Naval J. Antia

Evidence of Nickel Ion Requirement for Autotrophic Growth of a Marine Diatom with Urea serving as Nitrogen Source

The diatom Cyclotella cryptica Reimann, Lewin & Guillard was unable to grow on urea added to seawater enriched with phosphate, silicate, vitamins (Bag, thiamine, biotin), trace metal ions (Fe, Mn, Zn, Mo, Cu, Co) and buffered at pH 7.5 8-0. On addition of minute Ni 2 + concentrations to this medium, the diatom showed good growth on urea compared to that on equivalent nitrate without Ni 2+ addition. The growth on urea was optimal at Ni 2+ concentrations in the range 0.1-1 gM, but good growth was also obtained at 0-01 and 10 gM N i 2 +. Higher Ni 2 + concentrations were inhibitory to growth on both urea and nitrate, while the lower Ni 2 + concentration limit for measurable urea growth was reached at 4-5 riM. In the simultaneous presence of a chelating agent, the diatom failed to grow on urea, indicating a growth requirement for free Ni 2+ ions. This Ni 2+ requirement could not be replaced by Cu 2 +, Mn 2 +, Zn 2 +, Pd 2 +, while Co z + could substitute for N i 2 + to the extent of about 20~o. At a Ni z+ concentration of 0.1 g~, the diatom yield increased to a saturation maximum with increasing urea concentration up to 5 mM and was little depressed at higher urea levels. While a Ni 2 + requirement could be universal to urease-produeing phytoplankters, it was inferred that this requirement may be greater and more stringent for C. cryptica so that other algae could meet their requirement, without Ni 2+ supplementation, from the trace metal concentrations normally present in seawater. Alternatively, they might more readily substitute Ni 2+ by a trace metal analogue (such as Co 2+) from the nutrient enrichments normally made in preparing culture media.

COMPARATIVE STUDIES ON ALDOLASE ACTIVITY IN MARINE PLANKTONIC ALGAE, AND THEIR EVOLUTIONARY SIGNIFICANCE1

Fructose diphosphate aldolase activity was examined in acetone powders and cell‐free extracts of 15 photoautotrophically grown marine planktonic species belonging to 6 algal divisions as follows: Chlorophyta: Tetraselmis maculata, Dunaliella tertiolecta; Chrysophyta: Monochrysis lutheri, Isochrysis galbana, Prymnesium parvum, Coccolithus huxleyi; Bacillariophyta: Phaeodactylum tricornutum, Skeletonema costatum, Cyclotella nana; Cryptophyta: Cryptomonas sp., Rhodomonas lens, Hemiselmis virescens; Pyrrophyta: Amphidinium carteri; Cyanophyta: Anacystis marina, Agmenellum quadruplicatum. Indications of the types of aldolase (Rutters classes) present in each alga were obtained from comparative studies of the effects of pH and of the following reagents on the activity: ethylenediamine tetraacetate, dithiothreitol, p‐chloromercuriphenyl sulfonate. Type I (higher plant‐animal type) aldolase only was indicated in the 2 chlorophytes, in I chrys‐ophyte (M. lutheri), and in 1 bacillariophyte (P. tricornutum), while the remaining algae appeared to contain either exclusively or principally Type II (bacterial‐fungal type) aldolase. The evolutionary implications of these findings are discussed.

Some cytochemical studies on the cell surface ofAmphidinium carterae (Dinophyceae)

SummaryThe surface coat of the dinoflagellateAmphidinium carterae Hulburt was examined by fluorescence and transmission electron microscopy, using various fluorochromes and cationic dyes. The overall results showed cell-surface reactions typical of acid mucopolysaccharides. The cationic dye staining revealed an outer fine fibrillar layer (15–70 nm thick) overlying a dense anionic coat (40–60 nm thick) which appeared to thicken progressively with age. In general, the structure of the amphiesmal vesicles was similar to that previously described by other investigators. However, an acidic mucopolysaccharide layer was observed on the inner surface of these vesicles. Each of these structures is traversed by 1–3 pores and at least 2 types of extrusomes are formed, the spindle trichocysts and the mucocysts. Cell to cell adhesion through the surface coat was frequently observed. Evidence was also obtained for internalization of all the surface-coat markers used.

GROWTH OF A MARINE DIATOM AND A HAPTOPHYCEAN ALGA ON PHENYLALANINE OR TYROSINE SERVING AS SOLE NITROGEN SOURCE1

The marine diatom Navicula incerta Hustedt and the haptophyte Isochrysis galbana Parke, both accustomed to grow on nitrate, were tested for their adaptive capacity for growth in axenic culture on 0.025–2.0 mM l‐, d‐, and dl‐isomers of phenylalanine and tyrosine as sole nitrogen sources. The diatom showed immediate and excellent growth (superior to nitrate) on l‐phenylalanine and l‐tyrosine but required prolonged periods of adaptation before growing on the d‐isomers, which growth was considerably slower. Isochrysis showed good growth on l‐phenylalanine after short adaptation, fair growth on low but poor growth on high concentrations of l‐tyrosine and no growth on the d‐isomers of both amino acids. The racemate (dl‐mixture) of each amino acid produced growth responses, from both algae, inferior to those of the corresponding l‐isomer, indicating competitive inhibition from the d‐isomer in the presence of equal amounts of its counterpart. Increasing tyrosine concentration was beneficial to growth of Navicula but markedly inhibitory or toxic to that of Isochrysis. Phenylalanine concentration appeared to benefit growth of both algae. Ecological considerations suggest that N. incerta may be habituated to utilize both aromatic amino acids in its native milieu.

l-THREONINE DEAMINASE IN MARINE PLANKTONIC ALGAE. II. DISULFIDE AND SULFHYDRYL GROUP REQUIREMENTS OF ENZYME ACTIVITY IN TWO CRYPTOPHYTES1

The “biosynthetic”l threonine (deaminating) dehydratase of 2 cryptophytes (Chroomonas salina and Hemiselmis virescens) showed sensitive inhibition from all thiols tested (dithiothreitol, cysteine, etc.) but no effect from ascorbic acid or reduced NAD. By contrast, the enzyme activities from 5 noncryptophyceaen unicellular algae (2 cyanophytes, 1 rhodophyte, 1 diatom, 1 chlorophyte) were generally not affected by any of these reagents. The thiol reagent inhibition of the cryptophyte enzymes (1) achieved saturation with 60–70% reduction in activity, (2) was considerably reduced by pretreatment of the enzymes with l‐threonine and l‐isoleucine, and (3) was partially reversed by subsequent treatment with arsenite and exposure to air. It was deduced that such inhibitions were caused by thiol‐specific reduction of enzyme‐protein disulfide groups essential for the full expression of activity and that these groups were susceptible to ready reductive cleavage and oxidative restoration. This disulfide requirement, unique to the cryptophytes, may be the first recorded case of such a property of threonine dehydratase from all forms of life hitherto studied. The additional activity requirement of the cryptophyte enzymes for sulfhydryl groups (which requirement was common to all the algal enzymes) was confirmed (1) by the study of their sensitivity to inhibition from mercurials and disulfide‐sulfhydryl exchanging reagents, and (2) by the partial reversal of these inhibitions from subsequent treatment with dithio‐threitol. Both cryptophyte enzymes were densitized to feedback inhibition from l‐isoleucine by prior exposure to subinhibitory concentrations of HgCl2 or dithiodipyridine.

l-Threonine deaminase in marine planktonic algae

Summary1.The “biosynthetic” l-threonine (deaminating) dehydratase activity of 7 marine planktonic species from 5 classes of algae showed high substrate specificity toward l-threonine, with the exception of one alga. The algal extracts deaminated l-serine and l-allothreonine at rates which were 20–25 and 5–10%, respectively, of that of l-threonine, and these reaction were inhibited by l-isoleucine. d-Threonine, d-serine, l-homoserine, and l-O-methylthreonine were ineffective as substrates.2.Extracts of the cryptophyte Chroomonas salina were exceptional in showing about twice the activity with l-serine relative to l-threonine. The reaction with l-serine was insensitive to inhibition by l-isoleucine and differed, in several respects from that with l-threonine. It was inferred that this algal extract contains a specific l-serine deaminase in addition to the regular l-threonine deaminase.3.The rate of deamination of l-threonine by the algal extracts was not affected by the simultaneous presence of l-allothreonine but was markedly inhibited by l-serine and l-homoserine.4.Kinetic studies of the effects of graded concentrations of the substrate analogs at a fixed (saturating) substrate level or of substrate concentration at fixed analog levels indicated that l-serine inhibited the threonine deamination by irreversible competition with the substrate, whilst l-homoserine affected this reaction by an unknown (presumably allosteric) mechanism not involving substrate competition.

AGGLUTINATION OF THE CHLOROPHYCEAN FLAGELLATE DUNALIELLA TERTIOLECTA BY TREATMENT WITH LECTINS OR DIVALENT CATIONS AT ALKALINE pH1

Washed cells of Dunaliella tertiolecta Butcher became immobile and agglutinated upon exposure to 100–400 μ/mL lectins in NaCl solution. The agglutinations were strongest with Limulus polyphemus agglutinin and wheat‐germ agglutinin, moderate with soybean agglutinin and weakest with Concanavalin A. All lectin‐induced agglutinations were inhibited or mitigated by the simultaneous presence of specific lectin‐binding sugars. The differential sensitivity of the alga to these lectins suggested that sialic acid and/or N‐acetyl‐D‐glucosamine might be the predominant lectin‐receptor sugars in the algal surface coat, with N‐acetyl‐D‐galactosamine likely present as a lesser component. In the absence of lectins, the divalent cations Mg2+, Ca2+ or Mn2+ also caused agglutination, but this process required an alkaline pH of at least ca. 8.6–8.9. Such cation‐induced agglutination was reversibly inhibited by the cation complexing agent EDTA as well as by lowering the pH below 8.0. SEM observations of the agglutinations revealed random flagellar attachments as well as direct body contact between agglutinated cells.