Basant Bhandari

Stable isotope studies on the oxidation of ammonia to hydroxylamine by nitrosomonas europaea

Lees [l] and Hofman and Lees [2] observed that hydroxylamine accumulated when ammonia was oxidised by intact cells in the presence of hydrazine. Hydroxylamine was first oxidised with Iz to nitrite and determined calorimetrically. These results were confirmed by Yoshida and Alexander [3], who detected hydroxylamine by thin-layer chromatography [4]. Both these procedures, however, may not be specific for hydroxylamine. We now describe a method whereby hydroxylamine produced during the oxidation of ammonia by Nitrosomonas is isolated directly as an oxime. Using this technique we have employed stable isotopes to show that ‘sNH&l is oxidised to “NH,OH and that the oxygen of hydroxylamine is derived from ‘802 and not from HzlsO.

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max.

Nitrate, nitrite and nitrous oxide were denitrified to N2 gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N2 was produced from either K15NO3 or Na15NO2 by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N2O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of15NO3-or15NO2-and the produciton of15N2 was 2:1 and for N2O utilization and N2 production it was 1:1. Some of the15N2 gas produced by denitrification of15NO3-in bacteroids was recycled via nitrogenase into cell nitrogen.

ATP production coupled to the denitrification of nitrate in Rhizobium japonicum, grown in cultures and in bacteroids from Glycine max

The denitrification of nitrate in membrane fractions of Rhizobium japonicum CC705 grown in culture solutions with nitrate and those from bacteroids of Glycine max was coupled to ATP production. The ATP/2e− ratios for electron transfer from succinate and NADH to NO− 3 and O2 in membrane fractions of R. japonicum grown with nitrate were: succinate to O2, 2.0; succinate to nitrate, 1; NADH to O2, 3; NADH to nitrate, 2. In membrane fractions of bacteroids the value for succinate to nitrate was also 1.

Ammonia and O2 uptake in relation to proton translocation in cells of Nitrosomonas europaea

The uptake of ammonia and O2 by washed cells of Nitrosomonas has been followed simultaneously and continuously using electrode techniques. The stoichiometry of NH4+oxidation, O2 uptake and NO2-production was 1 : 1.5 : 1.0 and for NH2OH oxidation a ratio of 1 for O2 : NO2-. A variety of inhibitors of electron transport and metals as well as uncouplers restricted ammonia uptake more markedly than O2 utilization. There is good evidence for the involvement of copper in the NH4+uptake process.A quinacrine fluorescence technique has been used to study the proton extrusion by washed cells on adding NH4Cl and NH2OH respectively as substrates. The uptake of NH4+was followed by the extrusion of H+ and this process was depressed by those inhibitors which were also effective in the electrode experiments. A requirement for copper is also established for the translocation of protons into the medium, resulting from the uptake of NH4+by cells.

Glutamine synthetase, glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max

The growth yields of three strains of Rhizobium japonicum (CB 1809, CC 723, CC 705) in culture solutions containing L-glutamate were about twice those grown with ammonium. The activities of glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.4) were dependent on the nitrogen source in the medium and also varied with growth. Both NADPH-and NADH-dependent glutamate synthase (GOGAT; EC 1.4.1.13) and NADPH-dependent GDH were found in strains grown with either glutamate or ammonium but NADH-linked GDH was only detected in glutamate-grown cells. Glutamine synthetase was adenylylated in cells grown with NH4+(90%) and to lesser extent in those grown with L-glutamate (50%). In root nodules produced by the three strains in Glycine max (L.) Merr., the bulk of GS was located in the nodule cytosol (60–85%). The enzyme was adenylylated in bacteroids (43–75%) and in the nodule tissues (52–68%). The enzyme in cell-free extracts of Rh. japonicum (CC 705) grown in culture solutions containing glutamate and in bacteroids (CC 705) was deadenylylated by snake-venom phosphodiesterase. L-methionine-DL-sulfoximine restricted the incoporation of 15N-labelled (NH4)2SO4 into cells of strains CB 1809 and CC 705, as well as in bacteroids of strain CC 705. It is noteworthy that appreciable activities for GDH were found in the free-living rhizobia grown on glutamate. Thus the presence of an enzyme does not necessarily imply that a particular pathway is operative in assimilating ammonium into cell nitrogen. Based on 15N studies, the GS-GOGAT pathway of rhizobia (strains CB 1809 and CC 705) is important when grown in culture solutions as well as in bacteroids from root nodules of G. max.

Some properties of glutamine synthetase from Rhizobium japonicum strains CC705 and CC723

Glutamine synthetase (EC 6.3.1.2) has been purified from Rhizobium japonicum strains CC705 and CC723 respectively. The purified enzyme, which had a molecular weight of 720,000 (both strains) contained 12 subunits each of 60,000. The enzyme assayed by the γ-glutamyltransferase method had Km values for l-glutamine and hydroxylamine of 7.7 and 1.2 mM respectively. The inhibition of γ-glutamyltransferase by l-glutamate and ammonia was competitive for l-glutamine and non-competitive for hydroxylamine. The transferase activity was markedly (>65%) inhibited by l-methionine-d-sulfoximine and alanine and to a lesser extent (<45%) by glycine, arginine, aspartate, asparagine, lysine and serine. Different pairs of amino acids in various combinations resulted in a cumulative inhibition of enzyme activity. The enzyme was also inhibited by the following di- and tri-phosphate nucleotides — IDP, CDP, UDP, ATP, ITP, CTP and UTP. Malate, pyruvate, oxalate, succinate and α-ketoglutarate each at 10 mM depressed enzyme activity.

Properties of glutamine synthetase of Bacteroids from root nodules of Glycine max

Abstract Glutamine synthetase (GS) (EC 6.3.1.2) Isozyme I has been purified from bacteroids of root nodules of Glycine max . The purified GSI which had a molecular weight of 760 000 contained 12 subunits each of about 63 000 daltons. The GS had K m -values of 3.3 mM for hydroxylamine and 11.1 mM for l -glutamine for the γ-glutamyl transferase reaction. The K m -values for the biosynthetic reaction were 12.9 and 14.3 mM for l -glutamine and ATP, respectively. The K m -values of 2.8 and 8.9 mM were determined for NH 4 Cl from a biphasic plot. The inhibition of γ-glutamyl transferase activity by l -glutamate and NH 4 Cl was competitive for l -glutamine and non-competitive for hydroxylamine. When hydroxylamine and l -glutamine were the substrates, NH 4 Cl resulted in K i -values of 18 and 10 mM, respectively. Triphosphate nucleotides viz, ITP, UTP, CTP and ATP (48–92%) and diphosphate nucleotides (20–85%) viz. ADP, IDP, UDP, CDP and GDP, restricted either transferase or biosynthetic activities. Alanine, serine and glycine, respectively, inhibited GS activity by 78, 63 and 50%. Different pairs of amino acids in various combinations resulted in a cumulative inhibition of the enzyme.

Preparation of membrane vesicles in lithium chloride from cells of Nitrosomonas europaea

Abstract The preparation of membrane vesicles in lithium chloride is described which oxidizes NH 4 Cl via hydroxylamine to nitrite. The following properties of the vesicles were determined: the uptake of NH 4 Cl and O 2 by electrode methods, the production of NH 2 OH and NO 2 from NH 4 Cl as well as ATPase activity. The stoichiometry of NH 4 + :O 2 :NO 2 − was found to be 1:1.2:0.7 in vesicles compared with 1:1.5:1.0 in either spheroplasts or washed cells. It is shown that the membrane vesicles also contain a Cu and energy-dependent NH 4 − translocase as in spheroplasts and cells.