几种农田土壤中古菌、泉古菌和细菌的数量分布特征

真核生物、细菌和古菌三者共同构成了生命的三域系统.古菌作为第3种生命形式,不仅能在高温、强酸和高盐等极端环境下生存,而且在海洋、湖泊和土壤等生境中也广泛分布,预示其在整个生态系统中有着不可估量的作用.本文以2个农田剖面土壤和2个长期施肥试验站祁阳(QY)和封丘(FQ)的土壤为对象,以实时定量PCR方法为主要研究手段,对土壤中古菌(包括泉古菌)和细菌的16S rRNA基因拷贝数丰度变化进行了研究.结果表明:土壤泉古菌16S rRNA基因拷贝数要低于古菌l～2个数量级,两者与细菌相比,16S rRNA基因拷贝数大小顺序为土壤泉古菌＜古茵＜细菌,而古菌和泉古菌16S rRNA基因拷贝数与细菌的比值均随土壤深度加深而增大.不同施肥处理对土壤古菌和泉古茵的数量有显著影响.QY试验站土壤古菌和细菌的数量与土壤pH值显著相关(分别为r=0.850,P＜0.01和r=0.676,P＜0.05).FQ古菌、泉古菌和细菌与土壤pH值相关性不显著,与土壤有机质含量相关性均达显著水平(分别为r=0.783,P＜0.05;r=0.827,P＜0.05;r=0.767,P＜0.05).了解古菌包括泉古菌在农田土壤中的分布,可为评价其在生态系统和物质循环中的作用提供重要的理论依据.     

腾冲两热泉泉古菌多样性及系统发育的初步分析

通过构建16S rRNA基因片段的克隆文库对腾冲热海两温泉中泉古菌的多样性和系统发育关系进行了初步的研究.一共得到18个泉古茵克隆序列,可分为12个OTUs,两温泉的克隆序列与已知GenBank上关系最近序列的平均相似性较低,无名泉为92.56%,热爆区为93%.从基于16S rRNA基因片段序列构建的系统发育树来看,74℃的无名泉样点中既有属于超高温环境类群的泉古菌,同时又有属于和常温环境较接近的泉古菌;45℃的热爆区样点的泉古茵,相对来说则更接近于常温类群.本次研究表明,腾冲热泉与世界其它同类热泉之间的泉古茵类群存在着一定的差异;而且两实验样点代表了超高温和高温环境泉古菌逐渐向常温过度的两个重要环境.     

一个新的古菌类群———奇古菌门(Thaumarchaeota)

基于16S rRNA基因的系统发育关系,古菌域(Archaea)被分为两个主要类群:广古菌门(Euryarchaeota)和泉古菌门(Crenarchaeota)。近20年来,微生物分子生态学技术的快速发展和应用显示,在中温环境中广泛存在着大量的未培养古菌,而且它们可能在自然界重要元素(N、C)的生物地球化学循环中发挥着重要作用。最初,这些未培养古菌因在16S rRNA基因系统发育上与泉古菌关系较密切而被称作中温泉古菌(non-thermophilic Crenarchaeota)。而近年来,对更多新发现的中温古菌核糖体RNA基因序列和其它分子标记物进行的分析均不支持中温泉古菌由嗜热泉古菌进化而来的假设,而揭示其可能代表古菌域中一个独立的系统发育分支。基因组学、生理生态特征等分析也显示中温泉古菌与泉古菌具有明显不同的特征。因而专家建议将这些古菌(中温泉古菌)划分为一个新的门,成为古菌域的第三个主要类群—Thaumarchaeota(意译为奇古菌门)。这一新古菌门提出后得到其他研究证据的支持和认可。本文对目前已知的奇古菌门的分类地位演化、基因组学、多样性和生理代谢特征等作一简要综述。     

新疆沙湾冷泉沉积物中免培养古菌多样性初步研究

【目的】了解新疆沙湾冷泉沉积物的古菌组成及多样性。【方法】采用免培养法,液氮研磨提取冷泉沉积物总DNA,使用古菌通用引物进行16S rRNA基因扩增,构建16S rRNA基因文库。对阳性克隆进行HhaI限制性酶切分型,选出具有不同酶切图谱的序列进行测序,将所得序列与GenBank数据库中序列比对并构建16S rRNA基因系统发育树。【结果】从冷泉沉积物古菌16S rRNA基因文库中随机挑选了121个阳性克隆,共得到22个不同的可操作分类单元,BLAST结果表明全部克隆子归属于泉古菌门(Crenarchaeote)中免培养类群。系统发育分析归类为Soil-Freshwater-subsurface group和MarinegroupI,2个亚群并且各占整个文库的50%。其中40%左右的克隆子与具有无机碳和硝酸盐同化能力的泉古菌有高的相似性。此外还发现40%的克隆子与低温泉古菌类群具有很高的相似性。【结论】新疆沙湾冷泉沉积物中古菌类群多样性较低,但存有大量高度适应此低温、贫营养环境的泉古菌类群。     

俄罗斯堪察加地区热泉及其周边生境的泉古菌多样性

【目的】泉古菌为陆地热泉系统的主要古菌类群,可能在自然界生源元素的地球化学循环中发挥着重要作用。本研究旨在揭示俄罗斯堪察加地区热泉以及热泉周边区域的泉古菌多样性,同时基于之前已获得的我国云南地区热泉数据,比较两地区泉古菌群落差异。【方法】通过构建16S rRNA基因片段克隆文库获得序列信息和丰度,随后进行物种多样性、系统发育和群落结构差异分析。【结果】高温热泉Burlyashi Liza(BSL,89℃)中的泉古菌全部属于热变形菌纲(Thermoprotei)内的物种。中温热泉TF Vent 2(TFV,49℃)的群落结构主要由不确定的热变形菌纲类群、不确定的泉古菌类群、高温水环境泉古菌类群Ⅱ(HWCG-Ⅱ)和奇古菌下的Group1.1b类群组成。热泉周边常温环境的主要物种与热泉环境的代表性克隆pJP41一起聚成一个较大的遗传分枝。Jackknife聚类树和主坐标分析(Principal coordinates analysis,PCoA)的结果显示:温度相似的样点,其泉古菌群落结构相对来说更为相似。【结论】俄罗斯堪察加地区与我国云南地区热泉中的泉古菌存在着一定程度上的不同。陆地热泉系统影响着其周边环境的泉古菌类群。热泉中泉古菌群落结构受温度的明显影响。     

灌木林土壤古菌群落结构对地表野火的快速响应

为了评价地表野火对土壤古菌群落结构的即时影响,在灌木林火发生18h后,于火烧迹地和未烧地采集土壤样品,通过构建两个土壤古菌16S rDNA基因文库,研究火烧后小时尺度下土壤古菌群落结构的变化。从火烧和对照未烧土壤古菌文库中共得到19个古菌的操作分类单元 (Operation taxonomic units,OTUs),其中对照未烧林地土壤文库中存在9个OTUs是火烧土壤中缺失的类群,占克隆总数的5.91%。未烧林地中3个优势古菌克隆YMar-F25（GQ304791）、YMar-F32（GQ304792）及YMar-F21（GQ304789）系统发育归类为泉古菌门亚群GroupⅠ,分别占克隆总数的15.50%,18.18%,41.36%;而在火烧迹地中这3个古菌分类群仍为优势群,分别占克隆总数的15.59%,22.58%,46.24%。多样性指数分析显示对照土壤古菌文库Shannon-Wiener指数为1.87,而火烧土壤古菌文库为1.40。结果表明,在小时尺度上,高强度灌木林火使土壤古菌多样性立即降低,低丰度土壤古菌种群对火干扰敏感,高丰度古菌的种群结构火烧后无明显变化。     

土壤中温泉古菌研究进展

古菌一直被冠以嗜极端环境的特征,直到最近十几年,由于分子生物学技术的发展,越来越多的证据表明,在许多非极端环境,包括海洋、湖泊和土壤中,分布着一类特殊的古菌-非嗜热泉古菌(non-thermophilic Crenarchaeota).该类古菌不仅分布广泛,而且数量巨大.通过16S rRNA基因序列分析发现,中温泉古菌可能参与到全球碳、氮等生物地球化学循环,预示着其在整个生态系统中起着重要的作用.从古菌分类着手,阐述了中温泉古菌在土壤中的分布和数量特征、影响因素,进而对其在氮和碳循环过程中的潜在作用进行了简要介绍,并提出了今后的研究重点.     

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。     

西藏米拉山土壤古茵16S rRNA及amoA基因多样性分析

[目的]本研究旨在了解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.[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.利用DOTUR软件将古菌和氨氧化古菌序列按照相似性97%的标准分成若干个可操作分类单元(OTUs).[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.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.     

利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

The impact of grassland management on archaeal community structure in upland pasture rhizosphere soil

The community structure of rhizosphere soil Archaea from three grassland types, associated with different management practices, was examined at a site in the Borders region of Scotland, by analysis of 16S rRNA gene fragments amplified from 16S rDNA and from rRNA. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of amplified products indicated high relative abundance within the archaeal community of two distinct lineages of non-thermophilic (group 1) Crenarchaeota. Grassland management practices influenced archaeal community structure, as characterized by both 16S rRNA- and 16S rDNA-derived DGGE profiles. One band dominated DGGE profiles in all three grassland types examined, and reproducible differences in the presence and intensity of bands were observed between profiles from managed and natural grassland sites. Analysis of 16S rRNA-derived amplicons from managed and natural grasslands at sites in the north of England and the north of Wales also indicated high relative abundance of non-thermophilic crenarchaeotes within the archaeal community. The band dominating the Scottish grassland site also dominated DGGE profiles from the English and Welsh sites, and similar differences were seen between profiles derived from soils subjected to different management regimes. The study indicates that grassland archaeal communities are dominated by Crenarchaeota, with closely related members of this lineage ubiquitous in distribution in UK upland pasture, and indicate that management practices influence the nature of the crenarchaeotal community.     

Diverse archaeal community of a bat guano pile in Domica Cave (Slovak Karst,Slovakia)

The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bedrock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5–3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.     

Diversity of thermophilic and non-thermophilic Crenarchaeota at 80 degrees C

A hot spring in the solfataric field of Pisciarelli (Naples-Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min.     

Diversity and abundance of Crenarchaeota in terrestrial habitats studied by 16S RNA surveys and real time PCR

Novel phylogenetic lineages of as yet uncultivated crenarchaeota have been frequently detected in low to moderate-temperature, marine and terrestrial environments. In order to gain a more comprehensive view on the distribution and diversity of Crenarchaeota in moderate habitats, we have studied 18 different terrestrial and freshwater samples by 16S rDNA-based phylogenetic surveys. In seven different soil samples of diverse geographic areas in Europe (forest, grassland, ruderal) and Asia (permafrost, ruderal) as well as in two microbial mats, we have consistently found one particular lineage of crenarchaeota. The diversity of Crenarchaeota in freshwater sediments was considerably higher with respresentative 16S rDNA sequences distributed over four different groups within the moderate crenarchaeota. Systematic analysis of a 16S rDNA universal library from a sandy ecosystem containing 800 clones exclusively revealed the presence of the soil-specific crenarchaeotal cluster. With primers specific for non-thermophilic crenarchaeota we established a rapid method to quantify archaeal 16S rDNA in real time PCR. The relative abundance of crenarchaeotal rDNA was 0.5-3% in the bulk soil sample and only 0.16% in the rhizosphere of the sandy ecosystem. A nearby agricultural setting yielded a relative abundance of 0.17% crenarchaeotal rDNA. In total our data suggest that soil crenarchaeota represent a stable and specific component of the microbiota in terrestrial habitats.     

Phylogenetic Analysis of Nonthermophilic Members of the Kingdom Crenarchaeota and Their Diversity and Abundance in Soils

Within the last several years, molecular techniques have uncovered numerous 16S rRNA gene (rDNA) sequences which represent a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species. rDNA sequences of members of this novel crenarchaeotal group have been recovered from low- to moderate-temperature environments (−1.5 to 32°C), in contrast to the high-temperature environments (temperature, >80°C) required for growth of the currently recognized crenarchaeotal species. We determined the diversity and abundance of the nonthermophilic members of the Crenarchaeota in soil samples taken from cultivated and uncultivated fields located at the Kellogg Biological Station’s Long-Term Ecological Research site (Hickory Corners, Mich.). Clones were generated from 16S rDNA that was amplified by using broad-specificity archaeal PCR primers. Twelve crenarchaeotal sequences were identified, and the phylogenetic relationships between these sequences and previously described crenarchaeotal 16S rDNA sequences were determined. Phylogenetic analyses included nonthermophilic crenarchaeotal sequences found in public databases and revealed that the nonthermophilic Crenarchaeota group is composed of at least four distinct phylogenetic clusters. A 16S rRNA-targeted oligonucleotide probe specific for all known nonthermophilic crenarchaeotal sequences was designed and used to determine their abundance in soil samples. The nonthermophilic Crenarchaeota accounted for as much as 1.42% ± 0.42% of the 16S rRNA in the soils analyzed.     

Abundance and diversity of Archaea in heavy-metal-contaminated soils.

The impact of heavy-metal contamination on archaean communities was studied in soils amended with sewage sludge contaminated with heavy metals to varying extents. Fluorescent in situ hybridization showed a decrease in the percentage of Archaea from 1.3% +/- 0.3% of 4', 6-diamidino-2-phenylindole-stained cells in untreated soil to below the detection limit in soils amended with heavy metals. A comparison of the archaean communities of the different plots by denaturing gradient gel electrophoresis revealed differences in the structure of the archaean communities in soils with increasing heavy-metal contamination. Analysis of cloned 16S ribosomal DNA showed close similarities to a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species.     

Putative ammonia-oxidizing Crenarchaeota in suboxic waters of the Black Sea: a basin-wide ecological study using 16S ribosomal and functional genes and membrane lipids

Within the upper 400 m at western, central and eastern stations in the world's largest stratified basin, the Black Sea, we studied the qualitative and quantitative distribution of putative nitrifying Archaea based on their genetic markers (16S rDNA, amoA encoding for the alpha-subunit of archaeal ammonia monooxygenase), and crenarchaeol, the specific glycerol diphytanyl glycerol tetraether of pelagic Crenarchaeota within the Group I.1a. Marine Crenarchaeota were the most abundant Archaea (up to 98% of the total archaeal 16S rDNA copies) in the suboxic layers with oxygen levels as low as 1 microM including layers where previously anammox bacteria were described. Different marine crenarchaeotal phylotypes (both 16S rDNA and amoA) were found at the upper part of the suboxic zone as compared with the base of the suboxic zone and the upper 15-30 m of the anoxic waters with prevailing sulfide concentrations of up to 30 microM. Crenarchaeol concentrations were higher in the sulfidic chemocline as compared with the suboxic zone. These results indicate an abundance of putative nitrifying Archaea at very low oxygen levels within the Black Sea and might form an important source of nitrite for the anammox reaction.     

High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study

We surveyed the archaeal assemblage in a stratified sulfurous lake (Lake Vilar, Banyoles, Spain) over 5 consecutive years to detect potential seasonal and interannual trends in the free-living planktonic Archaea composition. The combination of different primer pairs and nested PCR steps revealed an unexpectedly rich archaeal community. Overall, 140 samples were analyzed, yielding 169 different 16S rRNA gene sequences spread over 14 Crenarchaeota (109 sequences) and six Euryarchaeota phylogenetic clusters. Most of the Crenarchaeota (98% of the total crenarchaeotal sequences) affiliated within the Miscellaneous Crenarchaeota Group (MCG) and were related to both marine and freshwater phylotypes. Euryarchaeota mainly grouped within the Deep Hydrothermal Vent Euryarchaeota (DHVE) cluster (80% of the euryarchaeotal sequences) and the remaining 20% distributed into three less abundant taxa, most of them composed of soil and sediment clones. The largest fraction of phylotypes from the two archaeal kingdoms (79% of the Crenarchaeota and 54% of the Euryarchaeota) was retrieved from the anoxic hypolimnion, indicating that these cold and sulfide-rich waters constitute an unexplored source of archaeal richness. The taxon rank-frequency distribution showed two abundant taxa (MCG and DHVE) that persisted in the water column through seasons, plus several rare ones that were only detected occasionally. Differences in richness distribution and seasonality were observed, but no clear correlations were obtained when multivariate statistical analyses were carried out.     

Analysis of the non-thermophilic Crenarchaeota phylogeny in the swamp soil of Zoige plateau wetland

Zoige wetland of Tibetan plateau is a model low temperature ecosystem in a low latitude (33°56′N, 102°52′E) and high altitude region. Its organism has a unique phylogeny. To better evaluate the resource of the non-thermophilic Crenarchaeota in such an ecosystem, both restriction fragment length polymorphism (RFLP) and clone techniques were employed to study the diversity and phylogenetics of the non-thermophilic Crenarchaeota in the wetland soil. Archaeal 16S rRNA genes were amplified with the archaea-specific primers, and a library consisting of 240 clones was established. The non-thermophilic Crenarchaeota phylogenetic tree was constructed using the ARB phylogenetic analysis software. Based on the results of the RFLP experiments, the clones of all three Zoige wetland swamp soil samples were grouped into 16 different restriction cleavage patterns, all the clone coverage indices were above 91%, showing high library coverage. The correlations analysis indicated that the biodiversity of the non-thermophilic Crenarchaeota be positively correlated with soil moisture. The phylogenetic analysis revealed that all of the 16 Crenarchaeota sequences were clustered into two groups: 13 sequences in the Group 1.1b and 3 in the Group 1.3, similar to those archaeal sequences obtained from grassland soil, freshwater reservoirs, and seawater above boreholes, and radioactive groundwater and hot springs.