Diversity of Archaea in Bottom Sediments of the Discharge Areas With Oil- and Gas-Bearing Fluids in Lake Baikal

Using massively parallel sequencing (the Roche 454 platform) we have studied the diversity of archaeal 16S rRNA gene sequences in oxic and anoxic sediments at six sites in Lake Baikal with oil- and gas-bearing fluids discharge. Archaeal communities appeared to be represented mainly by five phyla: Euryarchaeota, Crenarchaeota, Thaumarchaeota, Bathyarchaeota (miscellaneous Crenarchaeotic group), and Woesearchaeota (deep sea hydrothermal vent group 6). Among them we detected sequences of methanogens of the orders Methanomicrobiales, Methanosarsinales, Methanococcales, as well as representatives of the following uncultured archaeal lineages: Group C3, Marine Benthic Group D, and Terrestrial Miscellaneous Group. We have also identified sequences of ammonia-oxidizing archaea of the phyla Crenarchaeota and Thaumarchaeota. Phylogenetic analysis showed the presence ANME-2d-related sequences. However, the analysis of mcrA genes libraries has not revealed typical representatives of ANME groups. Comparison of amplicon libraries 16S rRNA gene fragments from different samples proved the widespread presence of previously detected Baikal archaeal lineages, which are members of the phylum Crenarchaeota and Thaumarchaeota (formerly Group C3 of Crenarchaeota).     

Population structure and phylogenetic characterization of marine benthic Archaea in deep-sea sediments.

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

Archaeal communities in mangrove soil characterized by 16S rRNA gene clones

An archaeal 16S rRNA gene library was constructed from mangrove soil. Phylogenetic analysis revealed archaea in mangrove soil including the Crenarchaeota (80.4%) and Euryarchaeota (19.6%) phyla. The archaeal community in mangrove soil appears to be a mixture of organisms found in a variety of environments with the majority being of marine origin.     

Archaeal diversity in a terrestrial acidic spring field revealed by a novel PCR primer targeting archaeal 16S rRNA genes

The phylogenetic diversity of archaeal 16S rRNA genes in a thermoacidic spring field of Ohwakudani, Hakone, Japan, was investigated by PCR-based analysis using a novel Archaea-specific primer designed in the present study. Clone libraries of archaeal 16S rRNA genes were constructed from hot water (78 °C) and mud (28 °C) samples by PCR using a newly designed forward primer and a previously reported forward primer with reverse primers. Most phylotypes found in the libraries from the hot water sample were related to cultured (hyper)thermophiles. The phylotypes and their detection frequencies from the hot water sample were similar for the libraries amplified with the two different primer sets. In contrast, phylotypes having a low similarity (<95%) to cultured Archaea were found in the libraries from the mud sample. Some of the phylotypes were relatively close to members of Thermoplasmata (80-93% similarity) and the others were not clearly affiliated with Crenarchaeota and Euryarchaeota, but related to Thaumarchaeota and Korarchaeota. The phylotypes and their detection frequencies were significantly different between the two libraries of the mud sample. Our results from the PCR-based analysis using the redesigned primer suggest that more diverse, uncultured Archaea are present in acidic environments at a low temperature than previously recognized.     

Microbial communities in iron-silica-rich microbial mats at deep-sea hydrothermal fields of the Southern Mariana Trough

The abundance, diversity and composition of bacterial and archaeal communities in the microbial mats at deep-sea hydrothermal fields were investigated, using culture-independent 16S rRNA and functional gene analyses combined with mineralogical analysis. Microbial mats were collected at two hydrothermal areas on the ridge of the back-arc spreading centre in the Southern Mariana Trough. Scanning electron microscope and energy dispersive X-ray spectroscopic (SEM-EDS) analyses revealed that the mats were mainly composed of amorphous silica and contained numerous filamentous structures of iron hydroxides. Direct cell counting with SYBR Green I staining showed that the prokaryotic cell densities were more than 10⁸ cells g⁻¹. Quantitative polymerase chain reaction (Q-PCR) analysis revealed that Bacteria are more abundant than Archaea in the microbial communities. Furthermore, zetaproteobacterial cells accounted for 6% and 22% of the prokaryotic cells in each mat estimated by Q-PCR with newly designed primers and TaqMan probe. Phylotypes related to iron-oxidizers, methanotrophs/methylotrophs, ammonia-oxidizers and sulfate-reducers were found in the 16S rRNA gene clone libraries constructed from each mat sample. A variety of unique archaeal 16S rRNA gene phylotypes, several pmoA , dsrAB and archaeal amoA gene phylotypes were also recovered from the microbial mats. Our results provide insights into the diversity and abundance of microbial communities within microbial mats in deep-sea hydrothermal fields.     

Phylogenetic diversity of planktonic archaea in the estuarine region of East China Sea

To examine the diversity and structure of archaeal communities in the Yangtze River estuarine region of East China Sea (ECS), the 16S rRNA gene clone libraries of two typical sites were constructed with the archaea-specific primers. In total, 71 clones randomly selected were screened by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and 21 clones with unique RFLP pattern were sequenced. All the sequences are clustered into the two groups of Marine Group I (MGI) and Marine Group II (MGII) which are affiliated with the phyla Crenarchaeota and Euryarchaeota, respectively. MGI clones dominate both libraries with 20 MGI sequences obtained. The majority of sequences are closely related to uncultured marine archaea except for two sequences of which the nearest neighbor is a newly identified isolate of nitrifying marine archaeon Nitrosopumilus maritimus (98% identity). The results indicate that ECS coastal waters are inhabited by archaeal community with low dominance and high diversity corresponding to the complex estuarine environments, suggesting that archaea may perform an important role in the estuarine ecosystem.     

Archaeal diversity along a soil salinity gradient prone to disturbance

We employed a cultivation-independent approach to examine archaeal diversity along a transient soil salinity gradient at Salt Spring in British Columbia, Canada that is routinely eroded due to heavy, recurrent rainfall. Archaeal 16S rRNA gene libraries were created using DNA extracted from three soil samples collected along this gradient. Statistical comparisons indicated similar archaeal richness across sites but, a significant shift in archaeal community composition along the salinity gradient. Seven distinct phylogenetic groups were represented in soil libraries. Haloarchaea were the most commonly sampled group. Other 16S rRNA sequences were related to uncultured Euryarchaeota and Crenarchaeota or halophilic methanogens. Haloarchaeal diversity was remarkably high in soil of elevated salinity compared with previously characterized haloarchaeal communities. Salt Spring haloarchaea were not closely related to known low-salt adapted/tolerant species, suggesting they may be frequently faced with local mortality as a result of frequent declines in soil salinity. We speculate that ecosystem disturbance -- in the form of salinity fluctuations -- is one mechanism for maintaining a diverse community of haloarchaea at Salt Spring.     

Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses

Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.     

Temporal changes in archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-microm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.     

Phylotype diversity of deep-sea hydrothermal vent prokaryotes trapped by 0.2- and 0.1-μm-pore-size filters

Eleven 16S rRNA gene clone libraries including 34 archaeal and 72 bacterial phylotypes were constructed from total 708 clones of hydrothermal vent prokaryotes trapped by 0.2- and 0.1-μm-pore-size filters. Crenarchaeota and Proteobacteria phylotypes dominated the archaeal and bacterial populations, respectively. Novel unaffiliated phylotypes occurred only in the 0.1-μm-trapped populations.     

Vertical structure of archaeal communities and the distribution of ammonia monooxygenase A gene variants in two meromictic High Arctic lakes

The distribution of archaeal amoA and 16S rRNA genes was evaluated in two marine-derived, meromictic lakes in the Canadian High Arctic: Lake A and Lake C1 on the northern coast of Ellesmere Island. The amoA gene was recorded in both lakes, with highest copy numbers in the oxycline. Sequence analysis showed that amoA from the two lakes shared 94% similarity, indicating at least two phylogenetically distinct clusters. Clone libraries of archaeal 16S rRNA genes from Lake A revealed strong vertical differences in archaeal community diversity and composition down the water column. The oxic layer was dominated by one group of Euryarchaeota affiliated to the Lake Dagow Sediment (LDS) cluster. This group was absent from the oxycline, which had an extremely low archaeal diversity of two phylotypes. Both belonged to the Crenarchaeota Marine Group I (MGI), the marine group that has been linked to archaeal amoA ; however, there was a low ratio of amoA to MGI copy numbers, suggesting that many MGI Archaea did not carry the amoA gene. The anoxic zone contained representatives of the RC-V (Rice Cluster-V) and LDS clusters of Euryarchaeota. These results show the strong vertical differentiation of archaeal communities in polar meromictic lakes, and they suggest archaeal nitrification within the oxycline of these highly stratified waters.     

Microbial diversity and stratification of South Pacific abyssal marine sediments

Abyssal marine sediments cover a large proportion of the ocean floor, but linkages between their microbial community structure and redox stratification have remained poorly constrained. This study compares the downcore gradients in microbial community composition to porewater oxygen and nitrate concentration profiles in an abyssal marine sediment column in the South Pacific Ocean. Archaeal 16S rRNA clone libraries showed a stratified archaeal community that changed from Marine Group I Archaea in the aerobic and nitrate-reducing upper sediment column towards deeply branching, uncultured crenarchaeotal and euryarchaeotal lineages in nitrate-depleted, anaerobic sediment horizons. Bacterial 16S rRNA clone libraries revealed a similar shift on the phylum and subphylum level within the bacteria, from a complex community of Alpha-, Gamma- and Deltaproteobacteria, Actinobacteria and Gemmatimonadetes in oxic surface sediments towards uncultured Chloroflexi and Planctomycetes in the anaerobic sediment column. The distinct stratification of largely uncultured bacterial and archaeal groups within the oxic and nitrate-reducing marine sediment column provides initial constraints for their microbial habitat preferences.     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

Response of Archaeal Community Structure to Environmental Changes in Lakes on the Tibetan Plateau,Northwestern China

To study how archaeal community responds to environmental changes, we investigated archaeal community structures in waters of three Tibetan saline lakes in northwestern China (Gahai, Xiaochaidan, and Charhan Lakes) with 16S rRNA gene phylogenetic analysis. Temperature, pH, and water chemistry (major anions and cations) of the lakes were measured. Three archaeal clone libraries were constructed with a total of 297 sequences. Incorporating our previous data obtained from other lakes on the Tibetan Plateau, we performed statistical analyses to identify dominant environmental parameters that could account for the observed variations in archaeal community structure. We concluded that salinity and water chemistry (Na and bicarbonate concentration in particular) played an important role in shaping archaeal community. In particular, the relative abundance of archaeal 16S rRNA genes affiliated with the Halobacteriales of the Euryarchaeota increased with salinity, whereas that of crenarchaeotal 16S rRNA gene sequences showed the opposite trend. Crenarchaeotal 16S rRNA gene sequences were retrieved from lake waters with salinity up to 28.3%. These results have important implications for our understanding of response of archaeal community to environmental changes in high-altitude lake ecosystems.     

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand

We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria , Acidobacteria , Chloroflexi , Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria , Firmicutes , Thermus/Deinococcus , Actinobacteria and Bacteroidetes , as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria , Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats.     

Archaea in the Gulf of Aqaba

Using a polyphasic approach, we examined the presence of Archaea in the Gulf of Aqaba, a warm marine ecosystem, isolated from major ocean currents and subject to pronounced seasonal changes in hydrography. Catalyzed reported deposition FISH analyses showed that Archaea make up to >20% of the prokaryotic community in the Gulf. A spatial separation between the two major phyla of Archaea was observed during summer stratification. Euryarchaeota were found exclusively in the upper 200 m, whereas Crenarchaeota were present in greater numbers in layers below the summer thermocline. 16S rRNA gene-based denaturing gradient gel electrophoresis confirmed this depth partitioning and revealed further diversity of Crenarchaeota and Euryarchaeota populations along depth profiles. Phylogenetic analysis showed pelagic Crenarchaeota and Euryarchaeota to differ from coral-associated Archaea from the Gulf, forming distinct clusters within the Marine Archaea Groups I and II. Endsequencing of fosmid libraries of environmental DNA provided a tentative identification of some members of the archaeal community and their role in the microbial community of the Gulf. Incorporation studies of radiolabeled leucine and bicarbonate in the presence of different inhibitors suggest that the archaeal community participates in autotrophic CO₂ uptake and contributes little to the heterotrophic activity.     

Bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong, China

We investigated the bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong in the Yunnan Province, China, using direct molecular analyses. The Langpu (LP) laminated mat was found by the side of a boiling pool with temperature of 60-65?°C and a pH of 8.5, while the Tengchong (TC) streamer mat consisted of white streamers in a slightly acidic (pH 6.5) hot pool outflow with a temperature of 72?°C. Four 16S rRNA gene clone libraries were constructed and restriction enzyme analysis of the inserts was used to identify unique sequences and clone frequencies. From almost 200 clones screened, 55 unique sequences were retrieved. Phylogenetic analysis showed that the LP mat consisted of a diverse bacterial population [Cyanobacteria, Chloroflexi, Chlorobia, Nitrospirae, 'Deinococcus-Thermus', Proteobacteria (alpha, beta and delta subdivisions), Firmicutes, Bacteroidetes and Actinobacteria], while the archaeal population was dominated by methanogenic Euryarchaeota and Crenarchaeota. In contrast, the TC streamer mat consisted of a bacterial population dominated by Aquificae, while the archaeal population also contained Korarchaeota as well as Crenarchaeota and methanogenic Euryarchaeota. These mats harboured clone sequences affiliated to unidentified lineages, suggesting that they are a potential source for discovering novel bacteria and archaea.     

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。     

Phylogenetic analysis of Group I marine archaeal rRNA sequences emphasizes the hidden diversity within the primary group Archaea

Archaea form one of the three primary groups of extant life and are commonly associated with the extreme environments which many of their members inhabit. Currently, the Archaea are classified into two kingdoms, Crenarchaeota and Euryarchaeota, based on phylogenetic analysis of ribosomal RNA (rRNA) sequences. Molecular techniques allowing the retrieval and analysis of rRNA sequences from diverse environments are increasing our knowledge of archaeal diversity. This report describes the presence of marine Archaea in north-east Atlantic waters. Quantitative estimates indicated that the marine Archaea constitute 8 per cent of the total prokaryotic rRNA in Irish coastal waters. Phylogenetic analysis of the archaeal rRNA gene sequences revealed sufficient genetic diversity within Archaea to indicate that the current two-kingdom classification of Crenarchaeota and Euryarchaeota is restrictive.