The pathway of ammonia assimilation in Bacteroides fragilis.

Abstract

The enzymes in ammonia assimilation pathways have been examined in a strictly anaerobic bacterium Bacteroides fragilis. The activities of NADPH-and NADH-linked glutamate dehydrogenase and glutamine synthetase were demonstrated in extracts of cells, but very low activity of NADPH-dependent glutamate synthase and no activity of alanine dehydrogenase were demonstrated. Both activities of the glutamate dehydrogenase were not distinguished electrophoretically, indicating the presence of a dual pyridine nucleotide-specific enzyme. At low concentrations of ammonia in batch cultures and at low dilution rates of continuous flow cultures, higher activities of glutamate dehydrogenase were found in the cells. The values of Km of NADPH-linked glutamate dehydrogenase were 0.8 mM, 0.15 mM, and 7 uM for ammonia, 2-oxoglutarate, and NADPH, respectively. Although glutamine synthetase activity was also higher in cultures with limited ammonia and at low dilution rates of continuous cultures, this enzyme may not be important for ammonia incorporation into amino acids, since cell growth was not affected by the addition to the culture of methionine sulfoximine, a glutamine synthetase inhibitor. This evidence suggests that ammonia assimilation is mainly carried out by the glutamate dehydrogenase in B. fragilis.

Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1

“…Sucrase, inulinase, glutamate dehydrogenase (cytoplasmic marker) (54), and succinate dehydrogenase (membrane marker) (22) activities were assayed in cell-free culture supernatants, sonicated cell extracts, and cytoplasmic, periplasmic, and membrane cell fractions. Alkaline phosphatase activity was assayed in cell extracts (5 (wt/vol).…”

The NADH-dependent glutamate dehydrogenase enzyme of Bacteroides fragilis Bf1 is induced by peptides in the growth medium

“…NADP(H)-specific GDH enzymes usually catalyse the assimilation of ammonia by reductive amination of a-ketoglutarate to form L-glutamate (anabolic), while NAD( H)-dependent GDH enzymes catalyse the reverse reaction (catabolic). Yamamoto et al (1984) have suggested that GDH activity may play the primary role in nitrogen assimilation in B. fragilis, since the levels of GDH were very much higher than GS and cells which had their GS activity inhibited with methiosulfoxamine did not show a decrease in cell growth rate.…”

“…GDH activity was assayed spectrophotometrically (DU65O spectrophotometer, Beckman) by following the decrease in A,,, during oxidation of NADPH or NADH (Yamamoto et al, 1984). The reactions were conducted at 25 "C in 1.0 ml reaction mix containing 100 mM Tris/HCl (pH 8.0), 40 mM NH,Cl, 5 mM a-ketoglutarate and 0.15 mM NADPH and were initiated by the addition of 50 pl CFE (75 pg total protein).…”

“…The two GDH enzymes were first identified by Yamamoto et al (1984) who concluded that one had dual specificity for pyridine nucleotides "AD (P)H], while the other was NADH-specific. Characterization of the activities of these enzymes showed that the NAD(P)H-dependent GDH was induced by low concentrations of ammonia, whereas high ammonia concentrations were thought to partially induce the NADHdependent GDH.…”

“…Ammonia is not replaced by amino acids as the source of nitrogen for growth (Varel & Bryant, 1974), while peptides can also be utilized. The enzymes involved in the assimilation of nitrogen are a structurally novel form of GS (GSIII) (Southern et al, 1986(Southern et al, ,1987Hill et al, 1989), two GDHs (Yamamoto et al, 1984(Yamamoto et al, , 1987aSaito et at., 1988) and GOGAT. These enzymes have been shown to be regulated by the levels of ammonia available.…”