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

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 growth constants (Ks) for B1 between 1.26 and 6.22 pmol B1 L , which is higher than reported seawater concentrations. Minimum B1 cell quotas for Ostreococcus and Micromonas spp. are high (2.20 3 10–4.46 3 10 pmol B1 cell) 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 provided 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 Pseudoalteromonas 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 required by all cells due to its role as a cofactor in the form of thiamine pyrophosphate (also called thiamine diphosphate [TDP]) for enzymes critical to carbon and amino acid metabolism (Jurgenson et al. 2009). Additionally, certain riboswitches, untranslated sections of messenger ribonucleic acid (RNA) capable of regulating gene expression, are activated by binding TDP, B1, and moieties (parts of the thiamine molecule, e.g., thiazole and pyrimidine; Moulin et al. 2013). Notably, this is one of the few documented riboswitch systems in eukaryotes, and appears to be important in regulating thiamine metabolism in marine microbes (McRose et al. 2014). In the euphotic upper ocean, thiamine concentrations appear to be low relative to macronutrients, e.g., pmol L or below (Sa~ nudo-Wilhelmy et al. 2012). Despite the scarcity of B1 in the upper ocean, especially the oligotrophic regions where published B1 concentrations are < 0.05 pmol L (Barada et al. 2013), several marine plankton (eukaryotes and prokaryotes) persist as B1 auxotrophs, unable to synthesize their own vitamin B1. Surveys of the occurrence of vitamin auxotrophy within phytoplankton noted that a large percentage ( 80%) of prymnesiophytes were B1 auxotrophs (Provasoli and Carlucci 1974; Croft et al. 2006). In contrast, a majority of diatoms produce B1 (Provasoli and Carlucci 1974), and both B1 producing and auxotrophic bacteria are present in the ocean (MacLeod et al. 1954; Burkholder and Lewis 1968). Many new algal strains have been isolated as the pioneering surveys of auxotrophy (Provasoli and Carlucci 1974), including multiple picoeukaryotic algal strains (less than three micrometer in cell diameter). There is increasing *Correspondence: [email protected] 215 LIMNOLOGY and OCEANOGRAPHY Limnol. Oceanogr. 60, 2015, 215–228 VC 2015 Association for the Sciences of Limnology and Oceanography doi: 10.1002/lno.10009 evidence, based largely on genome analysis, that several key picoeukaryotic algae are B1 auxotrophs (Palenik et al. 2007; Worden et al. 2009; Bertrand and Allen 2012; McRose et al. 2014) but also based on a small set of experiments with axenic cultures (e.g., Aureococcus anophagefferens CCMP1984, Micromonas spp.; Tang et al. 2010; Bertrand and Allen 2012; McRose et al. 2014). Picoeukaryotic phytoplankton are important contributors to marine primary production and phytoplankton biomass (Li 1994; Worden et al. 2004). Also some B1 auxotrophic picoeukaryotic and larger eukaryotic algae are harmful algal bloom (HAB)-associated species, e.g., A. anophagefferens and Phaeocystis globosa (Peperzak 2000; Tang et al. 2010). Thus, vitamin B1 availability may influence primary production rates, marine algal community structure, and water quality by influencing the growth and activity of picoeukaryotic phytoplankton. Several studies found positive correlations between primary productivity rates and additions of B1 or increases in natural B1 seawater concentrations. B1 additions to subarctic N. Pacific seawater stimulated bulk primary productivity (Natarajan 1970), a significant positive correlation between net primary productivity and B1 concentrations was found at one location off La Jolla, California (Carlucci 1970) and most recently, a positive correlation between B1 seawater concentrations and primary production (as well as nitrogen fixation) was reported for the upper water column of the western tropical Atlantic (Barada et al. 2013). The degree to which vitamin B1 availability affects growth of B1 auxotrophic algae will in part depend on multiple aspects of algal B1-related physiology, e.g., utilizable forms, uptake affinity, and per cell requirements. Early vitamin research showed that B1 moieties or chemical analogs (compounds sharing structural similarity to thiamine) satisfied the B1 requirement of auxotrophic marine algae, specifically flagellates, and benthic diatoms isolated from vitamin replete sediment and intertidal habitats (Provasoli and Carlucci 1974). Additionally, a representative strain of the ubiquitous marine bacterial family Pelagibacteraceae (also known as the SAR11 clade) exhibits an obligate requirement for 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) that cannot be satisfied by B1 (Carini et al 2014), and the coccolithophore Emiliania huxleyi is able to use both B1 and HMP to satisfy its B1 requirements (McRose et al. 2014), emphasizing the importance of understanding B1 moiety utilization patterns in marine microbes. In contrast, two strains of Micromonas only use B1 and not its precursors to satisfy thiamine requirements (McRose et al. 2014); it is currently unknown whether or not other B1 auxotrophic picoeukaryotic phytoplankton like Ostreococcus and Pelagomonas spp., representatives of picoeukaryotic algae that are cosmopolitan in the pelagic and neritic euphotic ocean (Li 1994; Worden et al. 2004) can utilize B1 moieties or chemical analogs to meet their B1 requirements. Vitamin B1 kinetic growth constants and minimum cell quota [called cell yield relation by Droop (2007)] are available for a small set of algae, specifically Pavlova lutheri (Carlucci and Silbernagel 1966), A. anophagefferens and larger dinoflagellate strains (Tang et al. 2010). No such data have been reported for Ostreococcus, Micromonas, or Pelagomonas spp. B1 kinetic growth constant and B1 cell quota data for auxotrophic picoeukaryotic phytoplankton would provide a better sense of; (1) whether or not they have the potential to be growthlimited by B1 availability in the euphotic ocean, (2) any dependence on intimate interactions with other microbes for B1, and (3) their influence on B1 cycling based on their B1 demand (pmol B1 cell). We conducted laboratory-based experiments with putative vitamin B1 auxotrophic and cosmopolitan picoeukaryotic phytoplankton, specifically, Ostreococcus lucimarinus CCE9901, Ostreococcus sp. CCE1301, Micromonas pusilla CCMP487, and Pelagomonas calceolata CCMP1756. Using axenic cultures, we confirmed these strains as B1 auxotrophs, determined their vitamin B1 half (Ks) and maximum (Kmax) saturation growth constants, their ability to utilize B1 chemical analogs (moieties and phosphorylated forms), and their respective minimal B1 cell quotas. Lastly, we examined if the presence of a simple microbial population (Gammaproteobacterium Pseudomonas sp. TW7) would enable a B1 auxotrophic algae (O. lucimarinus CCE9901) to utilize a phosphorylated B1 analog it cannot use in isolation.