Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of > 0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G + C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria ( Desulfosarcina group, Syntrophus and Geobacter / Pelobacter / Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.     

Biodiversity,community structural shifts,and biogeography of prokaryotes within Antarctic continental shelf sediment

16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region. Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions). The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs). At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes. At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria. Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core. Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment.     

东北太平洋深海沉积物细菌多样性

采用两种方法提取中国结核合同区东区沉积物不同层次总DNA,通过克隆测序构建了含有79个克隆子的细菌16S rRNA基因文库,分析了该海域沉积物中细菌的多样性.79个克隆在系统发育树中形成了11个大分支,包括Gamma proteobacteria(22.8%),Alpha proteobacteria(16.5%),Planctomycetacia(7.6%),Delta proteobacteria(6.3%), Nitrospira(6.3%),Actinobacteria(6.3%),Beta proteobacteria(5%),Acidobacteria(5.1%),Sphingobacteria(3.8%),Firmicutes(2.5%),Other bacteria(17.7%),其中Gamma proteobacteria在总文库中所占比例最高,该分支细菌在0～2cm、4～6cm层也是优势菌种.Gamma proteobacteria中假单胞菌(Pseudomonas)为优势属(22.2%).各个层次中所含细菌类群有所不同,Alpha proteobacteria、Gamma proteobacteria、Delta proteobacteria 、Planctomycetacia、Nitrospira 、Actinobacteria和Acidobacteria为三层样品共有类群.     

Bacterial diversity in deep-sea sediment from northeastern Pacific Ocean

16S rDNA sequencing method is one of the effectively used culture-independent techniques in recent years. In this study, 16S rDNA sequencing method was used to investigate the bacterial diversity in deep-sea sediment from northeastern Pacific polymetallic nodule province. Total DNAs were extracted by using 2 different methods (chemical method and DNA extracting kit method). After purification, genomic DNA was amplified by using 2 universal primers (27F and 1492R). Clones were selected and sequenced randomly. After the sequences were checked by using the Chimera Check Program of the RDP database, a bacterial 16S rRNA gene library of 79 clones was established. Phylogenetic analysis indicated that 79 clones could be divided into 11 phylotypes. Gamma Proteobacteria (22.8%) and alpha Proteobacteria (16.5%) were the dominant components of the sediment bacterial community, followed by Planctomycetacia (7.6%), delta Proteobacteria (6.3%), Nitrospira (6.3%), Actinobacteria (6.3%), beta Proteobacteria (5%), Acidobacteria (5.1%), Sphingobacteria (3.8%), Firmicutes (2.5%) and uncultured bacteria (17.7%). Gamma Proteobacteria also dominated at slices 0–2 cm and 4–6 cm. Different slices had different types of bacteria, alpha Proteobacteria, gamma Proteobacteria, delta Proteobacteria, Planctomycetacia, Nitrospira, Actinobacteria and Acidobacteria, however, appeared in all slices. Pseudomonas is common in many different deep-sea environments. In this study, it accounted for 22.2% of the total gamma Proteobacteria.     

Biodiversity, Community Structural Shifts, and Biogeography of Prokaryotes within Antarctic Continental Shelf Sediment

16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region. Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions). The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs). At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes. At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria. Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core. Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment.     

DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil

A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82 degrees 30'N, 62 degrees 19'W). The enrichment culture was grown on Jet A-1 fuel at 7 degrees C. Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector. Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones. The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp. (six sequences), and Pseudomonas synxantha (four sequences). Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above. Phylotype-specific PCR assays were developed and validated for the above three phylotypes. The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays. Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype. A representative strain of each phylotype was physiologically characterized. Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes. During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay. All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library. The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium. The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations.     

Prokaryotic diversity in continuous cropping and rotational cropping soybean soil

In spite of the techniques based on the amplification of 16S rRNA genes (16S rDNA) to compare bacterial communities that are now widely in use in microbial ecology, little is known about the composition of the soybean continuous cropping (CC) and rotational cropping (RC) soil microbial community. To address this, we compared the levels of bacterial community diversity in RC and 5-year CC rhizosphere soil samples. We selected 407 clones in RC and 490 clones in CC for restriction fragment length polymorphism analysis. A total of 123 phylotypes were identified among the 16S rDNA clones, while 78 unique and 21 common phylotypes were identified among the CC soil isolates. Analysis of sequences from a subset of the phylotypes showed that at least 11 bacterial divisions were represented in the clone libraries. The phylotype richness, frequency distribution (evenness), and composition of the two clone libraries were investigated using a variety of diversity indices. Although the analysis of diversity indices and LIBSHUFF comparisons revealed that the compared libraries were not significantly different ( P =0.05) between the RC vs. CC soils, some differences could be observed in terms of specific phyla and groups. We concluded that the group variance was not determined immediately by the cropping system's induction, but was a long-term and slow process.     

Diversity analysis of methanogens in rumen of Bubalus bubalis by 16S riboprinting and sequence analysis

The molecular diversity of rumen methanogens was investigated by 16S rDNA gene library prepared from the rumen contents obtained from Murrah buffaloes in India. Genomic DNA was isolated from adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the positive clones were selected assuming based on blue-white screening and sequenced. Positive clones were reamplified and the resulting PCR products were further subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) by using HaeIII enzyme. A total of 108 clones were examined, and the analysis revealed 16 phylotypes. Out of sixteen phylotypes, nine phylotypes belong to the uncultured group of methanogens, and the rest of seven phylotypes belong to the order Methanomicrobiales, Methanococcales and Methanobacteriales. Out of the 108 rDNA clones, 66 clones which constitute 61.1% of the total clone representing 9 phylotypes, show less than 97% sequence similarity with any of the cultured strain of methanogens. The second largest group of clones (24 clones) represented by four phylotypes show a sequence similarity ranging from 91% to 99% with Methanomicrobium mobile strain of methanogens. The third group of 16S rDNA clones clustered along with M. burtonii strain of methanogens. This group consists of 6 clones and constitutes about 5.5% of the total clones and represented by only single phylotype. Fourth and fifth clusters of 16S rDNA clones consist of 5 and 7 clones respectively, and these were matched with Methanobrevibacter gottschalkii and Methanobrevibacter rumanatium strain of methanogens and constitute about 4.6% and 6.4% of the total clones.     

Microbiology and geochemistry of great boiling and mud hot springs in the United States Great Basin

A coordinated study of water chemistry, sediment mineralogy, and sediment microbial community was conducted on four >73°C springs in the northwestern Great Basin. Despite generally similar chemistry and mineralogy, springs with short residence time (~5–20 min) were rich in reduced chemistry, whereas springs with long residence time (>1 day) accumulated oxygen and oxidized nitrogen species. The presence of oxygen suggested that aerobic metabolisms prevail in the water and surface sediment. However, Gibbs free energy calculations using empirical chemistry data suggested that several inorganic electron donors were similarly favorable. Analysis of 298 bacterial 16S rDNAs identified 36 species-level phylotypes, 14 of which failed to affiliate with cultivated phyla. Highly represented phylotypes included Thermus, Thermotoga, a member of candidate phylum OP1, and two deeply branching Chloroflexi. The 276 archaeal 16S rDNAs represented 28 phylotypes, most of which were Crenarchaeota unrelated to the Thermoprotei. The most abundant archaeal phylotype was closely related to “Candidatus Nitrosocaldus yellowstonii”, suggesting a role for ammonia oxidation in primary production; however, few other phylotypes could be linked with energy calculations because phylotypes were either related to chemoorganotrophs or were unrelated to known organisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution and phylogenetic diversity of cbbM genes encoding RubisCO form II in a deep-sea hydrothermal field revealed by newly designed PCR primers

To investigate the phylogenetic diversity of putative chemolithoautotrophs possessing the RubisCO form II gene (cbbM) in various environments, we designed a new PCR primer set targeting this gene. The primer set was designed to cover more diverse and longer sequences of cbbM genes than those reported previously. We analyzed various samples (i.e., benthic sands, basement rocks, sulfide chimneys, vent fluids and overlying bottom seawater) collected in a deep-sea hydrothermal field of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, by PCR-based analysis using the designed primer set. Most of the cbbM phylotypes recovered from the liquid samples were related to those of the SUP05 group that belongs to the Gammaproteobacteria and includes putative sulfide-oxidizing chemolithoautotrophs. In contrast, the cbbM phylotypes recovered from the solid samples were related to environmental clones with low similarity (74–90%) and not closely related to the SUP05 group (69–74%). The cbbM phylotypes recovered from the liquid samples were different from those of the solid samples. Furthermore, the cbbM phylotypes recovered from the solid samples were different from each other. Our results expand knowledge of the phylogenetic diversity and distribution of putative chemolithoautotrophs possessing RubisCO form II cbbM genes in deep-sea hydrothermal fields.     

Genetic diversity of archaea in deep-sea hydrothermal vent environments.

Molecular phylogenetic analysis of naturally occurring archaeal communities in deep-sea hydrothermal vent environments was carried out by PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the archaeal populations in deep-sea hydrothermal vent environments showed a great genetic diversity, and most members of these populations appeared to be uncultivated and unidentified organisms. In the phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were placed in deep lineages of the crenarchaeotic phylum prior to the divergence of cultivated thermophilic members of the crenarchaeota or between thermophilic members of the euryarchaeota and members of the methanogen-halophile clade. Whole cell in situ hybridization analysis suggested that some microorganisms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea hydrothermal vent environments. These findings expand our view of the genetic diversity of archaea in deep-sea hydrothermal vent environments and of the phylogenetic organization of archaea.     

Seasonal changes and diversity of bacteria in Bohai Bay by RFLP analysis of PCR-amplified 16S rDNA gene fragments

Information about seasonal bacterial composition and diversity is of great value for exploitation of marine biological resources and improvement of ecological environment. Here PCR-amplified restriction fragment length polymorphism (PCR–RFLP) of 16S rRNA genes was used to evaluate seasonal bacterial diversity and community composition in Bohai Bay. A total of 24 bacterial communities were sampled from seawater and sediment of three representative sites in a whole seasonal cycle: spring (April), summer (July), autumn (October), and winter (January). Bacterial Genomic DNA was extracted and PCR-amplified to obtain 16S rDNA fragments which were cloned to construct 24 16s rDNA libraries. Clones of each library were selected randomly for PCR–RFLP analysis of rDNA fragments, and eventually 101 genotypes were identified by RFLP fingerprintings. These 101 genotypes were sequenced and their respective phylotype was identified through the Blast tool of NCBI (similarity 96–100%) and phylogenetic analyses. Among our phylotypes, 80.2% belonged to the genera α-Proteobacteria, β-proteobacteria,γ-Proteobacteria, δ-Proteobacteria, ε-proteobacteria, Flavobacteria, Cytophaga-Flavobacteria-Bacteroides, Verrucomicrobia, Firmicutes and Actinobacteria. Sequence analyses revealed that 47.5% (48) of clone sequences were similar to those of uncultured marine bacteria in the environment. In addition, bacterial diversity and composition clearly displayed seasonal variety. More genera were discovered in summer than any other seasons, and some special species appeared only in specific season.     

Molecular diversity of symbiotic algae (zooxanthellae) in scleractinian corals of Kenya

In this first sequence analysis of ‘zooxanthellae’ (symbiotic algae of the genus Symbiodinium) in scleractinian corals in Africa, seven Kenyan species sampled in 2001–2002 were analysed by RFLP and sequencing of a PCR-amplified fragment of the LSU rRNA gene. Zooxanthellae of phylotypes A, C and D, all described previously in corals from other regions of the world, were detected. All sequences of phylotype D were identical, while phylotype C was variable, with 14 distinct sequences, seven of which clustered in a previously unreported subgroup of phylotype C, among the 22 samples. These data on the diversity of zooxanthellae in Kenyan corals 3–4 years after the 1998 bleaching event are of potential value for longitudinal studies of temporal changes in zooxanthella diversity in Kenyan corals, especially in relation to future large-scale bleaching episodes.     

Expansion of the geographic distribution of a novel lineage of epsilon-Proteobacteria to a hydrothermal vent site on the Southern East Pacific Rise

The diversity associated with a microbial mat sample collected from a deep-sea hydrothermal vent on the Southern East Pacific Rise was determined using a molecular phylogenetic approach based on the comparison of sequences from the small subunit ribosomal RNA gene (16S rDNA). The DNA was extracted from the sample and the 16S rDNA was amplified by PCR. Sixteen different phylotypes were identified by restriction fragment length polymorphism analysis; four phylotypes were later identified as putative chimeras. Analysis of the 16S rDNA sequences placed all the phylotypes within the Proteobacteria. The majority of the sequences (98%) were most closely related to a new clade of epsilon-Proteobacteria that were initially identified from an in situ growth chamber deployed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge in 1995. The similarity between phylotypes identified from Atlantic and Pacific deep-sea hydrothermal vent sites indicates that this new clade of Proteobacteria may be endemic to and widely distributed among deep-sea hydrothermal vents.     

Microbial Community of a Hydrothermal Mud Vent Underneath the Deep-Sea Anoxic Brine Lake Urania (Eastern Mediterranean)

The composition of a metabolically active prokaryotic community thriving in hydrothermal mud fluids of the deep-sea hypersaline anoxic Western Urania Basin was characterized using rRNA-based phylogenetic analysis of a clone library. The physiologically active prokaryotic assemblage in this extreme environment showed a great genetic diversity. Most members of the microbial community appeared to be affiliated to yet uncultured organisms from similar ecosystems, i.e., deep-sea hypersaline basins and hydrothermal vents. The bacterial clone library was dominated by phylotypes affiliated with the epsilon-Proteobacteria subdivision recognized as an ecologically significant group of bacteria inhabiting deep-sea hydrothermal environments. Almost 18% of all bacterial clones were related to delta-Proteobacteria, suggesting that sulfate reduction is one of the dominant metabolic processes occurring in warm mud fluids. The remaining bacterial phylotypes were related to alpha- and beta-Proteobacteria, Actinobacteria, Bacteroides, Deinococcus-Thermus, KB1 and OP-11 candidate divisions. Moreover, a novel monophyletic clade, deeply branched with unaffiliated 16S rDNA clones was also retrieved from deep-sea sediments and halocline of Urania Basin. Archaeal diversity was much lower and detected phylotypes included organisms affiliated exclusively with the Euryarchaeota. More than 96% of the archaeal clones belonged to the MSBL-1 candidate order recently found in hypersaline anoxic environments, such as endoevaporitic microbial mats, Mediterranean deep-sea mud volcanoes and anoxic basins. Two phylotypes, represented by single clones were related to uncultured groups DHVE-1 and ANME-1. Thus, the hydrothermal mud of hypersaline Urania Basin seems to contain new microbial diversity. The prokaryotic community was significantly different from that occurring in the upper layers of the Urania Basin since 60% of all bacterial and 40% of all archaeal phylotypes were obtained only from mud fluids. The uniqueness of the composition of the active prokaryotic community could be explained by the complex environmental conditions at the site. The interaction of oxygenated warm mud fluids with the cold hypersaline brine of the Urania Basin seems to simultaneously select for various metabolic processes, such as aerobic and anaerobic heterotrophy, sulfide- and methane-dependent chemotrophy along with anaerobic oxidation of methane, sulfate- and metal-reduction.     

Pan-oceanic distribution of new highly diverse clades of deep-sea diplonemids

Molecular rRNA gene surveys reveal a considerable diversity of microbial eukaryotes in different environments. Even within a single clade, the number of distinct phylotypes retrieved often goes beyond previous expectations. Here, we have used specific 18S rRNA PCR primers to investigate the diversity of diplonemids, a poorly known group of flagellates with only a few described species. We analysed surface and deep-sea plankton samples from different oceanic regions, including the water-column in the Marmara Sea. We retrieved a large diversity of diplonemid phylotypes, most of which formed two novel distinct clades without cultured representatives. Although most marine diplonemid phylotypes appeared to be cosmopolitan, they showed a marked stratified distribution through the water column, being very scarce or absent in surface waters. The small and specific diplonemid diversity found in surface samples and the fact that most sequences of uncultured diplonemids found in other studies came from deep-sea environments suggest that the two major uncultured diplonemid clades group species preferentially inhabit the deep ocean.     

Prokaryotic diversity,composition structure,and phylogenetic analysis of microbial communities in leachate sediment ecosystems

In order to obtain insight into the prokaryotic diversity and community in leachate sediment, a culture-independent DNA-based molecular phylogenetic approach was performed with archaeal and bacterial 16S rRNA gene clone libraries derived from leachate sediment of an aged landfill. A total of 59 archaeal and 283 bacterial rDNA phylotypes were identified in 425 archaeal and 375 bacterial analyzed clones. All archaeal clones distributed within two archaeal phyla of the Euryarchaeota and Crenarchaeota, and well-defined methanogen lineages, especially Methanosaeta spp., are the most numerically dominant species of the archaeal community. Phylogenetic analysis of the bacterial library revealed a variety of pollutant-degrading and biotransforming microorganisms, including 18 distinct phyla. A substantial fraction of bacterial clones showed low levels of similarity with any previously documented sequences and thus might be taxonomically new. Chemical characteristics and phylogenetic inferences indicated that (1) ammonium-utilizing bacteria might form consortia to alleviate or avoid the negative influence of high ammonium concentration on other microorganisms, and (2) members of the Crenarchaeota found in the sediment might be involved in ammonium oxidation. This study is the first to report the composition of the microbial assemblages and phylogenetic characteristics of prokaryotic populations extant in leachate sediment. Additional work on microbial activity and contaminant biodegradation remains to be explored.