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Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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
Algae are simple photosynthetic eukaryotes which—owing in 1937 discovered that some members of the chlorophyta and
to their colonization of the oceans—are responsible for up to cryptophyta require thiamine as a growth factor in culture (39).
50% of the planet’s atmospheric carbon fixation (22). They Over the following 40 years, many studies described algal spe-
comprise a diverse group that can broadly be defined as uni- cies that required different combinations of three B vitamins:
cellular (microalgae) or multicellular (macroalgae) photosyn- vitamin B12 (cobalamin), vitamin B1 (thiamine), and vitamin
thetic organisms that lack roots, stems, leaves, conducting ves- B7 (biotin) (50). A compilation of all of the available data (see
sels, and complex sex organs (55). A single endosymbiotic Table S1 in the supplemental material; summarized in Table 1)
event between a cyanobacterium-like organism and a non- reveals the widespread nature of vitamin auxotrophy within the
photosynthetic eukaryote is thought to have given rise to the algal kingdom. Of 306 species surveyed, more than half re-
three basal groups of algae: the chlorophyta (from which quired cobalamin, while 22% required thiamine and a smaller
higher plants arose), the glaucocystophyta, and the rhodo- proportion (5%) required biotin. Remarkably, for all three
phyta (Fig. 1). vitamins, the algal species that have an obligate requirement
The chlorophyta and the rhodophyta, via secondary and for the different cofactors do not appear to fall into any one
tertiary endosymbiotic events with different nonphotosynthetic lineage, but rather auxotrophy is present in several unrelated
eukaryotes, gave rise to algal groups with complex plastids phyla, indicating that it must have arisen independently several
(Fig. 1). Some groups, such as the apicomplexans (e.g., Plas- times through evolution. Even more surprisingly, this pattern is
modium falciparum) subsequently lost the ability to photosyn- also mirrored within individual genera. For example, Hemato-
thesize, although they still retain plastids. The number of sym- coccus in the chlorophyta, Peridinium in the dinophyta, Hym-
bioses that have occurred during the evolution of algae has enomonas in the haptophyta, and Nitzschia in the heterokon-
been heavily debated, and the details are discussed elsewhere tophyta all have species that require cobalamin and others that
(46), but there are likely to have been several events. Conse- do not (see Table S1 in the supplemental material). Similarly,
quently, it is not surprising that the physiology and metabolism the dinoflagellate Gymnodinium brevis requires all three vita-
of algae are extremely varied. For example, while the majority mins whereas G. spendens requires only cobalamin. For the
of green algae contain a highly structured cell wall comprising auxotrophy to have evolved so frequently in algae, the simplest
1175
1176 MINIREVIEWS EUKARYOT. CELL
TABLE 2. Genes involved in biotin thiamine and cobalamin metabolism in C. reinhardtii, T. pseudonana, C. merolae, and P. falciparuma
Model (length 关amino acids兴)
Gene
C. reinhardtii T. pseudonana C. merolae P. falciparum
Biotin biosynthesis
bioF e_gwW.18.89.1 2.889.1 (370) CML225C (433) chr12.glimmerm_1307 (630)
bioA e_gwH.100.1.1 (675) 128.16.1 (417) CMG023C (802)
bioB Chlre2_kg.scaffold_3000262 (PS) 10.577.1 (324) CML210C (379)
Thiamine biosynthesis
thiS CMV092C (67)
thiF e_gwW.11.33.1 (270) 54.113.1 (385) CMM289c (539) chr13.genefinder 27r (584)
iscS e_gwH.31.56.1 (396) 20.15.1 (432) CMT234C (453) chr7.phat_291 (1,250)
dxs estExt_fgenesh2_pg.C_710047 (744) 6.114.1 (703) CMF089C (772) chr13.phat_385 (1,177)
thiG 169.11.1 (254) CMV077C (258)
thiH/O MTC_estExt_fgenesh2_pg.C_120281 (567) 7.273.1 (353) CMM298C (781)
thiC estExt_fgenesh2_pg.C_360009 (637) 12.129.1 (636) CMG171C (673)
thiD mtc_168251 (711) 54.158.1 (267) CMO125C (297) chr5.glimmerm_537 (310)
thiE mtc_168251 (7110 54.123.1 (1,930) CMP214C (308) MAL6PI.285 (545)
TPK gwW.1.690.1 (358) 30.73.1 (217) CMH016C (251)
thiM estExt_gwp_1H.C_30409 (242) PFL1920c (302)
Cobalamin metabolism
metE Chlre2_kg.scaffold_82000007 (842) CMJ234C (767)
metH estExt_GenewiseW_1.C_30026 (1,357) 65.17.1 (1,248)
cblE e_gwH.14.9.1 (1,381) 80.8.1 (579)
cblA 2.1075.1 (301)
cblB 89.13.1 (190)
mmcM 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 E⫺20 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 presumably obtain biotin from its host, while D. discoideum is
enzyme. a slime mold that preys on soil microorganisms. The genomes
Using the data from the literature (see Table S1 in the of these two amoebae contain a gene with sequence similarity
supplemental material), we found that 14 out of 306 species to bioF, while D. discoideum also contains a gene with se-
surveyed were biotin auxotrophs (Table 1). All of these are quence similarity to bioA. Given our current knowledge of
confined to algal groups with complex plastids, such as Amphi- biotin biosynthesis in eukaryotes, it is not possible to conclude
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 䊐 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 dephosphor-
ylate thiamine monophosphate before pyrophosphorylating thiamine with TPK.
thetic enzymes from bacteria have been cloned, and in several thiamine or any of the intermediates in its biosynthesis for
cases the structures of the enzymes have been solved (60). growth, demonstrating that they can synthesize the vitamin de
We have a less complete understanding of the pathway in novo. BLAST searches with the thiamine biosynthetic genes
eukaryotes, and what knowledge there is comes mainly from from E. coli, S. enterica serovar Typhimurium, and B. subtilis
the yeast S. cerevisiae. The overall pathway is similar to that in against the genome of the red alga C. merolae demonstrates
bacteria, with thiamine monophosphate formed from thiazole that it has all of the genes necessary to synthesize thiamine
and pyrimidine moieties, but the enzymes involved appear to monophosphate via the bacterial route (Fig. 3 and Table 2).
not require the vitamin. BLAST searches of the C. reinhardtii, with many bacteria, there is more than one isoform of ribonu-
C. merolae, and P. falciparum genomes did not identify any cleotide reductase in this organism.
genes with sequence similarity to known cobalamin biosyn- An alternative explanation for the reduction in DNA bio-
thetic genes, and while a gene with sequence similarity to cbiP, synthesis during vitamin B12 deprivation is that it is a result of
encoding adenosyl-cobyric acid-a,c-diamide synthase, is a perturbation of folate metabolism which results from re-
present in the genome of T. pseudonana (new V2.0 genewise duced methionine synthase activity; this enzyme uses folate as
7.511.1), this organism does not possess any other genes re- a cofactor. Such a metabolic abnormality, which is termed
quired for cobalamin biosynthesis (11). Thus, algae do not “folate trapping,” is characteristic of vitamin B12 deficiency in
have the ability to synthesize cobalamin de novo, indicating humans (59). Vitamin B12 auxotrophy in the green alga
that cobalamin auxotrophy is likely to have arisen because of Lobomonas rostrata can only be rescued when both folate and
an obligate requirement for the cofactor in algal metabolism methionine are added to the culture medium together (11),
rather than from the inability to synthesize it. It is interesting demonstrating that folate trapping also occurs in algae and
that this is different from the situation observed for thiamine providing an explaining as to why earlier studies (29) could
and biotin auxotrophy, which appears to have arisen because of only partially rescue vitamin B12 auxotrophy in algae with the
the loss of one or more genes involved in the biosynthesis of addition of methionine alone.
the cofactors. Crude cell extracts of E. gracilis have been reported to con-
Soon after the isolation of vitamin B12 as the mammalian tain methylmalonyl-CoA mutase activity (67), leading the au-
anti-pernicious anemia factor (44), E. gracilis was shown to thors to suggest that this organism contains a third vitamin
require the vitamin for growth (52). Early studies showed that B12-dependent enzyme. This enzyme catalyzes the reversible
the requirement of many auxotrophic algae for vitamin B12 was conversion of succinyl-CoA to methylmalonyl-CoA. In mam-
reduced, but not completely removed, if methionine was added mals, methylmalonyl-CoA mutase is essential for the degrada-
to the culture medium (29). This observation can now be ex- tion of odd-chain fatty acids (40), but in other organisms, the
plained by the fact that cobalamin is a cofactor for methionine enzyme has a role in anaerobic metabolism during propionate
synthase. More-recent studies (30) have shown that E. gracilis fermentation, as well as in the biosynthesis of branched-chain
contains a vitamin B12-dependent methionine synthase (also fatty acids. E. gracilis is able to grow on propionate (67), pro-
called MetH), consistent with the idea that cobalamin plays a viding further evidence that an active methylmalonyl-CoA mu-
role in algal methionine biosynthesis. tase may be present in the cell. A methylmalonyl-CoA mutase
Higher plants do not require vitamin B12 for methionine gene is present in the genome of T. pseudonana, and there is
biosynthesis because they contain vitamin B12-independent also an expressed sequence tag with sequence similarity to this
methionine synthase (MetE) and not MetH. By contrast, ani- gene from the diatom Phaeodactylum tricornutum (PTMM04237).
mals contain MetH and not MetE and thus require cobalamin. Furthermore, the enzyme has recently been purified from the
The recent genome-sequencing projects have demonstrated vitamin B12-dependent haptophyte Pleurochrysis carterae (45).
that both MetH and MetE can be found in different algae. Interestingly, all of the algae that have been found to contain
While T. pseudonana contains MetH only and C. merolae con- methylmalonyl-CoA mutase have complex plastids.
tains MetE only, C. reinhardtii contains both enzymes (Table In mammalian cells, methylmalonyl-CoA mutase is located
2). In the presence of vitamin B12, C. reinhardtii uses MetH but in the mitochondrion. The proteins CblA and CblB are
in the absence of the vitamin it uses MetE (11). This phenom- thought to be responsible for the intracellular transport of
volved in methionine biosynthesis. The red alga C. merolae rapidly in the presence of bacteria and thus concluded that the
does not require vitamin B12 and, like higher plants, contains latter produce utilizable B vitamins for the algae (23, 36).
metE only. In contrast T. pseudonana, which does require vi- More-recent work has provided firm evidence for this, since
tamin B12, contains metH only, whereas C. reinhardtii possesses the cobalamin-dependent red alga Porphyridium purpureum
both enzymes. It seems likely that, as with many eubacteria, can be sustained in defined culture medium lacking exogenous
early eukaryotes contained both metE and metH and later lost vitamin B12 by the marine bacterium Halomonas sp. In return,
one of the genes. In animals and some algae such as T. pseudo- because there is no carbon source in the medium, the bacteria
nana, loss of metE in an environment that must have contained appear to be able to use the products of algal photosynthesis to
a readily available source of the vitamin resulted in the cre- grow (11). Halomonas sp. and others, such as Saccharophagus
ation of a vitamin B12-auxotrophic organism. degradans, have been shown to degrade complex algal carbo-
hydrates (18, 31). These symbiotic interactions between bacte-
ria and algae appear to be widespread since a number of
ACQUISITION OF VITAMINS
diverse algae are able to acquire vitamin B12 from bacteria
The requirement for biotin, thiamine, and cobalamin by so (11). The lack of cobalamin in the environment, combined with
many disparate algae indicates that the vitamins are available the fact that more than half of all algae require the vitamin
in the environment and that mechanisms exist for their uptake (Table 1), suggests that many algae form these symbiotic in-
into algal cells. These three vitamins are all water soluble and teractions in order to obtain the cofactor. Although there is no
comparatively stable, suggesting that they can be rescued by evidence that algae acquire thiamine directly from bacteria,
salvage. Indeed, thiamine-scavenging pathways are known in such an interaction would explain why the level of free vitamin
animals, fungi, and eubacteria (60). These vitamins are cofac- in natural waters does not limit algal growth. In support of this
tors for a limited number of enzymes and are thus required in theory, Menzel and Spaeth, following their studies in the Sar-
small quantities, reducing the pressure on biosynthetic flux and gasso Sea, found no evidence to suggest that vitamins limited
making salvage a viable option. algal productivity (43).
However, the uptake of these compounds is not as simple as A number of dinoflagellate, euglenoid, and heterokont algae
it may at first seem because their concentration in the natural are phagotrophic on bacterial prey, as is the amoeba D. dis-
environment is extremely low. Indeed, the minute amount of coideum. Furthermore, some dinoflagellates are known to con-
these organic micronutrients has made them difficult to mea- tain intracellular bacteria (48). In terms of organic micronu-
sure (50). The concentration of vitamin B12 in seawater is trient acquisition, this provides an obvious route by which
thought to vary between 0 and 3 ng/liter (9), and while higher these organisms are able to take up their vitamins. All of the
levels have been reported in some freshwaters (12, 34), these biotin-requiring algae fall into these groups, so the major route
levels are generally too low to support algal growth. Several to biotin acquisition may be phagotrophy, and in organisms
studies have shown that different vitamin B12-dependent algae that do not have the ability to ingest bacteria, there may be
require at least 10 ng/liter cobalamin in order to grow (50). strong evolutionary pressure to retain a functional biotin bio-
Similarly, the concentrations of both thiamine and biotin in the synthetic pathway. One other noteworthy point is that these
natural environment are below that normally required in cul- phagotrophic groups include species that contain cobalamin-
ture, with thiamine levels typically varying between 8 and 15 dependent methylmalonyl-CoA mutase (Fig. 1), suggesting
ng/liter at different points in the Pacific Ocean and biotin perhaps that, like humans, they use this enzyme for the deg-
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ACKNOWLEDGMENTS J. Woodward, T. Winckler, Y. Tanaka, G. Shaulsky, M. Schleicher, G. Wein-
stock, A. Rosenthal, E. C. Cox, R. L. Chisholm, R. Gibbs, W. F. Loomis, M.
We thank Emmanuel College, Cambridge, United Kingdom, and Platzer, R. R. Kay, J. Williams, P. H. Dear, A. A. Noegel, B. Barrell, and A.
the Biotechnology and Biological Sciences Research Council Kuspa. 2005. The genome of the social amoeba Dictyostelium discoideum.
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(BBSRC) of the United Kingdom for financial support. We also thank
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