Nitrite oxidase and nitrate reductase in Nitrobacter agilis

Nitrite oxidase and nitrate reductase in Nitrobacter agilis were shown to be separate enzymes. The best separation of the two systems was achieved by ammonium sulphate fractionation. The effects of various compounds, including antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide and chlorate, also clearly distinguish between the two enzyme reactions. The relationship between the two opposing reactions in Nitrobacter is discussed.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

Studies on the incorporation of labelled sulphate into cells and cell-free extracts of Nitrosomonas europaea

Summary The incorporation of [³⁵S]sulphate was followed into the washed cell suspensions of Nitrosomonas europaea. Thus bound sulphate, sulphite, sulphide, cysteine, glutathione, homocysteine and methionine were found in the ethanol soluble fraction as well as in the residual hydrolysed protein fraction. Cysteic acid, methionine sulphoxide and methionine sulphone were detected in the residual protein. The reaction between sulphydryl groups and N-ethylmaleimide has been successfully used to stabilize the thiol compounds in cell-extracts and the derivatives thus obtained were separated by paper chromatography. As in other microorganisms, sulphate is first activated by ATP in Nitrosomonas before it is reduced. The formation of APS and PAPS has been studied. A pathway for the incorporation of [³⁵S]sulphate is proposed.Abbreviations POPOP 1,4-bis-(5-phenyloxazolyl-2)-benzene - PPO 2,5-diphenyloxazole - APS adenosine-5-phosphosulphate - PAPS adenosine-3-phosphate 5-phosphosulphate - ATP adenosine triphosphate - DNA-ase deoxyribonuclease - NEM N-ethylmaleimide - TCA trichloro-acetic acid - GSH glutathione     

Survival of Clostridium botulinum Spores

Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a “tail” comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The “tail” was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation “inactivated” spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth.     

Effect of Temperature of Liquid Nitrogen on Radiation Resistance of Spores of Clostridium botulinum

An apparatus consisting of a Dewar flask and a relay system controlling the flow of liquid nitrogen permitted the irradiation of samples in tin cans or Pyrex tubes at temperatures ranging from 0 ± 1.5 C to -194 ± 2 C. An inoculated pack comprising 320 cans of ground beef containing 5 × 10⁴ spores of Clostridium botulinum 33A per can (10 cans per radiation dose) was irradiated with Co⁶⁰ at 0 and -196 C. Incubation was carried out at 30 C for 6 months. Approximately 0.9 Mrad more radiation was required to inactivate the spores at -196 C than at 0 C. Cans irradiated at -196 C showed partial spoilage at 3.6 Mrad and no spoilage at 3.9 Mrad; the corresponding spoilage-no spoilage doses at 0 C were 2.7 and 3.0, respectively. The majority of positive cans swelled in 2 to 14 days; occasional swelling occurred as late as 20 days. At progressively higher doses, swelling was delayed proportionally to the radiation dose received. The remaining nonswollen cans had no toxin after 6 months of storage, although occasional cans contained very low numbers of viable spores comprising on the average 0.1% of the original spore inoculum. The D₁₀ values in phosphate buffer were 0.290 Mrad for 0 C and 0.396 Mrad for -196 C; in ground beef, the corresponding D₁₀ values were 0.463 Mrad and 0.680 Mrad, respectively. These D₁₀ values indicate that the lethal effect of γ rays decreased at -196 C as compared with 0 C by 13.5% in phosphate buffer, and by 47% in ground beef.