Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO₃⁻ or NH₄⁺ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO₃⁻ concentration under microaerobic conditions. Products of NO₃⁻ reduction included NH₄⁺, N₂O, NO, and N₂. NO₂⁻ and NH₂OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO₃⁻. NO₃⁻ reduction to NH₄⁺ was completely repressed by the addition of 2 mM NH₄⁺ to the growth medium, whereas NO₃⁻ reduction to N₂O or to N₂ was not. C₂H₂ completely inhibited N₂O reduction to N₂ by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO₃⁻ by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O₂. The process of NO₃⁻ reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.     

Anaerobic Growth and Denitrification among Different Serogroups of Soybean Rhizobia

We screened soybean rhizobia originating from three germplasm collections for the ability to grow anaerobically in the presence of NO₃⁻ and for differences in final product formation from anaerobic NO₃⁻ metabolism. Denitrification abilities of selected strains as free-living bacteria and as bacteroids were compared. Anaerobic growth in the presence of NO₃⁻ was observed in 270 of 321 strains of soybean rhizobia. All strains belonging to the 135 serogroup did not grow anaerobically in the presence of NO₃⁻. An investigation with several strains indicated that bacteria not growing anaerobically in the presence of NO₃⁻ also did not utilize NO₃⁻ as the sole N source aerobically. An exception was strain USDA 33, which grew on NO₃⁻ but failed to denitrify. Dissimilation of NO₃⁻ by the free-living cultures proceeded without the significant release of intermediate products. Nitrous oxide reductase was inhibited by C₂H₂, but preceding steps of denitrification were not affected. Final products of denitrification were NO₂⁻, N₂O, or N₂; serogroups 31, 46, 76, and 94 predominantly liberated NO₂⁻, whereas evolution of N₂ was prevalent in serogroups 110 and 122, and all three were formed as final products by strains belonging to serogroups 6 and 123. Anaerobic metabolism of NO₃⁻ by bacteroid preparations of Bradyrhizobium japonicum proceeded without delay and was evident by NO₂⁻ accumulation irrespective of which final product was formed by the strain as free-living bacteria. Anaerobic C₂H₂ reduction in the presence of NO₃⁻ was observed in bacteroid preparations capable of NO₃⁻ respiration but was absent in bacteria that were determined to be deficient in dissimilatory nitrate reductase.     

Bacterium-Based NO2− Biosensor for Environmental Applications

A sensitive NO₂⁻ biosensor that is based on bacterial reduction of NO₂⁻ to N₂O and subsequent detection of the N₂O by a built-in electrochemical N₂O sensor was developed. Four different denitrifying organisms lacking NO₃⁻ reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO₂⁻ tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO₃⁻ and N₂O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N₂O reductase. The macroscale biosensors constructed exhibited a linear NO₂⁻ response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO₂⁻, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO₂⁻, and interference was observed only with NH₂OH, NO, N₂O, and H₂S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO₂⁻ biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO₂⁻ biosensor were constructed and used to measure NO₂⁻ profiles in marine sediment.     

Dissimilatory Reduction of NO2− to NH4+ and N2O by a Soil Citrobacter sp.

Dissimilatory reduction of NO₂⁻ to N₂O and NH₄⁺ by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N₂O production by this mechanism. In batch cultures with defined media, NO₂⁻ reduction to NH₄⁺ was favored by high glucose and low NO₃⁻ concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO₃⁻ concentrations. With succinate as the energy source, little or no NO₂⁻ was reduced to NH₄⁺ but N₂O was produced. Resting cell suspensions reduced NO₂⁻ simultaneously to N₂O and free extracellular NH₄⁺. Chloramphenicol prevented the induction of N₂O-producing activity. The K_m for NO₂⁻ reduction to N₂O was estimated to be 0.9 mM NO₂⁻, yet the apparent K_m for overall NO₂⁻ reduction was considerably lower, no greater than 0.04 mM NO₂⁻. Activities for N₂O and NH₄⁺ production increased markedly after depletion of NO₃⁻ from the media. Amendment with NO₃⁻ inhibited N₂O and NH₄⁺ production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH₄⁺ but not of N₂O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N₂O at rates equal to the wild type. These observations suggest that N₂O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO₂⁻ to NH₄⁺.     

Role of Chemotaxis in the Ecology of Denitrifiers

A modification of the Adler capillary assay was used to evaluate the chemotactic responses of several denitrifiers to nitrate and nitrite. Strong positive chemotaxis was observed to NO₃⁻ and NO₂⁻ by soil isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri, with the peak response occurring at 10⁻³ M for both attractants. In addition, a strong chemoattraction to serine (peak response at 10⁻² M), tryptone, and a soil extract, but not to NH₄⁺, was observed for all denitrifiers tested. Chemotaxis was not dependent on a previous growth on NO₃⁻, NO₂⁻, or a soil extract, and the chemoattraction to NO₃⁻ occurred when the bacteria were grown aerobically or anaerobically. However, the best response to NO₃⁻ was usually observed when the cells were grown aerobically with 10 mM NO₃⁻ in the growth medium. Capillary tubes containing 10⁻3 M NO₃⁻ submerged into soil-water mixtures elicited a significant chemotactic response to NO₃⁻ by the indigenous soil microflora, the majority of which were Pseudomonas spp. A chemotactic strain of P. fluorescens also was shown to survive significantly better in aerobic and anaerobic soils than was a nonmotile strain of the same species. Both strains had equal growth rates in liquid cultures. Thus, chemotaxis may be one mechanism by which denitrifiers successfully compete for available NO₃⁻ and NO₂⁻, and which may facilitate the survival of naturally occurring populations of some denitrifiers.     

Nitrous Oxide Production by Organisms Other than Nitrifiers or Denitrifiers

Heterotrophic bacteria, yeasts, fungi, plants, and animal breath were investigated as possible sources of N₂O. Microbes found to produce N₂O from NO₃⁻ but not consume it were: (i) all of the nitrate-respiring bacteria examined, including strains of Escherichia, Serratia, Klebsiella, Enterobacter, Erwinia, and Bacillus; (ii) one of the assimilatory nitrate-reducing bacteria examined, Azotobacter vinelandii, but not Azotobacter macrocytogenes or Acinetobacter sp.; and (iii) some but not all of the assimilatory nitrate-reducing yeasts and fungi, including strains of Hansenula, Rhodotorula, Aspergillus, Alternaria, and Fusarium. The NO₃⁻-reducing obligate anaerobe Clostridium KDHS2 did not produce N₂O. Production of N₂O occurred only in stationary phase. The nitrate-respiring bacteria produced much more N₂O than the other organisms, with yields of N₂O ranging from 3 to 36% of 3.5 mM NO₃⁻. Production of N₂O was apparently not regulated by ammonium and was not restricted to aerobic or anaerobic conditions. Plants do not appear to produce N₂O, although N₂O was found to arise from some damaged plant tops, probably due to microbial growth. Concentrations of N₂O above the ambient level in the atmosphere were found in human breath and appeared to increase after a meal of high-nitrate food.     

Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.     

Production of NO2- and N2O by Nitrifying Bacteria at Reduced Concentrations of Oxygen

Pure cultures of the marine ammonium-oxidizing bacterium Nitrosomonas sp. were grown in the laboratory at oxygen partial pressures between 0.005 and 0.2 atm (0.18 to 7 mg/liter). Low oxygen conditions induced a marked decrease in the rate for production of NO₂^-, from 3.6 × 10⁻¹⁰ to 0.5 × 10⁻¹⁰ mmol of NO₂^- per cell per day. In contrast, evolution of N₂O increased from 1 × 10⁻¹² to 4.3 × 10⁻¹² mmol of N per cell per day. The yield of N₂O relative to NO₂^- increased from 0.3% to nearly 10% (moles of N in N₂O per mole of NO₂^-) as the oxygen level was reduced, although bacterial growth rates changed by less than 30%. Nitrifying bacteria from the genera Nitrosomonas, Nitrosolobus, Nitrosospira, and Nitrosococcus exhibited similar yields of N₂O at atmospheric oxygen levels. Nitrite-oxidizing bacteria (Nitrobacter sp.) and the dinoflagellate Exuviaella sp. did not produce detectable quantities of N₂O during growth. The results support the view that nitrification is an important source of N₂O in the environment.     

Simulated Nitrogen Deposition Reduces CH4 Uptake and Increases N2O Emission from a Subtropical Plantation Forest Soil in Southern China

To date, few studies are conducted to quantify the effects of reduced ammonium (NH₄ ⁺) and oxidized nitrate (NO₃ ⁻) on soil CH₄ uptake and N₂O emission in the subtropical forests. In this study, NH₄Cl and NaNO₃ fertilizers were applied at three rates: 0, 40 and 120 kg N ha⁻¹ yr⁻¹. Soil CH₄ and N₂O fluxes were determined twice a week using the static chamber technique and gas chromatography. Soil temperature and moisture were simultaneously measured. Soil dissolved N concentration in 0–20 cm depth was measured weekly to examine the regulation to soil CH₄ and N₂O fluxes. Our results showed that one year of N addition did not affect soil temperature, soil moisture, soil total dissolved N (TDN) and NH₄ ⁺-N concentrations, but high levels of applied NH₄Cl and NaNO₃ fertilizers significantly increased soil NO₃ ⁻-N concentration by 124% and 157%, respectively. Nitrogen addition tended to inhibit soil CH₄ uptake, but significantly promoted soil N₂O emission by 403% to 762%. Furthermore, NH₄ ⁺-N fertilizer application had a stronger inhibition to soil CH₄ uptake and a stronger promotion to soil N₂O emission than NO₃ ⁻-N application. Also, both soil CH₄ and N₂O fluxes were driven by soil temperature and moisture, but soil inorganic N availability was a key integrator of soil CH₄ uptake and N₂O emission. These results suggest that the subtropical plantation soil sensitively responses to atmospheric N deposition, and inorganic N rather than organic N is the regulator to soil CH₄ uptake and N₂O emission.     

Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200

Although previous research has demonstrated that NO₃⁻ inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO₃⁻ biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO₃⁻ and synthetic, high-surface-area goethite. Results showed that the presence of NO₃⁻ inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO₃⁻ reduction, a 10-fold decrease in the rate of NO₂⁻ reduction, and a 20-fold increase in the amounts of N₂O produced. Nitrogen stable isotope experiments that utilized δ¹⁵N values of N₂O to distinguish between chemical and biological reduction of NO₂⁻ revealed that the N₂O produced during NO₂⁻ or NO₃⁻ reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO₃⁻ reduction produces NO₂⁻ and Fe(II), which then abiotically react to reduce NO₂⁻ to N₂O with the subsequent oxidation of Fe(II) to Fe(III).     

Light-Limited Growth Rate Modulates Nitrate Inhibition of Dinitrogen Fixation in the Marine Unicellular Cyanobacterium Crocosphaera watsonii

Biological N₂ fixation is the dominant supply of new nitrogen (N) to the oceans, but is often inhibited in the presence of fixed N sources such as nitrate (NO₃ ⁻). Anthropogenic fixed N inputs to the ocean are increasing, but their effect on marine N₂ fixation is uncertain. Thus, global estimates of new oceanic N depend on a fundamental understanding of factors that modulate N source preferences by N₂-fixing cyanobacteria. We examined the unicellular diazotroph Crocosphaera watsonii (strain WH0003) to determine how the light-limited growth rate influences the inhibitory effects of fixed N on N₂ fixation. When growth (µ) was limited by low light (µ = 0.23 d⁻¹), short-term experiments indicated that 0.4 µM NH₄ ⁺ reduced N₂-fixation by ∼90% relative to controls without added NH₄ ⁺. In fast-growing, high-light-acclimated cultures (µ = 0.68 d⁻¹), 2.0 µM NH₄ ⁺ was needed to achieve the same effect. In long-term exposures to NO₃ ⁻, inhibition of N₂ fixation also varied with growth rate. In high-light-acclimated, fast-growing cultures, NO₃ ⁻ did not inhibit N₂-fixation rates in comparison with cultures growing on N₂ alone. Instead NO₃ ⁻ supported even faster growth, indicating that the cellular assimilation rate of N₂ alone (i.e. dinitrogen reduction) could not support the light-specific maximum growth rate of Crocosphaera. When growth was severely light-limited, NO₃ ⁻ did not support faster growth rates but instead inhibited N₂-fixation rates by 55% relative to controls. These data rest on the basic tenet that light energy is the driver of photoautotrophic growth while various nutrient substrates serve as supports. Our findings provide a novel conceptual framework to examine interactions between N source preferences and predict degrees of inhibition of N₂ fixation by fixed N sources based on the growth rate as controlled by light.     

Denitrification in San Francisco Bay Intertidal Sediments

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO₃⁻ + NO₂⁻ concentrations (≤26 μM) were below the apparent K_m (50 μM) for nitrate. During the rainy season, when ambient NO₃⁻ + NO₂⁻ concentrations were higher (37 to 89 μM), an accurate estimate of the K_m could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N₂O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N₂O in the presence of C₂H₂ was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N₂O loss was caused by incomplete blockage of N₂O reductase by C₂H₂ at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N₂ m⁻² h⁻¹ (for undisturbed sediments) to 17 to 280 μmol of N₂ m⁻² h⁻¹ (for shaken sediment slurries).     

Microzonation of Denitrification Activity in Stream Sediments as Studied with a Combined Oxygen and Nitrous Oxide Microsensor

Microzonation of denitrification was studied in stream sediments by a combined O₂ and N₂O microsensor technique. O₂ and N₂O concentration profiles were recorded simultaneously in intact sediment cores in which C₂H₂ was added to inhibit N₂O reduction in denitrification. The N₂O profiles were used to obtain high-resolution profiles of denitrification activity and NO₃⁻ distribution in the sediments. O₂ penetrated about 1 mm into the dark-incubated sediments, and denitrification was largely restricted to a thin anoxic layer immediately below that. With 115 μM NO₃⁻ in the water phase, denitrification was limited to a narrow zone from 0.7 to 1.4 mm in depth, and total activity was 34 nmol of N cm⁻² h⁻¹. With 1,250 μM NO₃⁻ in the water, the denitrification zone was extended to a layer from 0.9 to 4.8 mm in depth, and total activity increased to 124 nmol of N cm⁻² h⁻¹. Within most of the activity zone, denitrification was not dependent on the NO₃⁻ concentration and the apparent K_m for NO₃⁻ was less than 10 μM. Denitrification was the only NO₃⁻-consuming process in the dark-incubated stream sediment. Even in the presence of C₂H₂, a significant N₂O reduction (up to 30% of the total N₂O production) occurred in the reduced, NO₃⁻-free layers below the denitrification zone. This effect must be corrected for during use of the conventional C₂H₂ inhibition technique.     

Capacity for Denitrification and Reduction of Nitrate to Ammonia in a Coastal Marine Sediment

The capacity for dissimilatory reduction of NO₃⁻ to N₂ (N₂O) and NH₄⁺ was measured in ¹⁵NO₃⁻-amended marine sediment. Incubation with acetylene (7 × 10⁻³ atmospheres [normal]) caused accumulation of N₂O in the sediment. The rate of N₂O production equaled the rate of N₂ production in samples without acetylene. Complete inhibition of the reduction of N₂O to N₂ suggests that the “acetylene blockage technique” is applicable to assays for denitrification in marine sediments. The capacity for reduction of NO₃⁻ by denitrification decreased rapidly with depth in the sediment, whereas the capacity for reduction of NO₃⁻ to NH₄⁺ was significant also in deeper layers. The data suggested that the latter process may be equally as significant as denitrification in the turnover of NO₃⁻ in marine sediments.     

Characterization of Nitrite Degradation by Lactobacillus casei subsp. rhamnosus LCR 6013

Nitrites are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. The nitrite degradation capacity of Lactobacillus casei subsp. rhamnosus LCR 6013 was investigated in pickle fermentation. After LCR 6013 fermentation for 120 h at 37°C, the nitrite concentration in the fermentation system was significantly lower than that in the control sample without the LCR 6013 strain. The effects of NaCl and Vc on nitrite degradation by LCR 6013 in the De Man, Rogosa and Sharpe (MRS) medium were also investigated. The highest nitrite degradations, 9.29 mg/L and 9.89 mg/L, were observed when NaCl and Vc concentrations were 0.75% and 0.02%, respectively in the MRS medium, which was significantly higher than the control group (p ≤ 0.01). Electron capture/gas chromatography and indophenol blue staining were used to study the nitrite degradation pathway of LCR 6013. The nitrite degradation products contained N₂O, but no NH₄ ⁺The LCR 6013 strain completely degraded all NaNO₂ (50.00 mg/L) after 16 h of fermentation. The enzyme activity of NiR in the periplasmic space was 2.5 times of that in the cytoplasm. Our results demonstrated that L. casei subsp. rhamnosus LCR 6013 can effectively degrade nitrites in both the pickle fermentation system and in MRS medium by NiR. Nitrites are degraded by the LCR 6013 strain, likely via the nitrate respiration pathway (NO₂ ⁻>NO⁻>N₂O⁻>N₂), rather than the aammonium formation pathway (dissimilatory nitrate reduction to ammonium, DNRA), because the degradation products contain N₂O, but not NH₄ ⁺.     

Effect of N Source on the Steady State Growth and N Assimilation of P-limited Anabaena flos-aquae

Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH₃, NO₃⁻, or N₂. Expressed as a function of relative growth rate, steady state cellular P contents and PO₄ assimilation rates did not vary with N-source. However, N-source did alter the maximal PO₄-limited growth rate achieved by the cultures: the NO₃⁻ and N₂ cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH₃ grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO₃⁻-grown cells were the smallest (627 ± 34 micromoles C · 10⁻⁹ cells), while NH₃-grown cells were largest (900 ± 44 micromoles C · 10⁻⁹ cells) and N₂-fixing cells were intermediate (726 ± 48 micromoles C · 10⁻⁹ cells) in size. In the NO₃⁻-and N₂-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH₃-grown cells. Heterocysts were absent in NH₃-grown cultures but were present in both the N₂ and NO₃⁻ cultures. In the NO₃⁻-grown cultures C₂H₂ reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N₂-fixing cultures the acetylene:N₂ ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal.

Compared with NH₃, the assimilation of NO₃⁻ and N₂ resulted either in a decrease in cellular C (NO₃⁻ and N₂ cultures) or in a lower maximal growth rate (N₂ culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO₃⁻ or N₂ compared with NH₃.

     

Nitrous Oxide Production in Sputum from Cystic Fibrosis Patients with Chronic Pseudomonas aeruginosa Lung Infection

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O₂ for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N₂O), an intermediate in the denitrification pathway. We measured N₂O and O₂ with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO₃ ⁻ and NO₂ ⁻ in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N₂O (range 1.4–157.9 µM N₂O). The concentration of N₂O in the sputum was higher below the oxygenated layers. In 4 samples the N₂O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO₃ ⁻ decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N₂O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.     

Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO₂⁻) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO₂⁻-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO₂⁻ tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO₂⁻. Nitrite decreased the cellular NADH/NAD⁺ ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO₂⁻-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO₂⁻. These findings demonstrate a link between NO₂⁻ tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO₂⁻-tolerating mechanism in this strain.