Elisabeth W. de Vrind-de Jong

GPA, a calcium-binding protein in the coccolithophorid Emiliania huxleyi (Prymnesiophyceae)

Intracellular polysaccharide fractions were isolated from calcifying B‐type cells of Emiliania huxleyi and separated by electrophoretic fractionation. In all fractions, the polysaccharide was immunologically related to the polysaccharide of (extracellular) B‐type coccoliths (CP‐B) and not to polysaccharides of A‐type coccoliths (CP‐A). Most polysaccharide fractions also contained protein material. The fraction with the largest proportion of protein was used to raise antibodies. The resulting antiserum, α‐BP, contained antibodies against both CP‐B‐ and protein‐epitopes. The antibodies specific for polysaccharide‐epitopes reacted with intracellular polysaccharide fractions of B‐type cells only. In contrast, the antibodies specific for protein‐epitopes reacted with the intracellular fractions of B‐type as well as A‐type cells. With immunolocalization, the presence of protein antigen in a layer surrounding both types of cells was demonstrated. A cDNA library of E. huxleyi was screened with α‐BP, and a gene called gpa was isolated. The open reading frame of gpa was found to encode a protein (GPA) of 36,608 D, containing, inter alia, 24% acidic residues (18% glutamic acid and 6% aspartic acid), 12% proline, and 23% alanine. GPA has two repeats, one containing a sequence resembling the Ca2+‐binding loop of EF‐hands. Overproduction of GPA in a prokaryotic system yielded a dimeric product capable of binding Ca2+. The possible role of GPA in the formation of coccoliths in E. huxleyi is discussed.

Composition of a methylated, acidic polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner

The water-soluble, acidic polysaccharide isolated from the coccoliths of the alga Emiliania huxleyi (Lohmann) Kamptner contains residues of the following sugars: l-galactose, d-glucose, d-mannose, l-mannose, l-rhamnose, l-arabinose, d-ribose, d-xylose, 6-O-methyl-d-mannose, 6-O-methyl-l-mannose, 2,3-di-O-methyl-l-rhamnose, 3-O-methyl-d-xylose, and d-galacturonic acid. l-Mannose, 6-O-methyl-d-mannose, 6-O-methyl-l-mannose, and 2,3-di-O-methyl-l-rhamnose are novel constituents of a polysaccharide. In addition, the presence of sulphate groups was found. Galacturonic acid and sulphate in the polysaccharide bind Ca2+ ions apparently in a ratio of one mol of Ca2+ per mol of acidic residue. This feature is relevant for the proposed matrix function of the polysaccharide in the formation of the calcified cell-wall plates (coccoliths) of the alga.

COCCOLITH-ASSOCIATED POLYSACCHARIDES FROM CELLS OF EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler, a unicellular calcifying alga, consist of calcite closely associated with an acidic, Ca2+‐binding polysaccharide. This polysaccharide is thought to play a regulatory role in coccolith synthesis by interfering with CaCO3 crystallization. Here we show that the polysaccharides from three different strains, A 92, L and 92 D, all inhibit the precipitation of CaCO3 in vitro to the same extent. The monosaccharide compositions of the A 92 and L polysaccharide are similar. The 92 D material, however, deviates from the other two: it contains significantly lower amounts of methylated sugars and ribose, and elevated levels of rhamnose and galactose. It also contains antigenic determinants not detected in the A 92 and L polysaccharides. In contrast to the latter two macromolecules the 92 D polysaccharide migrates as two bands upon polyacrylamide gel electrophoresis, possibly resulting from complexing with small amounts of protein. The coccolith polysaccharide from L cells, cultured at an elevated growth rate, also migrates as two bands. This phenomenon is due to an increase in molecular size distribution. The results suggest that certain properties of the molecule may be subject to variation without interfering with its function.

INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE (PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using45Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark‐incubated cells and in cells exposed to a repeating light‐dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.

Molar mass determination of the polysaccharide associated with coccoliths of emiliania huxleyi

Abstract Emiliania huxleyi is a marine alga which produces coccoliths, elaborate calcified structures consisting of CaCO 3 and a water-soluble acidic polysaccharide. The polysaccharide was subjected to light scattering measurements to determine the weight-average molar mass ( M w ) and osmometry to assess the number-average molar mass ( M n ). Values of M w of 88,600 ± 3200 g/mol and of M n of 49,100 ± 1500 g/mol were found. These experiments were performed with a polysaccharide mainly in the Na + form. Replacement of Na + by Ca 2+ did not significantly affect the weight-average molar mass or the second virial coefficient. The values of the refractive index increment of these salts were different [(d n /d c )Na + = 0.145 ± 0.004 ml/g; (d n /d c ) Ca 2+ = 0.090 ± 0.002 ml/g], indicating a possible conformational change in the molecule.

Manganese Transformations by Marine Bacillus Species

A wide variety of micro-organisms promote the oxidation or reduction of manganese, through indirect or direct mechanisms. An example of the latter category is Bacillus SGI, a marine organism isolated from a near-shore manganese sediment. Its mature dormant spores catalyze the oxidation of Mn 2+ to Mn 4+. The process requires molecular oxygen and is catalyzed by a spore coat component. The manganese oxide (Mn02) produced remains bound to the spore surface. The vegetative cells do not have the oxidizing capacity. They are able to reduce Mn4+ to Mn2+ under low-oxygen conditions. The reducing activity has a pH optimum of 7.5 and is abolished by preheating of the cells at 90° C for 5 minutes. Addition of mercuric chloride (HgCl2) (final concentration 0.01%) to cells which are actively reducing manganese oxide causes immediate cessation of the process. Manganese oxide reduction is also inhibited at high oxygen tensions and by inhibitors of the electron transport system. Bacillus cells contain b- and c-type cytochromes which are oxidized in situ when manganese oxide is added to an anoxic cell suspension. The results suggest that vegetative cells may use the manganese oxide formed by the spores from which they germinate as a terminal electron acceptor. The possible applications of manganese transforming micro-organisms in human society are discussed.