Unique Phylotypes Research Articles

Isolation of digital dermatitis treponemes from hoof lesions in Wild North American Elk (Cervus elaphus) in Washington State, USA.

Since 2008, a large increase in the numbers of cases of lameness have been seen in wild North American elk (Cervus elaphus) from Washington State, USA. The most recent cases manifested as foot lesions similar both clinically and pathologically to those seen in digital dermatitis (DD) in cattle and sheep, a disease with a bacterial etiopathogenesis. To determine whether the same bacteria considered responsible for DD are associated with elk lameness, lesion samples were subjected to bacterial isolation studies and PCR assays for three phylogroups of relevant DD treponemes. The DD treponemes were isolated from lesional tissues but not from control feet or other areas of the diseased foot (including the coronary band or interdigital space), suggesting that the bacteria are strongly associated with DD lesions and may therefore be causal. In addition, PCR analysis revealed that all three unique DD treponeme phylotypes were found in elk hoof disease, and in 23% of samples, all 3 DD-associated treponemes were present in lesions. Sequence analysis of the 16S rRNA gene showed that the elk lesion treponemes were phylogenetically almost identical to those isolated from cattle and sheep DD lesions. The isolates were particularly similar to two of the three culturable DD treponeme phylotypes: specifically, the Treponema medium/Treponema vincentii-like and Treponema phagedenis-like DD spirochetes. The third treponeme culturable phylogroup (Treponema pedis), although detected by PCR, was not isolated. This is the first report describing isolation of DD treponemes from a wildlife host, suggesting that the disease may be evolving to include a wider spectrum of cloven-hoofed animals.

The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics

ABSTRACTViruses have a profound influence on the ecology and evolution of plankton, but our understanding of the composition of the aquatic viral communities is still rudimentary. This is especially true of those viruses having RNA genomes. The limited data that have been published suggest that the RNA virioplankton is dominated by viruses with positive-sense, single-stranded (+ss) genomes that have features in common with those of eukaryote-infecting viruses in the order Picornavirales (picornavirads). In this study, we investigated the diversity of the RNA virus assemblages in tropical coastal seawater samples using targeted PCR and metagenomics. Amplification of RNA-dependent RNA polymerase (RdRp) genes from fractions of a buoyant density gradient suggested that the distribution of two major subclades of the marine picornavirads was largely congruent with the distribution of total virus-like RNA, a finding consistent with their proposed dominance. Analyses of the RdRp sequences in the library revealed the presence of many diverse phylotypes, most of which were related only distantly to those of cultivated viruses. Phylogenetic analysis suggests that there were hundreds of unique picornavirad-like phylotypes in one 35-liter sample that differed from one another by at least as much as the differences among currently recognized species. Assembly of the sequences in the metagenome resulted in the reconstruction of six essentially complete viral genomes that had features similar to viruses in the families Bacillarna-, Dicistro-, and Marnaviridae. Comparison of the tropical seawater metagenomes with those from other habitats suggests that +ssRNA viruses are generally the most common types of RNA viruses in aquatic environments, but biases in library preparation remain a possible explanation for this observation.

Arsenic(V) Reduction in Relation to Iron(III) Transformation and Molecular Characterization of the Structural and Functional Microbial Community in Sediments of a Basin-Fill Aquifer in Northern Utah

Basin-fill aquifers of the Southwestern United States are associated with elevated concentrations of arsenic (As) in groundwater. Many private domestic wells in the Cache Valley Basin, UT, have As concentrations in excess of the U.S. EPA drinking water limit. Thirteen sediment cores were collected from the center of the valley at the depth of the shallow groundwater and were sectioned into layers based on redoxmorphic features. Three of the layers, two from redox transition zones and one from a depletion zone, were used to establish microcosms. Microcosms were treated with groundwater (GW) or groundwater plus glucose (GW+G) to investigate the extent of As reduction in relation to iron (Fe) transformation and characterize the microbial community structure and function by sequencing 16S rRNA and arsenate dissimilatory reductase (arrA) genes. Under the carbon-limited conditions of the GW treatment, As reduction was independent of Fe reduction, despite the abundance of sequences related to Geobacter and Shewanella, genera that include a variety of dissimilatory iron-reducing bacteria. The addition of glucose, an electron donor and carbon source, caused substantial shifts toward domination of the bacterial community by Clostridium-related organisms, and As reduction was correlated with Fe reduction for the sediments from the redox transition zone. The arrA gene sequencing from microcosms at day 54 of incubation showed the presence of 14 unique phylotypes, none of which were related to any previously described arrA gene sequence, suggesting a unique community of dissimilatory arsenate-respiring bacteria in the Cache Valley Basin.

Molecular diversity reveals previously undetected air-dispersed protist colonists in a Mediterranean area

The molecular diversity of air-dispersed protists was examined through the 18S rRNA gene clone library construction in air samples and samples from experimental water containers passively collecting air-dispersed microorganisms, from July 2007 till October 2008 in three different sites of Northern Greece. The majority of the samplings took place in an urban industrialized coastal city (Thessaloniki). In all the samples, a total of 29 unique phylotypes were detected belonging to 10 known major taxonomic groups. The most abundant phylotypes were affiliated to known taxa of Ciliophora and Chlorophyceae, commonly found in various habitats. Additionally, various previously unnoticed and under-studied taxa, such as Bicosoecida, Oomycetes and Labyrinthulomycetes, were detected. These taxa are potentially important in ecological processes, through dispersal and colonization of various habitats. Multivariate statistical analysis associated the most abundant phylotypes with rainfall, suggesting that rain is a favorable means for reposition of air-dispersed protists. This is the first study investigating the molecular diversity of air-dispersed protists, including algae and heterotrophic protists.

Pyrosequencing Analysis of Oral Microbiota Shifting in Various Caries States in Childhood

Dental caries is one of the most prevalent childhood diseases worldwide, but little is known about the dynamic characteristics of oral microbiota in the development of dental caries. To investigate the shifting bacterial profiles in different caries states, 60 children (3-7-year-old) were enrolled in this study, including 30 caries-free subjects and 30 caries-active subjects. Supragingival plaques were collected from caries-active subjects on intact enamel, white spot lesions and carious dentin lesions. Plaques from caries-free subjects were used as a control. All samples were analyzed by 454 pyrosequencing based on 16S rRNA gene V1-V3 hypervariable regions. A total of 572,773 pyrosequencing reads passed the quality control and 25,444 unique phylotypes were identified, which represented 18 phyla and 145 genera. Reduced bacterial diversity in the cavitated dentin was observed as compared with the other groups. Thirteen genera (including Capnocytophaga, Fusobacterium, Porphyromonas, Abiotrophia, Comamonas, Tannerella, Eikenella, Paludibacter, Treponema, Actinobaculum, Stenotrophomonas, Aestuariimicrobium, and Peptococcus) were found to be associated with dental health, and the bacterial profiles differed considerably depending on caries status. Eight genera (including Cryptobacterium, Lactobacillus, Megasphaera, Olsenella, Scardovia, Shuttleworthia, Cryptobacterium, and Streptococcus) were increased significantly in cavitated dentin lesions, and Actinomyces and Corynebacterium were present at significant high levels in white spot lesions (P < 0.05), while Flavobacterium, Neisseria, Bergeyella, and Derxia were enriched in the intact surfaces of caries individuals (P < 0.05). Our results showed that oral bacteria were specific at different stages of caries progression, which contributes to informing the prevention and treatment of childhood dental caries.

Insights into the distribution and abundance of the ubiquitous Candidatus Saccharibacteria phylum following tag pyrosequencing

The phylum candidatus Saccharibacteria formerly known as Candidate Division TM7 is a highly ubiquitous phylum with 16S rRNA gene sequences reported in soils, sediments, wastewater and animals, as well as a host of clinical environments. Here, the application of two taxon-specific primers on environmental and human-associated samples using bar-coded tag pyrosequencing revealed two new clades for this phylum to exist and we propose that the division consists of 2 monophyletic and 2 polyphyletic clades. Investigation into TM7 ecology revealed that a high proportion (58%) of phylotypes were sample specific, few were widely distributed and of those most widely distributed all belonged to subdivision 3. Additionally, 50% of the most relatively abundant phylotypes observed were also subdivision 3 members. Community analysis showed that despite the presence of a high proportion of unique phylotypes, specific groups of samples still harbor similar TM7 communities with samples clustering together. The lack of relatively abundant phylotypes from subdivisions 1, 2 and 4 and the presence of very few cosmopolitan members' highlights not only the site specific nature of this phylum but provides insight into why the majority of studies into TM7 have been biased towards subdivision 3.

Anaerobic Oxidation of Ethene Coupled to Sulfate Reduction in Microcosms and Enrichment Cultures

Ethene is considered recalcitrant under anaerobic conditions, but biological reduction to ethane and oxidation to CO2 have been reported; however, little is known about these processes or the organisms carrying them out. In this report we describe sulfate dependent ethene consumption in microcosms prepared with sediments from a freshwater canal. A first dose of 0.6 mmol/L ethene was consumed within 77 days, and a second dose was largely consumed twelve days later. Material from this microcosm was transferred into growth medium with ethene as the only electron donor (except for trace amounts of vitamins) and sulfate as the electron acceptor. Four doses of ethene were consumed at increasing rates, and the cultures have been transferred at least eight times in this medium. Conversion of [(14)C]ethene primarily to (14)CO2 was demonstrated in fifth and sixth generation cultures, as well as production of sulfide in other cultures, confirming the ethene/sulfate couple. Ovoid cells 1-2 μm in diameter were found in cultures containing ethene and sulfate, and quantitative PCR showed large increases in bacterial 16S rRNA gene copy number. Over half of a 16S rRNA gene clone library from a sixth-generation culture was a phylotype with a sequence ca. 90% identical with a clade of Deltaproteobacteria that includes Desulfovirga adipica and several Syntrophobacter spp. These studies have solidified the concept that deficits in mass balances for chloroethene fate in sulfate reducing zones of contaminated groundwater sites may be due to ethene oxidation, and suggest a unique phylotype is involved in this process.

Differences in the Methanogen Population Exist in Sika Deer (Cervus nippon) Fed Different Diets in China

Understanding the methanogen structure from sika deer (Cervus nippon) in China may be beneficial to methane mitigation. In the present preliminary study, we investigated the methanogen community in the rumen of domesticated sika deer fed either tannin-rich plants (oak leaf, OL group) or corn stalk (CS group) using 16S rRNA gene clone libraries. Overall, we obtained 197 clone sequences, revealing 146 unique phylotypes, which were assigned to 36 operational taxonomic units at the species level (98% identity). Methanogens related to the genus Methanobrevibacter were the predominant phylotypes representing 83.9% (OL library) and 85.9% (CS library) of the clones. Methanobrevibacter millerae was the most abundant species in both libraries, but the proportion of M. millerae-related clones in the CS library was higher than in the OL library (69.5 and 51.4%, respectively). Moreover, Methanobrevibacter wolinii-related clones (32.5%) were predominant in the OL library. Methanobrevibacter smithii-related clones and Methanobrevibacter ruminantium-related clones accounted for 6.5 and 6.6% in the CS library, respectively. However, these clones were absent from the OL library. The concentrations of butyrate and total short-chain fatty acids (SCFAs) were significantly higher in the OL group, but the concentrations of acetate, propionate, and valerate and the acetate to propionate ratio in the OL group were not significantly different between the two groups. Tannin-rich plants may have affected the distribution of genus Methanobrevibacter phylotypes at the species level and the concentration and composition of SCFAs.

Multi‐factorial drivers of ammonia oxidizer communities: evidence from a national soil survey

The factors driving the abundance and community composition of soil microbial communities provide fundamental knowledge on the maintenance of biodiversity and the ecosystem services they underpin. Several studies have suggested that microbial communities are spatially organized, including functional groups and much of the observed variation is explained by geographical location or soil pH. Soil ammonia-oxidizing archaea (AOA) and bacteria (AOB) are excellent models for such study due to their functional, agronomic and environmental importance and their relative ease of characterization. To identify the dominant drivers of different ammonia oxidizers, we used samples (n = 713) from the National Soil Inventory of Scotland (NSIS). Our results indicate that 40-45% of the variance in community compositions can be explained by 71 environmental variables. Soil pH and substrate, which have been regarded as the two main drivers, only explained 13-16% of the total variance. We provide strong evidence of multi-factorial drivers (land use, soil type, climate and N deposition) of ammonia-oxidizing communities, all of which play a significant role in the creation of specific niches that are occupied by unique phylotypes. For example, one AOA phylotype was strongly linked to woodland/semi-natural grassland, rainfall and N deposition. Some soil typologies, namely regosols, have a novel AOA community composition indicating typology as one of the factors which defines this ecological niche. AOA abundance was high and strongly linked the rate of potential nitrification in the highly acidic soils supporting the argument that AOA are main ammonia oxidizers in acidic soils. However, for AOB, soil pH and substrate (ammonia) were the main drivers for abundance and community composition. These results highlight the importance of multiple drivers of microbial niche formation and their impact on microbial biogeography that have significant consequences for ecosystem functioning.

Niche Partition of Bacteriovorax Operational Taxonomic Units Along Salinity and Temporal Gradients in the Chesapeake Bay Reveals Distinct Estuarine Strains

The predatory Bacteriovorax are Gram-negative bacteria ubiquitous in saltwater systems that prey upon other Gram-negative bacteria in a similar manner to the related genus Bdellovibrio. Among the phylogenetically defined clusters of Bacteriovorax, cluster V has only been isolated from estuaries suggesting that it may be a distinct estuarine phylotype. To assess this hypothesis, the spatial and temporal distribution of cluster V and other Bacteriovorax phylogenetic assemblages along the salinity gradient of Chesapeake Bay were determined. Cluster V was expected to be found in significantly greater numbers in low to moderate salinity waters compared to high salinity areas. The analyses of water and sediment samples from sites in the bay revealed cluster V to be present at the lower salinity and not high salinity sites, consistent with it being an estuarine phylotype. Cluster IV had a similar distribution pattern and may also be specifically adapted to estuaries. While the distribution of clusters V and IV were similar for salinity, they were distinct on temperature gradients, being found in cooler and in warmer temperatures, respectively. The differentiation of phylotype populations along the salinity and temporal gradients in Chesapeake Bay revealed distinct niches inhabited by different phylotypes of Bacteriovorax and unique estuarine phylotypes.

Pyrosequencing analysis of the human microbiota of healthy Chinese undergraduates

BackgroundElucidating the biogeography of bacterial communities on the human body is critical for establishing healthy baselines from which to detect differences associated with disease; however, little is known about the baseline bacterial profiles from various human habitats of healthy Chinese undergraduates.ResultsUsing parallel barcoded 454 pyrosequencing targeting on the 16S rRNA gene V3 region, the bacterial diversity of the nasopharynx, saliva, dominant hands, and feces were investigated from 10 healthy Chinese junior boarding undergraduates at Zhejiang University. The participants were 21–24 years of age with a body mass index (BMI) < 24 kg/m2. A total of 156,717 high-quality pyrosequencing reads were obtained for evaluating bacterial diversity, which represented 29,887 unique phylotypes. The overall taxonomic distribution of the 16S rRNA gene-based amplicons demonstrated that these 4 habitats of the human body harbored distinct microbiota and could be divided into different clusters according to anatomic site, while the established patterns of bacterial diversity followed the human body habitat (feces, hands, saliva, and nasopharynx). Although significant inter-individual variation was observed, the healthy microbiota still shared a large number of phylotypes in each habitat, but not among the four habitats, indicating that a core microbiome existed in each healthy habitat. The vast majority of sequences from these different habitats were classified into different taxonmies that became the predominant bacteria of the healthy microbiota.ConclusionsWe first established the framework of microbial communities from four healthy human habitats of the same participants with similar living environments for the Chinese undergraduates. Our data represent an important step for determining the diversity of Chinese healthy microbiota, and can be used for more large-scale studies that focus on the interactions between healthy and diseases states for young Chinese adults in the same age range.

Baseline survey of the anatomical microbial ecology of an important food plant: Solanum lycopersicum (tomato)

BackgroundResearch to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop.ResultsDNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe “native” bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes.ConclusionsDistinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Dominant fungal taxa that could be identified to genus with 18S amplicons included Hypocrea, Aureobasidium and Cryptococcus. No definitive presence of Salmonella could be confirmed in any of the plant samples, although 16S sequences suggested that closely related genera were present on leaves, fruits and roots.

Diversity of methanogens in the hindgut of captive white rhinoceroses, Ceratotherium simum

BackgroundThe white rhinoceros is on the verge of extinction with less than 20,200 animals remaining in the wild. In order to better protect these endangered animals, it is necessary to better understand their digestive physiology and nutritional requirements. The gut microbiota is nutritionally important for herbivorous animals. However, little is known about the microbial diversity in the gastrointestinal tract (GIT) of the white rhinoceros. Methanogen diversity in the GIT may be host species-specific and, or, function-dependent. To assess methanogen diversity in the hindgut of white rhinoceroses, an archaeal 16S rRNA gene clone library was constructed from pooled PCR products obtained from the feces of seven adult animals.ResultsSequence analysis of 153 archaeal 16S rRNA sequences revealed 47 unique phylotypes, which were assigned to seven operational taxonomic units (OTUs 1 to 7). Sequences assigned to OTU-7 (64 out of 153 total sequencs – 42%) and OTU-5 (18%, 27/153) had 96.2% and 95.5% identity to Methanocorpusculum labreanum, respectively, making Methanocorpusculum labreanum the predominant phylotype in these white rhynoceroses. Sequences belonging to OTU-6 (27%, 42/153) were related (97.6%) to Methanobrevibacter smithii. Only 4% of the total sequences (6/153) were assigned to Methanosphaera stadtmanae (OTU-1). Sequences belonging to OTU-2 (4%, 6/153), OTU-3 (3%, 5/153) and OTU-4 (2%, 3/153) were distantly related (87.5 to 88,4%) to Methanomassiliicoccus luminyensis and were considered to be novel species or strains that have yet-to-be cultivated and characterized.ConclusionPhylogenetic analysis indicated that the methanogen species in the hindgut of white rhinoceroses were more similar to those in the hindgut of horses. Our findings may help develop studies on improving the digestibility of forage for sustainable management and better health of these endangered animals.

Characterization of eukaryotic microbial diversity in hypersaline Lake Tyrrell, Australia

This study describes the community structure of the microbial eukaryotic community from hypersaline Lake Tyrrell, Australia, using near full length 18S rRNA sequences. Water samples were taken in both summer and winter over a 4-year period. The extent of eukaryotic diversity detected was low, with only 35 unique phylotypes using a 97% sequence similarity threshold. The water samples were dominated (91%) by a novel cluster of the Alveolate, Apicomplexa Colpodella spp., most closely related to C. edax. The Chlorophyte, Dunaliella spp. accounted for less than 35% of water column samples. However, the eukaryotic community entrained in a salt crust sample was vastly different and was dominated (83%) by the Dunaliella spp. The patterns described here represent the first observation of microbial eukaryotic dynamics in this system and provide a multiyear comparison of community composition by season. The lack of expected seasonal distribution in eukaryotic communities paired with abundant nanoflagellates suggests that grazing may significantly structure microbial eukaryotic communities in this system.

南海陆坡沉积物细菌丰度预测

分别利用参数模型和无参数估计法预测南海陆坡沉积物柱MD05-2896中的细菌丰度。基于非培养的PCR-RFLP的16S rRNA基因分子技术,扩增了沉积物柱中的细菌16S rRNA基因序列,并构建16S rRNA基因文库。系统发育分析表明16S rRNA基因文库中,大多数序列属于17个已知的"门"。分别以99%、97%、90%和80%序列一致性作为分类单元分界点,将16S rRNA基因序列组群为分类单元。使用逆高斯分布模型、对数正态分布模型、负二项式分布模型、帕雷托分布模型、双指数分布模型以及ACE、ACE-1等估计方法预测不同分类单元分类水平下的细菌丰度。结果表明在"种"级分类水平上,负二项式分布为最优估计模型,估计细菌丰度为244±10(SE)。不过,受实验条件的限制,该估计值可能偏低。;Two families of statistical approaches, parametric models and nonparametric estimates, were developed and used to predict bacterial species richness in core MD05-2896 collected from the South China Sea during the Chinese-French joint MARCO POLO/IMAGES 147 cruise (chief operator Yvon Balut). MD05-2896 (08°49.50'N, 111°26.47'E) was an 11.03 m long core collected from the SCS south slope, where the water depth is 1 657 m. A total of twelve microbial subsamples were collected from the top to the bottom of the core at intervals of 1 m. For each sample, we extracted the bulk DNA and amplified the bacterial 16S rRNA gene sequences. All of the bacterial 16S rRNA gene sequences were used to construct a 16S rRNA gene library. A total of 194 unique phylotypes were identified based on the phylogenetic analysis, most of which clustered into 17 phyla that belong to Planctomycetes, Proteobacteria, Chloroflexi, Actinobacteria, Sprirochaetes, Verrucomicrobia, Acidobacteria, Bacteriodetes, Defferribacteres, Nitrospirae, and candidate divisions OP1, OP3, OP8, OP11, JS1, WS3, and TM6. All sequences were grouped into Operational Taxonomic Units (OTUs) based on 99%, 98%, 97%, 95%, 90%, and 80% similarity cut-off values. Sequences were aligned using the CLUSTALW software, after which the% sequence similarity was calculated and the sequences were grouped into OTUs using the unweighted pair group method. These frequency data were then analyzed by parametric models and coverage-based nonparametric estimates, ACE and ACE-1, respectively. Because nonparametric estimates usually underestimate the species richness, this study focused on application of parametric models. Five models, including the inverse Gaussian, log normal, negative binomial, Pareto and 2-mixed exponential, were adopted to fit asymptotically with the OTU's frequency data and predict the species richness. The parametric models were implemented step by step according to the procedures described by Hong et al., and the best fitted model was selected for the final parametric analysis. At the level of 99% rRNA sequence similarity, our data were best described by the 2-mixed exponential distribution model, which estimates the richness at 326±40(SE). At the level of 97% rRNA sequence similarity, the negative binomial distribution model describes our data best with an estimated richness of 244±10(SE). At the level of 95% rRNA sequence similarity, the negative binomial distribution model best described the data and estimated the richness at 220±6(SE). At the level of 90% rRNA sequence similarity, the 2-mixed exponential distribution model best described the data and estimated the richness at 127±4(SE). At the level of 80% rRNA sequence similarity, the Pareto distribution model described the data best and estimated the richness at 62±4(SE). The 99%, 97%, 95%, 90%, and 80% rRNA gene sequence similarities were adopted to identify bacterial strain, species, genera, families/classes, and phyla, respectively. Accordingly, core MD05-2896 contains at a minimum of 326 ± 40(SE) bacterial strains, 244 ± 10 (SE) bacterial species, 62 ± 4 (SE) bacterial phyla, 127 ± 4 (SE) bacterial families/classes, and 220 ± 6 (SE) bacterial genera. However, these numbers are conservative because of the limitations associated with laboratory experiments, such as coextracted interfering substances including humic and fulvic acids, and PCR bias.

Anaerobic degradation of propane and butane by sulfate-reducing bacteria enriched from marine hydrocarbon cold seeps.

The short-chain, non-methane hydrocarbons propane and butane can contribute significantly to the carbon and sulfur cycles in marine environments affected by oil or natural gas seepage. In the present study, we enriched and identified novel propane and butane-degrading sulfate reducers from marine oil and gas cold seeps in the Gulf of Mexico and Hydrate Ridge. The enrichment cultures obtained were able to degrade simultaneously propane and butane, but not other gaseous alkanes. They were cold-adapted, showing highest sulfate-reduction rates between 16 and 20 °C. Analysis of 16S rRNA gene libraries, followed by whole-cell hybridizations with sequence-specific oligonucleotide probes showed that each enrichment culture was dominated by a unique phylotype affiliated with the Desulfosarcina-Desulfococcus cluster within the Deltaproteobacteria. These phylotypes formed a distinct phylogenetic cluster of propane and butane degraders, including sequences from environments associated with hydrocarbon seeps. Incubations with (13)C-labeled substrates, hybridizations with sequence-specific probes and nanoSIMS analyses showed that cells of the dominant phylotypes were the first to become enriched in (13)C, demonstrating that they were directly involved in hydrocarbon degradation. Furthermore, using the nanoSIMS data, carbon assimilation rates were calculated for the dominant cells in each enrichment culture.

Organic Layer Serves as a Hotspot of Microbial Activity and Abundance in Arctic Tundra Soils

Tundra ecosystem is of importance for its high accumulation of organic carbon and vulnerability to future climate change. Microorganisms play a key role in carbon dynamics of the tundra ecosystem by mineralizing organic carbon. We assessed both ecosystem process rates and community structure of Bacteria, Archaea, and Fungi in different soil layers (surface organic layer and subsurface mineral soil) in an Arctic soil ecosystem located at Spitsbergen, Svalbard during the summer of 2008 by using biochemical and molecular analyses, such as enzymatic assay, terminal restriction fragment length polymorphism (T-RFLP), quantitative polymerase chain reaction (qPCR), and pyrosequencing. Activity of hydrolytic enzymes showed difference according to soil type. For all three microbial communities, the average gene copy number did not significantly differ between soil types. However, archaeal diversities appeared to differ according to soil type, whereas bacterial and fungal diversity indices did not show any variation. Correlation analysis between biogeochemical and microbial parameters exhibited a discriminating pattern according to microbial or soil types. Analysis of the microbial community structure showed that bacterial and archaeal communities have different profiles with unique phylotypes in terms of soil types. Water content and hydrolytic enzymes were found to be related with the structure of bacterial and archaeal communities, whereas soil organic matter (SOM) and total organic carbon (TOC) were related with bacterial communities. The overall results of this study indicate that microbial enzyme activity were generally higher in the organic layer than in mineral soils and that bacterial and archaeal communities differed between the organic layer and mineral soils in the Arctic region. Compared to mineral soil, peat-covered organic layer may represent a hotspot for secondary productivity and nutrient cycling in this ecosystem.

Molecular characterization of basidiomycetes associated with the decayed mangrove tree Xylocarpus granatum in Thailand

This study reports the frequency of decay (stem/butt rot) of Xylocarpus granatum trees at Hat Khanom-Mu Ko Thale Tai National Park, Nakhonsithammarat province, and other locations in Thailand, and the identification of purported causal basidiomycetes based on morphological and molecular analyses. Four survey plots at Hat Khanom-Mu Ko Thale Tai National Park were established and the incidence of butt rot determined. Percentage stem/butt rot incidence of X. granatum trees varied from 40.9 to 94.4 % with an average of 85.5 % along all four Plots. Trees in Plot 2 supported the heaviest incidence rate (94.4 %), with the lowest rate in Plot 3 (40.9 %). Ninety-two basidiomes were collected, and 46 fungal strains (50 %) isolated into axenic culture, the majority associated with tree roots (68.5 % of all collections) with 31.5 % from the tree trunks. Molecular results, based on LSU and ITS1, 2, 5.8 S rDNA analyses, confirmed that all samples belonged to the poroid genus Fulvifomes in the Hymenochaetaceae within the Hymenochaetales, Basidiomycota. The 43 specimens sequenced grouped into three clades; one clade comprised specimens isolated from only the trunks and branches (14 strains), while the remainders were from roots (29 strains) and shown to be salt tolerant. The stem/butt rot strains formed unique phylotypes which did not group with other known Fulvifomes species.

부산 연안역의 진핵플랑크톤 종다양성에 대한 메타게놈 분석 연구

The species composition of plankton is essential to understand the material and energy cycling within marine ecosystem. It also provides the useful information for understanding the properties of marine environments due to its sensitivity to the physicochemical characteristics and variability of water masses. In this study we adopted metagenomics to evaluate eukaryotic plankton species diversity from coastal waters off Busan. Characteristics of water masses at sampling sites is expected to be very complex due to the mixing of various water masses; Nakdong River runoff, Changjiang diluted water (CDW), South Sea coastal water, and Tsushima warm current. 18S rDNA clone libraries were constructed from surface waters at the three sites off Busan. Clone libraries revealed 94 unique phylotypes from 370 clones; Dinophyceae(42 phylotypes), Ciliophora(15 phylotypes), Bacillariophyta(7 phylotypes), Chlorophyta(2 phylotypes), Haptophyceae(1 phylotype), Metazoa(Arthropoda( 17 phylotypes), Chaetognatha(1 phylotypes), Cnidaria(2 phylotypes), Chordata(1 phylotype)), Rhizaria (Acantharea(2 phylotypes), Polycystinea(1 phylotype)), Telonemida(1 phylotype), Fungi(2 phylotypes). The difference in species diversity at the closely located three sites off Busan may be attributed to the various physicochemical properties of water masses at these sites by the mixture of water masses of various origins. Metagenomic study of species composition may provide useful information for understanding marine ecosystem of coastal waters with various physicochemical properties in the near feature.

Phylogenetic diversity of nitrogen-fixing bacteria and thenifHgene from mangrove rhizosphere soil

Nine types of nitrogen-fixing bacterial strains were isolated from 3 rhizosphere soil samples taken from mangrove plants in the Dongzhaigang National Mangrove Nature Reserve of China. Most isolates belonged to Gammaproteobacteria Pseudomonas, showing that these environments constituted favorable niches for such abundant nitrogen-fixing bacteria. New members of the diazotrophs were also found. Using a soil DNA extraction and PCR-cloning-sequencing approach, 135 clones were analyzed by restriction fragment length polymorphism (RFLP) analysis, and 27 unique nifH sequence phylotypes were identified, most of which were closely related to sequences from uncultured bacteria. The diversity of nitrogen-fixing bacteria was assessed by constructing nifH phylogenetic trees from sequences of all isolates and clones in this work, together with related nifH sequences from other mangrove ecosystems in GenBank. The nifH diversity varied among soil samples, with distinct biogeochemical properties within a mangrove ecosystem. When comparing different mangrove ecosystems, the nifH gene sequences from a specific site tended to cluster as individual groups. The results provided interesting data and novel information on our understanding of diazotroph community diversity in the mangrove ecosystems.