How haptophytes microalgae mitigate vitamin B12 limitation

Vitamin B12 (cobalamin) can control phytoplankton development and community composition, with around half of microalgal species requiring this vitamin for growth. B12 dependency is determined by the absence of cobalamin-independent methionine synthase and is unrelated across lineages. Despite their important role in carbon and sulphur biogeochemistry, little is known about haptophytes utilization of vitamin B12 and their ability to cope with its limitation. Here we report the first evaluation of B12 auxotrophy among this lineage based on molecular data of 19 species from 9 families. We assume that all species encode only a B12-dependent methionine synthase, suggesting ubiquitous B12 auxotrophy in this phylum. We further address the effect of different B12 limitations on the molecular physiology of the model haptophyte Tisochrysis lutea. By coupling growth assays in batch and chemostat to cobalamin quantification and expression analyses, we propose that haptophytes use three strategies to cope with B12 limitation. Haptophytes may assimilate dissolved methionine, finely regulate genes involved in methionine cycle and B12 transport and/or limit B12 transport to the mitochondrion. Taken together, these results provide better understanding of B12 metabolism in haptophytes and represent valuable data for deciphering how B12-producing bacteria shape the structure and dynamics of this important phytoplankton community.

cobalamin-deprived environments. In addition, accessory proteins CBLA and CBLB allow B 12 transport of MeCbl and conversion into AdoCbl in the mitochondrion for MMCM activity 23 (Fig. 1).
It has been proposed that loss of B 12 -independent methionine synthase arose multiple times in evolution 12,13,24 . A reason advanced would be that microalgae provided with a non-limiting supply of cobalamin would lost selective pressure on the energy-expensive 25 METE and retain only METH. As an example, a recent work on Chlamydomonas reinhardtii grown with a source of B 12 revealed a shift from cobalamin-independence to auxotrophy 24 . The conversion of methionine from homocysteine is essential in one-carbon metabolism as methionine undergoes several ways of use 26 . It is either assimilated into proteins, or converted by the enzyme methionine adenosyltransferase (MAT, gene metK) into S-adenosylmethionine (SAM), an important methyl donor and radical source 22,27 (Fig. 1). There are many reactions involving SAM demethylation, such as DNA methylation, synthesis of vitamin B 1 (thiamine) 22,28 and DMSP biosynthesis 29 . SAM demethylation leads to the formation of S-adenosylhomocysteine (SAH) which is finally hydrolyzed to regenerate homocysteine by the S-adenosylhomocysteine hydrolase (SAHH, gene sahH). All these reactions from methionine production to homocysteine regeneration are described as the methionine cycle ( Fig. 1).
Interestingly, the only way known for marine microalgae to produce DMSP implies both SAM demethylation and methionine transamination, which suggests that DMSP synthesis is an important sink of methionine 29 . The majority of DMSP production in the ocean is due to haptophytes and dinoflagellates and, as this molecule does not contain nitrogen, it is suggested that it acts in microalgae as a dissipating excess energy agent when sulphur assimilation exceeds nitrogen incorporation 29 . Numerous species from these lineages are considered to be cobalamin-dependent 11,13 . Therefore, vitamin B 12 may be particularly important in haptophytes and dinoflagellates cellular processes, especially in nitrogen-limited environments.
Previous studies based on culture assays showed that on the 22 haptophytes species tested, 8 were able to grow without B 12 addition and were considered as B 12 -independent 11,13 . In absence of culture assay in truthful axenic condition and of molecular evidence for the presence of METE in these species, the cobalamin dependence of haptophytes lineage stays unclear. Moreover, the question of how haptophytes acclimate and regulate key metabolic enzymes in B 12 limitation stays poorly documented. Considering that haptophytes are major contributors to nano and pico-plankton communities 17,30 and play a significant role in organic matter cycling, deciphering B 12 dependence and B 12 -associated metabolism of this lineage is of global importance.
Here, our analysis of genes metH and metE of 19 genome-sequenced or transcriptome-sequenced haptophyte species suggest that the auxotrophy for B 12 is ubiquitous in the haptophyte lineage. In a second part, by using batch and continuous cultures in controlled photobioreactors, we investigated the effect of different levels of B 12 limitation on the molecular physiology of the model haptophyte Tisochrysis lutea. Genes expression analyses showed that methionine cycle is finely regulated by B 12 availability in the environment.

Results
phylogenetic analysis of methionine synthase in haptophytes. A survey of methionine synthase isoforms in 19 haptophyte species based on transcriptomic and genomic datasets has been conducted (see Supplementary Table 1 on Supplementary Information for sequences details). All samples investigated contained the B 12 -dependent metH. The phylogeny of this phylum was reconstructed from this gene, with species from orders Coccolithales, Isochrysidales and Prymnesiales relevantly gathered (Fig. 2). This reconstruction was consistent with what is usually found for 18S sequence 31 , suggesting an absence of horizontal gene transfer. The cobalamin-independent isoform metE was not found in any of the samples, indicating that all 19 species are B 12 -auxotrophic (Fig. 2). The Marine Atlas of Tara Ocean Unigenes (MATOU) for eukaryotic data 32 was also investigated by searching similar genomic and proteomic sequences of METE from Phaeodactylum tricornutum and Chlamydomonas reinhardtii. The MATOU database gather large-scale environmental metatranscriptomic and metagenomic information. Since no haptophyte sequence was retrieved in these large datasets, this reinforces the hypothesis of absence of B 12 -independent methionine synthase in the haptophyte lineage.
Considering that comprehensive genomic and transcriptomic data of Tisochrysis lutea (Isochrysidaceae) were available, it was taken as model species for molecular physiology analyses depending on cobalamin quotas. In silico searches in T. lutea (strain CCAP 927/14) genome 31,33 allowed to identify several genes involved in vitamin B 12 metabolism, conversion and transport. Gene metH coding for cobalamin-dependent methionine synthase was found and the presence of the related protein was confirmed in our proteomic dataset. The B 12 -independent methionine synthase was not found in our proteomic nor genomic data, suggesting B 12 auxotrophy. Translated sequences of METH protein from other haptophytes were compared with the one of T. lutea when possible (see Supplementary Table 1

of Supplementary Information).
Assessment of B 12 requirement of Tisochrysis lutea. In order to validate biological dependency of T. lutea to vitamin B 12 , a growth assay was performed. The axenic microalgae were grown either in cobalamin-deprived medium, methionine adding or in complete medium. Cells grown with 40 ng L −1 cobalamin exhibited a maximal growth rate (μ max ) of 0.35 ± 0.04 d −1 and a maximal biomass increase (ΔC max ) of 0.48 ± 0.02 arbitrary units (A.U.) (Fig. 3). Cobalamin-free cultures showed a growth rate five times lower and statistically significant (p = 8.11 10 −8 ; two-tailed Student's t test) with μ max = 0.07 ± 0.03 d −1 and ΔC max = 0.05 ± 0.01 A.U. (Fig. 3). This was consistent with in silico analysis and clearly demonstrated T. lutea auxotrophy. The low growth observed for cobalamin-free cultures was due to the use of natural seawater which provided the cells with little naturally-present vitamin B 12 . Interestingly, microalgae grown with 0.50 mg L −1 methionine showed twice the growth of the negative control that was statistically significant (p = 4.04 10 −4 ; two-tailed Student's t test) with μ max = 0.17 ± 0.04 d −1 and ΔC max = 0.16 ± 0.04 A.U. (Fig. 3), meaning that T. lutea is able to uptake and assimilate dissolved methionine and use it instead of cobalamin. The assimilation of dissolved free amino acids by marine microalgae is not well documented. This result confirmed that cobalamin is vital for methionine synthesis and that a lack of B 12 may induce a lack of methionine. B 12 -limited batch experiment. Cobalamin-limited batch culture in triplicate was set up to analyze expression of genes involved in vitamin B 12 utilization, conversion and transport, and to compare their expression depending on cobalamin quota. Figure 4A presents the evolution of the average cell concentration against time and two sampling points for B 12 and qPCR measurements. Figure 4B presents results for intracellular B 12 measures, ranging from 20 ± 7 pg mg C −1 in early exponential phase to 8 ± 2 pg mg C −1 in late exponential phase, with a statistically significant two-fold decrease in intracellular cobalamin concentration due to vitamin starvation (p = 0.02; two-tailed Student's t test). In their cobalamin-limited batch experiment, Cruz-Lopez et al. 34 showed a two-fold decrease of B 12 quota for the dinoflagellate Lingulodinium polyedrum 34 , which is consistent with our result. The expression of genes metH, metK and sahH, involved in the methionine cycle, cblA, cblB and mmcm, involved in cobalamin transport, conversion and utilization in the mitochrondrion was followed during high cobalamin availability (early exponential phase) and cobalamin starvation (stationary phase). The expression of methionine cycle genes and mmcm did not show a clear trend (  Supplementary Fig. 2). This finding suggests that B 12 starvation decreases expression of genes involved in cobalamin transport and conversion. It must be pointed out that growth rate decrease at the end of the batch culture may lead to cellular processes influencing many biochemical pools. Therefore, the expression of genes analyzed here may be the result of a global physiological state not specifically related to B 12 starvation. A more accurate approach using cobalamin-limited chemostat was thus undertaken to confirm the effect of different vitamin B 12 status on genes expression.  To accurately describe the effect of vitamin B 12 status on the expression of genes involved in cobalamin use, a B 12 -limited chemostat experiment was implemented in controlled photobioreactors. Nitrogen-limited (N-limited) chemostats with the same dilution rate were taken as controls to verify whether the observed results were specific to B 12 limitation or rather related to a more general physiological status. Effort was made to prevent any bacterial contamination throughout the duration of the experiments. Particulate carbon and nitrogen and cellular concentration (see Supplementary  Fig. 3 in Supplementary Information) were monitored at high frequency and allowed to accurately describe culture phases. Biological duplicates exhibited similar trends during all the duration of the experiment (Fig. 6A,B). Based on stability of carbon and microalgal concentration, steady-state was reached in both chemostats at day 12 ( Fig. 6A,B; Supplementary Fig. 3). At day 25, a spike of limiting nutrient (vitamin B 12 or nitrates) resulted in an increase in carbon biomass in all cultures, confirming nutrient limitation during the steady-state phase (Fig. 6A,B). Samples were collected on days 14, 21, 25, 26 and 27 for B 12 content and qPCR analyses. For a same dilution rate, carbon content was slightly higher in B 12 limitation than in N-limited control chemostats. Based on N/C results, physiological status of N-limited chemostats were described: nitrogen limitation at steady-state; nutrient repletion one hour after nitrogen input during N/C increase and nutrient depletion 24 hours after nitrogen input, at N/C decrease. Intracellular B 12 content. Intracellular cobalamin content was measured at different times in B 12 -limited chemostats and nitrogen-limited control cultures. Three samples were collected during steady-state. Mean cobalamin quota in B 12 -limited cultures prior to the cobalamin spike was 0.03 ± 0.02 pg μg C −1 (Fig. 6C). In comparison, B 12 quota in N-limited chemostats was 1.73 ± 0.02 pg μg C −1 , value 50 times greater than the one observed in B 12 -limited cultures (Fig. 6D). One hour after cobalamin spike, mean quota of B 12 -limited chemostats was multiplied by 8, reaching 0.24 ± 0.14 pg μg C −1 , indicating an ability to quickly assimilate cobalamin (Fig. 6C). In N-limited cultures, B 12 quota was on average nine times higher one hour after a spike of nitrogen (2.31 ± 0.03 pg μg C −1 ) relative to the one of B 12 -limited cultures after a cobalamin pulse (Fig. 6D). One day after nutrient spike, mean B 12 -limited chemostats quota dropped below steady-state value of 0.02 ± 0.01 pg μg C −1 , suggesting rapid vitamin depletion (Fig. 6C), while after the nitrogen pulse the cobalamin quotas of the N-limited control cultures fell to 0.72 ± 0.17 pg μg C −1 (Fig. 6D), nearly 50 times higher than those of B 12 -limited cultures. This was likely attributable to the increase in cellular division, which was probably faster than vitamin acquisition. By combining B 12 quotas and N/C ratio, physiological states for B 12 -limited chemostats were described: nutrient limitation at steady-state; nutrient repletion 1 hour after B 12 input during N/C increase and nutrient depletion 24 hours after B 12 input, at the end of N/C increase.  Table 2 on Supplementary Information for primers). (2019) 9:8417 | https://doi.org/10.1038/s41598-019-44797-w www.nature.com/scientificreports www.nature.com/scientificreports/ Molecular analyses. Genes expression analyses were carried out on samples collected at each physiological state to relate genes expression patterns to nutrient quotas. Genes for which expression was followed were the same as those of the batch experiment. Results presented for steady-state correspond to the third sample collected, just before nutrient spike.
As can be seen in Fig. 7G, at steady-state all genes involved in methionine cycle were more expressed in B 12 limitation than in nitrogen limitation by 4-fold (metH) and 8-fold (metK and sahH) (  Supplementary Information). Genes cblA and cblB were around 2-fold less expressed in cobalamin limitation than in nitrogen limitation, whereas mmcm was expressed almost at the same level (Fig. 7G).
One hour after vitamin B 12 adding in cobalamin-limited reactors, during repletion phase, the expression of methionine cycle genes decreased by a factor 2 to 4 ( Fig. 7A-C) and that of cblA and cblB by a factor 2 (Fig. 7D,E). During subsequent nutrient depletion, their expression returned to that at steady-state ( Fig. 7A-E; Supplementary  Fig. 4). This pattern of expression reflects noticeably intracellular cobalamin rate contents (Fig. 6C), with methionine cycle genes overexpressed in B 12 -limited cells and repressed in B 12 -replete cells. Expression of mmcm seemed not to be affected by vitamin B 12 spike ( Fig. 7F; Supplementary Fig. 4).
One hour after nitrate spike in nitrogen-limited reactors, the expression of sahH showed a slight increase while the expression of metH, metK, cblA, cblB and mmcm did not seemed to be affected by nitrogen addition (see Supplementary Fig. 4 in Supplementary Information). These genes did not show clear changes of expression in the depletion phase ( Supplementary Fig. 4 in Supplementary Information). A decoupling between nitrogen status and the decrease in gene expression could explain the absence of regulation 24 hours after nitrogen spike. Overall, methionine cycle genes showed a clear trend directly related to vitamin B 12 quota, and did not respond in an evident way to nitrates spike. Genes encoding accessory proteins CBLA and CBLB also showed an explicit pattern of expression induced by cobalamin quota but were not influenced by nitrogen status. Gene mmcm did not show any clear regulation of expression during the experiment with its expression level being almost the same for all chemostats independently of the cultures physiological state. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Haptophytes microalgae play an important role in carbon 17,18 and sulphur cycling 20 but their ability to respond to cobalamin variations is poorly known, despite the previously described impact of B 12 limitation on phytoplankton growth and community composition 2,3,35 . The aim of this work was to (1) analyze B 12 dependency of haptophytes based on molecular data and (2) provide insights into cobalamin molecular physiology of haptophytes by studying the model marine microalgae Tisochrysis lutea. This work combined bioinformatic searches, growth tests in batch and chemostat at different levels of B 12 availability with the analyses of cobalamin quotas and expression of genes involved in B 12 metabolism.
Nineteen haptophyte species across six orders and nine families were assessed for the presence of methionine synthase isoforms. All species investigated encoded the B 12 -dependent metH only. It has been observed that cobalamin auxotrophy is determined by the presence of metH and the absence of the cobalamin-independent methionine synthase metE 11,12 . Our results, mainly based on transcriptomic datasets, suggest that all of these haptophytes are cobalamin auxotrophs. No metE sequence of haptophytes was retrieved from the MATOU database, supporting the idea that species of this phylum are B 12 -requiring for growth. On the other hand, Croft et al. 11 reported the occurrence of 8 haptophyte species among the 22 analyzed that did not require cobalamin 11 . It must be pointed out that 2 species over 22 were grown in their study and the remaining 20 were compiled from literature without any information about bacterial contamination. Recently, Helliwell et al. (2011) demonstrated the role of some bacteria so tightly attached to calcifying and non calcifying cells of E. huxleyi that they could not be disrupted with antibiotics, potentially providing the microalgae with vitamin B 12 12 . Among 8 species considered as B 12 -independent 6 belong to Coccolithales. Strongly-attached, antibiotic-resilient bacteria may have been a B 12 source for these species. The present paper is the first attempt to compile existing information based on molecular analyses for this phylum and, as no study found a species of Haptophyta phylum encoding metE nor a pseudogene, we assume that haptophytes are in majority cobalamin-dependent. This would be the first microalgae phylum gathering exclusively cobalamin auxotrophs, suggesting that their common ancestor did not encode metE, while in other phyla only certain species would have lost the B 12 -independent methionine synthase.
In order to explain the B 12 molecular physiology of haptophytes, we selected the model species T. lutea for which comprehensive genomic and transcriptomic data were made available. Growth assays in natural seawater without cobalamin enrichment confirmed results of in silico approach as T. lutea was B 12 -limited two days after inoculation. Moreover, the absence of calcified coccoliths on the cells prevented presence of non detectable bacteria after strain purification. Adding methionine instead of B 12 allowed T. lutea to develop, showing its ability to uptake and assimilate dissolved methionine to make up for cobalamin deprivation. This is consistent with another study 11 demonstrating that the B 12 -dependent freshwater chlorophyte Lobomonas rostrata could be grown for several subcultures with METH products (i.e. methionine and folic acid). Our control without B 12 exhibited 10 times lower maximal biomass compared with the control grown with 40 ng L −1 (24 pmol L −1 ) B 12 ,  Table 2 on Supplementary Information for primers).
www.nature.com/scientificreports www.nature.com/scientificreports/ suggesting around 4 ng L −1 (2.4 pmol L −1 ) cobalamin concentration in seawater. This is consistent with what was observed by Panzeca et al. 36 and Suffridge et al. 35 who estimated cobalamin concentrations ranging from 0.2 to 4 pmol L −1 in open oceans and 11 to 15 pmol L −1 in coastal ecosystems 35,36 and indicating that vitamin B 12 in natural seawater is limiting for this species. The maximal biomass obtained when T. lutea was grown with 500 g L −1 methionine was almost 3 times higher than the negative control but the concentration tested here was 625 times the maximal concentration found in seawater, that ranged from 0.27 ng L −1 offshore to 790 ng L −1 near the coast 37 . Recently, Suffridge et al. 35 reported particulate methionine concentrations in seawater along a Mediterranean transect ranging from 0.30 to 3 ng L −1 35 . These findings mean that methionine concentrations in natural environment are likely to be limiting for T. lutea development and support the idea that auxotrophic microalgae need to be supplemented with a readily available cobalamin source such as vitamin-producing bacteria 14 , cell lysate or B 12 -remodeling algae, that are able to convert the less bioavailable pseudocobalamin into a readily accessible vitamin B 12 form 38 .
We investigated the molecular physiology of T. lutea in batch and chemostat by focusing on the expression dynamics of genes involved in vitamin B 12 use, transport and conversion. Genes cblA and cblB, encoding proteins transporting cobalamin to the mitochondrion, were down-regulated under B 12 starvation in batch and B 12 limitation in chemostat compared with the N-limited controls. This suggests that when B 12 is limiting, cobalamin-dependent activities in the mitochondrion are reduced, possibly in favor of other cellular processes. Methionine cycle genes metH, metK and sahH and B 12 -dependent mmcm were not clearly affected by cobalamin starvation in batch. This differs from the results of Bertrand et al. 25,27 for the B 12 -requiring diatom Thalassiosira pseudonana, which exhibited an overexpression of methionine cycle genes in cobalamin starvation with respect to replete conditions 25,27 . As expression pattern in batch experiments could be the result of numerous cellular processes related to the absence of cell division, these results must be viewed with caution. To bypass this, we implemented cultures in chemostat.
In this experiment, methionine cycle genes, cblA and cblB exhibited dynamics remarkably mirroring cobalamin quotas, with an overexpression in B 12 limitation and downregulation in B 12 repletion. These results could mean that upregulation of metH, metK and sahH is needed in cobalamin-limited environments to maintain optimal biochemical kinetics for methionine production and SAM cycling. Interestingly, expression of mmcm, that catalyzes the conversion of succinyl-CoA to methylmalonyl-CoA in the mitochondrion with cobalamin as cofactor, remained identical independently of vitamin limitation in batch and chemostat. It has been suggested that B 12 -dependent MMCM is not vital for growth as not all cobalamin-requiring microalgae possess it 12 . Also, the reaction of MMCM is one of many entries in TCA cycle and there could be other mechanisms of regulation at this metabolic level which could explain the lack of modifications in mmcm expression. In the proteomic dataset from the N-limited chemostat described by Garnier et al. 39 , methionine cycle proteins and MMCM belonged to the top 400 highest accumulated proteins over the 4330 identified during steady-state. Proteins CBLA and CBLB were not detected in their experiment 39 . In general, our expression analysis trends are in accordance with their proteomic results, as genes cblA and cblB were the lowest expressed.
To our knowledge, this is the first time that an analysis of B 12 molecular physiology of a microalgae has been conducted in chemostat with accurately described nutrient states. More notably, this is the first time that expression dynamics of methionine cycle genes and B 12 transporters to the mitochondrion are correlated to slight changes in vitamin B 12 status in a marine microalgae, with rapid response no later than one hour after nutrient amendment. This fast regulation has been reported for T. lutea genes coding for nitrate and nitrite transporters (TlNrt2.1 and TlNrt2.3) after addition of different nitrogen substrates 40 . This suggests that haptophytes hold quick acclimation mechanisms to nutrient availability that might explain their ecological success. The fact that these three methionine cycle genes, although not all coding for B 12 -dependent enzymes, are regulated in the same way raises the question of a common regulation system. Transcription factors are among major players in regulating gene expression, and some of them have already been described for T. lutea and related to oxidative stress response, triacylglycerol synthesis and photosynthesis 41 . Genes cblA and cblB were found to belong to a same group regulated by a shared transcription factor but methionine cycle genes were not gathered in a same module 41 . McRose et al. 42 identified riboswitches affiliated with genes overexpressed in thiamine (vitamin B 1 ) starvation in haptophytes microalgae 42 . Therefore, it is likely that such regulation mechanism would play a role in regulating, directly or not, cobalamin-related genes.
In conclusion, this is the first time that B 12 dependency of haptophytes has been investigated. Based on 19 species surveyed, and since no haptophyte from the MATOU database was found encoding cobalamin-independent methionine synthase, we propose that haptophytes are cobalamin auxotrophs. Independence from vitamin B 12 has been described as a mosaic pattern across evolution [11][12][13] where Haptophyta would be the first microalgae phylum to gather only cobalamin-dependent species. The analysis of B 12 molecular physiology of the model haptophyte species T. lutea has been undertaken. A controlled approach using chemostat cultures was performed to define precisely ecophysiological states, demonstrating the common assertion that this type of approach is of great interest when analyzing fine and rapid molecular changes in microorganisms 43,44 . Based on these results, we propose that haptophytes use different strategies to make up for cobalamin deprivation that include methionine assimilation, short-term regulation mechanisms in case of sudden B 12 supply, such as cobalamin-producing bacteria excretion or cell lysis, and a preferential B 12 allocation in the methionine cycle for METH activity. These results point out the importance of this cofactor in haptophytes cellular processes and represent a first attempt to understand the response of these ecologically important communities in vitamin B 12 -limited environments. Searches for methionine synthase isoforms in other haptophytes were firstly realized by BlastX of T. lutea METH on NCBI database, allowing to retrieve METH from Chrysochromulina sp. CCMP 291, Thalassiosira pseudonana CCMP 1335 and Phaeodactylum tricornutum CCAP 1055/1. The iMicrobe database was then queried, yielding sixteen haptophyte transcriptomes samples corresponding to 9 haptophyte families (see Table 1 in Supplementary Material for sequence references). TblastN of METE from C. reinhardtii (XP_001702934.1, NCBI) and P. tricornutum (B7G1X4, Uniprot); and METH from T. lutea were realized on the transcriptomes. Transcripts were translated into proteins using NCBI ORFfinder. Protein sequences were then aligned with METH from T. lutea and T. pseudonana and METE of P. tricornutum. Protein sequences were also verified by sequences alignments and conserved domains analyses. Identity and similarity with T. lutea sequences were estimated with LALIGN tool. Sequences alignment was conducted with MUSCLE (full mode) on the 21 METH sequences with T. pseudonana and P. tricornutum taken as outgroup. On total, 1206 positions were conserved on the 1508 initial (80%). Curation step was done with Gblocks tool, allowing gap positions within the final blocks and phylogenetic tree was realized with PhyML (100 bootstraps). Sequences homologous to genomic and proteic METE from P. tricornutum and C. reinhardtii were searched in the Marine Atlas of Tara Ocean Unigenes (MATOU) for eukaryotic data 32 using BlastP and TBlastN (expected threshold 1E-1).

Methods
Algal strain and purification. To limit bacterial contamination, a purification step was carried out on Tisochrysis lutea CCAP 927/14 strain with an antibiotic treatment mix prepared following the method described by Cho et al. 45 . For the following experiments, an inoculum from purified T. lutea culture was transferred three times every 10 days. Ten percent volume were transfered each time in new Erlenmeyer flasks containing Conway medium enriched sterile seawater 46 with B 12 omitted in order for the cells to progressively run out their B 12 quota. Axenicity was verified in all the experiments by epifluorescence microscopy and cytometric analysis using SYBR ™ Green staining (Lonza, USA) and by plating on Marine Agar (BD Difco ™ , Becton Dickinson Company, USA). Petri dishes were then incubated 3 days at 25 °C before further observation. When no bacteria or colony were observed, strains were considered axenic.

Microalgal cultures.
Microtiter plate growth assay. A growth assay was carried out to assess T. lutea's cobalamin requirement and to investigate whether the microalgae can be grown with methionine, end product of METH activity. Two milliliters inoculum from the last cobalamin-limited batch were dispatched in test tubes (final concentration 1 10 6 cells mL −1 ) and enriched with Conway medium either containing 40 ng L −1 B 12 , or cobalamin free or cobalamin free enriched with 0.5 mg L −1 L-methionine (HPLC grade Sigma; >99% purity). Six replicates were inoculated for each condition in a microtiter plate that was incubated at 26 ± 1 °C and 90 μmol m −2 s −1 . O.D.680 was monitored by spectrophotometry (Quant, BIO-TEK Instruments inc, USA).
Batch experiment. To identify modifications in T. lutea's molecular physiology during vitamin B 12 consumption, a cobalamin-limited batch experiment was first performed. Three 1-liter autoclaved glass bottles were inoculated at 2.5 10 6 cells mL −1 and enriched with Conway medium with 40 ng L −1 B 12 . Cultures were homogenized by filtered air bubbling (Midisart 0.2 m, Sartorius) and were placed at 27 ± 1 °C with a continuous irradiance of 180 μmol m −2 s −1 photons. Cellular concentration was followed by counting Lugol stained cells with Malassez haemocytometer. Samples for quantitative analysis of B 12 and qPCR were taken at days 2 (early exponential phase) and 20 (stationary phase).
Chemostat experiment. Two inoculi were acclimatized at 27 ± 1 °C under a continuous irradiance of 180 μmol m −2 s −1 photons. After 10 days, they were divided into four autoclaved glass bottles filled with 4.5 L sterile seawater enriched with modified Conway medium, with a final concentration of either 40 ng L −1 B 12 or 25 mg L −1 NaNO 3 to ensure limitation in B 12 or nitrogen (N) respectively. Bottles were set up in chemostat supplied with a continuous input of the media described above. The experiment was carried out in duplicate for each condition at 27 ± 1 °C with a continuous irradiance of 400 μmol m −2 s −1 photons and pH maintained at 8.2 by CO 2 enrichment. Cultures were homogenized properly with a constant input of filtered air and a magnetic stirrer. Dilution rate (D) was adjusted at 0.5 d −1 and monitored daily by weighing system output. Populations were monitored by particulate carbon and nitrogen measurement and cell counting in Malassez haemocytometer. To confirm adequate nutrient limitation and to study physiological modifications after nutrient adding, discreet nutrient input was done at day 25 either with 25 g L −1 NaNO 3 or 40 ng L −1 B 12 for the N-limited and B 12 -limited chemostats respectively.
Biochemical analyses. Particulate carbon and nitrogen. Particulate organic nitrogen and carbon were measured by filtering 20 10 6 cells on 25 mm precombusted GF/F microfibers filters (0.7 m, Whatman, UK). Filters were then dried at 65 °C for at least 12 hours. Particulate organic nitrogen and carbon were analyzed with a CN elemental analyzer (Flash 2000, Thermo Fisher Scientific, Waltham, USA). B 12 measurements. Intracellular B 12 quantification was assessed with an ELISA test kit (Immunolab, Germany) with a sensitivity of 0.3 ng mL −1 . Zhu et al. showed that neither salinity nor dissolved organic matter do interfere with test quality 47 . This procedure allowed to measure the different chemical forms of vitamin B 12 (cyanocobalamin, methylcobalamin, adenosylcobalamin and hydroxycobalamin) with a cross-reactivity of 98-100% (2019) 9:8417 | https://doi.org/10.1038/s41598-019-44797-w www.nature.com/scientificreports www.nature.com/scientificreports/ among the chemical variants 47 . Cell pellets (80-150 10 6 cells) were resuspended in 100 μL PBS buffer (provided in the kit) and extraction was undertaken by boiling 15 minutes at 99 °C as previously described for microalgae 48 . Supernatant was collected after centrifugation (16 000 g, 5 minutes, 4 °C). Extracts were assayed following the method given in the kit, which provided cyanocobalamin solutions as standards. Absorbences at 450 and 620 nm (three technical replicates) were measured with a spectrophotometer (μQuant, BIO-TEK Instruments inc, USA). Standards and samples absorbency was defined as follows: O.D.450-O.D.620. Cobalamin concentration of samples was calculated using the calibration curve equation.
RNA extraction and Rt-qpCR. Cell lysis was obtained by adding 1 ml Trizol (Life Technologies) and 200 μL chloroform to cell pellets of 300 10 6 cells. Samples were then purified with RNeasy ™ kit (Qiagen) following the provided protocol. RNA purity and concentration were verified using a spectrophotometer (Infinite 200 PRO) at 260 and 280 nm. Diluted samples of 250 ng μL −1 were treated with DNase (Promega) 1 hour at 37 °C. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the provided protocol. Primer efficiency was quantified following protocol of Schmittgen et al. 49 (see Table 2 on Supplementary Information for primers sequences). Primer specificity was estimated with a denaturation cycle at PCR end. PowerUp ™ SYBR ™ Green mix (Applied Biosystems) was used for RT-qPCR. Thermocycler (Mx3000P, Agilent) parameters were set as follows: 1 cycle of 15 minutes at 95 °C, 40 cycles of 30 seconds at 95 °C and 30 seconds at 60 °C. Six genes coding for 18S, actin, EF1, GAPDH, tubulin and ubiquitin were tested as housekeeping genes. As GAPDH exhibited low cycle threshold (Ct) variations with the same order of magnitude than target genes it has been selected as reference gene (see Fig. 1 of Supplementary Information). Gene expression was calculated by raising negative cycle threshold values of each pair of primers and dividing it by mean expression of reference gene. Raw data of genes expression is available in Supplementary Datasets 1 and 2 for the batch and chemostat experiments respectively.