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R B Firestone

Publications and source records attributed to R B Firestone.

5 recordsLinked to original sources
PubMed1981

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13.

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, 13N as NO3-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of 13N internally was ammonium and amino acids; the amino acid labeling pattern indicated that 13NO3- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO3- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar Km values, 7 muM. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.

Ammonium Sulfate↗
PubMed1981

Denitrification and dissimilatory nitrate reduction to ammonium in digested sludge.

Acetylene inhibition and 13N methods were used to assay digested sludge for its potential to denitrify and to reduce nitrate to ammonium. At nitrate concentrations below 10 microM, the reduction of N2O to N2 was not inhibited by acetylene concentrations as high as 80 kPa, though at higher nitrate concentration acetylene was an effective inhibitor. NO, N2O, and N2 were produced immediately after addition of nitrate or nitrite, indicating that denitrifying enzymes were present. NO was maintained at a higher concentration of 2--5 nM, while nitrate or nitrite were being reduced, but this gas was depleted once the ionic N oxide substrates were exhausted. Acetylene had little effect on appearance and disappearance of NO. It was also noted that NO was readily consumed by chemical reactions in the anaerobic sludge. Added N2O was reduced without a lag, but pasteurized samples did not consume N2O although they produced it. Fresh digested sludge reduced 60--70% of the added 13NO3- to 13NH4+ with the rest of the NO3- -N presumably lost to denitrification. This agrees well with the nitrate partitioning observed by the acetylene inhibition method in which 30--40% of the NO3- -N was recovered as N2O. Denitrification capacity persisted in both digested sludge and a methanogenic enrichment culture which had been grown in a chemostat for 2.5 years with acetate and ammonium as the sole carbon and nitrogen source. This suggests that denitrifiers with capacities for alternative anaerobic energy metabolism may be more common than now known.

Bacteria↗
PubMed1980

Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens.

The influence of low redox potentials and H2S on NO and N2O reduction by resting cells of denitrifying Pseudomonas fluorescens was studied. Hydrogen sulfide and Ti(III) were added to achieve redox potentials near -200 mV. The control without reductant had a redox potential near +200 mV. Production of 13NO, [13N]N2O, and [13N]N2 from 13NO3- and 13NO2- was followed. Total gas production was similar for all three treatments. The accumulation of 13NO was most significant in the presence of sulfide. A parallel control with autoclaved cells indicated that the 13NO production was largely biological. The sulfide inhibition was more dramatic at the level of N2O reduction; [13N]N2O became the major product instead of [13N]N2, the dominant product when either no reductant or Ti(III) was present. The results indicate that the specific action of sulfide rather than the low redox potential caused a partial inhibition of NO reduction and a strong inhibition of N2O reduction in denitrifying cells.

Hydrogen Sulfide↗