Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica

Abstract

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 NOj--grown cells, and at maximum levels in nitrogenstarved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH4W have high NADPH-GDH activity, the levels of which increase with increasing NH4' supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH4V-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 ofthe alga in the presence of NW.Numerous studies have dealt with the uptake and utilization by marine microalgae of various nitrogen sources found in seawater (5,8,14,17,26). However, only a few of these have focused on metabolic pathways of nitrogen assimilation in these organisms (3,7,26). Furthermore, there appears to be a need to examine nitrogen assimilation in microalgae in relation to metabolic adaptation to salinity fluctuations. This consideration is particularly important for euryhaline microalgae which are subject to rapid changes in salinity in their natural environments (2). Metabolic activities such as photosynthesis have been shown to react to changes in external salt concentrations (4, 10, 13), but little is known about the effect of external salinity on nitrogen assimilation in microalgae.Eukaryotic green plants have the enzymic potential for the assimilation of NH4' by the reductive amination of 2-oxoglutarate either by GDH2 or by the combined action of GS and GOGAT (19,24,26). In higher plants, the GS-GOGAT cycle appears to be the primary pathway of NH4' assimilation, and GDH plays only a minor role (19,24). Also, for some microalgae such as Platymonas striata (7) and Chiamydomonas reinhardii (6), only the activities of GS and GOGAT were found to be sufficient to carry out the required rate of nitrogen assimilation. However, many microalgae contain high levels ofGDH activities (9,20,(22)(23)(24)27) provided by Everest and Syrett (9) for NO3-grown Stichococcus bacillaris which has an inducible NADPH-GDH with a low Km for NH4' (KmaPP, 1 mM). Several strains of Chlorella are also found to contain an inducible NADPH-GDH; however, in this case the enzyme is synthesized in NH4-grown cells (9,20,22,23,27). Shatilov and Kretovich (22) (9) have also shown that C. vulgaris cannot utilize NH4' in the presence of MSX. However, the lack of NH4' assimilation by MSX-treated cells of C. vulgaris shown in their study does not necessarily exclude a role for NADPH-GDH in NH41 assimilation of this alga, as the experimental conditions used were such that the activity of NADPH-GDH disappeared rapidly in both cells incubated with or without MSX.In this report, we present a study of nitrogen assimilation by the euryhali...

PATHWAYS OF AMMONIUM ASSIMILATION IN THE SOIL ALGA STICHOCOCCUS BACILLARIS NAEG.

“…One of the major objections to the proposal that NADPH-GDH plays a significant role in nitrogen assimilation in algae is its high apparent K^^ for ammonium (Mifiin & Lea, 1976;Stewart et al 1980). The apparent K^ for ammonium of NADPH-GDH isolated from Chlorella autotrophica was found to be very high (20 mM; Ahmad & Hellebust, 1984). However, Everest & Syrett (1983) recently reported that NADPH-GDH from Stichococcus bacillaris Naeg.…”

“…The synthetase activity of GS was determined by the procedure described previously (Ahmad & Hellebust, 1984, 1985. The activities of NADPH-and NADH-GDH were determined at pH 7 5 and 8-1, respectively, by the spectrophotometric method described previously (Ahmad & Hellebust, 1984, 1985.…”

“…For growth in the dark, glucose was supplied at 10 mM. In MSX studies, freshly prepared, filter-sterilized solutions of MSX were added to autoclaved growth media to a final concentration of 400/*M. Cell densities were determined as described previously (Ahmad & Hellebust, 1984). Growth constants {/i) were calculated as In 2/t^, where t^ is the doubling time in hours in the exponential growth phase.…”

“…The activities of NADPH-and NADH-GDH were determined at pH 7 5 and 8-1, respectively, by the spectrophotometric method described previously (Ahmad & Hellebust, 1984, 1985. Ferredoxin-dependent GOGAT activity was determined radio-chemically after Cullimore & Sims (1981a).…”

“…Other algae, such as Stichococcus bacillaris (Everest and Syrett, 1983) and several Chlorella species (Morris & Syrett, 1965;Tally, White & Schmidt, 1972;Israel etal, 1977;Shatilov & Kretovich 1977;Ahmad & Hellebust, 1984) possess high levels of NADPH-GDH, and may assimilate ammonium via this pathway. Recently, we demonstrated that Chlorella autotrophica is able to assimilate ammonium via NADPH-GDH, when grown in the presence of methionine sulphoximine (MSX), a specific inhibitor of GS (Ahmad & Hellebust, 1985).…”