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Limnol. Oceanogr., 55(5), 2010, 1959–1964 E 2010, by the American Society of and Oceanography, Inc. doi:10.4319/lo.2010.55.4.1959

Vitamin B12 excretion by cultures of the marine Crocosphaera and

Sophie Bonnet,a,b,* Eric A. Webb,b Caterina Panzeca,b David M. Karl,c Douglas G. Capone,b and Sergio A. San˜udo-Wilhelmyb a Institut de Recherche pour le Developpement (IRD), Laboratoire d’Oceanographie Physique et Biogeochimique, Marseille, France bDepartment of Biological Sciences, University of Southern California, Los Angeles, California c Department of Oceanography, University of Hawaii, Honolulu, Hawaii

Abstract

Axenic cultures of the N2-fixing marine cyanobacterium Crocosphaera watsonii exhibit very high B12 excretion rates (up to 40 times higher per cell), compared to the smaller non–N2-fixing strain of marine Synechococcus. The effect of N availability on vitamin synthesis is also evident in the non-diazotrophic strain, as they release five times more B12 under N-replete conditions relative to N-limiting conditions, although this value is still an order of magnitude smaller than the amount produced by the diazotroph. The cyanobacterial contribution to the oceanic B12 supply may be , 50 times higher than the contribution of heterotrophic . Oceanic cyanobacteria do not compete for exogenous B12 with other and , but instead are obligate producers, thereby defining a unique dual ecological role for N2-fixing cyanobacteria in the oligotrophic . They provide both a source of ‘‘new’’ bioavailable and the B12 needed to support the growth of larger auxotrophic eukaryotic important for the biological pump.

Vitamin B12 (cobalamin; B12 hereafter) is an essential production rates of two globally distributed oceanic growth factor for most marine eukaryotic phytoplankton cyanobacterial groups with different N acquisition capa- (Provasoli and Carlucci 1974; Droop 2007) that is required bilities: the unicellular, free-living N2-fixing (diazotrophic) for the activity of several vital in central cyanobacterium Crocosphaera watsonii WH 8501, and the (e.g., methylmalonyl-CoA mutase, methionine ubiquitous non–N2-fixing cyanobacterium Synechococcus synthase, and type II ribonucleotide reductase; Raux et al. sp. WH 7803. 2000; Martens et al. 2002). Many of the eukaryotic phytoplankton (over half of all species surveyed to date) Methods lack the biosynthetic pathway for this nitrogen (N)- containing B vitamin (C63H88CoN14O14P), and thus must Axenic strains of C. watsonii WH 8501 and Synecho- depend on an exogenous pool of dissolved B12 (Croft et al. sp. WH 7803 were grown in sterile YBCII medium 2005, 2006). Recent studies suggest this ambient pool may (Chen et al. 1996) in a temperature-controlled incubator be insufficient to support maximum in both (27uC) with light supplied on a 12 : 12 light : dark (LD) cycle coastal and open ocean environments, as phytoplankton (80 mmol quanta m22 s22). C. watsonii were grown under 21 growth is enhanced by picomolar B12 amendments (Pan- three initial B12 conditions (0, 400, and 800 pmol L ) and zeca et al. 2006; San˜udo-Wilhelmy et al. 2006; Bertrand et N depletion, except the treatment with no added vitamin al. 2007). Genomic data indicate that many prokaryotes B12, in which Crocosphaera were grown under both N- 21 z ( and ) could be significant deplete and N-replete (500 mmol L NH 4 ) conditions. sources of B12 to the (Palenik et al. 2003; Rocap Synechococcus sp. were grown under two B12 conditions (0 et al. 2003). However, there is currently little information and 400 pmol L21) and two N conditions (10 and translating this genomic potential into documented B 21 z 12 500 mmol L NH 4 ). Each condition was production rates or defining the chemical factors (e.g., N performed in triplicate. For all experiments, the final availability) controlling its in situ production. dissolved cobalt concentrations used were 50 pmol L21 (+ Cyanobacteria (including Crocosphaera, Prochlorococ- 2 mmol L21 ethylenediaminetetraacetic acid; i.e., a lower cus, Synechococcus,andTrichodesmium) are ubiquitous in concentration than the one traditionally used in YBCII the world’s oceans, where they often contribute significant- medium in order to avoid any cobalt toxicity and excess ly to (e.g., Partensky et al. 1999; cobalt release through vitamin B12 release). These cobalt Capone et al. 2005; Bonnet et al. 2009). In addition to this concentrations are about 3 times higher than those well-described role, representatives of each cyanobacterial considered limiting for B12 synthesis in the field (Panzeca contain the required to synthesize B12 (Palenik et al. 2008). Prior to experiments, the cells were acclimated et al. 2003; Rocap et al. 2003; E. A. Webb unpubl. data); to each nutrient condition for at least three generations. To however, their role in B12 production has not been assessed check that cultures were axenic, the medium was system- so far. In this study, we provide for the first time B12 atically examined for the presence of contaminating bacteria using 49-6-diamidino-2-phenylindole (DAPI) stain- * Corresponding authors: [email protected] ing during the course of the experiments. Growth rates 1959 1960 Bonnet et al.

Fig. 1. Nanomolar levels of B12 released by C. watsonii. (A) Growth curve of C. watsonii cultured under N- and B12-deplete conditions. Arrows indicate the time points of sampling and B12 measurements. (B) Coevolution of B12 concentrations and C. watsonii 21 cell density in the culture during the exponential growth phase. Black line denotes mean (n 5 3) B12 concentrations (nmol L ; error bars 21 6 SD). Grey bars denote mean (n 5 3) C. watsonii WH 8501 density (cells mL ; error bars 6 SD). T0: start of experiment (inoculation); 21 T1 and T2 correspond to time points sampled during the exponential phase (growth rate: 0.3 d ). were determined daily (for Synechococcus) and every other of two cyanobacterial groups. The diazotrophic cyanobac- day (for Crocosphaera) using relative fluorescence units. terium C. watsonii grown under N- and vitamin B12-deplete At 1 (for Synechococcus) or 2 (for Crocosphaera) selected conditions was able to produce and excrete nanomolar time points during the exponential growth phase, an levels of the vitamin to the surrounding environment after aliquot of 20–30 mL was extracted from triplicate cultures 12 d of growth (Fig. 1). This corresponds to an average 218 219 21 and filtered by hand through a 0.2-mm polycarbonate production of 1 3 10 (6 1.4 3 10 ) mol B12 using extremely low vacuum (, over the exponential phase of cell growth, and a daily 219 21 21 40 mm Hg) to avoid any cell breakage. Dissolved vitamin production rate of 1 3 10 mol B12 d cell . The same B12 concentrations were determined in the filtrate using the grown under a range of initial B12 concentrations protocol described in Okbamichael and San˜udo-Wilhelmy indicates that external B12 availability can repress the (2004). Cells were systematically counted at the same time vitamin synthesis pathway, as C. watsonii produced on points using flow cytometry (MoFlo cell sorter, Dako) in average two times less B12 per cell when grown under 21 order to determine vitamin B12 production rates per cell. At 400 pmol L initial B12 concentrations and stopped the same time points, microscopic analysis were performed production and began to consume the external pool when 21 under DAPI long-path filters in order to confirm that 100% grown under 800 pmol L initial B12 conditions (Fig. 2). of cells were in a good physiological state. Similar experiments conducted with the non-diazo- In order to confirm that the molecule released by the trophic cyanobacterium Synechococcus (WH 7803) indicate cells was actually vitamin B12, we tested whether it was that over the exponential phase this species produces 40 sufficient to allow the growth of a phytoplankton strain times less B12 per cell compared to the diazotroph when 220 known to be auxotrophic for B12, Synechococcus PCC7002. grown under N-limited conditions (2.5 3 10 6 1.1 3 220 218 219 21 Briefly, the indicator cells were grown in SN media (Chen 10 vs. 1 3 10 6 1.4 3 10 mol B12 cell ; Fig. 3) et al. 1996) to mid–log phase, incubated in soft agar SN and eight times less B12 when grown under N-replete 219 220 218 media (1% agar without B12), and allowed to grow for conditions (1.2 3 10 6 3.7 3 10 vs. 1 3 10 6 1.4 219 21 ,13 d at 27uC with a 14 : 10 LD cycle at 80 mmol quanta 3 10 mol B12 cell ). Synechococcus produced five times 22 22 m s . After this time, a B12 standard dilution was less B12 per cell when grown in low-N compared to N- spotted on the plate as well as the unknown compound replete conditions, whereas in the diazotroph C. watsonii, from Synechococcus and Crocosphaera cultures. After 3 d this effect was not observed (Fig. 3), probably because of additional incubation, there was clear growth above the Crocosphaera is able to satisfy its N requirements through background level in the B12 dilution series as well as the N2 fixation. putative purified B12 (data not shown). Discussion Results N availability—Vitamin B12 synthesis—Our study indi- Our results show that the synthesis and release rates of cates that the two cyanobacteria tested are possibly vitamin B12 vary substantially between the representatives important contributors to the supply of dissolved B12 in Vitamin B12 production by cyanobacteria 1961

Fig. 2. Apparent regulation of B12 production by C. watsonii Fig. 3. Comparison of B12 production capacity by a diazo- WH8501 by ambient B12. Black and grey bars represent the mean trophic and a non-diazotrophic cyanobacterium. The bars (n 5 3, error bars 6 SD) quantity of B12 produced per cell during represent the mean quantity of B12 produced per cell by C. the exponential growth phase (growth rate: 0.3 d21), respectively watsonii WH 8501 (black, n 5 3, error bars 6 SD) and after 10 and 12 d of growth. The x-axis represents initial B12 Synechococcus WH 7803 (grey, n 5 2, error bars 6 SD), under concentrations (pmol L21). 21 z N-replete conditions (500 mmol L NH 4 ) and N-deplete (for C. 21 z watsonii) and N-limited (10 mmol L NH 4 for Synechococcus) the euphotic zone of the world ocean. However, their conditions. relative importance in B12 supply may depend on the degree of oligotrophy and N limitation, which in turn determine the cyanobacteria community structure and the B12 times) than the currently estimated contribution of synthesis capacity. For example, although the amount of heterotrophic bacteria (Table 1). Because of the scarcity vitamin synthesized by the non–N2-fixing Synechococcus of published B12 production rates in , was significantly lower than that produced by the N2-fixing the values presented herein are an important first step for Crocosphaera, the former cyanobacterium is ubiquitous in predicting the contribution of autotrophic prokaryotes to temperate mesotrophic waters, reaching average abundanc- B12 flux in situ. Additional future data on B12 production es between 102 and 105 cells mL21 (Partensky et al. 1999). by other cultured representatives and natural marine At those abundances, and with a B12 synthesis capacity of bacterial assemblages will further increase the accuracy of 1.2 3 10219 mol cell21 over the exponential growth phase such estimates. In particular, it will be important to focus (in N-replete conditions), their contribution to the dis- on , the most abundant marine cyanobac- solved B12 pool is potentially significant, ranging from 0.01 terium in the ocean (Partensky et al. 1999) that also 21 to a maximum of 12 pmol L assuming that vitamin possesses the biosynthetic pathway for B12 production. uptake was not occurring (Table 1). However, in oligotro- phic N-depleted waters, the abundance of Synechococcus Why do cyanobacteria produce such high amounts and their concomitant B12 synthesis capacity decrease. In of B12?—Our laboratory results suggest that cyanobacteria those oligotrophic environments, unicellular diazotrophs could influence the amount of available B12 in surface can form significant blooms (Campbell et al. 2005; Le Moal waters of the world ocean. These results are consistent with and Biegala 2009), reaching up to 5 3 103 cells mL21. the aforementioned genomic data showing that marine Although not as dense as the Synechococcus blooms, the cyanobacteria contain the genes coding for B12 synthesis B12 synthesis capacity of unicellular diazotrophs such as C. (Palenik et al. 2003; Rocap et al. 2003). watsonii is 40 times higher and therefore their contribution Botanists have long suspected that cyanobacteria play a to the ambient B12 pool could be significant in these N- dual beneficial role in rice fields, by providing both N depleted environments, potentially reaching up to (through N2 fixation) and B12. Some authors have shown, 21 5 pmol L of B12 released to the surrounding environment for instance, that an algal inoculation on rice over the exponential growth period (Table 1). Our calcu- increased the yield of plants in the presence or absence of lations on the effects of cyanobacterial blooms on the inorganic fertilizer, suggesting that algae played an dissolved B12 pool are consistent with the range of B12 auxiliary role besides fixing atmospheric N (Sundara Rao values measured in different marine systems (0.2– et al. 1963). Shah and Vaidya (1977) later showed in axenic 21 4 pmol L ; Panzeca et al. 2006; San˜udo-Wilhelmy et al. cultures that N2-fixing cyanobacteria associated with rice 2006). It seems that in all cases, the maximal cyanobacterial plants were able to produce extremely high quantities of 21 contribution to the oceanic B12 supply is higher (, 50 B12 (12–35 nmol L depending on the culture age). 1962 Bonnet et al.

Table 1. Oceanic B12 supply contribution by cyanobacteria (diazotrophic and non-diazotrophic) and heterotrophic bacteria (calculation based on B12 production over the exponential phase of cell growth).

B12 synthesis capacity Oceanic density B12 supply (pmol cell21 over exp. phase) (cells mL21) (pmol L21) Cyanobacteria (non-diazotrophic) 2.5–12310220 (this study) 100–100,000* 0.002–12 Cyanobacteria (diazotrophic) 1310218 (this study) 0–5000{ 0–5 Heterotrophic bacteria 0.1–1.7310222{ 500,000–1,000,0001 0.005–0.17 * Partensky et al. 1999. { Le Moal and Biegala 2009. { Croft et al. 2005. 1 Azam et al. 1983.

One potential explanation for such high levels of B12 recently, the oceanic supply of B12 in the ocean was production is that in several cyanobacteria, the enzymes considered to be associated mainly with heterotrophic ribonucleotide reductase (which forms the deoxyribonucle- prokaryotes (Croft et al. 2005). These authors have shown otide precursors for deoxyribonucleic acid synthesis) and that many eukaryotes may satisfy their B12 requirements methionine synthase are B12-dependent (Gleason and through direct with heterotrophic prokaryotes Wood 1976; E. A. Webb pers. comm.), and actively (Croft et al. 2005), in which the algae supply fixed carbon in growing cultures of such algae are required to synthesize return for B12, or through direct phagotrophy on B12- B12 in substantial amounts. Moreover, our data seem to producing bacterial prey (Croft et al. 2006). We show that indicate that the cells have incomplete control over the maximal amount produced by studied heterotrophic 222 21 biosynthesis of this vitamin that could lead to the excessive bacteria (0.1–1.7 3 10 mol B12 cell ; Croft et al. 2005) synthesis and ultimate release into the external medium. is between 150 and 6000 times less than the average B12 Our data are consistent with this supposition, as produced by the cyanobacteria tested here. This suggests 21 800 pmol L exogenous B12 (i.e., two orders of magnitude that the cyanobacterial compartment now needs to be higher concentrations than those measured in the oceans; taken into account when studying the biogeochemical cycle Panzeca et al. 2006; San˜udo-Wilhelmy et al. 2006) was of B12 in the surface ocean, although a larger number of required to eliminate significant B12 excretion. Similarly to marine prokaryotes need to be tested for B12 synthesis and B12, diazotrophic cyanobacteria such as are excretion to draw stronger conclusions. known to release up to 80–90% of the N fixed from the In particular, diazotrophic cyanobacteria constitute key , in the form of dissolved organic N and phytoplanktonic species in N-depleted environments, as ammonium (Gilbert and Bronk 1994), but the explanation they provide a source of ‘‘new’’ N to fuel further primary for this is still unclear. Similarly, no conclusive explanation production (Capone et al. 2005). This culture study, even if is currently available to explain why cyanobacteria excrete performed at higher cell density compared to the field, such high amounts of N and B12. Our excretion results are shows that diazotrophs are also able to provide B12.Ifwe not completely unexpected, as similar large corrinoid ring consider an accumulation of Crocosphaera (1–5 3 103 cells 21 21 21 molecules have been shown to be excreted by some L ) fixing N2 at a rate of 0.02 pmol N cell d with an heterotrophic prokaryotes, such as Pseudomonas aerugi- atomic C : N ratio of 7, the ‘‘new’’ N provided by such a nosa, Pseudomonas putida,orPseudomonas fluorescens, bloom could sustain a rate of 0.14–0.7 mmol when intracellular levels are in excess (Harris et al. 1993), CL21 d21. If we apply a mean cell quota of 4.1 nmol and the concentration of the excreted B12 is an order of B12 : mol C (Carlucci and Bowes 1972), the ‘‘new’’ dissolved magnitude higher than those measured in our experiments B12 pool produced by these diazotrophs could support a (mmol L21; Mazumder et al. 1987). Our results show that phytoplankton carbon fixation rate of 0.5 mmol C L21 d21 the synthesis of B12 by the diazotrophic cyanobacterium (about the same amount of carbon fixation supported by Crocosphaera is disproportionately high when compared ‘‘new’’ N). This dual ecological function illustrates the with the non-diazotrophic cyanobacterium Synechococcus; importance of diazotrophs for potentially increasing however, we must consider that Crocosphaera has a higher phytoplankton by providing both ‘‘new’’ N and (5–70 times) biovolume compared to Synechococcus coenzymes that can facilitate the growth of large auxotro- (Waterbury et al. 1986; Webb et al. 2009), which could phic eukaryotic species such as , and thus possibly partly explain those differences, as nutrient requirements affecting increased export production in the ocean. As an are known to be size-dependent (Hein et al. 1995). example, the ecological success of –diazotroph associations, which sequester high quantities of atmospher- Potential biogeochemical implications of cyanobacterial ic carbon (Subramaniam et al. 2008), may be potentially B12 synthesis and excretion—This study focuses specifically explained by a dual supply of N and B12 from the on cyanobacteria that produce and excrete B12, and thus cyanobacterium to its host (Karl 2002). In contrast, high illustrates the dependency existing in the ocean between sporadic B12 inputs in the coastal environment from runoff prokaryotes and eukaryotes for primary production. Until have been hypothesized to cause phytoplanktonic succes- Vitamin B12 production by cyanobacteria 1963 sions (Yu and Harrison 2000), thus potentially resulting in GILBERT, P. M., AND D. A. BRONK. 1994. Release of dissolved the formation of harmful algal blooms. organic nitrogen by marine diazotrophic cyanobacteria, Further laboratory and field studies quantifying the Trichodesmium spp. Appl. Environ. Microb. 60: 3996–4000. internal quotas of B vitamins in eukaryotic phytoplankton GLEASON, F. K., AND J. M. WOOD. 1976. Ribonucleotide reductase as well as the extracellular release rates of vitamins in in blue-green algae; Dependance on adenosycyanocobalamin. Science 192: 1343–1344, doi:10.1126/science.818711 representative autotrophic and heterotrophic bacterial HARRIS, W. F., R. S. BURKHALER,W.LIN, AND R. TIMKOVICH. cultures are required to develop a robust quantitative 1993. Enhancement of bacterial porphyrin biosynthesis by model for the ecological importance of these important exogenous aminolevulicin and isomer specificity. Bioorg. cofactors. It would also be interesting to study in the field Chem. 21: 209–220, doi:10.1006/bioo.1993.1018 the interrelations existing between phytoplanktonic succes- HEIN, M., M. FOLAGER PEDERSEN, AND K. SAND-JENSEN. 1995. sions and B12 cycling, in order to determine the role of B12 Size-dependent nitrogen uptake in micro- and macroalgae. production in the so-called ‘‘.’’ Mar. Ecol. Prog. Ser. 118: 247–253, doi:10.3354/meps118247 KARL, D. M. 2002. Nutrient dynamics in the deep blue sea. Acknowledgments Trends Microbiol. 10: 410–418, doi:10.1016/S0966-842X This research was supported by the U.S. National Science (02)02430-7 Foundation (Biological Oceanography-Ocean Sciences-0623432 LE MOAL, M., AND I. C. BIEGALA. 2009. Diazotrophic pico and to S. San˜udo-Wilhelmy). S. Bonnet was supported by a University nano-cyanobacteria in the Mediterranean Sea: A seasonal of Southern California (USC) Women in Science and Engineering cycle. Limnol. Oceanogr. 54: 845–855. (WiSE) postdoctoral fellowship. 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