Communities of Archaea and Bacteria in a Subsurface Radioactive Thermal Spring in the Austrian Central Alps, and Evidence of Ammonia-Oxidizing Crenarchaeota

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the “Franz-Josef-Quelle” in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (α, β, γ, and δ), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (δ-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.     

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.     

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.     

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.     

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。     

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.     

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。     

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。     

Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of okhotsk

Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluated by performing both cultivation-dependent and cultivation-independent (molecular) analyses. The core, which extended 58.1 m below the seafloor, was composed of pelagic clays with several volcanic ash layers containing fine pumice grains. Direct cell counting and quantitative PCR analysis of archaeal and bacterial 16S rRNA gene fragments indicated that the bacterial populations in the ash layers were approximately 2 to 10 times larger than those in the clays. Partial sequences of 1,210 rRNA gene clones revealed that there were qualitative differences in the microbial communities from the two different types of layers. Two phylogenetically distinct archaeal assemblages in the Crenarchaeota, the miscellaneous crenarchaeotic group and the deep-sea archaeal group, were the most predominant archaeal 16S rRNA gene components in the ash layers and the pelagic clays, respectively. Clones of 16S rRNA gene sequences from members of the gamma subclass of the class Proteobacteria dominated the ash layers, whereas sequences from members of the candidate division OP9 and the green nonsulfur bacteria dominated the pelagic clay environments. Molecular (16S rRNA gene sequence) analysis of 181 isolated colonies revealed that there was regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with some gram-positive bacteria and actinobacteria. The porous ash layers, which ranged in age from tens of thousands of years to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloor clay sediment, which are capable of harboring microbial communities that are very distinct from the communities in the more abundant pelagic clays.     

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.     

Bacterial and archaeal communities inhabiting mussels,sediment and water in Indonesian anchialine lakes

Anchialine lakes are a globally rare and unique ecosystem consisting of saline lakes surrounded by land and isolated from the surrounding marine environment. These lakes host a unique flora and fauna including numerous endemic species. Relatively few studies have, however, studied the prokaryote communities present in these lakes and compared them with the surrounding ‘open water’ marine environment. In the present study, we used a 16S rRNA gene barcoded pyrosequencing approach to examine prokaryote (Bacteria and Archaea) composition in three distinct biotopes (sediment, water and the mussel Brachidontes sp.) inhabiting four habitats, namely, three marine lakes and the surrounding marine environment of Berau, Indonesia. Biotope and habitat proved significant predictors of variation in bacterial and archaeal composition and higher taxon abundance. Most bacterial sequences belonged to OTUs assigned to the Proteobacteria. Compared to sediment and water, mussels had relatively high abundances of the classes Mollicutes and Epsilonproteobacteria. Most archaeal sequences, in turn, belonged to OTUs assigned to the Crenarchaeota with the relative abundance of crenarchaeotes highest in mussel samples. For both Bacteria and Archaea, the main variation in composition was between water samples on the one hand and sediment and mussel samples on the other. Sediment and mussels also shared much more OTUs than either shared with water. Abundant bacterial OTUs in mussels were related to organisms previously obtained from corals, oysters and the deepsea mussel Bathymodiolus manusensis. Abundant archaeal OTUs in mussels, in contrast, were closely related to organisms previously obtained from sediment.     

Microbial community analysis of a coastal hot spring in Kagoshima, Japan, using molecular- and culture-based approaches

Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared <92% sequence similarity with their closest sequences in a public database, suggesting that the Ibusuki hot spring may harbor a unique and novel bacterial community. By contrast, 77 clones from the Site 2 archaeal library contained only three OTUs, most of which were affiliated with Thaumarchaeota.     

Diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: Bahía del Tóbari, Mexico

Nitrification within estuarine sediments plays an important role in the nitrogen cycle, both at the global scale and in individual estuaries. Although bacteria were once thought to be solely responsible for catalyzing the first and rate-limiting step of this process, several recent studies have suggested that mesophilic Crenarchaeota are capable of performing ammonia oxidation. Here we examine the diversity (richness and community composition) of ammonia-oxidizing archaea (AOA) and bacteria (AOB) within sediments of Bahía del Tóbari, a hypernutrified estuary receiving substantial amounts of ammonium in agricultural runoff. Using PCR primers designed to specifically target the archaeal ammonia monooxygenase alpha-subunit (amoA) gene, we found AOA to be present at five sampling sites within this estuary and at two sampling time points (January and October 2004). In contrast, the bacterial amoA gene was PCR amplifiable from only 40% of samples. Bacterial amoA libraries were dominated by a few widely distributed Nitrosomonas-like sequence types, whereas AOA diversity showed significant variation in both richness and community composition. AOA communities nevertheless exhibited consistent spatial structuring, with two distinct end member assemblages recovered from the interior and the mouths of the estuary and a mixed assemblage from an intermediate site. These findings represent the first detailed examination of archaeal amoA diversity in estuarine sediments and demonstrate that diverse communities of Crenarchaeota capable of ammonia oxidation are present within estuaries, where they may be actively involved in nitrification.     

太湖竺山湾沉积物中氨氧化原核生物的垂直分布与多样性

原核生物驱动的氨氧化过程对于富营养化湖泊的氮循环具有重要意义。为了解太湖藻型湖区沉积物中氨氧化原核生物的垂直分布和多样性特征,采用分子生态学方法,对竺山湾沉积物剖面中氨单加氧酶基因(amoA)或16S rRNA基因等特征分子标记的变化和序列特征进行了分析。结果表明,氨氧化细菌(ammonia-oxidizing bacteria,AOB)和氨氧化古菌(ammonia-oxidizing archaea,AOA)共存于沉积物各层。AOB的优势种在5cm深度以下发生明显改变,这可能与沉积物氧化还原电位及铵态氮的变化有关;所有细菌amoA序列均属亚硝化单胞菌(Nitrosomonas)。AOA群落结构自表层至7cm深度变化不大,所有古菌amoA序列分属泉古菌CG1.1b和CG1.1a两大类群,这可能与太湖形成历史上的海陆交替过程有关。此外,沉积物各层均未发现典型厌氧氨氧化(anaerobic ammonium oxidation,anammox)细菌16S rRNA基因序列。这些发现丰富了对太湖藻型湖区氨氧化原核生物分布、多样性及环境调控原理的认识,对理解富营养化湖泊氨氧化规律、认识湖泊生态系统氮循环功能具有借鉴意义。     

Microbial biodiversity of thermophilic communities in hot mineral soils of Tramway Ridge, Mount Erebus, Antarctica

Tramway Ridge, located near the summit of Mount Erebus in Antarctica, is probably the most remote geothermal soil habitat on Earth. Steam fumaroles maintain moist, hot soil environments creating extreme local physicochemical differentials. In this study a culture-independent approach combining automated rRNA intergenic spacer analysis (ARISA) and a 16S rRNA gene library was used to characterize soil microbial (Bacterial and Archaeal) diversity along intense physicochemical gradients. Statistical analysis of ARISA data showed a clear delineation between bacterial community structure at sites close to fumaroles and all other sites. Temperature and pH were identified as the primary drivers of this demarcation. A clone library constructed from a high-temperature site led to the identification of 18 novel bacterial operational taxonomic units (OTUs). All 16S rRNA gene sequences were deep branching and distantly (85–93%) related to other environmental clones. Five of the signatures branched with an unknown group between candidate division OP10 and Chloroflexi . Within this clade, sequence similarity was low, suggesting it contains several yet-to-be described bacterial groups. Five archaeal OTUs were obtained and exhibited high levels of sequence similarity (95–97%) with Crenarchaeota sourced from deep-subsurface environments on two distant continents. The novel bacterial assemblage coupled with the unique archaeal affinities reinvigorates the hypotheses that Tramway Ridge organisms are relics of archaic microbial lineages specifically adapted to survive in this harsh environment and that this site may provide a portal to the deep-subsurface biosphere.     

Variations in spatial and temporal distribution of Archaea in the North Sea in relation to environmental variables

The spatial and temporal distribution of pelagic Archaea was studied in the southern North Sea by rRNA hybridization, sequencing and quantification of 16S rRNA gene and membrane lipid analyses and related to physical, chemical and biological parameters to determine the factors influencing archaeal biogeography. A clear temporal variability was observed, with marine Crenarchaeota (Group I.1a) being relatively more abundant in winter and Euryarchaeota dominating the archaeal assemblage in spring and summer. Spatial differences in the lateral distribution of Crenarchaeota were also evident. In fact, their abundance was positively correlated with the copy number of the gene encoding the alpha subunit of crenarchaeotal ammonia monooxygenase (amoA) and with concentrations of ammonia, nitrate, nitrite and phosphorus. This suggests that most Crenarchaeota in the North Sea are nitrifiers and that their distribution is determined by nutrient concentrations. However, Crenarchaeota were not abundant when larger phytoplankton (>3 microm) dominated the algal population. It is hypothesized that together with nutrient concentration, phytoplankton biomass and community structure can predict crenarchaeotal abundance in the southern North Sea. Euryarchaeotal abundance was positively correlated with chlorophyll a concentrations, but not with phytoplankton community structure. Whether this is related to the potential of Euryarchaeota to perform aerobic anoxygenic phototrophy remains to be shown, but the conspicuous seasonal distribution pattern of Crenarchaeota and Euryarchaeota suggests that they occupy a different ecological niche.     

Diverse microbial communities inhabit Antarctic sponges

Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus. The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales. Of the 150 archaeal clones from L. apicalis, K. varialosa and M. acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota. Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones. Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity. Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group. Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species. Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives. In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K. varialosa.     

The phylogenetic affiliation of an uncultured population of ammonia-oxidizing bacteria harboring environmental sequences of amoA cluster-3

We investigated the phylogenetic diversity of ammonia-oxidizing bacteria (AOB) in Yellow Sea continental shelf sediment by the cloning and sequencing of PCR-amplified amoA and 16S rRNA genes. Phylogenetic analysis of the amoA-related clones revealed that the diversity of AOB was extremely low at the study site. The majority (92.7%) of amoA clones obtained belonged to a single cluster, environmental amoA cluster-3, the taxonomic position of which was previously unknown. Phylogenetic analysis on AOB-specific 16S rRNA gene sequences also demonstrated a very low diversity. All of the cloned 16S rRNA gene sequences comprised a single phylotype that belonged to the members of uncultured Nitrosospira cluster-1, suggesting that AOB belonging to the uncultured Nitrosospira cluster- 1 could carry amoA sequences of environmental amoA cluster-3.     

High 16S rDNA bacterial diversity in glacial meltwater lake sediment,Bratina Island,Antarctica

The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depth-related composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'G/CGC3'and Msp I. 5'C/CGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the alpha, gamma, and delta subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.