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in many of the world’s oceans (2), and Cyanidioschyzon merolaeis a unicellular thermophilic red alga isolated from sulfate-richhot springs (pH 1.5, 45°C) (42). In this article, we use thegenome sequence data available for these four species to in- vestigate the question of vitamin metabolism in algae, thusproviding the first clues as to why and how some algae have arequirement for these cofactors. Each vitamin will be discussedin turn before we focus on possible routes for their acquisitionby 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 growth-

promoting factor for yeast (69) and was finally isolated and

purified in 1941 (64). Biotin is a cofactor for several essentialcarboxylase enzymes (62), including acetyl coenzyme A (CoA)carboxylase, which is involved in fatty acid synthesis, and so isuniversally required. The molecule consists of an imidazolering fused to a sulfur-containing tetrahydrothiophene ring witha fatty acid side chain (Fig. 2). In eubacteria, the first precursor

for biotin synthesis is pimeloyl-CoA but the source of thisdiffers among different species. Thereafter, the concerted ac-tion of four enzymes, BioF, BioA, BioD, and BioB, convertspimeloyl-CoA to biotin (20) (Fig. 2). In the budding yeastSaccharomyces cerevisiae, homologues of bioA, bioD, and bioB,but not bioF , are present, so the source of 7-keto-8-aminopel-argonic acid remains unknown. The higher plant Arabidopsisthaliana contains genes for BioF, BioA (also called BIO1), andBioB (also called BIO2) (3, 49), but the genome does notappear to contain a gene with sequence similarity to known bioD genes. Since A. thaliana can synthesize biotin de novo, theabsence of a bioD gene from the genome suggests that inhigher plants the conversion of 7,8-diaminopelargonic acid to

FIG. 1. Summary of algal evolution. The three basal groups chlo-rophyta, rhodophyta, and glaucocystophyta are shown with green, red,and blue plastids, respectively. Those groups derived from the chloro-phyta and rhodophyta by secondary endosymbioses are shown with theappropriate colored plastids. Tertiary endosymbiotic events are notshown in this diagram. The boxed phyla contain at least one organism

with a sequenced genome.

FIG. 2. The biotin biosynthetic pathway as elucidated in eubacte-ria. There are several different pathways for the synthesis of pimeloyl-CoA. The gene names are followed by the symbol F indicating thepresence of the gene in C. merolae (Cm), the symbol E indicating thepresence of a gene in C. reinhardtii (Cr), the symbol ■ indicating that

the given gene is present in the genome of T . pseudonana (Tp), or thesymbol indicating that the gene is in the genome of P . falciparum(Pf). Genes with sequence similarity to bioF and bioA can also befound in the genome sequence of D. discoideum, while only bioF can befound in E. histolytica (see text for details).

TABLE 1. Vitamin requirements of the individual speciesdetailed in the supplemental material compiled

under the different algal groups a

Phylum No. of species

surveyed

No. of species requiring:

Cobalamin Thiamine Biotin

Chlorophyta 148 44 19 0 Rhodophyta 13 12 0 0Cryptophyta 6 5 5 1

Dinophyta 27 24 7 7 Euglenophyta 15 13 11 1 Haptophyta 17 10 14 0 Heterokontophyta 80 47 11 5

Total 306 155 67 14

a Only those species that have had their cobalamin, thiamine, and biotinrequirements assessed have been included in this survey, and for this reasonthose data do not include any glaucocystophytes, chloroarachnophytes, or api-complexans. A requirement for biotin is found only in species that containcomplex plastids, i.e., those that have arisen as the result of secondary andtertiary endosymbiosis with a eukaryotic alga. Furthermore, every species thatrequires biotin also requires cobalamin, thiamine, or both.

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D-desthiobiotin must be carried out by an as-yet-unidentifiedenzyme.

Using the data from the literature (see Table S1 in thesupplemental material), we found that 14 out of 306 speciessurveyed were biotin auxotrophs (Table 1). All of these areconfined to algal groups with complex plastids, such as Amphi- dinium carterae (dinophyte) and Ochromonas danica (het-erokontophyte). Interestingly, all biotin auxotrophs also have arequirement for either cobalamin, thiamine, or both (see TableS1 in the supplemental material). C. reinhardtii, T . pseudonana,and C. merolae are not biotin auxotrophs (see Table S1 in thesupplemental material), and as they contain several biotin-dependent carboxylases, they must have a functional biosyn-thetic pathway. Whether or not P . falciparum requires exoge-nous biotin is unknown. We used the BLAST algorithm (1) toquery the latest versions of their genome sequences with thefour bacterial biotin biosynthesis genes, bioF , bioA, bioD, and bioB. In C. reinhardtii, T . pseudonana, and C. merolae, we couldidentify 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 andnone of the other three genes, so it will be of interest todetermine whether or not it requires the vitamin for growth. If not, it suggests that there is a different biotin biosynthetic routein P . falciparum. Two known biotin auxotrophs whose genomeshave 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 isa slime mold that preys on soil microorganisms. The genomes

of these two amoebae contain a gene with sequence similarityto bioF , while D. discoideum also contains a gene with se-quence similarity to bioA. Given our current knowledge of biotin biosynthesis in eukaryotes, it is not possible to concludehow biotin auxotrophy arose initially in these lineages. Never-theless, the simplest explanation is that it was caused by theloss of a single biosynthetic gene, although this might not bethe same gene in every case.

THIAMINE

Like biotin, thiamine also plays a pivotal role in intermediarycarbon metabolism. The active form of the vitamin is thiaminepyrophosphate (TPP), which is essential for all organisms. Thecofactor associates with a number of enzymes involved in pri-mary carbohydrate and branched-chain amino acid metabo-lism, including pyruvate dehydrogenase, transketolase, -keto-acid decarboxylase, and -ketoacid oxidase (57). Recent workon the biosynthesis of thiamine has mainly concentrated onthree prokaryotic organisms, Escherichia coli, Salmonella en-terica serovar Typhimurium, and Bacillus subtilis (5). Thiamineconsists of a thiazole and a pyrimidine moiety, which are pro-duced in separate branches of the biosynthetic pathway beforebeing coupled together to produce thiamine phosphate. This isthen further phosphorylated to produce the active cofactorTPP (Fig. 3). Many of the genes encoding thiamine biosyn-

TABLE 2. Genes involved in biotin thiamine and cobalamin metabolism in C . reinhardtii, T . pseudonana, C. merolae, and P . falciparum a

GeneModel (length amino acids)

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 biosynthesisthiS 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) c hr5.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 theC. 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 algalgenomes 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 ispresent in the genome sequence. The thiH / O gene products are isozymes that catalyze the same reaction.

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thetic enzymes from bacteria have been cloned, and in severalcases 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 fromthe yeast S. cerevisiae. The overall pathway is similar to that inbacteria, with thiamine monophosphate formed from thiazoleand pyrimidine moieties, but the enzymes involved appear tobe different. None of the bacterial genes have homologues inthe yeast genome. In contrast, one enzyme of the thiazolebranch, thi4, and one pyrimidine biosynthetic gene, thi5, havebeen cloned from yeast, but neither shows any sequence sim-ilarity to the bacterial enzymes. Furthermore, thiL is absentand the terminal enzyme of the pathway is thiamine pyrophos-phokinase (TPK), which pyrophosphorylates thiamine to formthiamine pyrophosphate (Fig. 3).

Thiamine was the first vitamin found to be an algal growthfactor (39). Early studies on the specificity of this requirementshowed that in some cases thiamine auxotrophy could be re-lieved by addition of the thiazole moiety to the growth me-dium, in others cases the pyrimidine moiety was sufficient, while in the final group of auxotrophs the full thiamine mole-cule was essential for growth (50). These studies show that inalgae the thiamine biosynthetic pathway follows the same gen-eral pattern as in other organisms, with two separate branchesto make each of the moieties, which are then combined to-gether to make thiamine (Fig. 3). Furthermore, the presence of some parts of the pathway in thiamine auxotrophs suggests thatthey 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 forgrowth, demonstrating that they can synthesize the vitamin denovo. BLAST searches with the thiamine biosynthetic genes

from E. coli, S. enterica serovar Typhimurium, and B. subtilisagainst the genome of the red alga C. merolae demonstratesthat it has all of the genes necessary to synthesize thiaminemonophosphate via the bacterial route (Fig. 3 and Table 2).However, it does not contain a gene with similarity to bacterialthiamine monophosphate kinase (ThiL) and instead has a ho-mologue of the yeast TPK. The current versions of the C. reinhardtii and T . pseudonana genomes suggest they containthe genes for most of the enzymes in the pathway, but they donot contain genes with sequence similarity to the short bacte-rial thiS gene, and C. reinhardtii also lacks a gene with sequencesimilarity to thiG, which is involved in the synthesis of thiazolephosphate. Many of the enzymes in the thiazole branch aresimilar to those involved in molybdopterin biosynthesis, and soone must be careful when assigning a role to these proteinspurely on the basis of sequence similarity. Unlike T . pseudonana and C. merolae, C. reinhardtii contains a gene with se-quence similarity to thiM , which in eubacteria is involved inscavenging the thiazole moiety from the environment (60).However, in C. reinhardtii this gene appears to be essential forthiamine biosynthesis, since mutations in thiM lead to thiamineauxotrophy (21). This suggests that synthesis of the thiazolemoiety in C. reinhardtii follows a route different from that ineubacteria. Another difference in C. reinhardtii is that the ThiDand ThiE proteins are predicted to be part of the same largepolypeptide (mtc_168251), with the central region correspond-ing to thiD and the 3 end containing thiE. The N terminus of

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 indicatingthe presence of the gene in different algae (see Fig. 2 for the key). Glyceraldehyde-3-phosphate (Ga3P) and pyruvate (Pyr) are combined to formDXP in the thiazole branch, while 5-aminoimidazole ribonucleotide is converted to hydroxymethylpyrimidine diphosphate (HMP-PP) in thepyrimidine 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.


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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 biochemicalpathways from genome sequences is that the thiazole branch of the pathway initially involves the formation of deoxy-D-xylu-lose-5-phosphate (DXP) from glyceraldehyde-3-phosphateand pyruvate. In many organisms, DXP is also the precursor toisoprenoids. There are two known routes to isoprenoids, theDXP pathway and the mevalonate (MEV) pathway (37). An-imals use the MEV pathway, while eubacteria use either theDXP or the MEV pathway (53). Higher plants have the abilityto synthesize isoprenoids via both routes; the MEV pathway isin the cytosol and endoplasmic reticulum (as it is in animals), whereas the DXP pathway is confined to the plastids. Thechlorophytes, such as Scenedesmus obliquus, C. reinhardtii, andChlorella fusca (14), and P . falciparum (53) use the DXP pathway exclusively, whereas the euglenophyte Euglena gracilis usesonly the MEV pathway (14) and has an obligate requirementfor thiamine, suggesting that DXP is not used in the biosyn-thesis of either thiamine or isoprenoids in this organism. The

rhodophyte Cyanidium caldarum and the heterokontophyteOchromonas 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 dem-onstrates that it is not simply the ability to synthesize DXP thatdetermines whether or not an alga has a requirement for thi-amine.

The thiamine requirement of P . falciparum has not beencategorically established, although previous reports have sug-gested that it possesses the enzymes that catalyze the final stepsin the pathway (6). The P . falciparum genome has a comple-ment 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 ribo-nucleotide. It also lacks either a thiL or a TPK gene but doeshave a gene for ThiM. Given the parasitic lifestyle of P . falciparum, it is quite possible that it is able to acquire either thia-mine 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 genesspecific for thiamine biosynthesis are found in their genomes.Thus, although the currently available genome sequences donot allow us to determine the initial process leading to thia-mine auxotrophy, it appears that once this has arisen, there isno selection pressure for the retention of any of the biosyn-thetic genes and these are lost from the genome.

COBALAMIN

Cobalamin is a cobalt-containing tetrapyrrole related tochlorophyll and heme (Fig. 4A). Minot and Murphy first iden-tified this cofactor in the 1920s, when they described how they were able to cure the symptoms of pernicious anemia with liverextracts (44). The active factor was isolated (61) and crystal-lized (51) in 1948; it was given the name vitamin B12 or, as itcontained cobalt, cobalamin. Cobalamin acts as a cofactor forenzymes that catalyze either rearrangement-reduction reac-tions or methyl transfer reactions. In bacteria there are morethan 20 cobalamin-dependent enzymes (40), including thoseimportant for methanogenesis, but in eukaryotes there are

many fewer. In animals, there are two, methionine synthaseand methylmalonyl-CoA mutase, which is involved in the uti-lization of odd-chain fatty acids (40). Higher plants have nocobalamin-dependent enzymes and so neither utilize nor syn-thesize cobalamin.

Cobalamin biosynthesis has been well characterized in bac-teria. There are essentially two alternative routes, comprisingup to 20 enzymatic steps from the tetrapyrrole primogenitoruroporphyrinogen III (66). The first to be characterized was

the so-called late-insertion pathway (4, 63), which has an ab-solute requirement for molecular oxygen (58) and in which thecobalt ion is inserted into the tetrapyrrole macrocycle afterring contraction. The second route is called the early-insertionpathway (54), where the cobalt ion is chelated before ringcontraction and which can operate under anaerobic conditions. All archaea and many eubacteria are able to synthesize cobal-amin de novo, but several eubacteria lack the biosyntheticpathway. An example of the latter is E. coli, which utilizescobalamin from the environment if it is available but is able toalter its metabolism in the absence of the cofactor.

More than half of all microalgae surveyed (Table 1; seeTable S1 in the supplemental material) (11) have an obligaterequirement for exogenous vitamin B

12, leading to the remark-

able conclusion that auxotrophy is the norm rather than theexception in the algal kingdom, despite the fact that theseorganisms are photosynthetic. Of the algal species that did notrequire an exogenous supply for growth, some were found totake up cobalamin if it was available (11; see below). However, when grown in its absence, the cells did not contain measurableamounts of cobalamin. This demonstrates that, rather thanbeing able to synthesize it, these vitamin B12-independent algaehad no need for the cofactor in their metabolism, a situationsimilar to that found in E. coli.

Inspection of the available algal genome sequences con-firmed these observations. T . pseudonana has an obligate re-quirement for vitamin B12, but C. reinhardtii and C. merolae do

FIG. 4. Vitamin B12 metabolism in algal and human cells.(A) Structure of vitamin B12 (cobalamin). The molecule consists of ahighly modified tetrapyrrole ring to which a lower nucleotide loop isattached. 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 thesymbol E or ■ indicating the presence of the gene in different algae(see Fig. 2 for the key). Cobalamin derivatives (Cbl) and the oxidationstate of cobalt are shown. Cobalamin containing Co3 (CblIII) isconverted to methylcobalamin, while cobalamin containing Co (CblI)is converted to adenosylcobalamin.


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not require the vitamin. BLAST searches of the C. reinhardtii,C. merolae, and P . falciparum genomes did not identify anygenes with sequence similarity to known cobalamin biosyn-thetic genes, and while a gene with sequence similarity to cbiP ,encoding adenosyl-cobyric acid- a, c-diamide synthase, ispresent in the genome of T . pseudonana (new V2.0 genewise7.511.1), this organism does not possess any other genes re-quired for cobalamin biosynthesis (11). Thus, algae do nothave the ability to synthesize cobalamin de novo, indicatingthat cobalamin auxotrophy is likely to have arisen because of an obligate requirement for the cofactor in algal metabolismrather than from the inability to synthesize it. It is interestingthat this is different from the situation observed for thiamineand 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 mammaliananti-pernicious anemia factor (44), E. gracilis was shown torequire the vitamin for growth (52). Early studies showed thatthe requirement of many auxotrophic algae for vitamin B12 was

reduced, but not completely removed, if methionine was addedto the culture medium (29). This observation can now be ex-plained by the fact that cobalamin is a cofactor for methioninesynthase. More-recent studies (30) have shown that E. graciliscontains a vitamin B12-dependent methionine synthase (alsocalled MetH), consistent with the idea that cobalamin plays arole in algal methionine biosynthesis.

Higher plants do not require vitamin B12

for methioninebiosynthesis because they contain vitamin B12-independentmethionine synthase (MetE) and not MetH. By contrast, ani-mals contain MetH and not MetE and thus require cobalamin.The recent genome-sequencing projects have demonstratedthat both MetH and MetE can be found in different algae.

While T . pseudonana contains MetH only and C. merolae con-tains MetE only, C. reinhardtii contains both enzymes (Table2). In the presence of vitamin B

12, C. reinhardtii uses MetH but

in the absence of the vitamin it uses MetE (11). This phenom-enon is analogous to the situation in eubacteria such as E. coli, which also switch between MetE and MetH, depending uponthe availability of exogenous cobalamin (68). MetH has a muchhigher turnover rate than MetE, and so it is a preferred routefor methionine synthesis when cobalamin is present (27). In-terestingly, 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 completelyremove vitamin B

12 auxotrophy in algae prompted some inves-

tigators to look for other vitamin B12-dependent enzymes. A vitamin B

12-dependent ribonucleotide reductase has been par-

tially purified from E. gracilis (28), suggesting that this organ-ism may require cobalamin for DNA biosynthesis. This is con-sistent with the observation that DNA biosynthesis appears tobe inhibited during vitamin B12 deprivation. However, the vi-tamin B

12-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 studieshave shown that ribonucleotide reductase activity increases in E. gracilis during vitamin B12 deficiency (8), suggesting that, as

with many bacteria, there is more than one isoform of ribonu-cleotide reductase in this organism.

An alternative explanation for the reduction in DNA bio-synthesis during vitamin B12 deprivation is that it is a result of a perturbation of folate metabolism which results from re-duced methionine synthase activity; this enzyme uses folate asa cofactor. Such a metabolic abnormality, which is termed“folate trapping,” is characteristic of vitamin B12 deficiency inhumans (59). Vitamin B12 auxotrophy in the green alga Lobomonas rostrata can only be rescued when both folate andmethionine are added to the culture medium together (11),demonstrating that folate trapping also occurs in algae andproviding an explaining as to why earlier studies (29) couldonly partially rescue vitamin B12 auxotrophy in algae with theaddition of methionine alone.

Crude cell extracts of E. gracilis have been reported to con-tain methylmalonyl-CoA mutase activity (67), leading the au-thors to suggest that this organism contains a third vitaminB12-dependent enzyme. This enzyme catalyzes the reversibleconversion of succinyl-CoA to methylmalonyl-CoA. In mam-

mals, methylmalonyl-CoA mutase is essential for the degrada-tion of odd-chain fatty acids (40), but in other organisms, theenzyme has a role in anaerobic metabolism during propionatefermentation, as well as in the biosynthesis of branched-chainfatty acids. E. gracilis is able to grow on propionate (67), providing further evidence that an active methylmalonyl-CoA mu-tase may be present in the cell. A methylmalonyl-CoA mutasegene is present in the genome of T . pseudonana, and there isalso an expressed sequence tag with sequence similarity to thisgene from the diatom Phaeodactylum tricornutum (PTMM04237).Furthermore, the enzyme has recently been purified from the vitamin B

12-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 locatedin the mitochondrion. The proteins CblA and CblB arethought 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 canbe found in the genome of T . pseudonana (Table 2), suggestingthat the methylmalonyl-CoA mutase in this alga is likely to belocated in the mitochondrion and that the machinery for theintracellular transport of cobalamin is conserved between an-imals and algae. Not surprisingly for organisms that do notcontain methylmalonyl-CoA mutase, C. reinhardtii, C. merolae,and P . falciparum do not possess genes with sequence similarityto 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 or-ganisms containing MetH.

Why is it that so many algae have an absolute requirementfor vitamin B

12? The exact role of methylmalonyl-CoA mutase

in algae is not known, but the fact that E. gracilis can usepropionate as a carbon source suggests that it allows theseorganisms to grow heterotrophically when vitamin B12 is avail-able. The isolation of an expressed sequence tag encoding thisenzyme from P . tricornutum, a vitamin B12-independent alga,indicates that the presence of this enzyme in an algal cell doesnot in itself result in cobalamin auxotrophy. Instead, vitaminB12 auxotrophy appears to be determined by the enzymes in-


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volved in methionine biosynthesis. The red alga C. merolaedoes not require vitamin B

12 and, like higher plants, contains

metE only. In contrast T . pseudonana, which does require vi-tamin B12, contains metH only, whereas C. reinhardtii possessesboth enzymes. It seems likely that, as with many eubacteria,early eukaryotes contained both metE and metH and later lostone of the genes. In animals and some algae such as T . pseudonana, loss of metE in an environment that must have containeda readily available source of the vitamin resulted in the cre-ation of a vitamin B12-auxotrophic organism.

ACQUISITION OF VITAMINS

The requirement for biotin, thiamine, and cobalamin by somany disparate algae indicates that the vitamins are availablein the environment and that mechanisms exist for their uptakeinto algal cells. These three vitamins are all water soluble andcomparatively stable, suggesting that they can be rescued bysalvage. Indeed, thiamine-scavenging pathways are known inanimals, fungi, and eubacteria (60). These vitamins are cofac-

tors for a limited number of enzymes and are thus required insmall quantities, reducing the pressure on biosynthetic flux andmaking salvage a viable option.

However, the uptake of these compounds is not as simple asit may at first seem because their concentration in the naturalenvironment is extremely low. Indeed, the minute amount of these organic micronutrients has made them difficult to mea-sure (50). The concentration of vitamin B

12 in seawater is

thought to vary between 0 and 3 ng/liter (9), and while higherlevels have been reported in some freshwaters (12, 34), theselevels are generally too low to support algal growth. Severalstudies have shown that different vitamin B

12-dependent algae

require at least 10 ng/liter cobalamin in order to grow (50).

Similarly, the concentrations of both thiamine and biotin in thenatural environment are below that normally required in cul-ture, with thiamine levels typically varying between 8 and 15ng/liter at different points in the Pacific Ocean and biotin varying between 1 and 4 ng/liter in the same regions (9). In thecase of thiamine, the stability of the cofactor at the alkaline pHof seawater has been shown to be dependent on the temper-ature of the water, declining sharply between 10°C and 30°C(26). This makes acquisition of the free cofactor from solutionan unlikely route for many marine organisms.

The observation that only trace amounts of these vitamins were present in natural waters led several investigators to ex-amine whether these compounds influence the productivity,and succession, of different species. Menzel and Spaeth (43)reported that moderate diatom blooms occurred in the Sar-gasso Sea when cobalamin concentrations were at their high-est, and several other studies have shown a link between algalproductivity and vitamin concentrations (56, 65). Such observations led to suggestions that algae were significant contrib-utors to the pool of vitamins found in these waters (43). Whilethis may be true for thiamine and biotin, it cannot be the casefor cobalamin since the biosynthetic pathway is not present inany eukaryotic organism (11).

The fact that only prokaryotes have the ability to synthesizecobalamin implies that all of the vitamin B

12 found in algae,

and indeed animals, must originally have been produced bybacteria. Fogg and Kurata noted that many algae grew more

rapidly in the presence of bacteria and thus concluded that thelatter produce utilizable B vitamins for the algae (23, 36).More-recent work has provided firm evidence for this, sincethe cobalamin-dependent red alga Porphyridium purpureumcan be sustained in defined culture medium lacking exogenous vitamin B

12 by the marine bacterium Halomonas sp. In return,

because there is no carbon source in the medium, the bacteriaappear to be able to use the products of algal photosynthesis togrow (11). Halomonas sp. and others, such as Saccharophagus 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 diverse algae are able to acquire vitamin B

12 from bacteria

(11). The lack of cobalamin in the environment, combined withthe fact that more than half of all algae require the vitamin(Table 1), suggests that many algae form these symbiotic in-teractions in order to obtain the cofactor. Although there is noevidence that algae acquire thiamine directly from bacteria,such an interaction would explain why the level of free vitaminin natural waters does not limit algal growth. In support of this

theory, Menzel and Spaeth, following their studies in the Sar-gasso Sea, found no evidence to suggest that vitamins limitedalgal productivity (43).

A number of dinoflagellate, euglenoid, and heterokont algaeare phagotrophic on bacterial prey, as is the amoeba D. discoideum. Furthermore, some dinoflagellates are known to con-tain intracellular bacteria (48). In terms of organic micronu-trient acquisition, this provides an obvious route by whichthese organisms are able to take up their vitamins. All of thebiotin-requiring algae fall into these groups, so the major routeto biotin acquisition may be phagotrophy, and in organismsthat do not have the ability to ingest bacteria, there may bestrong evolutionary pressure to retain a functional biotin bio-

synthetic pathway. One other noteworthy point is that thesephagotrophic groups include species that contain cobalamin-dependent methylmalonyl-CoA mutase (Fig. 1), suggestingperhaps that, like humans, they use this enzyme for the deg-radation 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 thegrowth of the green alga Duniella balwardii under iron-deficient conditions, suggesting that the latter may be ableto utilize bacterial siderophores (7, 35). Zoospores of themacroalga Ulva pertusa have been shown to recognize thequorum-sensing N -acyl-L -homoserine lactone molecules re-leased by bacterial biofilms, thereby facilitating the adher-ence of the zoospores to the surface (32). Even more re-markably, the morphology of the related alga Monostroma oxyspermum is dependent on a growth factor, thallusin, synthe-sized by marine bacteria; in the absence of the bacteria, thealgal thallus does not form and instead the alga grows as aloose association of single cells (41).

CONCLUSIONS

Vitamins are defined as organic micronutrients that must beobtained 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, issurprising. We have used the emerging genome sequences to


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start to understand how this has arisen. For biotin and thia-mine, the requirement for an exogenous supply is likely due tothe loss of one or more key biosynthetic enzymes (Table 2). Incontrast, cobalamin biosynthesis is absent from algae alto-gether and auxotrophy has arisen because of the loss of acobalamin-independent methionine synthase.

The question now is why have these requirements arisen,and why is vitamin auxotrophy so widespread? Because thecofactors are complicated to synthesize and required in traceamounts only, it is possible that there is a selective advantagein dispensing with the need to produce them, but this can onlyoccur if there is a reliable external supply in their environment. At least for cobalamin, this comes from a symbiotic relation-ship with bacteria. There is now clear recognition that prokary-otic and eukaryotic organisms associate with each other (47) inorder to exchange metabolites (7, 35) or to exploit uniquebiological niches. Furthermore, most eukaryotes do not appearto live in isolation; land plants form interactions with mycor-rhizae to obtain phosphate and with bacteria to obtain nitro-gen, while animals rely on intestinal flora for their wellbeing. It

now seems likely that eukaryotic algae rely on other organismsfor a source of essential vitamins, at least in some cases via abeneficial symbiosis. In the coming decades, both the enzymol-ogy and the regulation of these metabolic processes are likelyto be explored in molecular detail.

ACKNOWLEDGMENTS

We thank Emmanuel College, Cambridge, United Kingdom, andthe Biotechnology and Biological Sciences Research Council(BBSRC) of the United Kingdom for financial support. We also thankthe European Union Viteomics Research Training Network (HPRN-CT-2002-00244) for funding and for providing a forum for helpfuldiscussions and the U.S. Department of Energy Joint Genome Insti-tute, 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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