The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples

Viruses are the most abundant biological entities on our planet. Interactions between viruses and their hosts impact several important biological processes in the world's oceans such as horizontal gene transfer, microbial diversity and biogeochemical cycling. Interrogation of microbial metagenomic sequence data collected as part of the Sorcerer II Global Ocean Expedition (GOS) revealed a high abundance of viral sequences, representing approximately 3% of the total predicted proteins. Cluster analyses of the viral sequences revealed hundreds to thousands of viral genes encoding various metabolic and cellular functions. Quantitative analyses of viral genes of host origin performed on the viral fraction of aquatic samples confirmed the viral nature of these sequences and suggested that significant portions of aquatic viral communities behave as reservoirs of such genetic material. Distributional and phylogenetic analyses of these host-derived viral sequences also suggested that viral acquisition of environmentally relevant genes of host origin is a more abundant and widespread phenomenon than previously appreciated. The predominant viral sequences identified within microbial fractions originated from tailed bacteriophages and exhibited varying global distributions according to viral family. Recruitment of GOS viral sequence fragments against 27 complete aquatic viral genomes revealed that only one reference bacteriophage genome was highly abundant and was closely related, but not identical, to the cyanomyovirus P-SSM4. The co-distribution across all sampling sites of P-SSM4-like sequences with the dominant ecotype of its host, Prochlorococcus supports the classification of the viral sequences as P-SSM4-like and suggests that this virus may influence the abundance, distribution and diversity of one of the most dominant components of picophytoplankton in oligotrophic oceans. In summary, the abundance and broad geographical distribution of viral sequences within microbial fractions, the prevalence of genes among viral sequences that encode microbial physiological function and their distinct phylogenetic distribution lend strong support to the notion that viral-mediated gene acquisition is a common and ongoing mechanism for generating microbial diversity in the marine environment.     

Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton

Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton.     

Exploring the diversity of bacterial communities in sediments of urban mangrove forests

Municipal sewage, urban runoff and accidental oil spills are common sources of pollutants in urban mangrove forests and may have drastic effects on the microbial communities inhabiting the sediment. However, studies on microbial communities in the sediment of urban mangroves are largely lacking. In this study, we explored the diversity of bacterial communities in the sediment of three urban mangroves located in Guanabara Bay (Rio de Janeiro, Brazil). Analysis of sediment samples by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments suggested that the overall bacterial diversity was not significantly affected by the different levels of hydrocarbon pollution at each sampling site. However, DGGE and sequence analyses provided evidences that each mangrove sediment displayed a specific structure bacterial community. Although primer sets for Pseudomonas, alphaproteobacterial and actinobacterial groups also amplified ribotypes belonging to taxa not intended to be enriched, sequence analyses of dominant DGGE bands revealed ribotypes related to Alteromonadales, Burkholderiales, Pseudomonadales, Rhodobacterales and Rhodocyclales. Members of these groups were often shown to be involved in aerobic or anaerobic degradation of hydrocarbon pollutants. Many of these sequences were only detected in the sampling sites with high levels of anthropogenic inputs of hydrocarbons. Many dominant DGGE ribotypes showed low levels of sequence identity to known sequences, indicating a large untapped bacterial diversity in mangrove ecosystems.     

Oxygen minimum zones harbour novel viral communities with low diversity

Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral‐to‐microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non‐redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non‐redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.     

Bacterial diversity in marine hydrocarbon seep sediments

Marine seeps introduce significant amounts of hydrocarbons into oceans and create unusual habitats for microfauna and -flora. In the vicinity of chronic seeps, microbes likely exert control on carbon quality entering the marine food chain and, in turn, hydrocarbons could influence microbial community composition and diversity. To determine the effects of seep oil on marine sediment bacterial communities, we collected sediment piston cores within an active marine hydrocarbon seep zone in the Coal Oil Point Seep Field, at a depth of 22 m in the Santa Barbara Channel, California. Cores were taken adjacent to an active seep vent in a hydrocarbon volcano, on the edge of the volcano, and at the periphery of the area of active seepage. Bacterial community profiles were determined by terminal restriction fragment length polymorphisms (TRFLPs) of 16S ribosomal genes that were polymerase chain reaction (PCR)-amplified with eubacterial primers. Sediment carbon content and C/N ratio increased with oil content. Terminal restriction fragment length polymorphisms suggested that bacterial communities varied both with depth into sediments and with oil concentration. Whereas the apparent abundance of several peaks correlated positively with hydrocarbon content, overall bacterial diversity and richness decreased with increasing sediment hydrocarbon content. Sequence analysis of a clone library generated from sediments collected at the periphery of the seep suggested that oil-sensitive species belong to the gamma Proteobacteria and Holophaga groups. These sequences were closely related to sequences previously recovered from uncontaminated marine sediments. Our results suggest that seep hydrocarbons exert a strong selective pressure on bacterial communities in marine sediments. This selective pressure could, in turn, control the effects of oil on other biota in the vicinity of marine hydrocarbon seeps.     

Metagenomic analysis of the viral communities in fermented foods

Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae, Podoviridae, and Siphoviridae). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil.     

Assessing the diversity and specificity of two freshwater viral communities through metagenomics

Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale.To help identify the characteristics and potential specificities of freshwater viral communities, such communities from two lakes differing by their ecological parameters were studied through metagenomics. Both the cluster richness and the species richness of the Lake Bourget virome were significantly higher that those of the Lake Pavin, highlighting a trend similar to the one observed for microorganisms (i.e. the specie richness observed in mesotrophic lakes is greater than the one observed in oligotrophic lakes). Using 29 previously published viromes, the cluster richness was shown to vary between different environment types and appeared significantly higher in marine ecosystems than in other biomes. Furthermore, significant genetic similarity between viral communities of related environments was highlighted as freshwater, marine and hypersaline environments were separated from each other despite the vast geographical distances between sample locations within each of these biomes. An automated phylogeny procedure was then applied to marker genes of the major families of single-stranded (Microviridae, Circoviridae, Nanoviridae) and double-stranded (Caudovirales) DNA viruses. These phylogenetic analyses all spotlighted a very broad diversity and previously unknown clades undetectable by PCR analysis, clades that gathered sequences from the two lakes. Thus, the two freshwater viromes appear closely related, despite the significant ecological differences between the two lakes. Furthermore, freshwater viral communities appear genetically distinct from other aquatic ecosystems, demonstrating the specificity of freshwater viruses at a community scale for the first time.     

Microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin

Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35 degrees C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.     

Global distribution of nearly identical phage-encoded DNA sequences

Phages, the most abundant biological entities on the planet, play important roles in biogeochemical cycling, horizontal gene transfer, and defining microbial community composition. However, very little is known about phage diversity or biogeography, and there has not yet been a systematic effort to compare the phages found in different ecosystems. Here, we report that T7-like Podophage DNA polymerase sequences occur in every major biome investigated, including marine, freshwater, sediment, terrestrial, extreme, and metazoan-associated. The majority of these sequences belong to a unique clade that is only distantly related to cultured isolates. Some identical T7-like phage-encoded DNA polymerase genes from this clade were >99% conserved at the nucleotide level in multiple different environments, suggesting that these phages are moving between biomes in recent evolutionary time and that the global genomic pool for T7-like phages may be smaller than previously hypothesized.     

The viriosphere, diversity, and genetic exchange within phage communities

Natural phage communities are reservoirs of the greatest uncharacterized genetic diversity on Earth. Yet, identical phage sequences can be found in extremely different environments, which implies that there is wide circulation of viral genes among distantly related host populations. Further evidence of genetic exchange among phage and host communities is the presence in phage of genes coding for proteins that are essential for photosynthesis. These observations support the idea that a primary role of host populations in phage ecology and evolution is to serve as vectors for genetic exchange.     

High diversity and potential origins of T4-type bacteriophages on the surface of Arctic glaciers

Tailed bacteriophages are the most abundant viruses in the biosphere. Here we examined the T4-type bacteriophage community inhabiting the surface of two glaciers in Svalbard. We used a molecular approach to target g23, the major capsid protein gene, to demonstrate that in the extreme cryoconite hole habitats the T4-type phages are surprisingly diverse. Phylogenetic analysis revealed that cryoconite hole sediments harbour a mixed phage community spanning multiple T4-type phage subgroups. The majority (71 %) of phage sequences clustered into three novel phylogenetically distinct groups, whilst the remainder clustered with known marine and soil derived phage sequences. The meltwater in cryoconite holes also contained a further distinct phage community which was related to previously detected marine phage variants. The ability of phages to move between marine and glacial habitats was tested in a transplantation experiment. Phages from the nearby marine fjord were found to be capable of initiating infection of supraglacial bacteria, suggesting suitable hosts could be found by non-native phages. Together this evidence suggests that the surface of glaciers contain both novel and cosmopolitan phages, some of which may have arrived in the cryosphere from other biomes.     

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments (Santos-São Vicente estuary, Brazil). The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.     

Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis

Abstract The diversity of bacterial communities in deep marine sediments, up to 503 metres below the sea floor of the Japan Sea, was investigated by sequence analysis of amplified 16S rRNA genes. The use of different sample handling procedures greatly affected the types and diversity of sequences obtained. DNA from sediment samples stored aerobically for up to 24 h before freezing was dominated by sequences belonging to the β- and γ-proteobacteria, many of which appeared to originate from aerobic bacteria. Sub-samples equilibrated anaerobically at 16°C, were then injected with a radiotracer and immediately frozen, to simulate the conditions of a typical control sample from a radiotracer based activity assay, contained mostly α-proteobacterial sequences. Pristine sediment samples taken anaerobically and frozen within 2 h contained the widest diversity of sequences from α-, γ-, δ-proteobacteria and Gram-positive bacteria, which appeared to have originated from predominantly anaerobic or facultative bacteria. It was clear that both samples that were not frozen immediately (within 2 h) showed signs of enrichment of specific bacterial groups. Our results strongly suggest that immediate freezing should always be employed when sediment samples are to be used to assess bacterial diversity by molecular methods.     

基于高通量测序对呼伦贝尔陶顺阿尔山细菌群落多样性的研究

对内蒙古呼伦贝尔陶顺阿尔山水体与沉积物的细菌群落结构和多样性，进行16S rRNA基因(V3～V5区)高通量测序。运用Illumina MiSeq高通量测序平台，共获得594 226条优化序列。结果显示，水体中最主要的细菌类群为变形菌门(Proteobacteria),而沉积物样品中的主要优势类群为盐厌氧菌门(Haloanaerobium);细菌在属分类水平上，水体中优势菌群为嗜盐单胞菌属(Halomonas)、冷弯菌属(Psychroflexus)、盐厌氧菌属(Haloanaerobium);沉积物样品中优势菌群为盐厌氧菌属、盐单胞菌属、未分类菌(no-rank-f-unclassified),其中存在大量的未知物种。陶顺阿尔山水体与沉积物中存在着丰富的微生物群落，且差异性大，未知菌种居多。本研究首次阐明了陶顺阿尔山细菌群落的多样性，为今后开发和利用陶顺阿尔山的功能菌群提供参考。     

Beyond biodiversity: fish metagenomes

Biodiversity and intra-specific genetic diversity are interrelated and determine the potential of a community to survive and evolve. Both are considered together in Prokaryote communities treated as metagenomes or ensembles of functional variants beyond species limits.Many factors alter biodiversity in higher Eukaryote communities, and human exploitation can be one of the most important for some groups of plants and animals. For example, fisheries can modify both biodiversity and genetic diversity (intra specific). Intra-specific diversity can be drastically altered by overfishing. Intense fishing pressure on one stock may imply extinction of some genetic variants and subsequent loss of intra-specific diversity. The objective of this study was to apply a metagenome approach to fish communities and explore its value for rapid evaluation of biodiversity and genetic diversity at community level. Here we have applied the metagenome approach employing the barcoding target gene coi as a model sequence in catch from four very different fish assemblages exploited by fisheries: freshwater communities from the Amazon River and northern Spanish rivers, and marine communities from the Cantabric and Mediterranean seas.Treating all sequences obtained from each regional catch as a biological unit (exploited community) we found that metagenomic diversity indices of the Amazonian catch sample here examined were lower than expected. Reduced diversity could be explained, at least partially, by overexploitation of the fish community that had been independently estimated by other methods.We propose using a metagenome approach for estimating diversity in Eukaryote communities and early evaluating genetic variation losses at multi-species level.     

黄海海域海洋沉积物细菌多样性分析

【背景】海洋独特的环境造就了海洋生物的多样性,海洋沉积物中细菌对海洋环境具有至关重要的作用。【目的】研究陆地土壤和海洋沉积物间细菌群落相似性和差异性,以便更好地认识海洋细菌多样性,深入了解沉积物细菌在海洋环境中的潜在作用。【方法】从中国黄海海域及大连市大黑山脚下分别采集样品,以陆地土壤为对照,采用16SrRNA基因高通量测序技术分析海洋沉积物的细菌群落结构。【结果】海洋沉积物样品中芽孢杆菌纲(Bacilli)、鞘氨醇单胞菌属(Sphingomonas)和芽孢杆菌属(Bacillus)丰度高于陆地土壤样品;海洋沉积物中亚硝化单胞菌(unculturedbacterium f. Nitrosomonadaceae)和厌氧绳菌(uncultured bacterium f. Anaerolineaceae)丰度虽低于陆地土壤,但丰度值也均高于1%;样品分类学统计显示酸杆菌门(Acidobacteria)在海洋沉积物和陆地土壤样品中的序列丰度比例都较大,鞘氨醇单胞菌属(Sphingomonas)在海洋沉积物样品中的序列丰度大于陆地土壤样品。【结论】海洋沉积物细菌多样性可作为海洋环境恢复情况的重要指标,研究为合理开发和利用海洋资源提供理论依据。     

Diversity and structure of bacterial communities in Fildes Peninsula, King George Island

To examine the bacterial community structure in the Fildes Peninsula, King George Island, Antarctica, we examined the bacterial diversity and community composition of samples collected from lacustrine sediment, marine sediment, penguin ornithogenic sediments, and soils using culture-dependent and culture-independent methods. The 70 strains fell into five groups: Actinobacteria, Bacteroidetes, Firmicutes, Gammaproteobacteria, and Betaproteobacteria. Bacterial diversity at the phylum level detected in Denaturing Gradient Gel Electrophoresis (DGGE) profiles comprised Proteobacteria (including the subphyla Alpha-, Beta-, Gamma-, Deltaproteobacteria), Bacteroidetes, Firmicutes, Chlorobi, and Deinococcus-Thermus. Gammaproteobacteria was identified to be the dominant bacterial subphylum by cultivation and DGGE method. By cluster analysis, the overall structure and composition of bacterial communities in the soil and lacustrine sediment were similar to one another but significantly different from bacterial communities in penguin ornithogenic sediment and marine sediment, which were similar to one another. The majority of 16S rDNA sequences from cultured bacteria were closely related to sequences found in cold environments. In contrast, a minority of 16S rDNA sequences from the DGGE approach were closely related to sequences found in cold environments.     

Phylogenetic diversities and community structure of members of the extremely halophilic Archaea (order Halobacteriales) in multiple saline sediment habitats

We investigated the phylogenetic diversity and community structure of members of the halophilic Archaea (order Halobacteriales) in five distinct sediment habitats that experience various levels of salinity and salinity fluctuations (sediments from Great Salt Plains and Zodletone Spring in Oklahoma, mangrove tree sediments in Puerto Rico, sediment underneath salt heaps in a salt-processing plant, and sediments from the Great Salt Lake northern arm) using Halobacteriales-specific 16S rRNA gene primers. Extremely diverse Halobacteriales communities were encountered in all habitats, with 27 (Zodletone) to 37 (mangrove) different genera identified per sample, out of the currently described 38 Halobacteriales genera. With the exception of Zodletone Spring, where the prevalent geochemical conditions are extremely inhospitable to Halobacteriales survival, habitats with fluctuating salinity levels were more diverse than permanently saline habitats. Sequences affiliated with the recently described genera Halogranum, Halolamina, Haloplanus, Halosarcina, and Halorientalis, in addition to the genera Halorubrum, Haloferax, and Halobacterium, were among the most abundant and ubiquitous genera, suggesting a wide distribution of these poorly studied genera in saline sediments. The Halobacteriales sediment communities analyzed in this study were more diverse than and completely distinct from communities from typical hypersaline water bodies. Finally, sequences unaffiliated with currently described genera represented a small fraction of the total Halobacteriales communities, ranging between 2.5% (Zodletone) to 7.0% (mangrove and Great Salt Lake). However, these novel sequences were characterized by remarkably high levels of alpha and beta diversities, suggesting the presence of an enormous, yet-untapped supply of novel Halobacteriales genera within the rare biosphere of various saline ecosystems.     

Microbial Diversity in Sediments Collected from the Deepest Cold-Seep Area, the Japan Trench

The Japan Trench land slope at a depth of 6,400 m is the deepest cold-seep environment with Calyptogena communities. Sediment samples from inside and beside the Calyptogena communities were collected, and the microbial diversity in the sediment samples was studied by molecular phylogenetic techniques. From DNA extracted directly from the sediment samples, 16S rDNAs were amplified by the polymerase chain reaction method. The sequences of the amplified 16S rDNAs selected by restriction fragment length polymorphism analysis were determined and compared with sequences in DNA databases. The results showed that 33 different bacterial 16S rDNA sequences from the two samples analyzed fell into similar phylogenetic categories, the α-, γ-, δ-, and ɛ-subdivisions of Proteobacteria, Cytophaga, and gram-positive bacteria; some of the 16S rDNA sequences were common to both samples. δ- and ɛ-Proteobacteria-related sequences were abundant in both sediments. These sequences are mostly related to sulfate-reducing or sulfur-reducing bacteria and epibionts, respectively. Eight different archaeal 16S rDNA sequences were cloned from the sediments. The majority of the archaeal 16S rDNA sequences clustered in Crenarchaeota and showed high similarities to marine group I archaeal rDNA. A Methanococcoides burtonii–related sequence obtained from the sediment clustered in the Euryarchaeota indicating that M. burtonii–related strains in the area of Calyptogena communities may contribute to production of methane in this environment. From these results, we propose a possible model of sulfur circulation within the microbial community and that of Calyptogena clams in the cold-seep environment. Received June 15, 1998; accepted November 10, 1998.     

九龙江河口区nirS型反硝化细菌多样性及系统发育学分析

【目的】结合16S rRNA基因克隆文库和nirS基因克隆文库的分析,揭示九龙江河口区nirS型反硝化细菌多样性。【方法】选取九龙江河口区一富营养化采样点,分别采集水样及沉积物样品,进行理化因子的测定并提取细菌总DNA。以水样DNA构建16S rRNA基因克隆文库,以沉积物DNA构建nirS基因克隆文库,分析微生物群落结构的多样性并构建系统发育树。【结果】从16S rRNA基因克隆文库中获得86条有效序列,按97%的序列相似性划分为53个OTU,分别属于Proteobacteria门、Planctomycetes门、Bacteroidetes门、Actinobacteria门、Firmicutes门和Chloroflexi门。其中属于Proteobacteria门OTU的克隆子占克隆数的62.9%,是最优势的类群,分属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Deltaproteobacteria纲等。从nirS基因克隆文库中获得190条有效序列,翻译为氨基酸序列后,按82%的序列相似性划分为60个OTU,并定位到属的水平。其中Proteobacteria门是最优势的类群,占文库克隆子总数的71.6%,包括Alphaproteobacteria纲(5.8%)、Betaproteobacteria纲(49.0%)和Gammaproteobacteria纲(16.9%)。nirS基因克隆文库中丰度最高的OTU与GenBank中的一株可培养反硝化菌Thauera sp. R-26906具有100%的序列相似性。【结论】九龙江河口区的微生物以及亚硝酸盐还原酶基因(nirS)具有丰富的多样性。大部分NirS序列在GenBank中的最相似序列来源于河口、海湾等相似的环境。