Nitrous Oxide Reductase Genes (nosZ) of Denitrifying Microbial Populations in Soil and the Earthworm Gut Are Phylogenetically Similar

Earthworms emit nitrous oxide (N₂O) and dinitrogen (N₂). It has been hypothesized that the in situ conditions of the earthworm gut activates ingested soil denitrifiers during gut passage and leads to these in vivo emissions (M. A. Horn, A. Schramm, and H. L. Drake, Appl. Environ. Microbiol. 69:1662-1669, 2003). This hypothesis implies that the denitrifiers in the earthworm gut are not endemic to the gut but rather are regular members of the soil denitrifier population. To test this hypothesis, the denitrifier populations of gut and soil from three different sites were comparatively assessed by sequence analysis of nosZ, the gene for the terminal enzyme in denitrification, N₂O reductase. A total of 182 and 180 nosZ sequences were retrieved from gut and soil, respectively; coverage of gene libraries was 79 to 100%. Many of the nosZ sequences were heretofore unknown, clustered with known soil-derived sequences, or were related to N₂O reductases of the genera Bradyrhizobium, Brucella, Dechloromonas, Flavobacterium, Pseudomonas, Ralstonia, and Sinorhizobium. Although the numbers of estimators for genotype richness of sequence data from the gut were higher than those of soil, only one gut-derived nosZ sequence did not group phylogenetically with any of the soil-derived nosZ sequences. Thus, the phylogenies of nosZ from gut and soil were not dissimilar, indicating that gut denitrifiers are soil derived.     

N2O-producing microorganisms in the gut of the earthworm Aporrectodea caliginosa are indicative of ingested soil bacteria

The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N(2)O) and (ii) to characterize the N(2)O-producing bacteria of the earthworm gut. The production of N(2)O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N(2)O were transient products when supplemental nitrate was reduced to N(2) by gut content homogenates. In contrast, nitrite and N(2)O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N(2)O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N(2)O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G+C contents, and 5 isolates were denitrifiers and reduced nitrate to N(2)O or N(2). The initial N(2)O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N(2)O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N(2)O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.     

Association of earthworm-denitrifier interactions with increased emission of nitrous oxide from soil mesocosms amended with crop residue

Earthworm activity is known to increase emissions of nitrous oxide (N(2)O) from arable soils. Earthworm gut, casts, and burrows have exhibited higher denitrification activities than the bulk soil, implicating priming of denitrifying organisms as a possible mechanism for this effect. Furthermore, the earthworm feeding strategy may drive N(2)O emissions, as it determines access to fresh organic matter for denitrification. Here, we determined whether interactions between earthworm feeding strategy and the soil denitrifier community can predict N(2)O emissions from the soil. We set up a 90-day mesocosm experiment in which (15)N-labeled maize (Zea mays L.) was either mixed in or applied on top of the soil in the presence or absence of the epigeic earthworm Lumbricus rubellus and/or the endogeic earthworm Aporrectodea caliginosa. We measured N(2)O fluxes and tested the bulk soil for denitrification enzyme activity and the abundance of 16S rRNA and denitrifier genes nirS and nosZ through real-time quantitative PCR. Compared to the control, L. rubellus increased denitrification enzyme activity and N(2)O emissions on days 21 and 90 (day 21, P = 0.034 and P = 0.002, respectively; day 90, P = 0.001 and P = 0.007, respectively), as well as cumulative N(2)O emissions (76%; P = 0.014). A. caliginosa activity led to a transient increase of N(2)O emissions on days 8 to 18 of the experiment. Abundance of nosZ was significantly increased (100%) on day 90 in the treatment mixture containing L. rubellus alone. We conclude that L. rubellus increased cumulative N(2)O emissions by affecting denitrifier community activity via incorporation of fresh residue into the soil and supplying a steady, labile carbon source.     

Denitrifying Bacteria in the Earthworm Gastrointestinal Tract and In Vivo Emission of Nitrous Oxide (N(inf2)O) by Earthworms

Earthworms (Lumbricus rubellus and Octolasium lacteum) and gut homogenates did not produce CH(inf4), and methanogens were not readily culturable from gut material. In contrast, the numbers of culturable denitrifiers averaged 7 x 10(sup7) and 9 x 10(sup6) per g (dry weight) of gut material for L. rubellus and O. lacteum, respectively; these values were 256- and 35-fold larger than the numbers of culturable denitrifiers in the soil from which the earthworms were obtained. Anaerobically incubated earthworm gut homogenates supplemented with nitrate produced N(inf2)O at rates exceeding that of soil homogenates. Furthermore, living earthworms emitted N(inf2)O under aerobic conditions, and N(inf2)O emission was stimulated by acetylene. For earthworms collected from a mildly acidic (pH 6) beech forest soil, the rates of N(inf2)O emission for earthworms and soil averaged 884 and 2 pmol per h per g (fresh weight), respectively. In contrast, for earthworms collected from a more acidic (pH 4.6) oak-beech forest soil, N(inf2)O emission by earthworms and soil averaged 145 and 45 pmol per h per g (fresh weight), respectively. Based on the extrapolation of this data, earthworms accounted for an estimated 16 and 0.25% of the total N(inf2)O produced at the stand level of these beech and oak-beech forest soils, respectively.     

Leachates from municipal solid waste disposal sites harbor similar, novel nitrogen-cycling bacterial communities

High emissions of nitrous oxide (N(2)O) have recently been documented at municipal solid waste (MSW) landfills. However, the biodiversity of the bacterial populations involved remains unexplored. In this study, we investigated communities of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria associated with the leachates from three MSW disposal sites by examining the diversity of the ammonia monooxygenase structural gene amoA and the nitrous oxide reductase gene nosZ, respectively. Cloning and phylogenetic analysis of the functional genes revealed novel and similar groups of prokaryotes involved in nitrogen cycling in the leachates with different chemical compositions. All amoA sequences recovered grouped within the Nitrosomonas europaea cluster in the Betaproteobacteria, with the vast majority showed only relatively moderate sequence similarities to known AOB but were exclusively most similar to environmental clones previously retrieved from wastewater treatment plants. All nosZ sequences retrieved did not cluster with any hitherto reported nosZ genes and were only remotely related to recognized denitrifiers from the Gammaproteobacteria and thus could not be affiliated. Significant overlap was found for the three denitrifying nosZ leachate communities. Our study suggests a significant selection of the novel N-cycling groups by the unique environment at these MSW disposal sites.     

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.     

Response of total and nitrate-dissimilating bacteria to reduced N deposition in a spruce forest soil profile

A field-scale manipulation experiment conducted for 16 years in a Norway spruce forest at Solling, Central Germany, was used to follow the long-term response of total soil bacteria, nitrate reducers and denitrifiers under conditions of reduced N deposition. N was experimentally removed from throughfall by a roof construction ('clean rain plot'). We used substrate-induced respiration (SIR) to characterize the active fraction of soil microbial biomass and potential nitrate reduction to quantify the activity of nitrate reducers. The abundance of total bacteria, nitrate reducers and denitrifiers in different soil layers was analysed by quantitative PCR of 16S rRNA gene, nitrate reduction and denitrification genes. Reduced N deposition temporarily affected the active fraction of the total microbial community (SIR) as well as nitrate reductase activity. However, the size of the total, nitrate reducer and denitrifier communities did not respond to reduced N deposition. Soil depth and sampling date had a greater influence on the density and activity of soil microorganisms than reduced deposition. An increase in the nosZ /16S rRNA gene and nosZ/nirK ratios with soil depth suggests that the proportion of denitrifiers capable of reducing N₂O into N₂ is larger in the mineral soil layer than in the organic layer.     

Actinobacterial nitrate reducers and proteobacterial denitrifiers are abundant in N2O-metabolizing palsa peat

Palsa peats are characterized by elevated, circular frost heaves (peat soil on top of a permanently frozen ice lens) and are strong to moderate sources or even temporary sinks for the greenhouse gas nitrous oxide (N(2)O). Palsa peats are predicted to react sensitively to global warming. The acidic palsa peat Skalluvaara (approximate pH 4.4) is located in the discontinuous permafrost zone in northwestern Finnish Lapland. In situ N(2)O fluxes were spatially variable, ranging from 0.01 to -0.02 μmol of N(2)O m(-2) h(-1). Fertilization with nitrate stimulated in situ N(2)O emissions and N(2)O production in anoxic microcosms without apparent delay. N(2)O was subsequently consumed in microcosms. Maximal reaction velocities (v(max)) of nitrate-dependent denitrification approximated 3 and 1 nmol of N(2)O per h per gram (dry weight [g(DW)]) in soil from 0 to 20 cm and below 20 cm of depth, respectively. v(max) values of nitrite-dependent denitrification were 2- to 5-fold higher than the v(max) nitrate-dependent denitrification, and v(max) of N(2)O consumption was 1- to 6-fold higher than that of nitrite-dependent denitrification, highlighting a high N(2)O consumption potential. Up to 12 species-level operational taxonomic units (OTUs) of narG, nirK and nirS, and nosZ were retrieved. Detected OTUs suggested the presence of diverse uncultured soil denitrifiers and dissimilatory nitrate reducers, hitherto undetected species, as well as Actino-, Alpha-, and Betaproteobacteria. Copy numbers of nirS always outnumbered those of nirK by 2 orders of magnitude. Copy numbers of nirS tended to be higher, while copy numbers of narG and nosZ tended to be lower in 0- to 20-cm soil than in soil below 20 cm. The collective data suggest that (i) the source and sink functions of palsa peat soils for N(2)O are associated with denitrification, (ii) actinobacterial nitrate reducers and nirS-type and nosZ-harboring proteobacterial denitrifiers are important players, and (iii) acidic soils like palsa peats represent reservoirs of diverse acid-tolerant denitrifiers associated with N(2)O fluxes.     

Phenotypic and genotypic heterogeneity among closely related soil-borne N2 - and N2 O-producing Bacillus isolates harboring the nosZ gene

Little is known about the genetic and phenotypic diversity of Gram-positive denitrifying bacteria. We compared the production of gaseous denitrification products for 14 closely related Bacillus soil isolates at pH 6 and 7 during 48-h batch incubations using a robotic gas-sampling apparatus. Primers targeting the nosZ gene encoding the nitrous oxide reductase were designed to confirm the presence of this gene in the isolates. The variation in the production of gaseous nitrogen products was compared with the genetic variation based on 16S rRNA gene sequences, genomic fingerprinting and nosZ sequences. The nosZ gene was detected in all isolates and all produced N(2) as the dominant end product at pH 7. Production of gaseous nitrogen products was more variable at pH 6, with different levels of NO and N(2) O production among the isolates, although minimal variation was observed among the 16S rRNA and nosZ gene sequences. One isolate was more divergent from the others based on genomic fingerprinting, and had two different nosZ gene copies, which coincided with the highest production of N(2) at pH 7 and the lack of intermediates at pH 6. Overall, our analysis suggests that genetic variation plays a role in the variation in N(2) O and N(2) production, but the variation in activity caused by acidification can be substantially greater than genotypic variation among closely related Bacillus.     

The earthworm gut: an ideal habitat for ingested N2O-producing microorganisms

The in vivo production of nitrous oxide (N(2)O) by earthworms is due to their gut microbiota, and it is hypothesized that the microenvironment of the gut activates ingested N(2)O-producing soil bacteria. In situ measurement of N(2)O and O(2) with microsensors demonstrated that the earthworm gut is anoxic and the site of N(2)O production. The gut had a pH of 6.9 and an average water content of approximately 50%. The water content within the gut decreased from the anterior end to the posterior end. In contrast, the concentration of N(2)O increased from the anterior end to the mid-gut region and then decreased along the posterior part of the gut. Compared to the soil in which worms lived and fed, the gut of the earthworm was highly enriched in total carbon, organic carbon, and total nitrogen and had a C/N ratio of 7 (compared to a C/N ratio of 12 in soil). The aqueous phase of gut contents contained up to 80 mM glucose and numerous compounds that were indicative of anaerobic metabolism, including up to 9 mM formate, 8 mM acetate, 3 mM lactate, and 2 mM succinate. Compared to the soil contents, nitrite and ammonium were enriched in the gut up to 10- and 100-fold, respectively. The production of N(2)O by soil was induced when the gut environment was simulated in anoxic microcosms for 24 h (the approximate time for passage of soil through the earthworm). Anoxia, high osmolarity, nitrite, and nitrate were the dominant factors that stimulated the production of N(2)O. Supplemental organic carbon had a very minimal stimulatory effect on the production of N(2)O, and addition of buffer or ammonium had essentially no effect on the initial N(2)O production rates. However, a combination of supplements yielded rates greater than that obtained mathematically for single supplements, suggesting that the maximum rates observed were due to synergistic effects of supplements. Collectively, these results indicate that the special microenvironment of the earthworm gut is ideally suited for N(2)O-producing bacteria and support the hypothesis that the in situ conditions of the earthworm gut activate ingested N(2)O-producing soil bacteria during gut passage.     

非典型氧化亚氮还原酶基因nosZ II研究进展

氧化亚氮(N_2O)是一种强力温室气体,能够破坏臭氧层。微生物含有的nosZ基因能够编码氧化亚氮还原酶,该酶可还原N_2O成为无害的N_2,因而对环境中nosZ基因的研究成为气候变化研究的一个热点。最近研究者对全基因组序列分析的结果揭示了一类新型nosZ基因(非典型nosZ Ⅱ基因)存在于更为广泛和多样的氮代谢微生物当中,这类nosZ编码的蛋白能够起到氧化亚氮还原酶的作用,并且广泛存在于多样的自然环境中。然而,针对含有非典型nosZ Ⅱ基因的微生物的相关研究还很不全面,这类微生物发挥作用的环境条件以及在N_2O还原过程中的特性仍然未知。本文主要综述了非典型nosZ Ⅱ基因与典型nosZ Ⅰ的主要差异、在环境中的分布情况以及未来研究方向的展望等。     

Abundance, diversity and functional gene expression of denitrifier communities in adjacent riparian and agricultural zones

Lands under riparian and agricultural management differ in soil properties, water content, plant species and nutrient content and are therefore expected to influence denitrifier communities, denitrification and nitrous oxide (N(2) O) emissions. Denitrifier community abundance, denitrifier community structure, denitrification gene expression and activity were quantified on three dates in a maize field and adjacent riparian zone. N(2) O emissions were greater in the agricultural zone, whereas complete denitrification to N(2) was greater in the riparian zone. In general, the targeted denitrifier community abundance did not change between agricultural and riparian zones. However, nosZ gene expression was greater in the riparian zone than the agricultural zone. The community structure of nirS-gene-bearing denitrifiers differed in June only, whereas the nirK-gene-bearing community structure differed significantly between the riparian and the agricultural zones at all dates. The nirK-gene-bearing community structure was correlated with soil pH, while no significant correlations were found between nirS-gene-bearing community structure and soil environmental variables or N(2) O emissions, denitrification or denitrifier enzyme activity. The results suggested for the nirK and nirS-gene-bearing communities different factors control abundance vs. community structure. The nirK-gene-bearing community structure was also more responsive than the nirS-gene-bearing community structure to change between the two ecosystems.     

The Earthworm Gut: an Ideal Habitat for Ingested N2O-Producing Microorganisms

The in vivo production of nitrous oxide (N₂O) by earthworms is due to their gut microbiota, and it is hypothesized that the microenvironment of the gut activates ingested N₂O-producing soil bacteria. In situ measurement of N₂O and O₂ with microsensors demonstrated that the earthworm gut is anoxic and the site of N₂O production. The gut had a pH of 6.9 and an average water content of approximately 50%. The water content within the gut decreased from the anterior end to the posterior end. In contrast, the concentration of N₂O increased from the anterior end to the mid-gut region and then decreased along the posterior part of the gut. Compared to the soil in which worms lived and fed, the gut of the earthworm was highly enriched in total carbon, organic carbon, and total nitrogen and had a C/N ratio of 7 (compared to a C/N ratio of 12 in soil). The aqueous phase of gut contents contained up to 80 mM glucose and numerous compounds that were indicative of anaerobic metabolism, including up to 9 mM formate, 8 mM acetate, 3 mM lactate, and 2 mM succinate. Compared to the soil contents, nitrite and ammonium were enriched in the gut up to 10- and 100-fold, respectively. The production of N₂O by soil was induced when the gut environment was simulated in anoxic microcosms for 24 h (the approximate time for passage of soil through the earthworm). Anoxia, high osmolarity, nitrite, and nitrate were the dominant factors that stimulated the production of N₂O. Supplemental organic carbon had a very minimal stimulatory effect on the production of N₂O, and addition of buffer or ammonium had essentially no effect on the initial N₂O production rates. However, a combination of supplements yielded rates greater than that obtained mathematically for single supplements, suggesting that the maximum rates observed were due to synergistic effects of supplements. Collectively, these results indicate that the special microenvironment of the earthworm gut is ideally suited for N₂O-producing bacteria and support the hypothesis that the in situ conditions of the earthworm gut activate ingested N₂O-producing soil bacteria during gut passage.     

Nitrous oxide reductase (nosZ) gene fragments differ between native and cultivated Michigan soils

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mean) = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.     

蚯蚓和三叉苦对亚热带人工林土壤N2O和CH4通量的短期效应

在广东鹤山大叶相思(Acacia auriculaeformis)人工林内设置外来蚯蚓西土寒宪蚓(Ocnerodrilus occidentalis)和乡土植物三叉苦(Evodia lepta)野外控制实验,用静态箱-气相色谱法对土壤N2O和CH4通量进行15 d的原位测定,研究蚯蚓和三叉苦对土壤N2O和CH4通量的影响。结果表明,三叉苦并未明显增加土壤N2O和CH4的通量,而假植物(模拟三叉苦的物理效应)则显著促进了土壤N2O的释放通量。整个实验阶段,蚯蚓效应分别使无植物对照和三叉苦处理土壤N2O通量增加了26.7%和66.3%,而在种假植物条件下,添加蚯蚓使土壤N2O通量降低了39.7%;同时,蚯蚓效应使对照处理土壤CH4吸收通量增加了10.3%,使假植物处理土壤CH4吸收通量降低了90.6%,而使三叉苦处理土壤CH4释放通量增加了301.8%。可见,蚯蚓能够促进人工林土壤N2O释放;同时促进人工林土壤从CH4“汇”向“源”转变。三叉苦的物理过程促进土壤N2O的释放,而三叉苦的生物过程抑制土壤N2O的排放。如何减缓人工林中土壤N2O和CH4的排放,必须综合考虑植物物理过程、生物过程以及蚯蚓对土壤N2O和CH4排放过程影响的独立效应和交互效应。     

Influence of maize mucilage on the diversity and activity of the denitrifying community

In order to understand the effect of the maize rhizosphere on denitrification, the diversity and the activity of the denitrifying community were studied in soil amended with maize mucilage. Diversity of the denitrifying community was investigated by polymerase chain reaction (PCR) amplification of total community DNA extracted from soils using gene fragments, encoding the nitrate reductase (narG) and the nitrous oxide reductase (nosZ), as molecular markers. To assess the underlying diversity, PCR products were cloned and 10 gene libraries were obtained for each targeted gene. Libraries containing 738 and 713 narG and nosZ clones, respectively, were screened by restriction fragment analysis, and grouped based on their RFLP (restriction fragment length polymorphism) patterns. In all, 117 and 171 different clone families have been identified for narG and nosZ and representatives of RFLP families containing at least two clones were sequenced. Rarefaction curves of both genes did not reach a clear saturation, indicating that analysis of an increasing number of clones would have revealed further diversity. Recovered NarG sequences were related to NarG from Actinomycetales and from Proteobacteria but most of them are not related to NarG from known bacteria. In contrast, most of the NosZ sequences were related to NosZ from alpha, beta, and gammaProteobacteria. Denitrifying activity was monitored by incubating the control and amended soils anaerobically in presence of acetylene. The N2O production rates revealed denitrifying activity to be greater in amended soil than in control soil. Altogether, our results revealed that mucilage addition to the soil results in a strong impact on the activity of the denitrifying community and minor changes on its diversity.     

Detection of a nitrous oxide reductase structural gene in Rhizobium meliloti strains and its location on the nod megaplasmid of JJ1c10 and SU47.

The gene encoding a denitrification enzyme, nitrous oxide reductase (EC 1.7.99.6), in Rhizobium meliloti and other gram-negative bacteria was detected by hybridization to an internal 1.2-kb PstI fragment of the structural gene (nosZ) cloned from Pseudomonas stutzeri Zobell (W.G. Zumft, A. Viebrock-Sambale, and C. Braun, Eur. J. Biochem. 192:591-599, 1990). Homology to the probe was detected in the DNAs of two N2-fixing strains of P. stutzeri, two denitrifying Pseudomonas species, one Alcaligenes eutrophus strain, and 36 of 56 R. meliloti isolates tested. Except for two isolates of R. meliloti, all showed nitrous oxide reduction activity (Nos+). Therefore, at least part of the nosZ sequence appears to be conserved and widely distributed among denitrifiers, which include free-living and symbiotic diazotrophs. By using Agrobacterium tumefaciens transconjugants harboring different megaplasmids of R. meliloti JJ1c10 and SU47, sequence homology with the nosZ probe was unequivocally located on the nod megaplasmid. A cosmid clone of JJ1c10 in which nosZ homology was mapped on a 4.2-kb BamHI fragment was selected. This cosmid, which conferred Nos+ activity to the R. meliloti wild-type strains ATCC 9930 and Balsac (Nos- and nondenitrifying, respectively) also restored Nos+ activity in the mutants of JJ1c10 and SU47 in which the 4.2-kb BamHI segment was deleted. Therefore, this segment contains sequences essential for nos gene expression in JJ1c10 and SU47 and thus confirms that the nod megaplasmid in JJ1c10 and SU47 which carries genes essential for symbiotic dinitrogen fixation also carries genes involved in the antagonistic process of denitrification.     

化肥对稻田土壤细菌多样性及硝化、反硝化功能菌组成的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,采用聚合酶链式反应(polymerase chain reaction,PCR)和DNA序列测定技术分析研究了3种长期施肥制度(对照不施肥-CK,单施氮肥-N,氮磷钾肥-NPK)对土壤细菌群落以及硝化、反硝化微生物种群的影响.通过系统分析细菌16S rDNA、细菌的硝化基因氨单加氧酶(ammonia monooxygenase,amoA)和反硝化基因氧化亚氮还原酶(nitrous oxide reductase,nosZ)等基因文库发现,长期单施氮肥导致细菌16S rDNA和amoA的多样性明显低于CK和NPK处理,而nosZ的多样性与之相反,即单施氮肥处理明显高于CK和NPK处理.LUBSHUFF软件统计分析显示:16S rDNA和amoA基因文库在CK与N,CK与NPK,NPK与N处理间均存在显著性差异.而对于nosZ基因文库,N和NPK与CK处理相比呈现出了显著性差异,N与NPK之间的差异没有达到显著水平.上述结果表明长期施用化肥对水稻土细菌的群落结构及硝化和反硝化细菌组成产生了明显的影响,但这种影响因基因类型而异.     

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.