biotin
varying between 1 and 4 ng/liter in the same regions (9). In the
case of thiamine, the stability of the cofactor at the alkaline pH
of seawater has been shown to be dependent on the temperature of the water, declining sharply between 10C and 30C
(26). This makes acquisition of the free cofactor from solution
an unlikely route for many marine organisms.
The observation that only trace amounts of these vitamins
were present in natural waters led several investigators to examine whether these compounds influence the productivity,
and succession, of different species. Menzel and Spaeth (43)
reported that moderate diatom blooms occurred in the Sargasso Sea when cobalamin concentrations were at their highest, and several other studies have shown a link between algal
productivity and vitamin concentrations (56, 65). Such observations led to suggestions that algae were significant contributors to the pool of vitamins found in these waters (43). While
this may be true for thiamine and biotin, it cannot be the case
for cobalamin since the biosynthetic pathway is not present in
any eukaryotic organism (11).
The fact that only prokaryotes have the ability to synthesize
cobalamin implies that all of the vitamin B12 found in algae,
and indeed animals, must originally have been produced by
bacteria. Fogg and Kurata noted that many algae grew more

1181

rapidly in the presence of bacteria and thus concluded that the

latter produce utilizable B vitamins for the algae (23, 36).
More-recent work has provided firm evidence for this, since
the cobalamin-dependent red alga Porphyridium purpureum
can be sustained in defined culture medium lacking exogenous
vitamin B12 by the marine bacterium Halomonas sp. In return,
because there is no carbon source in the medium, the bacteria
appear to be able to use the products of algal photosynthesis to
grow (11). Halomonas sp. and others, such as Saccharophagus
degradans, have been shown to degrade complex algal carbohydrates (18, 31). These symbiotic interactions between bacteria and algae appear to be widespread since a number of
diverse algae are able to acquire vitamin B12 from bacteria
(11). The lack of cobalamin in the environment, combined with
the fact that more than half of all algae require the vitamin
(Table 1), suggests that many algae form these symbiotic interactions in order to obtain the cofactor. Although there is no
evidence that algae acquire thiamine directly from bacteria,
such an interaction would explain why the level of free vitamin
in natural waters does not limit algal growth. In support of this
theory, Menzel and Spaeth, following their studies in the Sargasso Sea, found no evidence to suggest that vitamins limited
algal productivity (43).
A number of dinoflagellate, euglenoid, and heterokont algae
are phagotrophic on bacterial prey, as is the amoeba D. discoideum. Furthermore, some dinoflagellates are known to contain intracellular bacteria (48). In terms of organic micronutrient acquisition, this provides an obvious route by which
these organisms are able to take up their vitamins. All of the
biotin-requiring algae fall into these groups, so the major route
to biotin acquisition may be phagotrophy, and in organisms
that do not have the ability to ingest bacteria, there may be
strong evolutionary pressure to retain a functional biotin biosynthetic pathway. One other noteworthy point is that these
phagotrophic groups include species that contain cobalamindependent methylmalonyl-CoA mutase (Fig. 1), suggesting
perhaps that, like humans, they use this enzyme for the degradation of odd-chain fatty acids from their prey.
Algal-bacterial interactions are not limited to delivery of
vitamins. Halomonas sp. has also been shown to improve the
growth of the green alga Duniella balwardii under irondeficient conditions, suggesting that the latter may be able
to utilize bacterial siderophores (7, 35). Zoospores of the
macroalga Ulva pertusa have been shown to recognize the
quorum-sensing N-acyl-L-homoserine lactone molecules released by bacterial biofilms, thereby facilitating the adherence of the zoospores to the surface (32). Even more remarkably, the morphology of the related alga Monostroma
oxyspermum is dependent on a growth factor, thallusin, synthesized by marine bacteria; in the absence of the bacteria, the
algal thallus does not form and instead the alga grows as a
loose association of single cells (41).
CONCLUSIONS
Vitamins are defined as organic micronutrients that must be
obtained in the human diet. The observation that three of
these vitamins are also essential for many photosynthetic algae,
which are generally assumed to be completely autotrophic, is
surprising. We have used the emerging genome sequences to

1182

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EUKARYOT. CELL

start to understand how this has arisen. For biotin and thiamine, the requirement for an exogenous supply is likely due to
the loss of one or more key biosynthetic enzymes (Table 2). In
contrast, cobalamin biosynthesis is absent from algae altogether and auxotrophy has arisen because of the loss of a
cobalamin-independent methionine synthase.
The question now is why have these requirements arisen,
and why is vitamin auxotrophy so widespread? Because the
cofactors are complicated to synthesize and required in trace
amounts only, it is possible that there is a selective advantage
in dispensing with the need to produce them, but this can only
occur if there is a reliable external supply in their environment.
At least for cobalamin, this comes from a symbiotic relationship with bacteria. There is now clear recognition that prokaryotic and eukaryotic organisms associate with each other (47) in
order to exchange metabolites (7, 35) or to exploit unique
biological niches. Furthermore, most eukaryotes do not appear
to live in isolation; land plants form interactions with mycorrhizae to obtain phosphate and with bacteria to obtain nitrogen, while animals rely on intestinal flora for their wellbeing. It
now seems likely that eukaryotic algae rely on other organisms
for a source of essential vitamins, at least in some cases via a
beneficial symbiosis. In the coming decades, both the enzymology and the regulation of these metabolic processes are likely
to be explored in molecular detail.
ACKNOWLEDGMENTS
We thank Emmanuel College, Cambridge, United Kingdom, and
the Biotechnology and Biological Sciences Research Council
(BBSRC) of the United Kingdom for financial support. We also thank
the European Union Viteomics Research Training Network (HPRNCT-2002-00244) for funding and for providing a forum for helpful
discussions and the U.S. Department of Energy Joint Genome Institute, http://www.jgi.doe.gov/, for providing access to version 3 of the C.
reinhardtii genome sequence for use in this publication.
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