D. J. D. Nicholas

Definitive 15N NMR evidence that water serves as a source of ‘O’ during nitrite oxidation by Nitrobacter agilis

Using 18O isotope shifts in 15N NMR it has been shown that during oxidation of nitrite to nitrate by Nitrobacter agilis, the third ‘O’ in nitrate originates from water.

Purification and characterization of nitrous oxide reductase from Rhodopseudomonas sphaeroides f. sp. denitrificans

Abstract (1) A multi-copper N 2 O reductase has been purified to homogeneity from Rhodopseudomonas sphaeroides f.sp. denitricans grown under photodenitrifying conditions. Its molecular structure, types of copper and catalytic activity have been studied by various techniques, including polyacrylamide gel electrophoresis as well as visible and EPR spectroscopy. (2) N 2 O reductase is a monomeric enzyme and one or two disulphide bridges appear to be involved in its tertiary structure. The enzyme contains four copper atoms per mol and has an EPR spectrum of a type 2 Cu 2+ site(s). (3) An oxidized, EPR positive, form of N 2 O reductase has an unusual visible spectrum for a copper protein resolved into four bands: 481, 534, 635 and 740 nm.

Sulphide-linked Nitrite Reductase from Thiobacillus denitrificans with Cytochrome Oxidase Activity: Purification and Properties

A membrane-bound, sulphide-linked nitrite reductase from Thiobacillus denitrificans was solubilized and after further purification its properties were examined. The purified enzyme, mol. wt 120 000, contained cytochromes c and d in the ratio of 1:1. Both cytochromes were reduced by sulphide and re-oxidized with nitrite or air. Oxidation by nitrite resulted in the appearance of an absorption peak at 572 nm. The kinetics of the reduction of the enzyme with sulphide indicated that cytochrome c was reduced before cytochrome d. The redox potential of cytochrome d was 22 mV more positive than that of cytochrome c. Cytochromes c and d were dissociated from the purified enzyme by treatment with sodium dodecyl sulphate. The purified nitrite reductase also had cytochrome oxidase activity and both the activities were stimulated by cytochrome c-551 isolated from T. denitrificans. Reduced cytochrome c-551 was an effective electron donor for the purified enzyme with either nitrite or air as the terminal electron acceptor. Neither cytochrome c-554 (also isolated from T. denitrificans) nor mammalian cytochrome c was effective as reductant for the enzyme. NO and N2O were identified as the products of nitrite reduction by the purified sulphide-linked nitrite reductase.

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O2 (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 μg· ml-1) or chloramphenicol (50 μg·ml-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N2 fixation.

Characterization of a Soluble Cytochrome Oxidase/Nitrite Reductase from Nitrosomonas europaea

Summary: The soluble cytochrome oxidase/nitrite reductase of Nitrosomonas europaea, induced under low O2 tensions, has a high affinity for O2. EPR spectroscopy indicates that this enzyme contains “type I” (“blue”) and “type II” Cu2+-sites, and UV/visible spectroscopy suggests the presence of a “type III” Cu2+-pair. The enzyme binds exogenous copper in an unusual way, resulting in modified EPR spectra and a very high .M for the “blue” 598 nm absorption band when low levels of Cu2+ (10 μM) are present during purification.

Simultaneous Assimilation and Denitrification of Nitrate by Bradyrhizobium japonicum

Bradyrhizobium japonicum strain CB 1809 assimilated and denitrified 15NO- 3 simultaneously to 15NH+ 4 and 15N2, respectively, when incubated under anaerobiosis. Washed cells from cultures grown in air in the presence of 5 mM-nitrate, assimilated 70% of the 15NO- 3 into cell-nitrogen but only after a lag of 10 h during which time NO- 2 accumulated. In washed cells grown at 2% (v/v) O2 with 5 mM-nitrate, 15NO- 3 was denitrified to 15N2 (95%) in 1-2 h. The low O2 tension in the culture medium resulted in increased activity of nitrate and nitrite reductases in the cells. When 10 mM-glutamate was included with 5 mM-nitrate at 2% (v/v) O2, washed cells from these cultures had less nitrate and nitrite reductase activities and when 15NO- 3 was supplied to these cells there was a transient accumulation of NO- 2 (1 h) prior to maximum denitrification. Washed cells prepared from cultures grown at 2% (v/v) O2 with glutamate alone utilized 15NO- 3, only after a lag period of 20-40 h, assimilation by (60%) and denitrification (40%).

Annual uptake and distribution of N15-labelled ammonia and nitrate in young Jonathan/MM104 apple trees grown in solution cultures

SummaryBy using N15-labelled calcium nitrate and ammonium sulphate, the uptake of ammonia and nitrate by young Jonathan/MM104 apple trees grown in a glasshouse in water culture was determined. A batch of 4 trees was exposed to the appropriate tracer for seven days and then sampled. This procedure was repeated at 4 weekly intervals during a 12-month period. The results show that the uptake by apple trees of either ammonia or nitrate is continuous throughout the year with a relative high peak (expressed in µg N/100 mg dry weight) in December (summer period) and low values during August-September (winter period). The total uptake in the whole tree was steady for December to June inclusive and decreased from July, with the lowest uptake during August-September period. The young trees, previously receiving nitrogen as ammonium plus nitrate (1: 7), absorbed both ions in nearly equivalent amounts, except for the winter period when the uptake of ammonia was in excess of that for nitrate. This difference in uptake of both ions probably results from a lower activity of the enzymic nitrate reducing system during the time of low temperature. Low activity of the nitrate reductase enzyme results also in a restricted uptake of nitrate by apple trees previously receiving only ammonia nitrogen.A detailed analysis of total nitrogen content showed the lowest nitrogen values (0.77%) in February, near the end of the second growth period of the first year. From then on, the total nitrogen percentage of the apple trees steadily increased to reach a peak of 1.57% N during the flush of early spring growth of the second year. Leaves, fruit, soft twig growth, blossoms and roots had the highest N levels at this time. The total nitrogen percentage of the whole tree then gradually decreased.The established distribution pattern of absorbed nitrogen from both N15-labelled compounds showed that the relatively highest incorporation of absorbed nitrogen from both sources was in newly grown tissues during early spring growth flush of the second year, indicating the importance of currently absorbed nitrogen for early spring growth. With the increase in rate of growth in the later period, the value of the nitrogen stored within the apple trees became evident.

The adaptation of Rhodopseudomonas sphaeroides f. sp. denitrificans for growth under denitrifying conditions

SUMMARY: The adaptation of cells of Rhodopseudomonas sphaeroides f. sp. denitrificans for growth under denitrifying conditions in the light has been studied. The presence of nitrate in photosyntheti-cally grown bacterial cultures resulted in a drastic reduction of carotenoid and bacteriochloro-phyll contents as well as a loss of one of the polypeptides of the light harvesting complex, resulting in colour changes. Denitrifying cells had high activities of nitrate, nitrite and nitrous oxide reductases. The polypeptides corresponding to subunits of these enzymes were separated by PAGE. Synthesis of these enzymes was studied by pulse-chase labelling techniques. Nitrate and nitrite reductases are constitutive enzymes and it is likely that copies of mRNA for synthesis of these enzymes are “long lived” in the cells.

Acetylene reduction by rumen microflora

SummaryAcetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N2 fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N2 gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.

Symbiosis of Trifolium subterraneum with mycorrhizal fungi and Rhizobium trifolii as affected by ammonium sulphate and nitrification inhibitors.

Abstract The symbioses between Trifolium subterraneum , mycorrhizal fungi and Rhizohium are affected by (NH 4 ) 2 SO 4 and by the nitrification inhibitors 2-chloro-6 (trichloromethyl) pyridine (N-Serve) and 2-trichloromethyl pyridine (2TMP). At 50 μg · g −1 soil N-Serve and 2TMP had toxic effects on plant growth, measured as leaf expansion, root length and dry weight. Lower concentrations of N-Serve also produced some toxic symptoms. The addition of (NH 4 ) 2 SO 4 to the soil at 2 and 6 m-equiv NH + 4 per pot, resulted in reduced root length and nodulation. Shoot dry weight was reduced at 6 m-equiv NH + 4 per pot. In the presence of (NH 4 ) 2 SO 4 the toxic effects of the nitrification inhibitors on plant growth were less. Both nitrification inhibitors reduced development of mycorrhizal entry-points and extent of root colonization (% infection). Percentage infection of the root system was also reduced by (NH 4 ) 2 SO 4 . Development of nodules on the lateral roots was increased in the presence of N-Serve at 5 and 15 μ · g −1 . This effect, however, was accompanied by a marked reduction in N 2 ase activity. Smaller increases in nodulation were apparent with 2TMP and were associated with variable N 2 ase activity.