LIMNOLOGY and Limnol. Oceanogr. 00, 2015, 1–26 OCEANOGRAPHY VC 2015 Association for the Sciences of Limnology and Oceanography doi: 10.1002/lno.10009

Vitamin B1 of marine picoeukaryotic : Strain-specific differences and a new role for bacteria in vitamin cycling

R. W. Paerl,1* E. M. Bertrand,2,3 A. E. Allen,2,3 B. Palenik,1 F. Azam1 1Scripps Institution of Oceanography, Marine Biology Research Division, University of California at San Diego, La Jolla, California 2Scripps Institution of Oceanography, Integrative Oceanography Division, University of California at San Diego, La Jolla, California 3J. Craig Venter Institute, Microbial and Environmental Genomics Department, La Jolla, California Abstract We confirmed multiple picoeukaryotic algae, Ostreococcus, Micromonas, and Pelagomonas spp., as thiamine (vitamin B1) auxotrophs in laboratory experiments with axenic cultures. Examined strains have half saturation 21 growth constants (Ks) for B1 between 1.26 and 6.22 pmol B1 L , which is higher than reported seawater con- centrations. Minimum B1 cell quotas for Ostreococcus and Micromonas spp. are high (2.20 3 1028–4.46 3 1028 pmol B1 cell21) relative to other B1 auxotrophic phytoplankton, potentially making them B1 rich prey for zooplankton and significant B1 reservoirs in oligotrophic marine habitats. Ostreococcus and Micromonas genomes are nonuniformly missing portions of the B1 biosynthesis pathway. Given their gene repertoires, Ostreococcus lucimarinus CCE9901 and Ostreococcus tauri OTH95 are expected to salvage B1 from externally pro- vided 4-methyl-5-thiazoleethanol (HET) and 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). However, in culture, neither could use HET plus HMP instead of B1, highlighting current limitations of genome-based prediction of B1 salvaging by picoeukaryotic algae. HMP and phosphorylated B1 use varied amongst tested strains and notably all Prasinophytes tested could not use HMP. B1-limited O. lucimarinus CCE9901 could not grow on added thiamine diphosphate (TDP), a phosophorylated B1 form. However, in co-culture with Pseu- doalteromonas sp. TW7, a bacterium known to exhibit phosphatase activity, O. lucimarinus CCE9901 exhibited increased growth following TDP additions. This demonstrates that bacteria influence vitamin B1 availability beyond de novo synthesis and consumption; they can also serve as conduits that chemically alter, but not completely degrade or retain B1 analogs (e.g., TDP), and make them accessible to a broader range of microbes.

Vitamin B1 (called B1 herein), also known as thiamine, is or below (Sanudo-Wilhelmy~ et al. 2012). Despite the scarcity required by all cells due to its role as a cofactor in the form of B1 in the upper ocean, especially the oligotrophic regions of thiamine pyrophosphate (also called thiamine diphos- where published B1 concentrations are < 0.05 pmol L21 phate [TDP]) for enzymes critical to carbon and amino acid (Barada et al. 2013), several marine plankton (eukaryotes and metabolism (Jurgenson et al. 2009). Additionally, certain prokaryotes) persist as B1 auxotrophs, unable to synthesize riboswitches, untranslated sections of messenger ribonucleic their own vitamin B1. Surveys of the occurrence of vitamin acid (RNA) capable of regulating gene expression, are acti- auxotrophy within phytoplankton noted that a large per- vated by binding TDP, B1, and moieties (parts of the thia- centage ( 80%) of prymnesiophytes were B1 auxotrophs mine molecule, e.g., thiazole and pyrimidine; Moulin et al. (Provasoli and Carlucci 1974; Croft et al. 2006). In contrast, 2013). Notably, this is one of the few documented ribos- a majority of diatoms produce B1 (Provasoli and Carlucci witch systems in eukaryotes, and appears to be important in 1974), and both B1 producing and auxotrophic bacteria are regulating thiamine metabolism in marine microbes (McRose present in the ocean (MacLeod et al. 1954; Burkholder and et al. 2014). Lewis 1968). In the euphotic upper ocean, thiamine concentrations Many new algal strains have been isolated as the pioneer- appear to be low relative to macronutrients, e.g., pmol L21 ing surveys of auxotrophy (Provasoli and Carlucci 1974), including multiple picoeukaryotic algal strains (less than *Correspondence: [email protected] three micrometer in cell diameter). There is increasing

1 Paerl et al. Marine picoeukaryote thiamine physiology evidence, based largely on genome analysis, that several key cell yield relation by Droop (2007)] are available for a small picoeukaryotic algae are B1 auxotrophs (Palenik et al. 2007; set of algae, specifically Pavlova lutheri (Carlucci and Silberna- Worden et al. 2009; Bertrand and Allen 2012; McRose et al. gel 1966), A. anophagefferens and larger dinoflagellate strains 2014) but also based on a small set of experiments with (Tang et al. 2010). No such data have been reported for axenic cultures (e.g., Aureococcus anophagefferens CCMP1984, Ostreococcus, Micromonas,orPelagomonas spp. B1 kinetic Micromonas spp.; Tang et al. 2010; Bertrand and Allen 2012; growth constant and B1 cell quota data for auxotrophic McRose et al. 2014). Picoeukaryotic phytoplankton are picoeukaryotic phytoplankton would provide a better sense important contributors to marine and of; (1) whether or not they have the potential to be growth- phytoplankton biomass (Li 1994; Worden et al. 2004). Also limited by B1 availability in the euphotic ocean, (2) any some B1 auxotrophic picoeukaryotic and larger eukaryotic dependence on intimate interactions with other microbes algae are harmful algal bloom (HAB)-associated species, e.g., for B1, and (3) their influence on B1 cycling based on their A. anophagefferens and Phaeocystis globosa (Peperzak 2000; B1 demand (pmol B1 cell21). Tang et al. 2010). Thus, vitamin B1 availability may influ- We conducted laboratory-based experiments with putative ence primary production rates, marine algal community vitamin B1 auxotrophic and cosmopolitan picoeukaryotic structure, and water quality by influencing the growth and phytoplankton, specifically, Ostreococcus lucimarinus CCE9901, activity of picoeukaryotic phytoplankton. Ostreococcus sp. CCE1301, Micromonas pusilla CCMP487, and Several studies found positive correlations between pri- Pelagomonas calceolata CCMP1756. Using axenic cultures, we mary productivity rates and additions of B1 or increases in confirmed these strains as B1 auxotrophs, determined their natural B1 seawater concentrations. B1 additions to subarctic vitamin B1 half (Ks)andmaximum(Kmax) saturation growth N. Pacific seawater stimulated bulk primary productivity constants, their ability to utilize B1 chemical analogs (moi- (Natarajan 1970), a significant positive correlation between eties and phosphorylated forms), and their respective mini- net primary productivity and B1 concentrations was found mal B1 cell quotas. Lastly, we examined if the presence of a at one location off La Jolla, California (Carlucci 1970) and simple microbial population (Gammaproteobacterium Pseudo- most recently, a positive correlation between B1 seawater monas sp. TW7) would enable a B1 auxotrophic algae (O. luci- concentrations and primary production (as well as nitrogen marinus CCE9901) to utilize a phosphorylated B1 analog it fixation) was reported for the upper water column of the cannot use in isolation. western tropical Atlantic (Barada et al. 2013). The degree to which vitamin B1 availability affects growth of B1 auxotro- phic algae will in part depend on multiple aspects of algal Methods B1-related physiology, e.g., utilizable forms, uptake affinity, M. pusilla CCMP487, Ostreococcus spp., and P. lutheri cul- and per cell requirements. tures were grown on modified F/2 medium (Guillard 1975) Early vitamin research showed that B1 moieties or chemi- plus sodium selenite and sodium citrate (as formulated by cal analogs (compounds sharing structural similarity to thia- Palenik unpubl.). The seawater base was twice 0.22 lm fil- mine) satisfied the B1 requirement of auxotrophic marine tered, microwaved and treated with activated charcoal to algae, specifically flagellates, and benthic diatoms isolated remove organics as previously described (Carlucci and Silber- from vitamin replete sediment and intertidal habitats (Prova- nagel 1966). This seawater base was then supplemented with soli and Carlucci 1974). Additionally, a representative strain autoclaved F/2 nitrate and phosphate solutions as well as 0.2 of the ubiquitous marine bacterial family Pelagibacteraceae lm filter-sterilized stocks of the trace metal mix, sodium sel- (also known as the SAR11 clade) exhibits an obligate require- enite (87 nmol L21 final), and sodium citrate (50 lmol L21 ment for 4-amino-5-hydroxymethyl-2-methylpyrimidine final). P. calceolata CCMP1756 cultures were grown in Aquil (HMP) that cannot be satisfied by B1 (Carini et al 2014), and synthetic seawater supplemented with F/2 concentrations of the coccolithophore Emiliania huxleyi is able to use both B1 filter-sterilized nitrate, phosphorus, and trace metal mix. For and HMP to satisfy its B1 requirements (McRose et al. 2014), all cultures, filter-sterilized vitamin B12 (cobalamin) and B7 emphasizing the importance of understanding B1 moiety (biotin; 184 pmol L21 and 1 nmol L21 final respective con- utilization patterns in marine microbes. In contrast, two centrations) solutions were added individually so that B1 strains of Micromonas only use B1 and not its precursors to could be added later at varying concentrations (medium satisfy thiamine requirements (McRose et al. 2014); it is cur- without B1 is noted as F/2 2 B1). Before use, the growth rently unknown whether or not other B1 auxotrophic medium lacking B1 was tested for bacterial contaminants by picoeukaryotic phytoplankton like Ostreococcus and Pelago- adding one to two milliliter of ZoBell bacterial growth monas spp., representatives of picoeukaryotic algae that are medium (ZoBell 1941). Micromonas, Ostreococcus,andP. cosmopolitan in the pelagic and neritic euphotic ocean (Li lutheri cultures were grown in five milliliter polystyrene fal- 1994; Worden et al. 2004) can utilize B1 moieties or chemi- con tubes (Becton Dickinson) at 20C, without shaking cal analogs to meet their B1 requirements. Vitamin B1 and under 160 lmol quanta m22 s21 of constant light kinetic growth constants and minimum cell quota [called from T5 28 W fluorescent white light bulbs, unless noted

2 Paerl et al. Marine picoeukaryote thiamine physiology otherwise. CCE9901 cultures grown under a 14 h : 10 h were maintained in exponential growth on minimal B1 light : dark regime were exposed to 72 lmol quanta m22 medium for at least five transfers then transferred 1 : 10 to F/ s21 of the same white light. Cultures were vortexed and 2 2 B1 medium until growth declined relative to a parallel inverted at least once a day. Pelagomonas cultures were positive control culture amended with 1 nmol B1 L21. The grown in 50 mL acid washed glass tubes, vortexed daily, culture exhibiting early B1 growth limitation was transferred and exposed to a 14 h : 10 h light : dark regime with day- into a series of cultures containing a range of B1 concentra- time light levels of 70 lmol quanta m22 s21. tions (8–10 total cultures, from 0 to 1000 pmol B1 L21 final Ostreococcus tauri OTH95, M. pusilla CCMP487, and P. concentration). Instantaneous growth (l) was determined lutheri CCMP1325 were obtained from the National Center using the formula l 5 ln (Bf : Bi)/T where Bf 5 final biomass, for Marine Algae and Microbiota (NCMA). Axenic cultures of Bi 5 initial biomass, and T 5 time. Analysis of maximum P. lutheri CCMP1325 and P. calceolata CCMP1756 were instantaneous growth rate vs. B1 concentration was done obtained from NCMA. O. lucimarinus CCE9901 was originally using the Michealis–Menton analysis option within the soft- isolated from waters off La Jolla, California (Price et al. 1989; ware package Prism (GraphPad Software). Maximum growth Kirchman 2000; Worden et al. 2004). Ostreococcus sp. constants were estimated based on the maximum growth CCE1301 was isolated from 100 m waters off La Jolla, Califor- rate calculated by the Michealis–Menton analysis. The mini- nia and is closer in relation to Ostreococcus sp. CCE9901 and mal B1 cell quota was determined using the difference RCC356 than O. tauri OTH95 based on 18S ribosomal ribonu- between maximum yields of mid-range B1 amendments cleic acid gene phylogeny (Palenik, data not shown). Axenic (0.5–100 pmol L21), which reached stationary phase well cultures of M. pusilla CCMP487, O. lucimarinus CCE9901, and before higher B1 additions. Ostreococcus sp. CCE1301 were obtained using an antibiotic For cell biovolume estimates, first M. pusilla CCMP487, O. cocktail of streptomycin, chloramphenicol, and cefotaxime at lucimarinus CCE9901, Ostreococcus sp. CCE1301, and P. lutheri respective final concentrations of 10 lgmL21,1lgmL21, CCMP1325 cells taken from exponentially growing cultures and 80 lgmL21 or only cefotaxime treatments at a final con- were fixed with 2% final formaldehyde, and filtered by hand centration of 80 lgmL21. Antibiotic treatments were discon- pump on 0.2 lm pore sized polycarbonate filters. Biovolume tinued or deemed unnecessary after confirming cultures as was subsequentially determined by measuring cell dimen- bacteria free by epifluorescence microscopy using 40-6- sions with a Nikon C1 microscope, equipped with ultraviolet diamindino-2-phenylindole staining, and by an absence of light source and NIS Elements software (Nikon). Volume (V) bacterial growth in flasks containing two milliliter ZoBell bac- of an ellipsoid was used to calculate biovolume, V 5 (4p/ terial liquid medium and one milliliter of the tested algal cul- 3)abc, where a, b, and c represent the length of three elliptic ture. Axenic cultures were always transferred to fresh radii. The length and width of 13–50 random cells was meas- medium at a ratio of 1 : 10, algal culture to fresh medium. ured by epifluorescence microscopy and used to calculate a Vitamins and chemical analogs (moieties or phosphoryl- and b (half of the length or width). Values for a or b were ated forms) used in experiments were obtained from Sigma, used for c in the formula to obtain maximum and minimum Spectrum, Alfa Aesar, Tokyo Chemical Industry, MP Biomed- values calculated for biovolume and B1 quota per cell biovo- icals or Calbiochem at high performance liquid chromatog- lume. P. calceolata CCMP1756 biovolumes were estimated by raphy (HPLC) grade, > 98% pure, except for HMP which was measuring the radii of live, exponentially growing cells and > 90% pure based on nuclear magnetic resonance and was assuming and oblate spheroid shape, where (4p/3)a2c 5 bio- purchased from VitasMLab. Working stocks of chemicals volume and a is the equatorial (longer) cell radius and c is were dissolved in autoclaved Milli-Q water except for chlor- the polar (short) cell radius. Both a and c were obtained amphenicol, which was dissolved entirely in ethanol. from 25 random cells using overlaid epifluorescence and Algal biomass in cultures was monitored based on in vivo light micrographs obtained on a Leica DU6000 CS confocal Chlorophyll a (Chl a) fluorescence measured using an Aquafluor microscope using the Leica Applications Suite. portable fluorometer (Model 8000-010), or bench top fluorome- Co-cultures of O. lucimarinus CCE9901 and TW7 (a Gam- ter AU-10, (Turner Designs). In vivo Chl a fluorescence and algal maproteobacterium; Bidle and Azam 2001) were maintained cell abundances were linearly correlated for O. lucimarinus and transferred using aseptic technique in a laminar flow CCE9901, Ostreococcus sp. CCE1301, M. pusilla CCMP487, and P. hood. An initial co-culture was started by transferring O. luci- calceolata CCMP1756 over the measured fluorescence range marinus CCE9901 cells to F/2 2 B1 medium (anticipating B1 (data not shown). Prior to limitation experiments, all algal cul- limitation). Simultaneously, TW7 cultures were grown in tures were maintained in exponential phase on F/2 2 B1 ZoBell medium to late exponential phase then centrifuged 21 21 medium as described above plus 1nmolB1L . All experi- and washed two times in F/2 1 NH4 (50 lmol L final) 2 ments were conducted in triplicate, unless noted otherwise. B1 medium and 3 3 105 TW7 cells mL21 were added to an Growth kinetic constants and minimal B1 cell quota initial B1-limited O. lucimarinus CCE9901 culture. All co-cul- (pmol B1 per cell) were determined for each alga from two tures were incubated under continuous light, under the or three separate gradient addition experiments. Cultures same conditions as described for axenic algal cultures. When

3 Paerl et al. Marine picoeukaryote thiamine physiology

known reference proteins (e.g., ThiM from Bacillus subtilis, Uniprot ID E8VEP7_BACST) against Ostreococcus genomes via the National Center for Biotechnology Information (NCBI) website. Positive hits (an alignment score > 20) were then verified using blastp searches against the UniProtKB protein database via the Uniprot website.

Results B1 auxotrophy, growth kinetics, and minimum cell quota of tested strains All axenic algal cultures exhibited growth limitation fol- lowing multiple transfers to F/2 2 B1 medium without vita- min B1 addition. In the growth kinetics experiments, Fig. 1. A vitamin B1 growth kinetics curve for O. lucimarinus CCE9901. cultures exhibited maximum growth rates ranging from 0 The data plotted are from three independent gradient addition experi- d21 to 1.13 d21 depending on the amendment of B1. Plots ments. Error bars represent one standard deviation, and where not visi- of maximum growth rate vs. vitamin B1 amendment yielded ble, they fall within the bounds of the symbol. A hyperbolic Michaelis– 2 a typical Michealis–Menten hyperbolic curve for each algal Menten model is plotted (r 5 0.98, degrees freedom (df) 5 28). The Ks lies between 1.01 pmol B1 L21 and 1.52 pmol B1 L21 (a best fit value 5 strain (Fig. 1; data shown only for O. lucimarinus CCE9901). 1.26 pmol B1 L21) at a 95% confidence interval (CI). Data points for A Michealis–Menten model strongly represented changes in 21 21 500 pmol B1 L and 1000 pmol B1 L are not plotted but were growth rate vs. B1 amendment concentrations (e.g., r2 equivalent to 100 pmol L21 (l 5 1.0 d21). 0.84). The half-saturation growth constants (Ks) were 1.26– 6.22 pmol B1 L21 for all strains tested. The maximum satura- 21 O. lucimarinus CCE9901 growth in co-cultures reached expo- tion growth constants (Kmax) were 150 pmol B1 L or less 2 nential to early stationary phase based on Chl a fluores- (Table 1), with 1 nmol B1 L 1 being the maximum addition cence, the single co-cultures were transferred at a ratio of (data not shown). 1 : 10 (e.g., 200 lL of culture to 1800 lL of medium) to new The minimum vitamin B1 cell quota for strains ranged 21 28 21 28 medium (either F/2 1 NH4 2 B1 or F/2 1 NH4 1 1 nmol L from 2.20 3 10 pmol B1 cell 6 1.89 3 10 pmol B1 2 2 2 2 TDP where appropriate). In vivo Chl a fluorescence of all co- cell 1 to 4.46 3 10 8 pmol B1 cell 1 6 1.09 3 10 8 pmol 2 cultures over seven transfers was measured daily as described B1 cell 1. O. lucimarinus CCE9901 cultures grown under a 14 above using an Aquafluor portable fluorometer. After six h : 10 h light : dark regime exhibited a comparable quota to transfers of the B1-limited co-culture, aliquots (50 lL) of the that of cultures exposed to continuous higher irradiance ( 2 2 2 2 co-culture were transferred and spread on multiple ZoBell 160 lmol quanta m 2 s 1), 2.20 3 10 8 pmol B1 cell 1 6 2 2 2 2 agar plates (1% agarose). Lawns of TW7 were visible on all 1.89 3 10 8 pmol B1 cell 1 vs. 2.72 3 10 8 pmol B1 cell 1 2 2 ZoBell plates after two to three days, while no colonies 6 1.70 3 10 8 pmol B1 cell 1 (Table 1). appeared on a negative (medium only added) control ZoBell plate. Utilization of vitamin B1 chemical analogs Hidden Markov Model (HMM) search (Eddy 1998) was Based on complete genome sequences, O. lucimarinus used to identify TDP-dependent enzymes in peptide sequence CCE9901 and O. tauri OTH95 each possess partial vitamin B1 files. HMM models from each of the eight TDP-dependent biosynthesis pathways (Bertrand and Allen 2012; McRose et superfamilies were downloaded from the Thiamine diphos- al. 2014). Both lack ThiC or Thi5, proteins required for de phate dependent Enzyme Engineering Database (TEED; novo HMP production, but possess ThiM and Th1 (Fig. 2), http://www.teed.uni-stuttgart.de) and concatenated into one which are involved in thiazole and pyrimidine modification HMM file for searching against peptide data. Peptide data as well as condensation of moieties to yield thiamine mono- were downloaded from the Joint Genome Institute (JGI), the phosphate (TMP; Rapala-Kozik et al. 2007; Jurgenson et al. Marine Microbial Eukaryote Transcriptome Sequencing Pro- 2009). As a result, we hypothesized that both could generate ject (MMETSP) website (http://camera.calit2.net/mmetsp/)or TMP and TDP from thiazole and pyrimidine precursors 4- Genoscope (https://bioinformatics.psb.ugent.be/gdb/bathy- methyl-5-thiazoleethanol (HET) and HMP (Fig. 2). We tested coccus/). Protein sequences with a domain score > 20 were in culture experiments if HMP and HET (and each individual considered positive hits and then dereplicated based on pro- compound) could satisfy the B1 requirement of O. lucimari- tein sequence identifier (ID). Replicates with the highest nus CCE9901 and O. tauri OTH95, as well as other B1 auxo- domain score were used in the analysis. trophic algae whose genome sequences are not currently B1 biosynthesis proteins in O. lucimarinus CCE9901 and available. Single or combined additions of HET and HMP, O. tauri OTH95 were identified using blastp and searching did not support the growth of Ostreococcus spp. or M. pusilla

4 ar tal. et Paerl Table 1. Minimum vitamin B1 cell quotas, half saturation growth constants (Ks), maximum saturation growth constants (Kmax ), and maximum growth rates (lmax ) for several B1 auxotrophic eukaryotic phytoplankton. All numeric data pertain to vitamin B1 additions except for P. calceolata CCMP1756 data noted with an HMP superscript, in which case, HMP was the added substrate instead of B1 and it is assumed that each HMP molecule is completely converted to a B1 molecule within the cell. OTH95 data are noted with two asterisks, as cultures were found postexperiment to contain bacterial contaminants. P. lutheri bio- volume was determined in this study by microscopy (8.27 3 1028 lL minimum, 7.12 3 1028 lL maximum, see Methods). Data from this study are in bold. Additional P. lutheri data are from Carlucci and Silbernagel (1966) and all other data are from Tang et al. (2010). For quota estimates the average value plus minus one standard deviation is presented. L : D 5 data for O. lucimarinus CCE9901 cultures grown under a light : dark regime. Util. 5 utilization of a provided B1 substrate indicated by growth (N 5 no, Y 5 yes). B1 1 P 5 phosphorylated B1 compounds thiamine monophosphate and thiamine diphosphate. HMP 5 4-amino-5-hydroxymethyl-2-methylpyrimidine. HET 5 hydroxyethylthiazole. nd 5 not determined.

HMP B1 per Class, B1 1 P and/or biovolume B1 per cell Ks Kmax l max Organism Order Util.? HET Util.? (pmol B1 lL21 ) (pmol B1 cell21 ) (pmol L21 ) (pmol L21 ) (d21 ) O. lucimarinus Prasinophyceae, N N 67.1657.6 2.202 08 61.892 08 1.2660.13 ~50 1.0660.02 CCE9901 Mamiellales (n59) (n59) (best fit) (best fit) O. lucimarinus Prasinophyceae, NN nd 2.722 08 61.702 08 nd nd nd CCE9901 (L : D) Mamiellales (n512) O. tauri OTH95 Prasinophyceae, N** N** nd nd nd nd nd Mamiellales Ostreococcus sp. Prasinophyceae, Y N 89.7616.5 3.58310208 6 6.2260.97 ~150 1.0760.03 CCE1301. Mamiellales (n512) 6.58310209 (n512) (best fit) (best fit) M. pusilla CCMP487 Prasinophyceae, Y N 72.82616.5 2.55310208 6 4.88 6 0.65 ~150 0.7760.02 5 3 209 5 5 Mamiellales (n 14) 6.16 10 (n 14) (best fit) (best fit) P. calceolata Pelagophyceae, Y HMP 6.5261.56 4.463 2.3860.57 ~100 0.6760.03 CCMP1756 Pelagomonadales (n512) 10208 61.093 (best fit) (best fit) 102 08 (n512) P. calceolata Pelagophyceae, Y HMP 5.2461.07 3.5931028 6 3.4660.85 ~100HMP 0.7260.03 CCMP1756HMP Pelagomonadales (n512)HMP 7.30310209 (best fit)HMP (best fit)HMP (n512)HMP P. lutheri Pavlovophyceae, Y HMP 10.04–11.65 8.30310207 471 nd nd CCMP1325 Pavlovales 209

A. anophagefferens Pelagophyceae, nd nd 1.16 6.52310 5.9461.36 nd nd physiology thiamine picoeukaryote Marine CCMP1984 Pelagomonadales Prorocentrum Dinophyceae, nd nd 0.545 6.27310207 86.363.76 nd nd minimum Prorocentrales CCMP696 Scrippsiella Dinophyceae, nd nd 0.285 2.41310206 131630.2 nd nd trochoidea MS1 Peridiniales Gyrodinium aureolum KA6 Dinophyceae, nd nd 5.65 1.94310205 96.964.01 nd nd Dinotrichales Rhodomonas salina Cryptophyceae, nd nd 3.43 3.02310207 184681.5 nd nd CCMP1319 Pyrenomonadales Paerl et al. Marine picoeukaryote thiamine physiology

Fig. 2. A schematic of core vitamin B1 biosynthesis and scavenging pathways in O. lucimarinus CCE9901 and O. tauri OTH95 based on their complete genome sequences. In bold text are the names of proteins present in both strains. Chemical transformations attributed to proteins identified in Ostreo- coccus genomes are represented by bold arrows. The Th1 protein is equivalent to the ThiD 1 ThiE complexes listed in Bertrand and Allen (2012). Pro- teins absent from the two Ostreococcus genomes are presented here in standard font. In parenthesis following protein names are protein IDs; in the case of absent proteins, IDs of proteins found in Cyanoidioschyzon merolae or Saccharomyces cerevisiae (ID names start with “C” or “Y,” respectively) are given. Pathways associated with the missing proteins are capped with a line (instead of an arrow) and their known substrates are circled. Chemical ana- logs provided in culture experiments are boxed, with B1 being double-boxed as it was utilizable by all strains tested in this study and was the only B1- related compound utilizable by O. lucimarinus CCE9901 and O. tauri OTH95. Phosphatases (P-tase) and kinases involved in the phosophorylation and dephosphorylation of B1 (lower right corner of diagram) are not known and hence noted with question marks; they are assumed to be present in the cell as these organisms grow on provided vitamin B1 and have known TDP-dependent enzymes (Table 2). ˆ 5 ThiG is chloroplast encoded in other algae (Bertrand and Allen 2012) and plastid genome sequence information for O. lucimarinus CCE9901 is currently unavailable; SW 5 seawater; HMP 5 4-amino-5-hydroxymethyl-2-methylpyrimidine; HMP-P 5 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate; HMP-PP 5 4-amino-5- hydroxymethyl-2-methylpyrimidine diphosphate; HET 5 hydroxyethylthiazole; HET-P 5 hydroxyethylthiazole phosphate; AIR 5 50-phosphoribosyl-5- aminoimidazole; DXP 5 1-deoxy-D-xylulose 5-phosphate; NAD 5 nicotinamide adenine dinucleotide; TPK 5 thiamine pyrophosphokinase.

CCMP487; HMP alone supported the growth of P. calceolata phate amendment, containing only phosphorous from the CCMP1756 and the larger alga P. lutheri CCMP1325 (Fig. 3). seawater base, and TDP as a B1 source. Phosphate concentra- Phosphorylated B1 is generated within cells and presum- tions from surface waters off the end of the Scripps Pier, La ably becomes available in the water column due to cell Jolla, California, are typically < 0.8 lmol L21 (Tai and Pale- mortality, excretion, or diffusion. The majority of strains nik 2009). Reduced phosphate concentrations did not enable examined (all except O. lucimarinus CCE9901 and O. tauri O. lucimarinus CCE9901 to grow on TDP (Fig. 5A). OTH95) grew on TMP or TDP as sole B1 sources (Fig. 4). The

F/2 growth medium is phosphate replete ( 36 lmol PO4 Growth of CCE9901 in co-cultures with Pseudomonas sp. L21), which we hypothesized could inhibit phosphatase TW7 following TDP amendment activity, and furthermore, dephosphorylation of TMP or TDP In co-culture with TW7, B1-limited O. lucimarinus to B1. Comparable levels of phosphate cause inhibition of CCE9901 grew in response to the addition of 1 nmol TDP alkaline phosphatase activity in certain algae (Dyhrman and L21 and ultimately reached 103 its maximum biomass (in Ruttenberg 2006). To test for this inhibition, O. lucimarinus vivo Chl a fluorescence) observed in un-amended control co- CCE9901 was grown on F/2 medium without typical phos- cultures (Fig. 5B). O. lucimarinus CCE9901 exhibited net

6 Paerl et al. Marine picoeukaryote thiamine physiology

Fig. 3. Maximum yields of B1-limited algal cultures grown on B1 or B1 moieties HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine) and HET (4- methyl-5-thiazole ethanol). (A) Growth of cultures on individual or combined HMP and HET additions. (B) Growth of cultures that responded to HMP addition alone (as noted on the x-axis). P. lutheri cultures in (B) were exposed to 72 lmol quanta m22 s21 of white light (hence the larger y-axis scal- ing), all others were exposed to 160 lmol quanta m22 s21. B1 was added at a final concentration of 1 nmol L21. HMP was added at a final concentra- tion of 1 nmol L21 and HET at a final concentration of 2 nmol L21. Means of triplicate cultures are presented; error bars represent one standard deviation from the mean and where not visible, fall within the bounds of the line. FL (AU) 5 in vivo Chl a fluorescence in fluorometer-specific arbitrary units. positive growth in B1-limited co-cultures with TW7 over It is unknown what proteins are critical to high affinity multiple transfers (to B1-deplete medium), but maximum O. transport of B1 in our tested picoalgae, nor any other algae lucimarinus CCE9901 biomass remained low relative to co- to the best of our knowledge. Notably, A. anophagefferens 21 cultures amended with TDP (Fig. 5B). Axenic O. lucimarinus CCMP1984 also has a relatively low Ks value, 6 pmol L , CCE9901 cultures transferred successively to B1-deplete r2 5 0.71; Tang et al. 2010), suggesting other picoeukaryotic medium or medium supplemented with TDP showed no net algae beyond those tested in this study possess high affinity growth, and in vivo Chl a fluorescence became undetectable B1 uptake systems. A high affinity, adenosine triphosphate after one to two transfers (data not shown). (ATP)-driven thiamine transport system is a logical candidate for bringing B1 into the cell against the dramatic B1 concen- tration gradient present between seawater (< 0.05 pmol Discussion B1 L21 to 100 pmol B1 L21) and the inside of a picoeu- Physiological and ecological implications of karyotic phytoplankton cell (24.6–32.2 lmol B1 L21; based picoeukaryotic algae vitamin B1 Ks values on the minimum yield quota for CCE9901, Table 1). Possible Picoeukaryotic algal strains examined in this study must candidate proteins have been identified via sequence analysis possess high-affinity B1 uptake systems to explain their (Worden et al. 2009), but are yet to be characterized in the very low Ks (again, half-saturation growth constant) values laboratory. 21 (1.26–6.22 pmol B1 L ; Table 1). They also possess rela- The low Ks values of picoeukaryotic phytoplankton make 21 tively low Kmax values ( < 150 pmol B1 L ), indicating them highly competitive for B1 at low concentrations. How- they have singularly prioritized maintenance of a high- ever, their Ks values are high relative to published B1 con- affinity acquisition lifestyle, rather than also possessing a centrations from oligotrophic euphotic waters (Fig. 6), low-affinity system, which is often concomitant with a making it feasible that some picoeukaryotic phytoplankton higher maximum growth rate. Other larger bloom forming experience growth limitation due to B1 availability in such B1-auxotrophic algae presumably posses lower affinity sys- habitats. There is broad interest in characterizing nutrient tems based on their higher Ks values (Table 1; Tang et al. limitation of picophytoplankton populations (eukaryotes 2010). and cyanobacteria) in the pelagic ocean. Primarily, “classic”

7 Paerl et al. Marine picoeukaryote thiamine physiology

Fig. 4. Maximum yields of B1-limited algal cultures grown on B1 or TMP or TDP (all added at a final concentration of 1 nmol L21). Means of tripli- cate cultures are presented; error bars represent one standard deviation from the mean and where not visible, fall within the bounds of the line. All cultures were exposed to 160 lmol quanta m22 s21 white light.

Fig. 5. O. lucimarinus CCE9901 is unable to utilize TDP under reduced PO4 concentrations, but begins to utilize TDP in co-culture with Pseudomonas sp. TW7. (A) Maximum Chl a fluorescence of O. lucimarinus CCE9901 cultures grown on F/2 medium with varying additions of B1, TDP and phosphate concentrations. B1 or TDP was added at a final concentration of 1 nmol L21. Filter-sterilized (0.2 lm) sodium dihydrogen phosphate monohydrate stock 21 was added at a final concentration of 36 lmol L (noted as 1 PO4) or at varying concentrations (as noted). An O. lucimarinus CCE9901 culture in F/2 medium 2 PO4 2 B1 in exponential growth was used to inoculate the first grow-out experiment (larger plot). Maximum yield data for cultures trans- ferred a second time (1 : 10, culture to medium) to respective media (ensuring phosphate concentrations and B1 concentrations were sufficiently low in the starting F/2 2 PO4 2 B1 medium) are presented on the inset plot. All cultures exhibited positive net growth before the second transfer. Data pre- sented are from triplicate cultures; error bars are plotted (although not visible in most cases as they fall within the bounds of the line) and representone standard deviation from the mean. (B) O. lucimarinus CCE9901 growth increases in co-cultures with TW7 following the addition of TDP. TDP (1 nmol TDP L21) was added (where applicable) immediately following the transfer of co-cultures. Values for 2 B1 1 TW7 and 1 TDP 1 TW7 treatments are the means of maximum Chl a fluorescence for individual co-cultures across seven consecutive transfers (n 5 8). The data for 1 TDP 2 TW7 is from Fig. 4, where TDP was added to axenic O. lucimarinus CCE9901 cultures (n 5 3). Error bars represent one standard deviation from the mean.

8 Paerl et al. Marine picoeukaryote thiamine physiology macronutrients (N, P) and Fe have been considered as growth-limiting nutrients for picophytoplankton (Mann and Chisholm 2000; Davey et al. 2008). Representative picocya- nobacterial (Synechococcus and Prochlorococcus) genomes pos- sess complete B1 biosynthesis pathways (Sanudo-Wilhelmy~ et al. 2014) and are presumed not to depend on environ- mental B1 sources. In stark contrast, multiple picoeukaryotic phytoplankton depend on exogenous B1 (Table 1; Croft et al. 2006; Bertrand and Allen 2012; McRose et al. 2014). Picoeukaryotic phytoplankton cells are present throughout the oligotrophic pelagic ocean (Li 1994; Zubkov et al. 1998), where B1 concentrations are reported to be as low as < 0.05 21 pmol L (Barada et al. 2013). Considering our presented Ks data alongside published B1 concentrations from euphotic open-ocean waters, B1-associated growth limitation of pico- phytoplankton in B1-deplete euphotic waters deserves fur- ther investigation.

Ostreococcus and Micromonas spp. have relatively high minimum B1 quotas 21 The minimum B1 cell quotas (pmol B1 lL biovolume) Fig. 6. A comparison of published B1 concentrations from seawater for Ostreococcus and Micromonas spp. examined in this study (SW) alongside B1 Ks (half-saturation growth constant) values for tested were notably higher than that of P. calceolata CCMP1756, A. picoeukaryotic phytoplankton strains (abbreviated names are presented anophagefferens CCMP1984, and dinoflagellates (Table 1). for brevity). Ks data are the unfilled boxes (covering the 95% confidence interval). All B1 estimates were determined by Liquid Chromatography Why there is a large difference based on biovolume remains and Mass Spectrometry (LCMS) except those noted with the following unclear. Thiamine itself exhibits antioxidant activity in vitro symbols: a 5 estimated by bioassay; b 5 estimated by HPLC. Data for (Lukienko et al. 2000) and is linked to hydrogen peroxide La Jolla, California (CA) is from (Carlucci 1970; < 80 m depth). LIS 5 accumulation in plants (Ahn et al. 2007). Possibly thiamine Long Island Sound, New York (Koch et al. 2012; surface; lowest 21 is a more important component of cellular oxidative stress recorded values are below detection limit of 0.1 pmol L ); SCCS 5 Southern California Current System off Baja, Mexico (Sanudo-Wilhelmy~ defense in these green algae than in other phytoplankton. et al. 2012; four or ten meters depth); SBHC 5 Stony Brook Harbor Alternatively, there may be differences in the demand for B1 (Okbamichael and Sanudo-Wilhelmy~ 2005; surface); WTNA 5 Western as an enzyme cofactor that may help explain these quota dif- North Atlantic (Barada et al. 2013; 40 m depth); NA 5 North Atlan- ferences. A variety of TDP-requiring enzymes, e.g., oxidore- tic (Panzeca et al. 2008; surface). ductases, dehydrogenases, and decarboxylases, belong to described superfamilies based on amino acid sequence and planktonic grazers based on B1 per biovolume (Table 1). predicted structure (Widmann et al. 2010). O. lucimarinus Picoeukaryotic algae are desirable prey in the ocean and CCE9901, P. calceolata CCMP1756, and A. anophagefferens thought to be periodically grazed at rates > 1d21(Worden et CCMP1984 possess a similar total number of TDP-requiring al. 2004). Primarily picoeukaryotic algae have been consid- enzymes (11, 10, and 10, respectively), but there is a differ- ered in terms of their carbon contribution to higher trophic ence in the number belonging to each superfamily (Table 2). levels (Worden et al. 2004), but likely they also serve as key O. lucimarinus CCE9901 possesses a more consistent number sources of vitamins for predators, particularly vitamin auxo- of TDP-requiring enzymes across superfamilies where as P. trophic grazers that lack high-affinity vitamin transporters. calceolata CCMP1756 and A. anophagefferens CCMP1984 have The relatively high picoeukaryotic algal demand may at more ketoacid dehydrogenases (PK2) than a-ketoglutarate times significantly decrease B1 concentrations in oligotro- dehydrogenases (KDH) and decarboxylases (DC; Table 2). phic coastal waters like those off southern CA, as described These patterns are largely consistent among related Mamiel- below. However, availability of B1 analogs may change the lales and Pelagomonadales based on currently available potential for B1 limitation of the overall community. At the genome and transcriptome data (Table 2). However, to thor- San Pedro Ocean Time Series (SPOTS), off Los Angeles, Cali- oughly evaluate cofactor associated B1 demands of these fornia), Ostreocococus abundances reach upward of 8.2 3 104 small algae (especially vs. antioxidant demand), abundances cells mL21 in surface waters, 3 3 105 cells mL21 near the of TDP-requiring enzymes need to be estimated per cell deep Chl a max, and other B1 auxotrophic picoeukaryotic between strains. phytoplankton besides Ostreocococus sp. are expected to Using our minimum quota estimates (Table 1), Ostreococ- occur at SPOTS (Countway and Caron 2006). A concentra- cus and Micromonas spp. seem to be vitamin B1 rich prey for tion of 8.2 3 104 B1 auxotrophic picoeukaryotic cells mL21

9 Paerl et al. Marine picoeukaryote thiamine physiology

accounts for 2.6 pmol B1 L21, using the minimum B1 208 0.52 0.96 quotas determined in this study (mean of 3.20 3 10 pmol 6 6 21 Mamiel- B1 cell for Ostreococcus, Micromonas, and Pelagomonas spp. examined). This demand (notably a minimum estimate) , the Marine appears small but could significantly deplete B1 concentra- tions in oligotrophic waters like those reported off S. Baja 0 1.75

0.74 3.38 2 6 California (e.g., 0.34 pmol B1 L 1;Sanudo-Wilhelmy~ et al. 6 2012) and could influence picoplankton community compo- sition depending on the accessibility and concentrations of other B1 analogs in seawater. 0.5 2.00

0.76 2.38 Confirmation of B1 auxotrophy in picoeukaryotic algae 6 6 and predicting salvage based on genome sequences

-ketoglutarate dehydrogenase, K (type 1 O. lucimarinus O.

a Direct confirmation of CCE9901 and

5 tauri OTH95 as B1 auxotrophs gives credence to genome based prediction of B1 auxotrophy in algae based on the rom the Joint Genome Institute (JGI)

https://bioinformatics.psb.ugent.be/gdb/bathycoc- absence of biosynthesis genes (Table 1; Bertrand and Allen 2012; McRose et al. 2014). In contrast, B1 salvaging capabil-

0.5ities 0 remains 5.75 difficult to predict, as O. lucimarinus CCE9901 1.39 0 2.5 6 one standard deviation (SD) for total TDP-requiring (req.) 6 and O. tauri OTH95 appear to have sufficient proteins to syn- 6 thesize TMP from HMP and HET (Fig. 2). Interestingly, how- ever, additions of HMP and HET did not support the growth )orGenoscope( of B1-limited O. lucimarinus CCE9901 or O. tauri OTH95 (Fig.

0.5 0.75 3), as seen for two Micromonas sp. strains (McRose et al. 6 2.07 2.75

6 2014). Possibly homologs of Th1 or other essential thiamine Enzymes KDH K1 K2 TK DC

TDP Req. biosynthesis proteins in O. lucimarinus CCE9901, O. tauri OTH95, and other microbes with similar gene complements

lit2.net/mmetsp/ deviate from their function in model and bacterial sys- SD 10.25

6 tems. For instance, Th1 analogs in O. lucimarinus CCE9901 scriptomes or genomes were obtained f SD 11

6 and O. tauri OTH95 (OSTLU_17535 and Q00Y64_OSTTA) lack avg. chloroplast transit peptide sequences that are found in Th1 avg. of higher A. thaliana and Z. mays based on searches Ostta4 10 2 0 3 2 3 Auran1 10 1 0 6 2 1 decarboxylases. The average (avg.) j j MicpuC3 10 2 0 3 2 3 MicpuN3 10 2 0 3 2 3 j j Ost9901_3 11 3 0 3 2 3

j using the ChloroP 1.1 prediction server (Emanuelsson et al. 5 http://camera.ca 1999; http://www.cbs.dtu.dk/services/ChloroP/). This suggests genome version Transcriptome or Mamiellales Th1 is not localized to the chloroplast in Ostreococcus strains, Pelagomonadales which may have functional consequences. Alternatively, O. lucimarinus CCE9901 and O. tauri OTH95 may not be capable of transporting the tested precursors or analogs into the cell to ultimately synthesize TDP. Notably, O. lucimarinus CCE9901 and O. tauri OTH95 both possess transketolase and DC sodium ion (SSS) family transporters (OSTLU_24399; 5 Q01B34_OSTTA) with upstream TDP-riboswitch elements that were recently hypothesized to be involved in pyrimi-

e Sequencing Project(MMETSP) ( dine (e.g., HMP) or thiazole (e.g., HET) transport (Worden et al. 2009). The function of these transporters remains unclear, and their presence in algal genomes, although widespread

. Individual TDP-requiring enzyme superfamilies classified by the TEED database are as follows: KDH (McRose et al. 2014), has not yet been directly linked to sal-

RCC1105 Mamiellales bathy14mar2011vaging of HMP, 10 HET, or 2 another 0 B1 analog. 3 2 3 CCMP1850CCMP1984 Pelagomonadales Pelagomonadales MMETSP0914 jgi 11 1 0 6 2 2 Variability in B1 analog utilization by B1 auxotrophic Clade-A-BCC118000 Mamiellales MMETSP0939 11 3 0 1 3 4 CCE9901 Mamiellales jgi picoalgae CCMP1756RCC969 Pelagomonadales Pelagomonadales MMETSP0887 MMETSP1328 10 10 0 1 0 0 5 6 2 2 3 1 Abundances and types of TDP-requiring enzymes in small (approximately less than three micrometers in diameter) eukaryotic algae belonging to the sp. CCMP1545sp. RCC299sp. CCMP1646 Mamiellales Mamiellales Mamiellales jgi MMETSP1080 jgi 16 6 0 2 4 4 BCC99000 Mamiellales MMETSP0933 10 2 0 2 2 4

2-ketoacid dehydrogenases, TK Picoeukaryotic algae clearly demonstrate variability in OTH95 Mamiellales jgi Pelagomonadales

5 their affinity for and utilization of B1 analogs (Figs. 3, 4; or ) websites. Table 1). Some of the Prasinophytes (Ostreococcus and Micro- O. tauri O. prasinos O. lucimarinus Micromonas Micromonas Micromonas Bathyococcus prasinos P. calceolata P. calceolata A. anophagefferens A. anophagefferens Table 2. O. lucimarinus lales Microbial Eukaryote Transcriptom or 2) cus/ Organism Order enzymes and each superfamily are included in bold, in the last two rows. Tran monas spp.) surveyed in this study utilized phosphorylated

10 Paerl et al. Marine picoeukaryote thiamine physiology

B1 (B1 1 P) as a B1 source. In contrast, none utilized the in seawater (Carini et al. 2014). Although, the advantages provided B1 moieties to satisfy their B1 requirements (Table obtained by picoeukaryotic phytoplankton using B1 analogs 1); it may be that these strains utilize alternative pyrimidine are yet to be elucidated, but presuming that analogs exist at and thiazole compounds rather than HET and HMP to sal- environmentally relevant concentration in seawater, like vage B1. The inability of O. lucimarinus CCE9901 (and possi- HMP, we speculate about their ecological effects. bly other Prasinophytes) to use TMP or TDP is not simply The HMP demand by picoeukaryotic algae (like P. calceo- due to phosphatase inhibition by high phosphate concentra- lata CCMP1756 and E. huxleyi) potentially affect the growth 21 tions in our test medium ( 36 lmol PO4 L in F/2; Fig. 5). of B1 auxotrophic populations with obligate HMP require- O. lucimarinus CCE9901 presumably lacks extracellular phos- ments, like Pelagibacter ubique HTCC1062 and other repre- phatases that can dephosphorylate TDP or the appropriate sentatives of the abundant SAR11 bacterioplankton clade proteins for direct TDP uptake. Furthermore, TMP and TDP (Carini et al. 2014). Additional estimates of HMP concentra- utilization was not inhibited relative to B1 utilization at tions in the euphotic ocean, HMP turnover rates, and further high phosphate concentrations in M. pusilla CCMP487 and characterization of HMP uptake and/or growth kinetics of P. calceolata CCMP1756 suggesting that dephosphorylation is algal and bacterial populations will provide an improved not a prerequisite for uptake of TMP and TDP in these understanding of competition for HMP amongst plankton in strains. Contrasting with the Prasinophytes, P. calceolata the upper ocean. We did find that P. calceolata CCMP1756 CCMP1756, and P. lutheri CCMP1325 (belonging to classes utilized HMP at a comparable capacity to that of B1, making Pelagophyceae and Pavlovophyceae, respectively) utilized it an equivalent resource to B1 for this organism (Table 1). HMP and B1 1 P (Table 1). As these strains are able to use In B1-deplete waters, B1 analogs could promote the HMP, it is expected that they possess a complete thiazole growth of select portions of the microbial community, e.g., biosynthesis pathway; this has yet to be genetically identi- promoting phytoplankton like P. calceolata CCMP1756 over fied or characterized. Given the recent demonstration of phytoplankton like O. lucimarinus CCE9901, as the two HMP utilization by E. huxleyi, these data suggest that HMP appear to not salvage the same compounds. It is currently use by marine algae may be widespread (McRose et al. 2014). unknown if HAB-associated B1 auxotrophic species, e.g., A. Additionally, P. lutheri CCMP1325 was used previously to anophagefferens spp. and P. globosa, are capable of salvaging estimate B1 concentrations in seawater samples (Carlucci analogs for B1 biosynthesis. If so, the success and persistence and Silbernagel 1966). P. lutheri-associated B1 concentration of HAB-associated B1 auxotrophic populations could depend estimates can now be understood to include (at least) B1 1 P on their ability to use B1 analog pools. and HMP and thus overestimate B1 concentrations. Clearly B1 analogs deserve further consideration as B1 TDP is considered to be an important active form of B1 sources for auxotrophic plankton (algae, bacteria, and zoo- that serves as an enzyme cofactor and/or riboswitch ligand plankton) in the ocean. B1 has been a primary focus of (Jurgenson et al. 2009). Presumably, obtaining B1 1 P, espe- recent research largely due to the development of chemical cially TDP, from the environment is advantageous, enabling methods to estimate concentrations of the B1 molecule in conservation of intracellular resources required for de novo seawater (Sanudo-Wilhelmy~ et al. 2012; Barada et al. 2013). TDP biosynthesis or salvaging (e.g., enzyme synthesis, ATP, More recently, HMP concentrations were estimated and it P). Some model and marine bacteria can use TDP as a B1 appears to serve a critical role in sustaining the growth of source (Burkholder and Lewis 1968; Webb et al. 1998). Con- several representatives of the SAR11 bacterioplankton clade sidering intracellular concentrations of TDP can reach (Carini et al. 2014). B1 analogs beyond HMP exist in nature, x1.3–2 that of B1 in larger phytoplankton (Pinto et al. 2003) e.g., TMP, TDP, and could also play key roles in B1 cycling, and marine plankton mortality is expected to be high (Sherr microbial interactions, and the ecology of B1 auxotrophic and Sherr 1994; Suttle 1994), B1 1 P might be present in sea- plankton. water at significant concentrations despite potentially fast degradation or dephosphorylation rates, either of which Coculturing of O. lucimarinus CCE9001 with TW7 remain incompletely characterized. implicates bacteria as conduits in B1 cycling The utilization and availability of B1 analogs could be key Knowing that O. lucimarinus CCE9901 could not utilize in determining the persistence of particular auxotrophic TDP as a sole B1 source, we determined that a simple consor- picoeukaryotic algae in regions of the euphotic ocean, espe- tium with a B1 prototrophic (synthesizing) bacterium (TW7), cially as B1 concentrations are apparently very low in oligo- known to exhibit extracellular or periplasmic phosphatase trophic euphotic waters (e.g., < 0.05 pmol B1 L21;Sanudo-~ activity (Chichester et al. 2008) enabled O. lucimarinus

Wilhelmy et al. 2012; Barada et al. 2013). B1 Ks values are CCE9901 to grow on the phosophorylated B1 analog TDP comparable to HMP Ks values for at least one B1 auxotrophic (Fig. 5B). It is likely that the added TDP was dephosphoryl- algae (P. calceolata CCMP1756; Table 1) and initial measure- ated to B1 by TW7-derived phosphatases, then utilized by O. ments of one moiety (HMP) indicate it is present at concen- lucimarinus CCE9901. This result demonstrates that bacteria trations equivalent to or greater than concentrations of B1 can increase vitamin availability beyond de novo synthesis

11 Paerl et al. Marine picoeukaryote thiamine physiology and also it suggests that B1 auxotrophic plankton like O. Carlucci, A. F. 1970. Part II: Vitamin B12, Thiamine, and Bio- lucimarinus CCE9901, that are unable to utilize B1 1 P (and tin. In The ecology of the plankton off La Jolla, California, HMP, HET) alone, ultimately depend on other microorgan- in the period April through September, 1967. Bull. Scripps isms to modify B1 1 P to an utilizable form. These are newly Inst. Oceanogr. 17: 23–31. doi:10.1002/9781118669235 described functional interactions within marine microbial Carlucci, A. F., and S. B. Silbernagel. 1966. Bioassay of sea- consortia that may be critical in marine B1 cycling, depend- water II. Methods for the determination of concentrations ing on modification and turnover rates of B1 analogs in sea- of dissolved vitamin B1 in seawater. Can. J. Microbiol. 12: water and the B1 physiologies of ecologically important B1 1079–1089. auxotrophs, e.g., SAR11 and SAR86 clade representatives Chichester, K. D., M. Sebastian, J. W. Ammerman, and C. L. (Dupont et al. 2011; Carini et al. 2014). Colyer. 2008. Enzymatic assay of marine bacterial phos- Bacteria are critical remineralizers of organic compounds phatases by capillary electrophoresis with laser-induced in the ocean (Kirchman 2000) and they are traditionally fluorescence detection. Electrophoresis 29: 3810–3816. viewed as “producers” of vitamins (especially B12; Haines doi:10.1002/elps.200800173 and Guillard 1974). More recently, they have been proposed Countway,P.D.,andD.A.Caron.2006.Abundanceanddistribu- as top competitors in terms of uptake for available vitamins tion of Ostreococcus sp. in the San Pedro Channel, California, in seawater (specifically B1 and B12; Koch et al. 2012), as revealed by quantitative PCR. Appl. Environ. Microbiol. although how this relates to net vitamin availability is 72: 2496–2506. doi:10.1128/AEM.72.4.2496-2506.2006 unknown. Our results suggest an additional role for bacteria Croft, M. T., M. J. Warren, and A. G. Smith. 2006. Algae as conduits that do not extensively remineralize a vitamin need their vitamins. Eukaryot. Cell 5: 1175–1183. doi: analog (TDP) but rather chemically modify it and make it 10.1128/EC.00097-06 available to microbes that cannot use the analog in isolation. Davey, M., G. A. Tarran, M. M. Mills, and C. Ridame. 2008. We anticipate B1 and analogs play key roles in interactions Nutrient limitation of picophytoplankton between B1 auxotrophic algae and B1 prototrophic microbes and growth in the tropical North Atlantic. Limnol. Oce- and in the survival of B1 auxotrophic picoeukaryotic algae anogr. 53: 1722–1733. doi:10.4319/lo.2008.53.5.1722 in the sea. Here we have demonstrated one instance where a Droop, M. R. 2007. 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ZoBell, C. E. 1941. Studies on marine bacteria. I. The cultural ribonucleic acid gene sequencing, two anonymous reviewers for requirements of heterotrophic aerobes. J. Mar. Res. 4: 42–75. improvements to the manuscript, as well as, A. Carlucci and H. Paerl for helpful discussions. Zubkov, M. V., M. A. Sleigh, G. A. Tarran, P. H. Burkill, and R. EMB was supported by National Science Foundation Antarctic Division J. G. Leakey. 1998. Picoplanktonic community structure (ANT) grant 1103503. on an Atlantic transect from 50Nto50S. Deep Sea Res. I This work was funded in part by the Gordon and Betty Moore 45: 1339–1355. doi:10.1016/S0967-0637(98)00015-6 Foundation through grant GBMF2758 and grant GBMF3828 to FA and AEA. Acknowledgements We thank members of the Brahamsha, Palenik, and Azam labs at the Submitted 17 April 2014 Scripps Institution of Oceanography for assistance during experiments Revised 17 September 2014 and culture maintenance. Additional thanks are extended to S. Podell Accepted 7 October 2014 and E. Allen for assistance with Hidden Markov Model (HMM) searches, W. Lambert for help with Ostreococcus sp. CCE1301 18S ribosomal Associate editor:

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