Effects of NH4+ assimilation on dark carbon fixation and β-1,3-glucan metabolism in the marine diatom Skeletonema costatum (Bacillariophyceae)

The effects of NH4+ assimilation on dark carbon fixation and beta-1,3-glucan metabolism in the N-limited marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) were investigated by chemical analysis of cell components and incorporation of C-14-bicarbonate. The diatom was grown in pH-regulated batch cultures with a 14:10 h LD cycle until N depletion. The cells were then incubated in the dark with C-14-bicarbonate, but without a source of N for 2 h, then in the dark with 63 mu mol.L-1 NH4+ for 3 h. Without N, the cellular concentration of free amino acids was almost constant (similar to 4.5 fmol.cell(-1)). Added NH4+ was assimilated at a rate of 12 fmol.cell(-1).h(-1), and the cellular amino acid pool increased rapidly (doubled in <1 h, tripled in <3 h). The glutamine level increased steeply (45 x within 3 h), and the Gln/Glu ratio increased from 0.1 to 2.4 within 3 h. The rate of dark C fixation during N depletion was only 1.0 fmol.cell(-1).h(-1). The addition of NH4+ strongly stimulated dark C fixation, leading to an assimilation rate of 4.0 fmol.cell(-1).h(-1), corresponding to a molar C/N uptake ratio of 0.33. Biochemical fractionation of organic C-14 showed no significant C-14 fixation into amino acids during N depletion, but during the first 1-2 h of NH4+ assimilation, amino acids were rapidly radiolabeled, accounting for virtually all net C-14 fixation. These results indicate that anaplerotic beta-carboxylation is activated during NH4+ assimilation to provide C, intermediates for amino acid biosynthesis. The level of cellular beta-1,3-D-glucan was constant (16.5 pg.cell(-1)) during N depletion, but NH4+ assimilation activated a mobilization of 28 % of the reserve glucan within 3 h. The results indicate that beta-1,3-glucan in diatoms is the ultimate substrate for beta-carboxylation, providing precursors for amino acid biosynthesis in addition to energy from respiration.