The microbiome of Brazilian mangrove sediments as revealed by metagenomics

Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H(2)S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments.     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

Bacterial diversity in deep-sea sediments from different depths

Seven sediment samples have been examined, taken from different depths of the deep-sea in the range of 1159m to 6482m. A total of 75 different 16S rDNA sequences (149 clones) analyzed clustered into the Proteobacteria, Gram-positive bacteria, Cytophaga, Planctomyces, and Actinomycetes and many sequences were from microorganisms that showed no phylogenetic affiliation with known bacteria. Clones identical to 16S rDNA sequences of members of the genus Pseudomonas were observed in all of the sediments examined. The second group of common sequences cloned from six sediment samples was related to the 16S rDNA sequence of a chemoautotrophic bacterium, the Solemya velum symbiont. Five 16S rDNA sequences from three sediments were related to those of the Alvinella pompejana epibiont which is a member of the -Proteobacteria. Only one sequence was obtained that was closely related to the 16S rDNA of the barophilic bacterium, Shewanella benthica, which might be a minor population in the deeper sediments. -Proteobacteria-related sequences were cloned from sediments obtained from sites near man-made garbage deposits and a Calyptogena community. These environments obviously would be richer in nutrients than other sites, and might be expected to show more types of bacteria than other deep-sea sediments. A large number of cloned sequences in this study showed very low identity to known sequences. These sequences may represent communities of as-yet-uncultivated microorganisms in the sediments.     

Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics

Quaternary ammonium compounds (QACs) represent widely used cationic biocides that persist in natural environments. Although microbial degradation, sensitivity and resistance to QACs have been extensively documented, a quantitative understanding of how whole communities adapt to QAC exposure remain elusive. To gain insights into these issues, we exposed a microbial community from a contaminated river sediment to varied levels of benzalkonium chlorides (BACs, a family of QACs) for 3 years. Comparative metagenomic analysis showed that the BAC‐fed communities were dramatically decreased in phylogenetic diversity compared with the control (no BAC exposure), resulting presumably from BAC toxicity, and dominated by Pseudomonas species (> 50% of the total). Time‐course metagenomics revealed that community adaptation occurred primarily via selective enrichment of BAC‐degrading Pseudomonas populations, particularly P. nitroreducens, and secondarily via amino acid substitutions and horizontal transfer of a few selected genes in the Pseudomonas populations, including a gene encoding a PAS/PAC sensor protein and ring‐hydroxylating dioxygenase genes. P. nitroreducens isolates were reproducibly recoverable from communities after prolonged periods of no‐BAC exposure, suggesting that they are robust BAC‐degraders. Our study provides new insights into the mechanisms and tempo of microbial community adaptation to QAC exposure and has implications for treating QACs in biological engineered systems.     

南海深海沉积物放线菌多样性分析

【目的】免培养和纯培养相结合分析南海深海沉积物放线菌多样性。【方法】免培养方法通过提取沉积物宏基因组DNA,利用放线菌门特异性引物扩增放线菌16S r RNA基因序列,构建放线菌16S r RNA基因克隆文库,文库经RFLP(Restriction fragment length polymorphism)分析后挑选代表序列测序并进行多样性指数分析和系统发育分析。可培养方法利用8种培养基进行菌株分离,对排重后的菌株进行16S r RNA基因序列多样性分析。【结果】构建的两个深海位点的16S r RNA基因克隆文库在放线菌门的放线菌纲(Actinobacteria)、酸微菌纲(Acidimicrobiia)、腈基降解菌纲(Nitriliruptoria)和嗜热油菌纲(Thermoleophilia)4个纲中均有分布;两个位点中的种群结构有差异,N40-4位点的优势种群是放线菌纲的链霉菌目(Streptomycetales);N63-4位点的优势种群是腈基降解菌纲的腈基降解菌目(Nitriliruptorales)。8种培养基共分离出41株放线菌,根据形态特征排重后得到的19株菌分布于10个不同的属,12个不同的种,其中稀有放线菌属比例较高,菌株OAct400为潜在的微杆菌属(Microbacterium)新种。【结论】南海深海沉积物蕴含着丰富的放线菌物种资源及大量未知种群,具有进一步研究的价值。     

Fungal communities from the calcareous deep-sea sediments in the Southwest India Ridge revealed by Illumina sequencing technology

The diversity and ecological significance of bacteria and archaea in deep-sea environments have been thoroughly investigated, but eukaryotic microorganisms in these areas, such as fungi, are poorly understood. To elucidate fungal diversity in calcareous deep-sea sediments in the Southwest India Ridge (SWIR), the internal transcribed spacer (ITS) regions of rRNA genes from two sediment metagenomic DNA samples were amplified and sequenced using the Illumina sequencing platform. The results revealed that 58–63 % and 36–42 % of the ITS sequences (97 % similarity) belonged to Basidiomycota and Ascomycota, respectively. These findings suggest that Basidiomycota and Ascomycota are the predominant fungal phyla in the two samples. We also found that Agaricomycetes, Leotiomycetes, and Pezizomycetes were the major fungal classes in the two samples. At the species level, Thelephoraceae sp. and Phialocephala fortinii were major fungal species in the two samples. Despite the low relative abundance, unidentified fungal sequences were also observed in the two samples. Furthermore, we found that there were slight differences in fungal diversity between the two sediment samples, although both were collected from the SWIR. Thus, our results demonstrate that calcareous deep-sea sediments in the SWIR harbor diverse fungi, which augment the fungal groups in deep-sea sediments. This is the first report of fungal communities in calcareous deep-sea sediments in the SWIR revealed by Illumina sequencing.     

Global distribution of a wild alga revealed by targeted metagenomics

Eukaryotic phytoplankton play key roles in atmospheric CO(2) uptake and sequestration in marine environments [1,2]. Community shifts attributed to climate change have already been reported in the Arctic ocean, where tiny, photosynthetic picoeukaryotes (≤3 μm diameter) have increased, while larger taxa have decreased [3]. Unfortunately, for vast regions of the world's oceans, little is known about distributions of different genera and levels of genetic variation between ocean basins. This lack of baseline information makes it impossible to assess the impacts of environmental change on phytoplankton diversity, and global carbon cycling. A major knowledge impediment is that these organisms are highly diverse, and most remain uncultured [2]. Metagenomics avoids the culturing step and provides insights into genes present in the environment without some of the biases associated with conventional molecular survey methods. However, connecting metagenomic sequences to the organisms containing them is challenging. For many unicellular eukaryotes the reference genomes needed to make this connection are not available. We circumvented this problem using at-sea fluorescence activated cell sorting (FACS) to separate abundant natural populations of photosynthetic eukaryotes and sequence their DNA, generating reference genome information while eliminating the need for culturing [2]. Here, we present the complete chloroplast genome from an Atlantic picoeukaryote population and discoveries it enabled on the evolution, distribution, and potential carbon sequestration role of a tiny, wild alga.     

Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies

Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed "vector-enabled metagenomics" (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors (in this case, whiteflies) from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies (Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases.     

滇牡丹遗传多样性的ISSR分析

应用ISSR标记对中国西南地区特有植物滇牡丹(Paeonia delavayi)的遗传多样性进行了研究。从100个引物中筛选出10个用于正式扩增,在取自16个自然居群和1个迁地保护居群的511个个体中,检测到92个多态位点。在居群水平上,多态位点百分率（PPB）为44.61%,Nei′s基因多样性指数（H）和Shannon信息指数（I）分别为0.1657和0.2448。在物种水平上,多态位点百分率（PPB）为79.31%,Nei′s基因多样性指数（H）和Shannon信息指数（I）分别为0.2947和0.4355。居群间的遗传分化系数(G_ST)达0.4349。结果表明：滇牡丹遗传多样性水平较高,居群间遗传分化较大。结合以前的研究结果,对滇牡丹的现状进行评估的结果显示,滇牡丹并不濒危。     

印度洋深海沉积物石油烃降解菌分离、鉴定与多样性分析

烃类物质在海洋环境中广泛分布,深海可能含有特殊的烃降解微生物。本研究通过对西南印度洋中脊与印度洋中部深海沉积物中石油降解菌的富集培养和分离鉴定,从6个站点的样品中共分离得到800株菌,通过BOX-PCR去重复菌株后,对其中183株菌进行了16S rRNA基因序列分析,发现这些菌分属于23个属;其中,γ-变形菌纲的食烷菌属(Alcanivorax)和放线菌纲的微杆菌属(Microbacterium)占优势。此外,还发现了食烷菌属2个潜在的新种、假海栖菌属(Pseudooceanicola)1个潜在新种。高通量测序结果证明,富集菌群中γ-变形菌纲是优势菌,主要包括食烷菌属、盐单胞菌属(Halomonas)、海杆菌属(Marinobacter)等。结合可培养菌与高通量测序结果,食烷菌属、盐单胞菌属、海杆菌属、交替假单胞菌(Pseudoalteromonas)、海源菌属(Idiomarina)与微杆菌属(Microbacterium)是沉积物样品中常见的石油烃降解菌,迪茨氏菌属(Dietzia)、红球菌属(Rhodococcus),假单胞菌属(Pseudomonas)、赤杆菌属(Erythrobacter)与副球菌属(Paracoccus)等可能也参与了烃的降解。     

东太平洋多金属结核区两个站位深海沉积物细菌多样性北大核心CSCD

【目的】为了解东太平洋中国多金属结核勘探合同区西区2个站位(WBC1305和WBC1316A)深海沉积物细菌群多样性。【方法】直接提取环境样品总基因组,通过PCR和TA克隆策略构建了2个站位6个层次16S r RNA基因文库,对2个站位沉积物表层泥样中细菌多样性和群落结构特征进行分析,并通过构建系统发育树,进行系统发育学分析。【结果】2个站位6个文库共获得有效克隆533个,其中472个克隆包括α-变形菌纲、β-变形菌纲、γ-变形菌纲、δ-变形菌纲、浮霉菌门、酸杆菌门、硝化螺旋菌门、放线菌门、绿弯菌门、厚壁菌门、拟杆菌门、迷踪菌门、芽单胞菌门、Hydrogenedentes、Chlorobi和Nitrospinae16个细菌类群,而另外61个克隆为不可分类细菌类群。【结论】结果表明γ-变形菌纲和厚壁菌门分别是WBC1305和WBC1316A站位的优势种群;WBC1316A站位细菌群落结构更加丰富和复杂。     

Eutrophication processes in Spanish reservoirs as revealed by biological records in profundal sediments

Eutrophication of reservoirs can be detected by changes in the abundance of insect and crustacean remains in the sediments. In recently constructed reservoirs, the time of impoundment can be determined through the presence of chironomid head capsules. The initial phase is characterised by highly enriched water as can be seen by the abundance of Bosmina remains. If further nutrient input into the reservoir remains low, the upper part of the sediment column is almost completely lacking in invertebrate remains.     

Molecular diversity among marine picophytoplankton as revealed by psbA analyses

Photosynthetic microorganisms play a crucial role in the marine environment. In vast areas of the oceans, marine primary productivity is performed by cells smaller than 2-3 micro m (picoplankton). Here, we report on molecular analyses of the conserved photosynthetic psbA gene (coding for protein D1 of photosystem II reaction centre) as a diversity indicator of naturally occurring marine oxygenic picophytoplankton. The psbA genes proved to be good indicators of the presence of a wide variety of photosynthetic marine microbial groups, including new cyanobacterial groups and eukaryotic algae (prasinophytes). Furthermore, using environmental bacterial artificial chromosome (BAC) libraries, we were able to correlate psbA genes with small subunit rRNAs and, therefore, to confirm their phylogenetic affiliation.     

Genetic diversity of Carica papaya as revealed by AFLP markers.

Genetic relationships among Carica papaya cultivars, breeding lines, unimproved germplasm, and related species were established using amplified fragment length polymorphism (AFLP) markers. Seventy-one papaya accessions and related species were analyzed with nine EcoRI-MseI primer combinations. A total of 186 informative AFLP markers was generated and analyzed. Cluster analysis suggested limited genetic variation in papaya, with an average genetic similarity among 63 papaya accessions of 0.880. Genetic diversity among cultivars derived from the same or similar gene pools was smaller, such as Hawaiian Solo hermaphrodite cultivars and Australian dioecious cultivars with genetic similarity at 0.921 and 0.912, respectively. The results indicated that self-pollinated hermaphrodite cultivars were as variable as open-pollinated dioecious cultivars. Genetic diversity between C. papaya and six other Carica species was also evaluated. Carica papaya shared the least genetic similarity with these species, with an average genetic similarity of 0.432; the average genetic similarity among the six other species was 0.729. The results from AFLP markers provided detailed estimates of the genetic variation within and among papaya cultivars, and supported the notion that C. papaya diverged from the rest of Carica species early in the evolution of this genus.     

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above-ground community structure. We collected 3800 root fragments from four randomly positioned, 1-m-deep soil profiles (two vertical transects per plot), located in an old-field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below-ground was correlated with their frequency above-ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above-ground quadrats. Multivariate analyses indicated that diversity was highly structured below-ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above-ground. Furthermore, analyses of species co-occurrence indicates strong species segregation overall but random co-occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below-ground, and also demonstrate the utility of barcoding for the identification of plant roots.     

Genetic diversity among wild and cultivated barley as revealed by RFLP

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed an intermediate level. The proportion of gene diversity residing among geographical groups (F_ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups.