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Chlorella strain (UTEX 27) maintains optimal photosynthetic capacity when growing photoautotrophically in the presence of ammonium. Nitrate-grown photoautotrophic cells, however, show a drastic loss of chlorophyll content and ribulose-1,6-bisphosphate carboxylase/oxygenase activity, resulting in a greater than 10-fold decrease in photosynthetic capacity and growth rate. Nitrate-grown cells are not deficient in protein content, and under mixotrophic and heterotrophic conditions, the alga can utilize nitrate as well as it does ammonium. The alga metabolizes both glucose and acetate in the dark with a doubling time of 5 to 6 hours. However, its growth on acetate is inhibited by light. Ribulose-1,6-biphosphate carboxylase/oxygenase activity correlates well with photosynthetic capacity, and glucose 6-phosphate dehydrogenase and hexokinase activities are altered in a manner consistent with the availability of glucose in growing cells. The alga appears to assimilate ammonium under photoautotrophic conditions primarily via the glutamine synthetase pathway, and shows an induction of both NADH and NADPH dependent glutamate dehydrogenase pathways under mixotrophic and heterotrophic conditions. Multiple isoforms are present only for hexokinase and glucose 6-phosphate dehydrogenase. Etiolated nitrate-grown cells resume greening and increase their photosynthetic capacity after about 6 hours of incubation in the presence of ammonium under photoautotrophic conditions. Similarly, the loss of photosynthetic capacity in ammonium-grown photoautotrophic cells commence about 9 hours after their transfer to heterotrophic nitrate containing media.

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Although the existence of isozymes of ornithine carbamoyltransferase (carbamoylphosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) in higher plants has been reported, and the possibility exists that one or more of these operates catabolically to produce ornithine and carbamoylphosphate from citrulline and inorganic phosphate, no proof has been forthcoming. In view of the fact that many unicellular algae degrade arginine via arginine deiminase to citrulline and ammonium, and that the pathway of utilization of citrulline is unknown, we decided to investigate the possibility of the presence of a catabolic form of ornithine carbamoyltransferase in three microalgae known to have arginine deiminase activity. These were Chlorella autotrophica, Chlorella saccharophila, and Dunaliella tertiolecta. Our results show that the properties of OCT from these three algae are similar to OCTs from many higher plants with respect to general kinetics (K(m) values for ornithine and carbamoylphosphate), substrate inhibition by ornithine at high pHs, apparent sequential ordered kinetic mechanisms and paucity of apparent regulatory properties. Our data indicate an exclusively anabolic role of ornithine carbamoyltransferase in these algae.

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Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent K(m) values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments.

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A new spectrophotometric procedure is described for determining glutamate-dependent activities of aspartate aminotransferase, alanine aminotransferase, and ornithine aminotransferase with NADPH-linked glutamate dehydrogenase (GDH) from nitrate-grown Stichococcus bacillaris. The algal NADPH-GDH is highly specific for oxoglutarate and can catalyze the reduction of this keto acid in the presence of high glutamate concentrations, and thus is suitable for the measurement of oxoglutarate produced in glutamate-dependent amino-transferase reactions. The alga produces large amounts of NADPH-GDH which can be adequately purified in a few simple steps. The purified enzyme can be stored at 4 degrees C for several weeks without any detectable loss of activity. The algal NADPH-GDH can also be used for the estimation of small amounts of oxoglutarate in aqueous extracts.

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Chlorella autotrophica, a euryhaline marine alga, and Stichococcus bacillaris, a salt-tolerant soil alga, grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining high levels of NADPH-glutamate dehydrogenase. Nitrate reductase showed no change in MSX-adapted cells. For both species, MSX-adapted cells retained their capacity to accumulate proline in response to salinity, and in S. bacillaris no major shift was observed in the presence of MSX toward the accumulation of sorbitol. Following transfer from 33 to 150% artificial seawater (ASW), both algae exhibited increases in organic solute levels without a lag. Within 6 h of this sudden increase in salinity, the levels of proline in C. autotrophica and of proline and sorbitol in S. bacillaris were similar to those found in steady state 150% ASW cultures. Following transfer from 33 to 150% ASW, S. bacillaris continued [(14)C] bicarbonate photoassimilation at a normal rate and maintained active enzymes of nitrogen assimilation. The incorporation of [(14)C]phenylalanine into proteins was inhibited for about 30 minutes in MSX-free cells and 90 minutes in MSX-adapted cells following transfer from 33 to 150% ASW; the recovery after these lag periods was almost complete.

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Two forms of glutamine synthetase (GS(1) and GS(2)) have been separated from cells of Stichococcus bacillaris by fast protein liquid chromatography. The activities of the two isoenzymes were influenced by the composition of the media employed; thiol reagents were essential for stabilizing GS(2) but they suppressed GS(1) activity. The activity of each isoenzyme was, therefore, determined following separate purification procedures. Growth conditions influenced both isoenzymes; GS(2) showed maximum activity under photoautotrophic conditions, whereas GS(1) showed maximum activity under heterotrophic conditions.

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The green euryhaline flagellate Chlamydomonas pulsatilla Wollenweber, isolated from a coastal marine environment, was grown exponentially over the salinity range of 10 to 200% artificial seawater (ASW). The cellular volume and aqueous space of the alga, measured by [(14)C] mannitol and (3)H(2)O tracer analyses of centrifuged cell pellets, ranged between 2.3 and 3.1 picoliters and between 1.5 and 2.1 picoliters, respectively. The nonaqueous space determined in those analyses (28-35%) was consistent with the cell composition of the alga. The glycerol content of the alga increased almost linearly with increasing salinity; its contribution to intracellular osmolality at 200% ASW was about 57%. The contribution of amino acids and soluble carbohydrates to the cell osmotic balance was small. Intracellular ion concentrations determined by analyzing centrifuged cell pellets of known [(14)C]mannitol space by atomic absorption spectrophotometry, and by neutron activation analyses of washed cells were similar. At 10% ASW, potassium and magnesium were the major cations, and chloride and phosphate were the major anions. The sodium and chloride content of the alga increased with increasing salinity; at 200% ASW the intracellular concentration of both sodium and chloride was about 400 millimolar. The intracellular osmolality (pi(int)) matched closely the external osmolality (pi(ext)) over the entire salinity range except at 10% ASW where pi(int) exceeded pi(ext) by 120 to 270 milliosmoles per kilogram H(2)O.

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Stichococcus bacillaris Naeg., a green soil alga, can grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining a high level of NADPH-glutamate dehydrogenase activity. MSX-grown cells can utilize both NH(4) (+) and NO(3) (-) as nitrogen source for growth. [(14)C]Methylammonium is not metabolized by S. bacillaris, and is transported by a carrier system that obeys Michaelis Menten kinetics, and is insensitive to MSX.

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The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO(3) (-)-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH(4) (+) have high NADPH-GDH activity, the levels of which increase with increasing NH(4) (+) supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH(4) (+)-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH(4) (+).

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Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.

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Methylamine, ethylamine, and dimethylamine (10 micromolar) are taken up and concentrated 600 to 6,000-fold by Cyclotella cryptica. Methylamine is concentrated most strongly, and its accumulation and retention are relatively insensitive to external pH but strongly inhibited by 30 millimolar external K(+). Accumulation and retention of ethyl- and dimethylamine, on the other hand, are strongly affected by external pH and less sensitive to external [K(+)]. Intracellular pH, as estimated from neutral red staining and quenching of 9-aminoacridine fluorescence, was between 4 and 5, with the central vacuole being the major acidic compartment. The accumulation of ethyl- and dimethylamine could result from diffusion of the uncharged amine across the membrane(s) and passive equilibration of the charged form (R-NH(3) (+)) inside and outside the cell. Differences in the accumulation ratio and the ion dependence for methylamine uptake relative to ethyl- and dimethylamine uptake suggests that a different mechanism is responsible for the concentration of the simpler amine.

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Navicula pelliculosa and an associated Flavobacterium sp. were isolated from the epiphyton of Scirpus maritimus, an emergent macrophyte growing in a brackish drainage dyke. Both micro-organisms possessed active transport systems for glucose uptake. In N. pelliculosa the transport system was fully induced in the dark in the absence of glucose, and subsequently inactivated when transferred to the light in the absence of the substrate. The presence of glucose during the dark induction period prevented the achievement of maximum specific activity of the transport system, while incubation at a high light intensity with or without the presence of the substrate resulted in a very marked inhibition of glucose uptake. Inhibition in the light was partially offset by blocking photosynthetic electron flow with 3'(3,4 dichlorophenyl)1'1' dimethyl urea. The transport system accumulated 3-O-methyl glucose against a concentration gradient and was highly specific for glucose as there was no competition by most of the other sugars tested. However, 6-deoxyglucose was taken up instead of glucose and this suggested that glucose was transported in a non-phosphorylated state, whereas inhibition of glucose transport activity with dicyclohexylcarbodimide implicated the involvement of an adenosine triphosphatase on the cell membrane. Inhibitors of oxidative phosphorylation tetrachlorosalicylaniline and carbonylcyanide m-chlorophenylhydrazone also inhibited glucose transport activity. The affinity of the diatom for glucose was greater than that shown by the bacterium, but the Km for glucose transport, 1.5x10-5M was too high to allow effective removal of glucose at in situ concentrations.

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Navicula pavillardi Hustedt, a marine, littoral, pennate diatom, can grow in the dark on glutamate or on the complex organic supplements tryptone or yeast extract. Growth on glutamate in the dark took place without an initial lag phase, whereas growth on tryptone began only after a 2-day lag phase that could be abolished by the simultaneous presence of glucose. Lactate inhibited growth in the dark on glutamate, but not photoautotrophic growth. Relatively low concentrations of glutamine inhibited photoautotrophic growth. The observed doubling time for heterotrophic growth on glutamate or tryptone was about 70 h, compared with a doubling time of 24 h under optimal photoautotrophic conditions. Glucose did not decrease the doubling time in the dark on tryptone. The assimilation efficiency for glutamate was 41%. The estimated necessary uptake rate for glutamate to account for the observed heterotrophic doubling time on glutamate was close to those measured with isotope techniques. The kinetic parameters for glutamate uptake, which followed Michelis-Menten kinetics, were Ks = 0.018 mM, and Vmax = 7.0 X 10(-10) mumol per cell per minute. Although several amino acids served as sole nitrogen sources for photoautotrophic growth and were demonstrated by the use of isotope techniques to enter the cells, they could not be used as substrates for growth in the dark. Glucose was not taken up to a significant extent except by cells grown in the presence of tryptone. Lactate was taken up only by dark-grown cells. Results of preliminary studies on the metabolic fate of several uniformly labeled amino acids are presented.

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Melosira nummuloides, clone Mel-3, shows a very high specificity with regard to its ability to take up organic substrates. Amino acids supplied in the medium at 1 X 10(-4) M are taken up at initial rates of the same order of magnitude as that of photoassirnilation of COj. However, sugars, sugar alcohols, or organic acids supplied at the same concentration are not taken up. The mechanism for uptake of amino acids appears to require energy, since tlie uptake of the amino acid analog α-aminoisobutyric acid is strongly inhibited by 2 f-dinitrophenul. The uptake mechanism does not appear to be inducible. The ability of M. numinuloides to utilize amino acids as a nitrogen source is quite restricted. Arginine, ghttamine, asparagine, proline, and glutamic acid were good nitrogen sources. Seventeen other amino acids, including α-aminoisobutyric acid, were unsatisfactory for growth, although they were rapidly taken up from the medium.

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Campbell, Ann E. (Woods Hole Oceanographic Institution, Woods Hole, Mass.), Johan A. Hellebust, and Stanley W. Watson. Reductive pentose phosphate cycle in Nitrosocystis oceanus. J. Bacteriol. 91:1178-1185. 1966.-Assays in cell-free extracts of Nitrosocystis oceanus, a marine chemoautotrophic bacterium, have demonstrated the presence of all of the enzymes of the reductive pentose phosphate cycle, with activities high enough to account for the normal growth rate of the cells. Studies on ribulosediphosphate carboxylase activity in these extracts showed that it is inhibited by MgCl(2) (30% at 0.01 m), MnCl(2) (70% at 0.01 m), NaCl and KCl (100% at 0.5 m, 63% at 0.2 m), and by sulfate (35% at 0.01 m); phosphate, glutathione, and ethylenediaminetetraacetic acid had no effect. The bacterial enzyme differs from the spinach enzyme with respect to its affinity for bicarbonate and its pH optimum. Whole cells were incubated with C(14)O(2), and the acid-soluble fraction was analyzed by paper chromatography and autoradiography. Phosphoglyceric acid and the sugar phosphates were the earliest labeled compounds; several amino acids and organic acids were also labeled. It is concluded that N. oceanus incorporates CO(2) primarily via the reductive pentose phosphate cycle.

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