Nitrous oxide reductase of Flexibacter canadensis: a unique membrane-bound enzyme

Abstract The subcellular distribution of nitrous oxide reductase was studied in the gliding soil bacterium Flexibacter canadensis . Nitrous oxide reductase activity, as measured by the methyl viologen-nitrous oxide oxidoreductase assay, was associated entirely with the membrane fraction of cell-free extracts. The enzyme was liberated from the membranes with use of detergents but not by high-salt concentrations, thus implying that nitrous oxide reductase is an integral membrane protein. The nitrous oxide reductase of F. canadensis is the first reported example of a membrane-bound form of this respiratory enzyme.     

Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil.

The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction. Most-probable-number estimates of naturally occurring F. canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F. canadensis (Is-11). Our survey suggests that F. canadensis is common in the natural environment.     

Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil.

The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction. Most-probable-number estimates of naturally occurring F. canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F. canadensis (Is-11). Our survey suggests that F. canadensis is common in the natural environment.     

Sulfide Alleviation of the Acetylene Inhibition of Nitrous Oxide Reduction in Soil

The alleviation of the acetylene blockage of nitrous oxide reduction by sulfide was studied in anaerobically incubated Brookston soil to better characterize the process. Removal of nitrate-derived nitrous oxide from soil amended with acetylene and sulfide occurred earlier in the presence of glucose than it did in its absence. This was attributed to the influence of glucose on nitrous oxide production rather than reduction during the early stages of the soil incubation. Glucose was found to have no effect on reduction of injected nitrous oxide in the presence of acetylene- and sulfide-amended soil, whereas carbon dioxide significantly stimulated reduction. It is suggested that the microorganism(s) involved may use carbon dioxide as a cellular carbon source. The sulfide added to the soil probably did not act solely as an electron donor, as the number of electrons required to reduce the added nitrous oxide in our systems was greater than the amount supplied by the sulfide. The soil pH at which the alleviation occurred was 6.7 and was not affected by the sulfide treatment.     

Effects of sulfide and low redox potential on the inhibition of nitrous oxide reduction by acetylene in Pseudomonas nautica

Membrane introduction mass spectrometry was used to investigate the inhibitory effect of acetylene on the nitrous oxide reductase activity of intact cells of Pseudomonas nautica. We studied the effects of the concentrations of nitrate and sulfide, and the redox potential, which have all been implicated in causing a decrease in the inhibitory effects of acetylene during measurements of denitrification in natural environments. There was no evidence that the concentration of nitrate influenced the effect of acetylene. Lowering the redox potential with the reductant Ti(III)-nitrilotriacetate caused a slight alleviation of acetylene inhibition. Much greater effects at the same redox potential were obtained with concentrations of sulfide in the range 1-10 microM.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Acetylene inhibition technique underestimates in situ denitrification rates in intact cores of freshwater sediment

Abstract Denitrification in intact sediment cores was measured by the acetylene inhibition technique and compared with the nitrate flux between water and sediment. Less than half of the nitrate-N consumed by the sediment could be recovered as nitrous oxide-N. The low recovery rate of nitrous oxide from intact sediment cores indicated losses of nitrous oxide by diffusion down to nitrate-free sediment layers, where reduction of nitrous oxide may take place. In sediment slurries 100% of nitrate-N could be recovered as nitrous oxide-N as long as the nitrate concentration in the liquid phase was above 10 μM. Nitrous oxide added to nitrate-free sediment slurries was reduced regardless of whether acetylene was present or not. Therefore denitrification may be significantly underestimated by this method.     

Reduction of nitrous oxide by a soil Cytophaga in the presence of acetylene and sulfide

Abstract A denitrifying Cytophaga was isolated from soil enriched by anaerobic incubation with glucose, sulfide (S²⁻), nitrous oxide (N₂O), and acetylene (C₂H₂). Such soil enrichments and pure cultures of the isolated Cytophaga reduced N₂O rapidly even in the presence of a normally inhibitory concentration of C₂H₂ (4 kPa) providing S²⁻ was present (8 μmol/g soil or 0.4 μmol/ml culture). Since C₂H₂ inhibition of the reduction of N₂O is used as a tool in the assay of denitrification, the presence in large numbers of such a Cytophaga may influence the effectiveness of this assay especially in sulfidic environments.     

Nitrous oxide emissions from a gully mire in mid-Wales, UK, under simulated summer drought

Certain general circulation models predict that a doubling of atmospheric carbon dioxide concentrations will increase the frequency of summer drought in northern wetlands due to hotter, drier summers. There is currently much uncertainty as to how drought will affect emissions of the greenhouse gas, nitrous oxide, from wetlands. We have demonstrated that an eight centimetre drawdown of the water table in a gully mire does not significantly affect nitrous oxide emissions from this site. However, under a more extreme drought scenario carried out on peat monoliths, nitrous oxide emissions increased exponentially with a linear decrease in water table height. Drought caused a significant increase in nitrous oxide productionbelow the water table but most of the total increase could be attributed to increased emissionsabove the water table. Results from an acetylene block experiment suggested that increased emissions were caused by increased nitrous oxide production from denitrification, rather than by increased production from nitrification. In the laboratory study, drought severity had no effect on peatwater nitrate concentrations below the water table, however, increasing drought severity decreased ammonium concentrations.     

Development of techniques to genetically manipulate members of the genera Cytophaga, Flavobacterium, Flexibacter, and Sporocytophaga.

The Bacteroides-Cytophaga-Flavobacterium branch of the eubacterial phylogenetic tree contains a diverse group of bacterial species. Techniques for the genetic manipulation of Bacteroides spp. are well developed (A. A. Salyers, N. B. Shoemaker, and E. P. Guthrie, Crit. Rev. Microbiol. 14:49-71, 1987). Recently we developed techniques to genetically manipulate the gliding bacterium Cytophaga johnsonae (M. J. McBride and M. J. Kempf, J. Bacteriol. 178:583-590, 1996). We now demonstrate that some of these techniques allow genetic manipulation of a number of environmentally or medically significant bacteria in this group. The Bacteroides transposon Tn4351 was introduced into Cytophaga hutchinsonii, Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadensis, Flexibacter sp. strain FS1, and Sporocytophaga myxococcoides by conjugation. Tn4351 integrated itself into the host chromosomes and conferred erythromycin resistance. We isolated several auxotrophic mutants of Flavobacterium meningosepticum following Tn4351 mutagenesis. The C. johnsonae-Escherichia coli shuttle vector pCP11 functioned in C. succinicans but not in the other bacteria. pLYL03 did not replicate in any of these bacteria and should function as a convenient suicide vector. The identification of a system of gene transfer, a selectable marker, a suicide vector, and a transposon that functions in these diverse bacteria allows genetic manipulations to be performed.     

Measurement of Denitrification in Two Freshwater Sediments by an In Situ Acetylene Inhibition Method

An acetylene inhibition method was satisfactorily used for the in situ measurement of denitrification in two sediment-water systems incubated for not more than 22 h. In the presence of added nitrate, denitrification acted as a source of nitrous oxide in a drainage pond, but acted as a sink in its absence. The averaged rates of nitrous oxide accumulation with nitrate enrichment in the absence and presence of acetylene were 0.15 and 0.30 mg of N m⁻²h⁻¹, respectively. Acetylene reduction at an average rate of 0.07 mmol of C₂H₄ formed m⁻²h⁻¹ was simultaneously measured in the absence of added nitrate. In a small eutrophic lake where nitrogen was nonlimiting, the in situ rates of sediment denitrification were 0.09 and 0.11 mg of N m⁻²h⁻¹ in the presence and absence of macrophytes, respectively, and no acetylene reduction activity was found.     

Nitrous oxide production, its source and distribution in urine patches on grassland on peat soil

Urine patches are considered to be important sites for nitrous oxide (N₂O) production through nitrification and denitrification due to their high concentration of nitrogen (N). The aim of the present study was to determine the microbial source and size of production of N₂O in different zones of a urine patch on grassland on peat soil. Artificial urine was applied in elongated patches of 4.5 m. Four lateral zones were distinguished and sampled for four weeks using an intact soil core incubation method. Incubation of soil cores took place without any additions to the headspace to determine total N₂O production, with acetylene addition to determine total denitrification (N₂O+N₂), and with methyl fluoride to determine the N₂O produced through denitrification.Nitrous oxide production was largest in the centre and decreased towards the edge of the patch. Maximum N₂O production was about 50 mg N m^–2 d^–1 and maximum denitrification activity was 70 mg N m^–2 d^–1. Nitrification was the main N₂O producing process. Nitrous oxide production through denitrification was only of significance when denitrification activity was high. Total N loss through nitrification and denitrification over 31 days was 4.1 g N per patch which was 2.2% of the total applied urine-N.     

The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment

Abstract Experiments were carried out with slurries of saltmarsh sediment to which varying concentrations of nitrate were added. The acetylene blocking technique was used to measure denitrification by accumulation of nitrous oxide, while reduction of nitrate to nitrite and ammonium was also measured. There was good recovery of reduced nitrate and at the smallest concentration of nitrate used (250 μM) there was approximately equal reduction to either ammonium or nitrous oxide (denitrification). Nitrite was only a minor end-product of nitrate reduction. As the nitrate concentration was increased the proportion of the nitrate which was denitrified to nitrous oxide increased, to 83% at the greatest nitrate concentration used (2 mM), while reduction to ammonium correspondingly decreased. This change was attributed either to a greater competitiveness by the denitrifiers for nitrate as the ratio of electron donor to electron acceptor decreased; or to the increased production of nitrite rather than ammonium by fermentative bacteria under high nitrate, the nitrite then being reduced to nitrous oxide by denitrifying bacteria.     

Inhibition of existing denitrification enzyme activity by chloramphenicol.

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (greater than 50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log-phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.     

Inhibition of existing denitrification enzyme activity by chloramphenicol.

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (greater than 50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log-phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.     

The response of denitrifiers in a sandy loam soil affected by a long-term fertilization to organic carbon and nitrate

The response of denitrifiers to carbon in the form of glucose (Glc-C) and nitrate (NO ₃ ⁻ -N) amendments was studied in four differently fertilized plots of sandy-loam soil. Two basically different characteristics of denitrification activity were determined: (1) potential denitrification measured as nitrous oxide production during 1-d incubation in the presence of acetylene, and (2) denitrifying enzyme activity determined in soil slurries as a N₂O production in the presence of acetylene and chloramphenicol during 1 h of incubation. Potential denitrification was strongly influenced by both Glc-C and NO ₃ ⁻ -N amendments in their various combinations, but was also affected by the fertilization practice. The response of denitrifiers to Glc-C and NO ₃ ⁻ -N was generally lower in unfertilized and surprisingly also in highly fertilized soils than in organically and moderately fertilized soils. Denitrifying enzyme activity was stimulated by the fertilization and was, in contrast to potential denitrification, the highest in highly fertilized soil. The results indicate that although active denitrifiers were present in a highly fertilized soil, their ability to develop under optimal conditions was decreased (being similar to that of denitrifiers in unfertilized soil). This suggests long-term changes in soil microbial community in a highly fertilized soil, presumably connected to changes in soil chemistry caused by fertilization.     

Detection,with a pH indicator,of bacterial mutants unable to denitrify

In order to facilitate isolation of mutants with alterations in the denitrification pathway, a new screening procedure using phenol red incorporated into agar overlay has been defined. Alkalinization in the neighbourhood of denitrifying colonies respiring nitrate or nitrite gives rise to a red circular halo. Antimycin blocked these colour changes, which suggests their association with the periplasmic reduction of nitrite. Inhibition of nitrous oxide reductase by acetylene had no significant effect on alkalinization elicited by nitrate or nitrite. Several mutants negative by the phenol red staining test were generated by transposon Tn5 mutagenesis of Paracoccus denitrificans. All these mutants were defective in the activities of nitrite and nitric oxide reductases while the other denitrification activities were present at the wild-type level.     

Effects of specific inhibitors on anammox and denitrification in marine sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2 production were investigated by using short anoxic incubations of marine sediment with a 15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10+/-0.02 microM-1, and there was 95% inhibition at approximately 30 microM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 microM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049+/-0.004 microM-1; 95% inhibition at approximately 70 microM). Methanol specifically inhibited anammox with a decay constant of 0.79+/-0.12 mM-1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by approximately 50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2 production in marine sediments and the use of 15N isotope methods for their quantification.     

Impacts of nitrogen application rates on the activity and diversity of denitrifying bacteria in the Broadbalk Wheat Experiment

Bacterial denitrification results in the loss of fertilizer nitrogen and greenhouse gas emissions as nitrous oxides, but ecological factors in soil influencing denitrifier communities are not well understood, impeding the potential for mitigation by land management. Communities vary in the relative abundance of the alternative dissimilatory nitrite reductase genes nirK and nirS, and the nitrous oxide reductase gene nosZ; however, the significance for nitrous oxide emissions is unclear. We assessed the influence of different long-term fertilization and cultivation treatments in a 160-year-old field experiment, comparing the potential for denitrification by soil samples with the size and diversity of their denitrifier communities. Denitrification potential was much higher in soil from an area left to develop from arable into woodland than from a farmyard manure-fertilized arable treatment, which in turn was significantly higher than inorganic nitrogen-fertilized and unfertilized arable plots. This correlated with abundance of nirK but not nirS, the least abundant of the genes tested in all soils, showing an inverse relationship with nirK. Most genetic variation was seen in nirK, where sequences resolved into separate groups according to soil treatment. We conclude that bacteria containing nirK are most probably responsible for the increased denitrification potential associated with nitrogen and organic carbon in this soil.