基于amoA基因扩增子高通量测序的氨氧化古菌多样性分析方法

【背景】对于环境样品中氨氧化古菌(Ammonia-oxidizing archaea,AOA)多样性的研究,利用amoA功能基因作为分子标记会比16SrRNA基因有更强的特异性和更高的分辨率,能更准确地反映环境样品中氨氧化古菌的种群结构和分布特征。然而,目前对amoA基因扩增子高通量测序的分析存在两大限制因素:一是缺乏相应的amoA基因参考数据库;二是AOA amoA基因在种水平上的相似性阈值未知,分析过程中没有明确的划分种水平操作分类单元(Operational taxonomic unit,OTU)的阈值。【目的】构建基于amoA功能基因序列分析氨氧化古菌多样性的方法,为基于高通量测序的功能微生物多样性分析提供参考。【方法】基于目前已通过分离纯化或富集培养获得的34株氨氧化古菌及功能基因数据库中收录的环境样品amoA基因序列,构建氨氧化古菌amoA基因参考数据库。通过菌株间两两比对获得的amoA基因相似度与16SrRNA基因相似度的相关性分析,确定amoA基因在种水平上的相似性阈值。基于MOTHUR软件平台,利用建立的参考数据库和确定的阈值对南海一个垂直水体剖面样品的amoA基因序列进行多样性分析。【结果】构建了含有26 091条序列信息的古菌amoA基因参考数据库,确定了89%作为分析过程中古菌amoA基因划分种水平OTU的阈值,对南海水体样品氨氧化古菌的多样性分析结果很好地显示了南海不同深度水层水体中氨氧化古菌的种群结构和系统发育关系,有效揭示了南海氨氧化古菌的垂直分布差异。【结论】建立了基于amoA基因高通量测序的氨氧化古菌多样性分析方法,此方法可以有效分析环境样品中氨氧化古菌的多样性。     

Community composition of ammonia-oxidizing bacteria and archaea in soils under stands of red alder and Douglas fir in Oregon

This study determined nitrification activity and nitrifier community composition in soils under stands of red alder (Alnus rubra) and Douglas fir (Pseudotsuga menziesii) at two sites in Oregon. The H.J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon, has low net N mineralization and gross nitrification rates. Cascade Head Experimental Forest, in the Coast Range, has higher net N mineralization and nitrification rates and soil pH is lower. Communities of putative bacterial [ammonia-oxidizing bacteria (AOB)] and archaeal [ammonia-oxidizing archaea (AOA)] ammonia oxidizers were examined by targeting the gene amoA, which codes for subunit A of ammonia monooxygenase. Nitrification potential was significantly higher in red alder compared with Douglas-fir soil and greater at Cascade Head than H.J. Andrews. Ammonia-oxidizing bacteria amoA genes were amplified from all soils, but AOA amoA genes could only be amplified at Cascade Head. Gene copy numbers of AOB and AOA amoA were similar at Cascade Head regardless of tree type (2.3-6.0 x 10(6)amoA gene copies g(-1) of soil). DNA sequences of amoA revealed that AOB were members of Nitrosospira clusters 1, 2 and 4. Ammonia-oxidizing bacteria community composition, determined by terminal restriction fragment length polymorphism (T-RFLP) profiles, varied among sites and between tree types. Many of the AOA amoA sequences clustered with environmental clones previously obtained from soil; however, several sequences were more similar to clones previously recovered from marine and estuarine sediments. As with AOB, the AOA community composition differed between red alder and Douglas-fir soils.     

Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors

We report molecular evidence that ammonia-oxidizing archaea (AOA) occur in activated sludge bioreactors used to remove ammonia from wastewater. Using PCR primers targeting archaeal ammonia monooxygenase subunit A (amoA) genes, we retrieved and compared 75 sequences from five wastewater treatment plants operating with low dissolved oxygen levels and long retention times. All of these sequences showed similarity to sequences previously found in soil and sediments, and they were distributed primarily in four major phylogenetic clusters. One of these clusters contained virtually identical amoA sequences obtained from all five activated sludge samples (from Oregon, Wisconsin, Pennsylvania, and New Jersey) and accounted for 67% of all the sequences, suggesting that this AOA phylotype may be widespread in nitrifying bioreactors.     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。     

Abundance and composition dynamics of soil ammonia-oxidizing archaea in an alpine fir forest on the eastern Tibetan Plateau of China

Real-time qPCR and clone library sequencing targeting amoA genes were used to investigate the seasonal dynamics of an ammonia-oxidizing archaea (AOA) community in an alpine fir forest in western China. AOA were detected at all sampling dates, and there were significant variations in archaeal amoA gene copy numbers (7.63?×?10(5) to 8.35?×?10(8) per gram of dry soil) throughout the nongrowing season. Compared with ammonia-oxidizing bacteria (AOB), the AOA displayed a higher abundance on the majority of sampling dates during the freeze-thaw period. All of the AOA sequences fell within soil and sediment lineages and were affiliated with 7 clusters. Compared with the other clusters, cluster 1 was more sensitive to low temperature and was the dominant group in August. In contrast, cluster 3 dominated the AOA community in winter and probably represents a group of cold-adapted archaea. Redundancy analysis (RDA) revealed that the seasonality of the AOA community was mainly attributed to changes in soil temperature and nutrient availability (e.g., dissolved organic nitrogen and carbon). Our results indicate that AOA exist in frozen soils in the alpine coniferous forest ecosystem of the eastern Tibetan Plateau. Moreover, soil temperature may directly and (or) indirectly affect AOA abundance and composition and may further influence the soil N cycle during the winter.     

Occurrence of ammonia-oxidizing Archaea in activated sludges of a laboratory scale reactor and two wastewater treatment plants

Aims:  Characterization of the ammonia-oxidizing archaea (AOA) community in activated sludge from a nitrogen removal bioreactor and wastewater treatment plants (WWTPs).
Methods and Results:  Three primer sets specific for ammonia mono-oxygenase α -subunit ( amoA ) were used to construct clone libraries for activated sludge sample from a nitrogen removal bioreactor. One primer set resulted in strong nonspecific PCR products. The other two clone libraries retrieved both shared and unique AOA amoA sequences. One primer set was chosen to study the AOA communities of activated sludge samples from Shatin and Stanley WWTPs. In total, 18 AOA amoA sequences were recovered and compared to the previous reported sequences. A phylogenetic analysis indicated that sequences found in this study fell into three clusters.
Conclusions:  Different primers resulted in varied AOA communities from the same sample. The AOA found from Hong Kong WWTPs were closely similar to those from sediment and soil, but distinct from those from activated sludge in other places. A comparison of clone libraries between Shatin WWTP and bioreactor indicated the AOA community significantly shifted only after 30-day enrichment.
Significance and Impact of the Study:  This study confirmed the occurrence of AOA in a laboratory scale nitrogen removal bioreactor and Hong Kong WWTPs treating saline or freshwater wastewater. AOA communities found in this study were significantly different from those found in other places. To retrieve diverse AOA communities from environmental samples, a combination of different primers for the amoA gene is needed.     

Links between ammonia oxidizer community structure, abundance, and nitrification potential in acidic soils

Ammonia oxidation is the first and rate-limiting step of nitrification and is performed by both ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the environmental drivers controlling the abundance, composition, and activity of AOA and AOB communities are not well characterized, and the relative importance of these two groups in soil nitrification is still debated. Chinese tea orchard soils provide an excellent system for investigating the long-term effects of low pH and nitrogen fertilization strategies. AOA and AOB abundance and community composition were therefore investigated in tea soils and adjacent pine forest soils, using quantitative PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of respective ammonia monooxygenase (amoA) genes. There was strong evidence that soil pH was an important factor controlling AOB but not AOA abundance, and the ratio of AOA to AOB amoA gene abundance increased with decreasing soil pH in the tea orchard soils. In contrast, T-RFLP analysis suggested that soil pH was a key explanatory variable for both AOA and AOB community structure, but a significant relationship between community abundance and nitrification potential was observed only for AOA. High potential nitrification rates indicated that nitrification was mainly driven by AOA in these acidic soils. Dominant AOA amoA sequences in the highly acidic tea soils were all placed within a specific clade, and one AOA genotype appears to be well adapted to growth in highly acidic soils. Specific AOA and AOB populations dominated in soils at particular pH values and N content, suggesting adaptation to specific niches.     

白洋淀湖滨湿地岸边带氨氧化古菌与氨氧化细菌的分布特性

摘要：本研究通过分子生物学分析方法,以amoA基因为标记,考察了氨氧化古菌（Ammonia-Oxidizing Archaea, AOA）和氨氧化细菌（Ammonia-Oxidizing Bacteria,AOB）在华北平原的白洋淀这一典型湖泊的湖滨湿地岸边带系统中的生物多样性和丰度分布。在前人的研究中,氨氧化古菌在海洋、原生态土壤和人为干扰土壤等环境中主导氨氧化过程的完成。但本研究发现,在湿地岸边带系统中氨氧化过程并不是完全由氨氧化古菌主导完成,即氨氧化古菌和氨氧化细菌在不同区域分别占据主导地位。根据主导微生物的不同,可以将湿地岸边带区域划分为陆相区、中间区和湖相区。在湿地岸边带陆相区,氨氧化古菌主导氨氧化过程,氨氧化古菌的amoA基因丰度是氨氧化细菌的526倍（AOA：1.23?108每克干土;AOB：2.34?105每克干土）;在岸边带湖相区,氨氧化细菌主导氨氧化过程,氨氧化古菌的amoA基因丰度只有氨氧化细菌的1/50倍（AOA：3.17?106每克干土;AOB：1.39?108每克干土）;在岸边带中间区,两种微生物对氨氧化过程的贡献相当,二者的amoA基因丰度也相当 (AOA：9.83?106, AOB：4.08?106)。研究还发现,湿地中间区的微生物生物多样性高于陆相区和湖相区。在湿地中间区,氨氧化古菌和氨氧化细菌的生物多样性都最高,分别有5和7个操作分类单元（OTUs）;相比之下,岸边带陆相区和湖相区的多样性依次降低,陆相区的氨氧化古菌和氨氧化细菌分别有3和6个操作分类单元,湖相区的氨氧化古菌和氨氧化细菌分别有2和6个分类单元。本研究的两个结论进一步反映了湿地岸边带极强的空间异质性。     

Abundance and composition of epiphytic bacterial and archaeal ammonia oxidizers of marine red and brown macroalgae

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are important for nitrogen cycling in marine ecosystems. Little is known about the diversity and abundance of these organisms on the surface of marine macroalgae, despite the algae's potential importance to create surfaces and local oxygen-rich environments supporting ammonia oxidation at depths with low dissolved oxygen levels. We determined the abundance and composition of the epiphytic bacterial and archaeal ammonia-oxidizing communities on three species of macroalgae, Osmundaria volubilis, Phyllophora crispa, and Laminaria rodriguezii, from the Balearic Islands (western Mediterranean Sea). Quantitative PCR of bacterial and archaeal 16S rRNA and amoA genes was performed. In contrast to what has been shown for most other marine environments, the macroalgae's surfaces were dominated by bacterial amoA genes rather than those from the archaeal counterpart. On the basis of the sequences retrieved from AOB and AOA amoA gene clone libraries from each algal species, the bacterial ammonia-oxidizing communities were related to Nitrosospira spp. and to Nitrosomonas europaea and only 6 out of 15 operational taxonomic units (OTUs) were specific for the host species. Conversely, the AOA diversity was higher (43 OTUs) and algal species specific, with 17 OTUs specific for L. rodriguezii, 3 for O. volubilis, and 9 for P. crispa. Altogether, the results suggest that marine macroalgae may exert an ecological niche for AOB in marine environments, potentially through specific microbe-host interactions.     

Grassland management regimens reduce small-scale heterogeneity and species diversity of beta-proteobacterial ammonia pxidizer populations.

The impact of soil management practices on ammonia oxidizer diversity and spatial heterogeneity was determined in improved (addition of N fertilizer), unimproved (no additions), and semi-improved (intermediate management) grassland pastures at the Sourhope Research Station in Scotland. Ammonia oxidizer diversity within each grassland soil was assessed by PCR amplification of microbial community DNA with both ammonia oxidizer-specific, 16S rRNA gene (rDNA) and functional, amoA, gene primers. PCR products were analysed by denaturing gradient gel electrophoresis, phylogenetic analysis of partial 16S rDNA and amoA sequences, and hybridization with ammonia oxidizer-specific oligonucleotide probes. Ammonia oxidizer populations in unimproved soils were more diverse than those in improved soils and were dominated by organisms representing Nitrosospira clusters 1 and 3 and Nitrosomonas cluster 7 (closely related phylogenetically to Nitrosomonas europaea). Improved soils were only dominated by Nitrosospira cluster 3 and Nitrosomonas cluster 7. These differences were also reflected in functional gene (amoA) diversity, with amoA gene sequences of both Nitrosomonas and Nitrosospira species detected. Replicate 0.5-g samples of unimproved soil demonstrated significant spatial heterogeneity in 16S rDNA-defined ammonia oxidizer clusters, which was reflected in heterogeneity in ammonium concentration and pH. Heterogeneity in soil characteristics and ammonia oxidizer diversity were lower in improved soils. The results therefore demonstrate significant effects of soil management on diversity and heterogeneity of ammonia oxidizer populations that are related to similar changes in relevant soil characteristics.     

PCR profiling of ammonia-oxidizer communities in acidic soils subjected to nitrogen and sulphur deposition

Communities of ammonia-oxidizing bacteria (AOB) were characterized in two acidic soil sites experimentally subjected to varying levels of nitrogen and sulphur deposition. The sites were an acidic spruce forest soil in Deepsyke, Southern Scotland, with low background deposition, and a nitrogen-saturated upland grass heath in Pwllpeiran, North Wales. Betaproteobacterial ammonia-oxidizer 16S rRNA and ammonia monooxygenase (amoA) genes were analysed by cloning, sequencing and denaturing gradient gel electrophoresis (DGGE). DGGE profiles of amoA and 16S rRNA gene fragments from Deepsyke soil in 2002 indicated no effect of nitrogen deposition on AOB communities, which contained both Nitrosomonas europaea and Nitrosospira. In 2003, only Nitrosospira could be detected, and no amoA sequences could be retrieved. These results indicate a decrease in the relative abundance of AOB from the year 2002 to 2003 in Deepsyke soil, which may be the result of the exceptionally low rainfall in spring 2003. Nitrosospira-related sequences from Deepsyke soil grouped in all clusters, including cluster 1, which typically contains only sequences from marine environments. In Pwllpeiran soil, 16S rRNA gene libraries were dominated by nonammonia oxidizers and no amoA sequences were detectable. This indicates that autotrophic AOB play only a minor role in these soils even at high nitrogen deposition.     

Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices

The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated by using quantitative real-time polymerase chain reaction, cloning and sequencing approaches based on amoA genes. The soil, classified as agri-udic ferrosols with pH (H(2)O) ranging from 3.7 to 6.0, was sampled in summer and winter from long-term field experimental plots which had received 16 years continuous fertilization treatments, including fallow (CK0), control without fertilizers (CK) and those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): N, NP, NK, PK, NPK and NPK plus organic manure (OM). Population sizes of AOB and AOA changed greatly in response to the different fertilization treatments. The NPK + OM treatment had the highest copy numbers of AOB and AOA amoA genes among the treatments that received mineral fertilizers, whereas the lowest copy numbers were recorded in the N treatment. Ammonia-oxidizing archaea were more abundant than AOB in all the corresponding treatments, with AOA to AOB ratios ranging from 1.02 to 12.36. Significant positive correlations were observed among the population sizes of AOB and AOA, soil pH and potential nitrification rates, indicating that both AOB and AOA played an important role in ammonia oxidation in the soil. Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from different treatments were affiliated with Nitrosospira or Nitrosospira-like species and grouped into cluster 3, and little difference in AOB community composition was recorded among different treatments. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). Cluster M dominated exclusively in the N, NP, NK and PK treatments, indicating a pronounced difference in the community composition of AOA in response to the long-term fertilization treatments. These findings could be fundamental to improve our understanding of the importance of both AOB and AOA in the cycling of nitrogen and other nutrients in terrestrial ecosystems.     

Comparative analysis of archaeal 16S rRNA and <Emphasis Type="Italic">amoA</Emphasis> genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments

Considering their abundance and broad distribution, non-extremophilic Crenarchaeota are likely to play important roles in global organic and inorganic matter cycles. The diversity and abundance of archaeal 16S rRNA and putative ammonia monooxygenase alpha-subunit (amoA) genes were comparatively analyzed to study genetic potential for nitrification of ammonia-oxidizing archaea (AOA) in the surface layers (0-1 cm) of four marine sediments of the East Sea, Korea. After analysis of a 16S rRNA gene clone library, we found various archaeal groups that include the crenarchaeotal group (CG) I.1a (54.8%) and CG I.1b (5.8%), both of which are known to harbor ammonia oxidizers. Notably, the 16S rRNA gene of CG I.1b has only previously been observed in terrestrial environments. The 16S rRNA gene sequence data revealed a distinct difference in archaeal community among sites of marine sediments. Most of the obtained amoA sequences were not closely related to those of the clones retrieved from estuarine sediments and marine water columns. Furthermore, clades of unique amoA sequences were likely to cluster according to sampling sites. Using real-time PCR, quantitative analysis of amoA copy numbers showed that the copy numbers of archaeal amoA ranged from 1.1 x 10(7) to 4.9 x 10(7) per gram of sediment and were more numerous than those of bacterial amoA, with ratios ranging from 11 to 28. In conclusion, diverse CG I.1a and CG I.1b AOA inhabit surface layers of marine sediments and AOA, and especially, CG I.1a are more numerous than other ammonia-oxidizing bacteria.     

Seasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species in oligotrophic alpine lakes

The annual changes in the composition and abundance of ammonia-oxidizing archaea (AOA) were analyzed monthly in surface waters of three high mountain lakes within the Limnological Observatory of the Pyrenees (LOOP; northeast Spain) using both 16S rRNA and functional (ammonia monooxygenase gene, amoA) gene sequencing as well as quantitative PCR amplification. The set of biological data was related to changes in nitrogen species and to other relevant environmental variables. The whole archaeal assemblage was dominated by phylotypes closely related to the crenarchaeal 1.1a group (58% ± 18% of total 16S rRNA gene sequences), and consistent structural changes were detected during the study. Water temperature was the environmental variable that better explained spring, summer, and winter (ice-covered lakes) archaeal assemblage structure. The amoA gene was detected year round, and seasonal changes in amoA gene composition were well correlated with changes in the archaeal 16S rRNA gene pool. In addition, copy numbers of both the specific 1.1a group 16 rRNA and archaeal amoA genes were well correlated, suggesting that most freshwater 1.1a Crenarchaeota had the potential to carry out ammonia oxidation. Seasonal changes in the diversity and abundance of AOA (i.e., amoA) were better explained by temporal changes in ammonium, the substrate for nitrification, and mostly nitrite, the product of ammonia oxidation. Lacustrine amoA gene sequences grouped in coherent freshwater phylogenetic clusters, suggesting that freshwater habitats harbor typical amoA-containing ecotypes, which is different from soils and seas. We observed within the freshwater amoA gene sequence pool a high genetic divergence (translating to up to 32% amino acid divergence) between the spring and the remaining AOA assemblages. This suggests that different AOA ecotypes are adapted to different temporal ecological niches in these lakes.     

Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam

The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) communities under different long-term (17 years) fertilization practices were investigated using real-time polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE). A sandy loam with pH (H(2)O) ranging from 8.3 to 8.7 was sampled in years 2006 and 2007, including seven fertilization treatments of control without fertilizers (CK), those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): NP, NK, PK and NPK, half chemical fertilizers NPK plus half organic manure (1/2OMN) and organic manure (OM). The highest bacterial amoA gene copy numbers were found in those treatments receiving N fertilizer. The archaeal amoA gene copy numbers ranging from 1.54 x 10(7) to 4.25 x 10(7) per gram of dry soil were significantly higher than those of bacterial amoA genes, ranging from 1.24 x 10(5) to 2.79 x 10(6) per gram of dry soil, which indicated a potential role of AOA in nitrification. Ammonia-oxidizing bacteria abundance had significant correlations with soil pH and potential nitrification rates. Denaturing gradient gel electrophoresis patterns revealed that the fertilization resulted in an obvious change of the AOB community, while no significant change of the AOA community was observed among different treatments. Phylogenetic analysis showed a dominance of Nitrosospira-like sequences, while three bands were affiliated with the Nitrosomonas genus. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). These results suggest that long-term fertilization had a significant impact on AOB abundance and composition, while minimal on AOA in the alkaline soil.     

西藏米拉山土壤古菌16S rRNA及amoA基因多样性?分析

摘要：【目的】硝化作用在全球土壤氮循环中具有重要的作用,虽然细菌一度被认为单独负责催化这个过程的限速步骤,但是最近一些研究结果表明泉古菌具有氨氧化的能力。本文通过构建古菌16S rRNA 基因克隆文库和氨氧化古菌amoA基因文库,分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,为揭示青藏高原高寒草甸土壤古菌的多样性提供理论基础。【方法】采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA 基因和氨氧化古菌amoA基因克隆文库。【结果】通过构建系统发育树,表明古菌16S rRNA 基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲。氨氧化古菌amoA基因克隆文库中序列均为泉古菌。通过DOTUR软件分析,古菌16S rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和 75个OTUs。【结论】西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用。所获得的一些序列与已知环境中土壤、淡水及海洋沉积物中获得的一些序列具有很高的相似性,其古菌及氨氧化古菌来自不同环境的可能性比较大,可能与青藏高原的地质历史变迁过程有关。米拉山古菌及氨氧化古菌与陆地设施土壤中相似性最高,说明与西藏米拉山高寒草甸土壤的退化有关。     

Crenarchaeota and their role in the nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps

Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the "Franz-Josef-Quelle" (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (+/-4.5%) in the total 4',6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45 degrees C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation "thermal spring" cluster and which may be older than most AOA from soils on earth.     

古尔班通古特沙漠生物土壤结皮对氨氧化微生物生态位的影响

生物结皮作为荒漠地表的重要覆被类型, 在荒漠生态系统的氮素循环中扮演重要角色。融雪期为古尔班通古特沙漠生物结皮的复苏和生长提供了充足的水分, 也成为该沙漠氮素固定和转化的重要时期, 但该时期生物结皮如何影响驱动氨氧化转化的微生物群落动态尚未明确。因此, 我们利用荧光定量PCR (fluorescent quantitative PCR, qPCR)方法分析融雪期生物结皮与去除结皮不同土层(0-2, 2-5, 5-10和10-20 cm)氨氧化菌群丰度特征, 结合潜在硝化速率和土壤理化参数, 探究融雪期生物结皮对荒漠土壤氮素转化作用。结果表明: 氨氧化古菌(ammonia-oxidizing archaea, AOA)是古尔班通古特沙漠土壤优势氨氧化菌, 生物结皮对0-2 cm层土壤中AOA、氨氧化细菌(ammonia-oxidizing bacteria, AOB) amoA基因丰度具有显著抑制作用(P < 0.01), 对10-20 cm层土壤中AOA amoA基因丰度具有显著促进作用(P < 0.01)。冗余分析(redundancy analysis, RDA)表明, AOA、AOB amoA基因丰度主要受土壤含水量和铵态氮含量的影响, 占总条件效应的54.90%。氨氧化速率分析发现, 去除生物结皮显著降低古尔班通古特沙漠土壤硝化作用潜力(P < 0.001), 证实生物结皮对荒漠土壤氮素转化具有重要的调控作用。综上所述, 古尔班通古特沙漠氨氧化微生物的分布规律受环境因子调控, 特别是生物结皮可以通过调节土壤含水量和铵态氮含量影响AOA和AOB的空间生态位分化, 促进沙漠土壤的硝化作用。     

Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures

The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils. Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10 degrees C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22 degrees C). The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37 degrees C). OMS soil was also incubated with less fertilizer than the other soils. The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of the amoA gene, which codes for the alpha subunit of ammonia monooxygenase. All amoA gene sequences found belonged to the genus Nitrosospira. The analysis showed community change due to temperature both in moist soil and in the soil slurry. Two patterns of community change were observed. One pattern was a change between the different Nitrosospira clusters, which was observed in moist soil and slurry incubations of GMS and OMS. Nitrosospira AmoA cluster 1 was mainly detected below 30 degrees C, while Nitrosospira cluster 4 was predominant at 25 degrees C. Nitrosospira clusters 3a, 3b, and 9 dominated at 30 degrees C. The second pattern, observed in KMS, showed a community shift predominantly within a single Nitrosospira cluster. The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively to Nitrosospira cluster 3a. We conclude that ammonia oxidizer populations are influenced by temperature. In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers. Thus, Nitrosospira cluster 1 was absent in OMS soil treated with less fertilizer, while Nitrosospira cluster 9 was only found in the sample given less fertilizer.     

Spatial variability in nitrification rates and ammonia-oxidizing microbial communities in the agriculturally impacted Elkhorn Slough estuary, California

Ammonia oxidation-the microbial oxidation of ammonia to nitrite and the first step in nitrification-plays a central role in nitrogen cycling in coastal and estuarine systems. Nevertheless, questions remain regarding the connection between this biogeochemical process and the diversity and abundance of the mediating microbial community. In this study, we measured nutrient fluxes and rates of sediment nitrification in conjunction with the diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing betaproteobacteria (β-AOB). Sediments were examined from four sites in Elkhorn Slough, a small agriculturally impacted coastal California estuary that opens into Monterey Bay. Using an intact sediment core flowthrough incubation system, we observed significant correlations among NO(3)(-), NO(2)(-), NH(4)(+), and PO(4)(3+) fluxes, indicating a tight coupling of sediment biogeochemical processes. (15)N-based measurements of nitrification rates revealed higher rates at the less impacted, lower-nutrient sites than at the more heavily impacted, nutrient-rich sites. Quantitative PCR analyses revealed that β-AOB amoA (encoding ammonia monooxygenase subunit A) gene copies outnumbered AOA amoA gene copies by factors ranging from 2- to 236-fold across the four sites. Sites with high nitrification rates primarily contained marine/estuarine Nitrosospira-like bacterial amoA sequences and phylogenetically diverse archaeal amoA sequences. Sites with low nitrification rates were dominated by estuarine Nitrosomonas-like amoA sequences and archaeal amoA sequences similar to those previously described in soils. This is the first report measuring AOA and β-AOB amoA abundance in conjunction with (15)N-based nitrification rates in estuary sediments.