Additivity in the sensitizing effects of nitrous oxide and oxygen

In earlier work, we proposed that nitrous oxide (N2O) and low concentrations of oxygen (10(-6) less than [O2] less than 10(-4) mol dm-3) share a common sensitizing mechanism. We also proposed that the basis for sensitization by N2O is different from that by high concentrations of oxygen ([O2] greater than 10(-4) mol dm-3). We have now tested these proposals with several Escherichia coli strains using mixtures of O2 and N2O. In the strains that are sensitized by N2O, we found that damage from low concentrations of O2 does not add to that from N2O. In contrast, we did find additivity in the sensitizing effects of N2O and high concentrations of O2. In those E. coli strains that are not sensitized by N2O, the effects of any concentration of O2 are the same in either N2 or N2O. These results are qualitatively the same as those from our previous study with E. coli B/r, and they support our proposals concerning similarities and differences in sensitizing mechanisms of N2O and O2.     

The participation of cytochromes in the reduction of N20 to N2 by a denitryfying bacterium.

The oxidation of cytochromes during the reduction of N2O to N2 by a denitrifying bacterium was studied spectrophotometrically. The reduced b- and c-type cytochromes are partially oxidized when N2O is added to intact cells reduced with lactate under anaerobic conditions. The oxidation of cytochromes is inhibited non-competitively by azide, cyanide, 2,4-dinitrophenol and CuSO4, which inhibit the reduction of N2O to N2. In the presence of each inhibitor at a high concentration, at which the reduction of N2O to N2 is perfectly inhibited, cytochromes are not oxidized by N2O, while when an adequate, low concentration of inhibitor is added, b-type cytochrome is partially oxidized but c-type cytochrome is apparently not oxidized. In cell-free extracts, prepared by the sonic disruption of cells, that have entirely lost their activity in the reduction of N2O to N2, cytochromes are not oxidized by N2O. From the above results, it was concluded that b-type and c-type cytochromes should participate in the electron transport mechanism of the reduction of N2O to N2.     

Effects of varying O2 concentration on the X-ray sensitivity of transforming DNA.

The X-ray-induced inactivation of the biological activity of Bacillus subtilis transforming DNA in dilute aqueous solution has been studied over a wide range of O2 concentrations in an attempt to elucidate the mechanisms involved in O2 action. When the DNA is irradiated in the presence of 100 per cent O2 there is a protection of the transforming DNA compared to the sensitivity in N2-saturated or in N2O-saturated solutions. When the equilibrating gas contains intermediate concentrations of O2 (1 per cent--90 per cent) in N2 or N2O, the DNA sensitivity is equivalent to that in pure N2 or N2O respectively. At low O2 concentrations (approximately 0.14 per cent O2 in N2 or in N2O) there is a sensitization of the DNA and this sensitization can be prevented by .OH scavengers. Possible mechanisms for these actions of O2 on the radiation sensitivity of transforming DNA are discussed.     

Plant-mediated nitrous oxide emissions from beech (Fagus sylvatica) leaves

Nitrous oxide (N2O) emission estimates from forest ecosystems are based currently on emission measurements using soil enclosures. Such enclosures exclude emissions via tall plants and trees and may therefore underestimate the whole-ecosystem N2O emissions. Here, we measured plant-mediated N2O emissions from the leaves of potted beech (Fagus sylvatica) seedlings after fertilizing the soil with 15N-labelled ammonium nitrate (15NH4(15)NO3), and after exposing the roots to elevated concentrations of N2O. Ammonium nitrate fertilization induced N2O + 15N2O emissions from beech leaves. Likewise, the foliage emitted N2O after beech roots were exposed to elevated concentrations of N2O. The average N2O emissions from the fertilization and the root exposure experiments were 0.4 and 2.0 microg N m(-2) leaf area h(-1), respectively. Higher than ambient atmospheric concentrations of N2O in the leaves of the forest trees indicate a potential for canopy N2O emissions in the forest. Our experiments demonstrate the existence of a previously overlooked pathway of N2O to the atmosphere in forest ecosystems, and bring about a need to investigate the magnitude of this phenomenon at larger scales.     

Determination of anesthetic molecule environments by infrared spectroscopy. II. Multiple sites for nitrous oxide in proteins, lipids, and brain tissue

The presence of molecules of the general anesthetic nitrous oxide (N2O) in oils, esters, proteins, red cells, cream, lipid vesicles, and brain tissue upon exposure to the gas was observed by infrared spectroscopy. Analysis of the N-N-O antisymmetric stretch band reveals a distribution of N2O molecules among several sites of differing polarity in these solutions and tissues. The sensitivity of the band intensity and frequency to the number and strength of the dipoles in the solvating molecules is demonstrated by the resolution of N2O-ester and N2O-alkane interactions in acetic acid ethyl ester and oleic acid methyl ester. In all aqueous solutions and in all tissues a population of N2O molecules in water is observed. At least two sites of N2O-protein interaction are observed in purified hemoglobin A and packed red cells; multiple N2O sites may also be present in bovine serum albumin. Two sites of N2O-lipid interaction are observed in whipping cream and in an aqueous suspension of phosphatidylcholine vesicles. The sites providing the least polar immediate environment to N2O in hemoglobin, cream, and vesicles give similar band frequencies to those found in pure alkane solvents. Infrared spectra of bovine brain tissue, upon exposure to N2O, show N2O molecules present in water and in two less-polar environments. Analysis of spectra of N2O in cerebellum tissue removed from a dog under halothane-N2O anesthesia reveals, in addition to N2O in water, a single population of N2O molecules in an alkane-like environment. Infrared spectroscopy provides a unique means of probing the structure of the environment of N2O and should prove useful in correlating anesthetic potency with anesthetic environment under physiological conditions.     

A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis.

Soil microorganisms are important sources of the nitrogen trace gases NO and N2O for the atmosphere. Present evidence suggests that autotrophic nitrifiers such as Nitrosomonas europaea are the primary producers of NO and N2O in aerobic soils, whereas denitrifiers such as Pseudomonas spp. or Alcaligenes spp. are responsible for most of the NO and N2O emissions from anaerobic soils. It has been shown that Alcaligenes faecalis, a bacterium common in both soil and water, is capable of concomitant heterotrophic nitrification and denitrification. This study was undertaken to determine whether heterotrophic nitrification might be as important a source of NO and N2O as autotrophic nitrification. We compared the responses of N. europaea and A. faecalis to changes in partial O2 pressure (pO2) and to the presence of typical nitrification inhibitors. Maximal production of NO and N2O occurred at low pO2 values in cultures of both N. europaea (pO2, 0.3 kPa) and A. faecalis (pO2, 2 to 4 kPa). With N. europaea most of the NH4+ oxidized was converted to NO2-, with NO and N2O accounting for 2.6 and 1% of the end product, respectively. With A. faecalis maximal production of NO occurred at a pO2 of 2 kPa, and maximal production of N2O occurred at a pO2 of 4 kPa. At these low pO2 values there was net nitrite consumption. Aerobically, A. faecalis produced approximately the same amount of NO but 10-fold more N2O per cell than N. europaea did. Typical nitrification inhibitors were far less effective for reducing emissions of NO and N2O by A. faecalis than for reducing emissions of NO and N2O by N. europaea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter

During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.     

Generation of nitric oxide by enzymatic oxidation of N-hydroxy-N-nitrosamines

The nitric oxide (N = O) free radical exhibits potent cytocidal, mutagenic and vasodilatory properties. We have examined the hypothesis that the hydroxynitrosamino functionality (see sequence in text), which occurs naturally in antineoplastic and antihypertensive agents, will directly generate N = O following peroxidatic 1-electron oxidation. Cupferron (see sequence in text) is indeed an excellent (k greater than 10(7) m-1 s-1) substrate for horseradish peroxidase. The products are N = O and nitrosobenzene (phi - N = O) which are generated and consumed as follows. First, cupferron is oxidized by the classical peroxidatic mechanism to form an unstable nitroxide free radical (see sequence in text) which then forms N = O and phi - N = O spontaneously (see sequence in text). The N = O then reacts with phi - N = O to reform cupferron (see sequence in text) or with the enzyme to generate the characteristic peroxidase--N = O chromophore. Simultaneously, in a competitive reaction with O2, the N = O is converted to NO-2 (4N = O + O2 + 2H2O------------4NO-2 + 4H+). The reactivity of hydroxynitrosamino compounds with horseradish peroxidase is in the order cupferron greater than hydroxynitrosaminomethane greater than alanosine. These model reactions, involving direct oxidation of the hydroxynitrosamino moiety, comprise a novel pathway for the biological production of N = O.     

Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase

N(2)O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N(2)O uptake and conversion of (15)N-N(2)O into (15)N-N(2). Free-living cells of USDA110 showed N(2)O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N(2)O reductase activity. When detached soybean nodules formed with USDA110 were fed with (15)N-N(2)O, they rapidly emitted (15)N-N(2) outside the nodules at a ratio of 98.5% of (15)N-N(2)O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N(2)O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N(2)O (0.34 ppm). These results indicate that the conversion of N(2)O to N(2) depends exclusively on the respiratory N(2)O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N(2)O.     

微生物驱动的滨海湿地N2O产生及其机制研究进展

滨海湿地位于海陆交界,具有初级生产力高、生物多样性丰富以及微生物驱动的营养元素循环活跃等特点,同时也是大气中一氧化二氮(N_2O)的重要排放源。N_2O是仅次于二氧化碳(CO2)和甲烷(CH4)的第三大温室气体,而全球90%以上的N_2O排放由微生物主导,并与滨海湿地氮循环的微生物群落多样性及功能密切相关。因此,滨海湿地系统中N_2O的产生与转化逐渐受到关注。本文综述了滨海湿地生态系统中微生物驱动下N_2O的产生过程,以及氮元素及其与碳、硫和金属元素耦合过程中产生N_2O的代谢途径,N_2O排放的时空变化与微生物调控,并对未来相关研究方向进行了展望,旨在揭示微生物驱动的N_2O产生及环境调控机制,为减缓全球变暖提供科学依据。     

干旱胁迫对小麦幼苗根系生长和叶片光合作用的影响

采用水培试验方法,以2个耐旱性不同的小麦品种(敏感型望水白和耐旱型洛旱7号)为材料,研究了干旱胁迫对小麦幼苗根系形态、生理特性以及叶片光合作用的影响,以期揭示小麦幼苗对干旱胁迫的适应机制.结果表明： 干旱胁迫下,2个小麦品种幼苗的根系活力显著增大,而根数和根系表面积受到抑制;干旱胁迫降低了望水白的叶片相对含水量,提高了束缚水/自由水,而对洛旱7号无显著影响;干旱胁迫降低了2个小麦品种叶片的叶绿素含量、净光合速率、蒸腾速率、气孔导度和胞间CO2浓度,但随胁迫时间的延长,洛旱7号的叶绿素含量和净光合速率与对照差异不显著;干旱胁迫降低了2个小麦品种幼苗的单株叶面积,以及望水白的根系、地上部和植株生物量,而对洛旱7号无显著影响.水分胁迫下,耐旱型品种可以通过提高根系活力、保持较高的根系生长量来补偿根系吸收面积的下降,保持较高的根系吸水能力,进而维持较高的光合面积和光合速率,缓解干旱对生长的抑制.     

Evidence for involvement of gut-associated denitrifying bacteria in emission of nitrous oxide (N(2)O) by earthworms obtained from garden and forest soils.

Earthworms (Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) obtained from nitrous oxide (N(2)O)-emitting garden soils emitted 0.14 to 0.87 nmol of N(2)O h(-1) g (fresh weight)(-1) under in vivo conditions. L. rubellus obtained from N(2)O-emitting forest soil also emitted N(2)O, which confirmed previous observations (G. R. Karsten and H. L. Drake, Appl. Environ. Microbiol. 63:1878-1882, 1997). In contrast, commercially obtained Lumbricus terrestris did not emit N(2)O; however, such worms emitted N(2)O when they were fed (i.e., preincubated in) garden soils. A. caliginosa, L. rubellus, and O. lacteum substantially increased the rates of N(2)O emission of garden soil columns and microcosms. Extrapolation of the data to in situ conditions indicated that N(2)O emission by earthworms accounted for approximately 33% of the N(2)O emitted by garden soils. In vivo emission of N(2)O by earthworms obtained from both garden and forest soils was greatly stimulated when worms were moistened with sterile solutions of nitrate or nitrite; in contrast, ammonium did not stimulate in vivo emission of N(2)O. In the presence of nitrate, acetylene increased the N(2)O emission rates of earthworms; in contrast, in the presence of nitrite, acetylene had little or no effect on emission of N(2)O. In vivo emission of N(2)O decreased by 80% when earthworms were preincubated in soil supplemented with streptomycin and tetracycline. On a fresh weight basis, the rates of N(2)O emission of dissected earthworm gut sections were substantially higher than the rates of N(2)O emission of dissected worms lacking gut sections, indicating that N(2)O production occurred in the gut rather than on the worm surface. In contrast to living earthworms and gut sections that produced N(2)O under oxic conditions (i.e., in the presence of air), fresh casts (feces) from N(2)O-emitting earthworms produced N(2)O only under anoxic conditions. Collectively, these results indicate that gut-associated denitrifying bacteria are responsible for the in vivo emission of N(2)O by earthworms and contribute to the N(2)O that is emitted from certain terrestrial ecosystems.     

N2O as a substrate and as a competitive inhibitor of nitrogenase

We have investigated the inhibitory effect of N2O on NH3 formation by purified component proteins from Klebsiella pneumoniae and have confirmed that the inhibition is competitive with respect to N2 and that N2O is reduced to N2, which in turn is further reduced to NH3. In addition, we have shown that N2O is unable to support HD formation from D2 and H2O. N2-supported HD formation from D2 and H2O was found to be inhibited by N2O. In contrast to N2, N2O was found to suppress nitrogenase-mediated H2 evolution completely at infinitely high pN2O. H2 was found to inhibit N2O-supported NH3 production but not N2O-supported N2 production. The steady-state kinetics of N2O reduction showed a good fit to Michaelis-Menten kinetics with a Km for N2O of 5 mM at 30 degrees C, corresponding to 24 kPa of N2O. A model is proposed that fits the observed results.     

Loss of N2O reductase activity as an explanation for poor growth of Pseudomonas aeruginosa on N2O

N2O uptake activity of cells and N2O reductase activity of the soluble fraction from denitrifying bacteria were assayed. Pseudomonas aeruginosa strains PAO1 and P1 lost most of their N2O uptake activity and the ability to grow well on N2O within 2 to 5 h after exposure to N2O. Extensive loss of N2O reductase activity accompanied the nearly complete loss of N2O uptake activity under N2O. Paracoccus denitrificans retained much, but not all, of both activities and the ability to grow vigorously on N2O. The pattern with P. aeruginosa strain P2 resembled that for PAO1 and P1 except that loss of the activities proceeded at a slower rate and growth could continue for up to 12 h after exposure to N2O. The inability of a number of P. aeruginosa strains to grow well on N2O is therefore a direct consequence of the nearly complete loss of N2O reductase activity. Turnover-dependent inactivation of N2O reductase and its reactivation under reducing conditions occurred in vitro for the enzyme from P. aeruginosa and Paracoccus denitrificans. These events may be significant in determining the activity level of N2O reductase in denitrifying bacteria during N2O respiration.     

A comparison between rates of cell death and DNA damage during irradiation of Saccharomyces cerevisiae in N2 and N2O

Yeast and several other organisms are more sensitive to the lethal effects of ionizing irradiation if exposed in the presence of N2O as compared to N2. It has been suggested that this increased sensitivity is due to the cooperative effects of OH and H2O2 generated external to the cell wall. Using diploid yeast, wild type for radiation resistance, we have compared the rates of cell death due to gamma irradiation in N2 and N2O with the rates of DNA damage measured by gene conversion of trp- to trp+ (a recombinational repair event). We find that DNA damage as measured by gene conversion increases at a faster rate, per unit dose, during irradiation in N2O as compared to N2, just as lethality was higher in N2O. When DNA damage was compared in N2 and N2O at equal levels of survival, however, there was no significant difference between the two irradiation conditions. Therefore, increased lethality during irradiation in N2O seems to be directly due to increased DNA damage. If the observed increased lethality results from external OH and H2O2, the effect of these highly reactive species is expressed by increased internal damage at the level of DNA.     

Global oceanic production of nitrous oxide

We use transient time distributions calculated from tracer data together with in situ measurements of nitrous oxide (N(2)O) to estimate the concentration of biologically produced N(2)O and N(2)O production rates in the ocean on a global scale. Our approach to estimate the N(2)O production rates integrates the effects of potentially varying production and decomposition mechanisms along the transport path of a water mass. We estimate that the oceanic N(2)O production is dominated by nitrification with a contribution of only approximately 7 per cent by denitrification. This indicates that previously used approaches have overestimated the contribution by denitrification. Shelf areas may account for only a negligible fraction of the global production; however, estuarine sources and coastal upwelling of N(2)O are not taken into account in our study. The largest amount of subsurface N(2)O is produced in the upper 500 m of the water column. The estimated global annual subsurface N(2)O production ranges from 3.1 ± 0.9 to 3.4 ± 0.9 Tg N yr(-1). This is in agreement with estimates of the global N(2)O emissions to the atmosphere and indicates that a N(2)O source in the mixed layer is unlikely. The potential future development of the oceanic N(2)O source in view of the ongoing changes of the ocean environment (deoxygenation, warming, eutrophication and acidification) is discussed.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

植物排放N2O和CH4的研究

N2O和CH4是2种重要的温室气体,但其排放源尚未得到充分鉴别.1990年和2006年先后报道植物能排放N2O和CH4,并日益受到广泛的关注.然而,迄今为止对植物排放这2种气体的研究均是分开单独进行的.该文以8种陆生草本植物为研究对象,首次同步考察了新鲜离体植物地上部排放N2O和CH4的通量.研究结果表明:8种植物均能排放这2种气体.其中,黑麦草(Lolium perenne)、抱茎苦荬菜(Ixendium sonchifolium)和菠菜(Spinacia oleracea)的CH4通量较高,分别为165.38、52.28和21.64 ngCH4.g-1dw·h-1;抱茎苦荬菜、蒙古蒿(Artemisia mongolica)、大豆(Glycine max)和菠菜的N2O通量较高,分别为7.19、6.92、5.44和4.05 ngN2O·g-1dw.h-1.研究结果不仅为植物本身既能排放N2O又能排放CH4在植物中可能具有普遍性提供了进一步的实验依据,而且为深入研究其机理找到了几种适宜的植物种(如抱茎苦荬菜、菠菜).     

Loss of N2O reductase activity as an explanation for poor growth of Pseudomonas aeruginosa on N2O.

N2O uptake activity of cells and N2O reductase activity of the soluble fraction from denitrifying bacteria were assayed. Pseudomonas aeruginosa strains PAO1 and P1 lost most of their N2O uptake activity and the ability to grow well on N2O within 2 to 5 h after exposure to N2O. Extensive loss of N2O reductase activity accompanied the nearly complete loss of N2O uptake activity under N2O. Paracoccus denitrificans retained much, but not all, of both activities and the ability to grow vigorously on N2O. The pattern with P. aeruginosa strain P2 resembled that for PAO1 and P1 except that loss of the activities proceeded at a slower rate and growth could continue for up to 12 h after exposure to N2O. The inability of a number of P. aeruginosa strains to grow well on N2O is therefore a direct consequence of the nearly complete loss of N2O reductase activity. Turnover-dependent inactivation of N2O reductase and its reactivation under reducing conditions occurred in vitro for the enzyme from P. aeruginosa and Paracoccus denitrificans. These events may be significant in determining the activity level of N2O reductase in denitrifying bacteria during N2O respiration.     

Nitrous oxide production by the ectomycorrhizal fungi Paxillus involutus and Tylospora fibrillosa

Nitrous oxide (N(2)O) production by filamentous fungi has been demonstrated in pure culture and has been estimated indirectly in soils. However, it is unknown whether ectomycorrhizal fungi can also produce N(2)O. We demonstrate for the first time the ability of nitrogen (N)-tolerant ectomycorrhizal fungi (Paxillus involutus and Tylospora fibrillosa), found in forest soils under moderate to high rates of N deposition, to produce N(2)O from nitrate reduction. The N(2)O concentrations from the ectomycorrhizal fungal treatments after a 10-day pure culture experiment were 0.0117±0.00015 (P. involutus) and 0.0114±0.0003 (T. fibrillosa), and 0.0114±0.00043 μmol N(2)O L(-1) from a known fungal denitrifier (Fusarium lichenicola). No N(2)O was detected in the control treatment. Our results indicate the potential for these two N-tolerant ectomycorrhizal fungi to contribute to N(2)O production. Given that these species are abundant in many forest soils, the strength and regulation of fungal N(2)O production should now be verified in situ.