Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Isotope pool dilution studies are increasingly reported in the soils and ecology literature as a means of measuring gross rates of nitrogen (N) mineralization, nitrification, and inorganic N assimilation in soils. We assembled data on soil characteristics and gross rates from 100 studies conducted in forest, shrubland, grassland, and agricultural systems to answer the following questions: What factors appear to be the major drivers for production and consumption of inorganic N as measured by isotope dilution studies? Do rates or the relationships between drivers and rates differ among ecosystem types? Across a wide range of ecosystems, gross N mineralization is positively correlated with microbial biomass and soil C and N concentrations, while soil C:N ratio exerts a negative effect on N mineralization only after adjusting for differences in soil C. Nitrification is a log-linear function of N mineralization, increasing rapidly at low mineralization rates but changing only slightly at high mineralization rates. In contrast, NH 4 1 assimilation by soil microbes increases nearly linearly over the full range of mineralization rates. As a result, nitrification is proportionately more important as a fate for NH4 1 at low mineralization rates than at high mineralization rates. Gross nitrification rates show no relationship to soil pH, with some of the fastest nitrification rates occurring below pH 5 in soils with high N mineralization rates. Differences in soil organic matter (SOM) composition and concentration among ecosystem types in- fluence the production and fate of mineralized N. Soil organic matter from grasslands appears to be inherently more productive of ammonium than SOM from wooded sites, and SOM from deciduous forests is more so than SOM in coniferous forests, but differences appear to result primarily from differing C:N ratios of organic matter. Because of the central importance of SOM characteristics and concentrations in regulating rates, soil organic matter depletion in agricultural systems appears to be an important determinant of gross process rates and the proportion of NH4 1 that is nitrified. Addition of 15 N appears to stimulate NH4 1 consumption more than NO3 2 consumption processes; however, the magnitude of the stim- ulation may provide useful information regarding the factors limiting microbial N trans- formations.

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Controls on nitrogen cycling in terrestrial ecosystems: a synthetic analysis of literature data

We conducted a 456-d laboratory incubation of an old-growth coniferous forest soil to aid in the elucidation of C controls on N cycling processes in forest soils. Gross rates of N mineralization, immobilization, and nitrification were measured by 'IN isotope dilution, and net rates of N mineralization and nitrification were calculated from changes in KCl-extractable inorganic N and NOE-EN pool sizes, respectively. Changes in the availability of C were assessed by monitoring rates of CO, evolution and the sizes of extractable organic C and microbial biomass pools. Net and gross rates of N mineralization (r2 = 0.038, P =.676) and nitrification (r2 = 0.403, P = .125) were not significantly correlated over the course of the incubation, suggesting that the factors controlling N consumptive and productive processes do not equally affect these processes. A significant increase in the NO, pool size (net nitrification) only occurred after 140 d, when the NO3- pool size increased suddenly and massively. However, gross nitrification rates were substantial throughout the entire incubation and were poorly correlated with these changes in NO3 pool sizes. Concurrent decreases in the microbial biomass suggest that large increases in NO3 pool sizes after prolonged incubation of coniferous forest soil may arise from re- ductions in the rate of microbial immobilization of NO3, rather than from one of the mechanisms proposed previously (e.g., sequestering of NH,+ by microbial heterotrophs, the deactivation of allelopathic compounds, or large increases in autotrophic nitrifier pop- ulations). Strong correlations were found between rates of CO2 evolution and gross N mineralization (r2 = 0.974, P < .0001) and immobilization (r2 = 0.980, P < .0001), but not between CO, evolution and net N mineralization rates. Microbial growth efficiency, determined by combining estimates of gross N immobilization, CO2 evolution, and micro- bial biomass C and N pool sizes, declined exponentially over the incubation. These results suggest the utilization of lower quality substrates as C availability declined during incu- bation. Results from this research indicate the measurement of gross rates of N transfor- mations in soil provides a powerful tool for assessing C and N cycling relationships in forests.

https://www.jstor.org/stable/pdfplus/1939413.pdf

Dynamics of Gross Nitrogen Transformations in an Old-Growth Forest: The Carbon Connection

The regulation of NO−3 assimilation by NH+4 and amino acids was studied to better define the conditions under which raicrobial NO−3 assimilation could be expected to take place in nature. The assay system was microbial isolates from soil and slurries of two soils enhanced in NO−3 assimilating capacity by previous exposure to glucose for 18–24 h. The NO−3 assimilation rate was monitored by a NO−3 electrode and the inhibition determined by the change in rate following addition of NH+4 or amino acids. NH+4 inhibited (by 60%) NO−3 assimilation immediately (< 1 min) and at very low NH+4 concentrations (O.1 μg N g−1 soil). At higher NH+4 concentrations (10 μg N g− soil) a second state of inhibition (80%) occurred after 5 min. The speed of the first inhibition stage suggests that the site of inhibition is the transport of NO−3 into the cell. Asparagine was the only amino acid to act as rapidly and effectively as NH+4 in the soil, and thus the amino acids are unlikely to be important regulators of NO−3 assimilation in soils. Of the three soil isolates studied, Saccharomyces cerevisiae and Pseudomonas fluorescens responded to NH+4 and amino acids in a manner similar to the soil but NO−3 assimilation in the third organism, Azotobacter vinelandii, was generally insensitive to these N substrates in this short-term assay. Only high concentrations of NH+4 caused a partial inhibition of NO−3 assimilation in A. vinelandii. Due to the effectiveness of low NH+4 concentrations in inhibiting NO−3 assimilation in soils, microbial immobilization is not expected to be an important fate of NO−3 unless there are NH+4-free microsites in the soil.

Regulation of nitrate assimilation by ammonium in soils and in isolated soil microorganisms

Shifts in plant community structure in shrub and grass-dominated ecosystems are occurring over large land areas in the western US. It is not clear what effect this vegetative change will have on rates of carbon and nitrogen cycling, and thus long-term ecosystem productivity. To study the effect of different plant species on the decomposability of soil organic substrates and rates of C- and N-cycling, we conducted laboratory incubations of soils from a 15-yr-old experimental plot where big sagebrush (Artemisia tridentata Nutt.) and crested wheatgrass (Agropyron desertorum [Fisch.] Schult.) plants had been planted in a grid pattern. Soil samples collected from beneath crested wheatgrass had significantly greater total N and NO3− concentrations and lower C-to-N ratios than samples collected beneath sagebrush plants or from interspaces. Soil carbonate concentrations beneath crested wheatgrass were intermediate between sagebrush soils and interspace soils. During the 16-week laboratory incubation, soil C mineralization rates, gross and net N mineralization rates, gross NH4+ consumption rates, net nitrification rates and soil NO3− concentrations were significantly different, with slightly higher values in the crested wheatgrass soil and lower values in the interspace soil. Gross NH4+ assimilation, gross nitrification, gross NO3− assimilation, microbial biomass C and N and microbial growth efficiency showed no significant differences. During the 16-week incubation, microbial biomass C, microbial respiration and gross N assimilation rates declined by more than 50%, suggesting that the microbial biomass was C-limited. However, addition of NH4+ appeared to stimulate NH4+ assimilation. In addition, the form of N assimilated by micro-organisms shifted from predominantly NH4+ (95%) when NO3− was relatively unavailable at the beginning of the incubation, to predominantly NO3− (88–96%) as NO3− concentrations increased. Both of these latter observations suggest that microbes were NH4+-limited. Either co-limitation by C and NH4+ or the presence of separate C-limited and NH4+-limited microsites could explain these results. Nitrifying bacteria consumed an increasing proportion of the NH4+ pool as the incubation progressed, suggesting that increased microsite structure was responsible for shifts in C and N dynamics over time. The results of this study demonstrate that different plant species can significantly influence soil C and N cycling rates; however, after 15 yr the magnitude of the effect was still fairly small.

Plant Species Effects and Carbon and Nitrogen Cycling in a Sagebrush -- Crested Wheatgrass Soil

Pseudomonas aeruginosa can reduce nitrate to nitrite and evenutally to nitrogen gas by the denitrification pathway, thereby providing the organism with a mode of respiration and ATP generation in the absence of oxygen. P. aeruginosa can also reduce nitrate to nitrite through an assimilatory pathway that provides the cell with reduced nitrogen for biosyntheses. In order to establish whether this organism synthesizes a single nitrate reductase protein that functions in both pathways, or produces one for each pathway, we isolated mutants blocked in the assimilation of nitrate. These mutants are unaffected in the reduction of nitrate be the denitrification pathway, although they produce low or undectable levels of assimilatory nitrate reductase. On the basis of transductional analysis, the mutations were found to be distributed among four genes designated nasA, nasB, nasC, and nasD. Shifting a nasA mutant from anaerobic to aerobic growth eliminated the culture's ability to reduce nitrate, i.e. the anaerobic nitrate reductase cannot function in the presence of oxygen. Thus P. aeruginosa can synthesize two distinct proteins which reduce nitrate to nitrite: an assimilatory nitrate reductase and a dissimilatory nitrate reductase. If conditions of growth are fully aerobic, the latter is not synthesized and does not function. The former, synthesized under the control of at least four genes, is repressed by readily available nitrogen sources.

Stacey R. Sias

Papers

Isolation and analysis of mutants of Pseudomonas aeruginosa unable to assimilate nitrate.