Intragenomic Spread of Plastid-Targeting Presequences in the Coccolithophore Emiliania huxleyi

Nucleus-encoded plastid-targeted proteins of photosynthetic organisms are generally equipped with an N-terminal presequence required for crossing the plastid membranes. The acquisition of these presequences played a fundamental role in the establishment of plastids. Here, we report a unique case of two non-homologous proteins possessing completely identical presequences consisting of a bipartite plastid-targeting signal in the coccolithophore Emiliania huxleyi. We further show that this presequence is highly conserved in five additional proteins that did not originally function in plastids, representing de novo plastid acquisitions. These are among the most recent cases of presequence spreading from gene to gene and shed light on important evolutionary processes that have been usually erased by the ancient history of plastid evolution. We propose a mechanism of acquisition involving genomic duplications and gene replacement through non-homologous recombination that may have played a more general role for equipping proteins with targeting information.     

Accumulation and Utilization of Dissolved Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi

The mechanism for utilization of dissolved inorganic carbon(DIC) was investigated in the marine unicellular calcareousalga Emiliania huxleyi, grown with constant aeration. The apparentK_0.5 (DIC), the concentration of DIC which attains one-halfof the maximum velocity of apparent photosynthesis, for photosyntheticevolution of O₂, measured under saturating light, was 5.5 mM(55 µM for CO₂) at pH 8.0 and 25°C. The value of K_0.5was not affected by inhibitors of carbonic anhydrase (CA), andan electrometric assay of CA showed that the enzyme was notinvolved in photosynthesis in this alga. The rate of photosyntheticfixation of ¹⁴C-DIC into acid-stable products was about 20 timeshigher than that into CaCO₃, irrespective of the external concentrationof DIC. In short-term experiments, ¹⁴C-DIC was usually incorporatedinto the internal pool of DIC (IIC) to concentrations up to13 to 16 times higher than that of the external DIC. CO₂ addedexternally was utilized mainly for fixation of CO₂ and accumulationof IIC. By contrast, HCO^-₃ was utilized mainly for productionof CaCO₃ and accumulation of IIC. Incorporation of ¹⁴C intoIIC was partially suppressed by DCMU or in darkness but itstransfer to CaCO₃ was unaffected. These results suggest thataccumulation of IIC in this alga, even under ordinary circumstances,is only partially responsible for increasing the efficiencyof utilization of DIC by photosynthetic fixation but may bemost useful for the production of CaCO₃. (Hydroxyethylidene) bisphosphonic acid, an inhibitor of thegrowth of CaCO₃ crystals, completely suppressed production ofCaCO₃. The accumulation of IIC was also partially suppressed,but photosynthetic fixation of CO₂ was enhanced. In a pulse-chaseexperiment with ¹⁴CDIC, ¹⁴C incorporated into IIC and CaCO₃in darkness was transferred to acid-stable products of photosynthesisin the light. These results suggest that ¹⁴C-DIC in IIC andpre-formed CaCO₃ may be useful sources of carbon for fixationof CO₂. (Received July 2, 1993; Accepted January 10, 1994)     

Cold Stress Stimulates Intracellular Calcification by the Coccolithophore, Emiliania huxleyi (Haptophyceae) Under Phosphate-Deficient Conditions

Intracellular calcification by the coccolith-producing haptophyte Emiliania huxleyi (NIES 837) is regulated by various environmental factors. This study focused on the relationship between cold and phosphate-deficient stresses to elucidate how those factors control coccolith production. (45)Ca incorporation into coccoliths was more than 97% of the total (45)Ca incorporation by whole cells. In a batch culture, orthophosphate in the medium (final concentration, 28.7 muM) was rapidly depleted within 3 days, and then extracellular alkaline phosphatase (AP) activity, an indicator of phosphate deprivation, increased during the stationary growth phase. The increase in AP activity was slightly higher at 20 degrees C than at 12 degrees C. The calcification started to increase earlier than AP activity, and the increase was much higher at 12 degrees C than at 20 degrees C. Such enhancement of calcification was suppressed by the addition of phosphate, while AP activity was also suppressed after a transient increase. These results suggest that phosphate deprivation is a trigger for calcification and that a rather long induction period is needed for calcification compared to the increase in AP activity. While calcification was greatly stimulated by cold stress, other cellular activities such as growth, phosphate utilization, and the induction of AP activity were suppressed. The stimulation of coccolith production by cold stress was minimal under phosphate-sufficient conditions. The high calcification activity estimated by (45)Ca incorporation was confirmed by morphological observations of coccoliths on the cell surface under bright-field and polarization microscopy. These results indicate that phosphate deprivation is the primary factor for stimulating coccolith production, and cold stress is a secondary acceleration factor that stimulates calcification under conditions of phosphate deprivation.     

Evidence for the Involvement of Mitochondrial Respiration in Calcification in a Marine Coccolithophorid, Emiliania huxleyi

In the marine coccolithophorid, Emiliania huxleyi, CaCO₃ productionunder illumination showed a lag phase for about 3 h and thenincreased greatly. During the lag phase the rate of CaCO₃ productionin the light was similar to that in the dark. The productionof CaCO₃ in the dark was inhibited by the addition of 170 µMCCCP, 1 mM KCN and 1 mM SHAM. These results suggest that a littleproduction of CaCO₃ is supported by energy from mitochondrialrespiration, but that large amount of CaCO₃ production requiresphotosynthesis. ¹Present address: SDS Biotech K.K., Tsukuba Technology Center,Midorigahara 2-1, Tsukuba, Ibaraki, 300-26 Japan     

Induction of Phase Variation Events in the Life Cycle of the Marine Coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

Liquid-Saturated Hydrocarbons Resulting from Pyrolysis of the Marine Coccolithophores Emiliania huxleyi and Gephyrocapsa oceanica

Two nanoplanktonic marine coccolithophores, Emiliania huxleyi and Gephyrocapsa oceanica, were grown at 23°C with a 16-hour light and 8-hour darkness regimen. The cells were dried at room temperature and then subjected to pyrolysis at 100° to 500°C under anoxygenic conditions to produce hydrocarbons. Temperature-dependent profiles of the liquid-saturated hydrocarbons (saturates) produced during pyrolysis were very similar for the two strains, although the total amount was higher in E. huxleyi than in G. oceanica. The amount of saturates produced was only 0.05% to 0.15% below 200°C, but about 2.1% to 2.8% at 300°C. Their major components were normal alkanes in a series ranging from nC₁₁ to nC₃₅ with the predominant peak at nC₁₅. At 400° and 500°C most of saturates transformed into gaseous compounds. The major saturates identified in all pyrolysates were normal C₃₁ monounsaturated and diunsaturated alkenes, a series of normal alkanes, phytenes, C₂₈ sterenes, and steranes. Profiles of saturates in gas chromatography–mass spectroscopy varied with increasing pyrolysis temperature and also differed between E. huxleyi and G. oceanica. The two coccolithophores are useful candidates for the production of renewable liquid fuel through pyrolysis—especially E. huxleyi, which has higher production. The results also provide information for further studies on the characterization, source, and paleogeographic distribution of marine sediment. Received October 28, 1998; accepted February 15, 1999     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)     

Characterization of ectoenzyme activity and phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi

Three phosphate-regulated proteins in the coccolithophorid Emilianiahuxleyi were detected by the biotinylation of cell-surface proteins.Two of these phosphate-regulated proteins have reduced denaturedmolecular weights near 110 000 Da (118 078 and 110 541, respectively),while the third, and most abundant, is 69 087 Da. Inductionof the three proteins and the common marker of phosphate stress,alkaline phosphatase activity, occur in the presence of <0.25µM inorganic phosphate in batch culture. Phosphate-regulatedproteins and enzyme activity differed among E. huxleyi strains.Alkaline phosphatase is an enzyme commonly induced by phytoplanktonin response to phosphate stress in order for cells to scavengeinorganic phosphate from organic sources. In E. huxleyi, thisenzyme activity and the phosphate-regulated proteins are rapidlylost when phosphate is added back to phosphate-stressed cultures.This contrasts with the slower loss of alkaline phosphataseactivity in the dinoflagellate Prorocentrum minimum. The presenceof the three phosphate-regulated proteins and enzyme activityappear to differ somewhat among E. huxleyi strains. Based onthese differences between strains, kinetic data, growth experimentsand enzyme activities, the 69 087 Da protein may be a phosphatasewith a high specificity for 5'-nucleotides.     

The Requirement of Selenium for the Growth of Marine Coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp. (Prymnesiophyceae)

Marine coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanicaand Helladosphaera sp. were found to require selenium for theirgrowth. The optimum concen trations were 1–10 nM for SeO₂and SeO₃^2– and above 1 µM for SeO₂^4– Duringthe depletion of selenium algal growth was strongly suppressedaccompanied with the decrease in net photosynthesis. (Received November 18, 1998; Accepted April 16, 1999)     

Bloom of Emiliania huxleyi (Prymnesiophyceae) in the western South Atlantic Ocean

A monospecific bloom of the coccolithophorid Emiliania huxleyi(Lohmann) Hay & Mohler (Prymnesiophyceae) was detected offRio de La Plata at 36°S and 54°W in November 1989. Thecell densities observed were up to 6x10⁵ cells I^–1. Thisis the first record of a bloom of E.huxleyi in the area.     

Characterization of the selenite uptake mechanism in the coccolithophore Emiliania huxleyi (Haptophyta)

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ??Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H?-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H? concentration gradient may be a motive force for selenite transport. [??Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO??, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H?-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.     

Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

The effect of growth temperature on the long-chain alkenes composition in the marine coccolithophorid Emiliania huxleyi

The hydrocarbon fraction of a pure culture of Emiliania huxleyi, composed of a mixture of C31, C33, C37 and C38 polyunsaturated n-alkenes, appeared strongly dependent on the growth temperature of the alga between 8 degrees C and 25 degrees C. The total hydrocarbon content increased linearly with decreasing temperatures. C37 and C38 alkenes (which accounted for more than 90% of the total hydrocarbons) showed distinct changes in distribution compared to C31 and C33 alkenes, suggesting different biological syntheses and/or functions for these two groups of compounds. C37 and C38 alkenes and C37 methyl ketones (alkenones) all showed a trend to lower proportions of the two diunsaturated isomers and to higher proportions of the corresponding trienes with decreasing temperature. Unlike the alkenone unsaturation ratio (U37k'), ratios based on the C37 and C38 alkadi- and trienes could be linearly related to the growth temperature of E. huxleyi only between 15 degrees C and 25 degrees C. The modifications in the distribution of alkenes induced by varying temperature appeared, however, to be twice as fast as the modifications undergone by the alkenones. Although structurally and biochemically related, the distinct evolutions of alkenes and alkenones in response to changes in growth temperature might indicate that these two classes of compounds play two distinct physiological functions. The non-systematic linearity of relationships to temperature of parameters based on alkenes distribution suggested that these compounds are of limited use as paleotemperature indicator in the marine environment in contrast with the alkenones.