Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization

We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from gamma-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from alpha-proteobacteria, which dominated the clone libraries. The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4', 6'-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for alpha-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. alpha-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.     

Community structure and diversity of biofilms from a beer bottling plant as revealed using 16S rRNA gene clone libraries

The microbial composition of biofilms from a beer bottling plant was analyzed by a cultivation independent analysis of the 16S rRNA genes. Clone libraries were differentiated by amplified 16S rRNA gene restriction analysis and representative clones from each group were sequenced. The diversity of the clone libraries was comparable with the diversity found for environmental samples. No evidences for the presence of strictly anaerobic taxa or important beer spoilers were found, indicating that biofilms developed for more than 6 months at the plant formed no appropriate habitat for those microorganisms. The genus Methylobacterium was one of the dominating groups of the clone libraries. The size of this population was assessed by fluorescence in situ hybridization and fatty acid analysis. In addition, considerable numbers of clones were assigned to uncultivated organisms.     

PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries

ABSTRACT. Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray–Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.     

Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end illumina reads

Microbial communities host unparalleled taxonomic diversity. Adequate characterization of environmental and host-associated samples remains a challenge for microbiologists, despite the advent of 16S rRNA gene sequencing. In order to increase the depth of sampling for diverse bacterial communities, we developed a method for sequencing and assembling millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; ～200 nucleotides) by using an Illumina genome analyzer. To confirm reproducibility and to identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNAs from known cultured bacterial isolates (>1 million postassembly sequences) and an Arctic tundra soil sample (>6 million postassembly sequences). The Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e.g., 454 pyrosequencing), while the assembly of paired-end 125-base reads offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane, may be modified readily to target other variable regions or genes, and demonstrates unprecedented and economical access to DNAs from organisms that exist at low relative abundances.     

A meta-analysis of 16S rRNA gene clone libraries from the polymicrobial black band disease of corals

Black band disease (BBD) is a polymicrobial disease affecting corals on reefs worldwide. Since 2002, researchers have constructed clone libraries from the BBD consortium using 16S rRNA gene primers targeting a variety of phyla. In the present study, a meta-analysis was conducted of published libraries from 2002 through the present that contain bacterial sequence data associated with individual clones and BBD samples. The libraries analyzed were from 87 BBD samples, collected from 16 species of scleractinian corals in 10 different geographic locations, and included 327 unique operational taxonomic units (OTUs). One OTU (cyanobacterial) was present in 62 (71%) samples, and three (one Cytophaga-Flavobacter-Bacteriodetes and two alphaproteobacterial) were present in 11 (13%) of the samples. The remaining 323 unique OTUs were present in <10% of the samples. The Alphaproteobacteria was the most diversely represented group. Analysis of clone libraries using nonmetric multidimensional scaling indicated strong regional specificity of BBD microbial populations, but limited host coral specificity. The results of this analysis support the hypotheses that: (1) a specific cyanobacterium may be the primary pathogen of BBD; (2) additional functional groups, required for BBD pathobiology, are represented by variable opportunistic species; and (3) opportunistic BBD microorganisms are primarily derived from the environment.     

Seasonal and regional diversity of maple sap microbiota revealed using community PCR fingerprinting and 16S rRNA gene clone libraries

An arbitrary primed community PCR fingerprinting technique based on capillary electrophoresis was developed to study maple sap microbial community characteristics among 19 production sites in Québec over the tapping season. Presumptive fragment identification was made with corresponding fingerprint profiles of bacterial isolate cultures. Maple sap microbial communities were subsequently compared using a representative subset of 13 16S rRNA gene clone libraries followed by gene sequence analysis. Results from both methods indicated that all maple sap production sites and flow periods shared common microbiota members, but distinctive features also existed. Changes over the season in relative abundance of predominant populations showed evidence of a common pattern. Pseudomonas (64%) and Rahnella (8%) were the most abundantly and frequently represented genera of the 2239 sequences analyzed. Janthinobacterium, Leuconostoc, Lactococcus, Weissella, Epilithonimonas and Sphingomonas were revealed as occasional contaminants in maple sap. Maple sap microbiota showed a low level of deep diversity along with a high variation of similar 16S rRNA gene sequences within the Pseudomonas genus. Predominance of Pseudomonas is suggested as a typical feature of maple sap microbiota across geographical regions, production sites, and sap flow periods.     

The effect of protozoa on the composition of rumen bacteria in cattle using 16S rRNA gene clone libraries

The effect of the presence of protozoa on the composition of rumen bacteria was investigated in cattle. Seven castrated Holstein cattle were divided into two groups: four faunated and three unfaunated, and 16S ribosomal RNA gene (rDNA) clonal libraries were constructed. A total of 312 clones were sequenced across 1,500 bp. The 151 sequences of the faunated cattle were classified into 98 operational taxonomic units (OTUs) having at least 97% similarity. The sequences derived from the faunated cattle were classified into Firmicutes (59.7%), Bacteroidetes (34.4%), Spirochaetes (2.6%), Actinobacteria (2.0%), and Proteobacteria (1.3%). Bacteroides and Prevotella (34.4%) were the major groups in the faunated cattle. The 161 sequences in the unfaunated cattle were classified into 72 OTUs. The sequences derived from the unfaunated libraries were classified into Firmicutes (65.7%), Bacteroidetes (31.1%), Proteobacteria (1.9%), and Spirochaetes (1.2%). The Clostridium botulinum group and its relatives (36.0%) were the major groups in the unfaunated cattle.An analysis by the computer program LIBSHUFF clarified that the presence of ruminal protozoa markedly affected the composition of rumen bacteria.     

Distribution patterns of marine planktonic cyanobacterial assemblages in transitional marine habitats using 16S rRNA phylogeny

The community composition of marine planktonic cyanobacteria in transitional marine habitats can influence its overall contribution to aquatic primary production. To understand distribution patterns of marine planktonic cyanobacterial assemblages, phylogenetic and statistical analyses were undertaken on planktonic cyanobacterial 16S rRNA gene sequences from four transitional marine habitats [Baltic Sea (BL), Monterey Bay (MB), South China Sea (SCS) and Sundarbans (SB)]. Out of 3255 sequences analyzed, only 546 sequences were found to be planktonic cyanobacteria and were considered in this study. Among these, 338 sequences representative of Sundarbans, the world's largest mangrove were generated based on Sanger and Illumina sequencing approaches. Based on 16S rRNA phylogeny, four major taxonomic orders of marine planktonic cyanobacteria were recovered in varying proportions with several novel 16S rRNA sequences in each of the four targeted sites. Members of the order Synechococcales were dominant in all the sites (?94% sequences) while the orders Chroococcales and Oscillatoriales were only detected in SB and SCS sites, respectively. In the phylogenetic tree, sequences representing the major marine picocyanobacterial genus Synechococcus showed overwhelming dominance in SB and they were found in three other sites. Prochlorococcus ‐like sequences were found in sizeable number in MB and SCS but were absent in SB and coastal BL. Synechococcus ‐like sequences were represented by three major marine clusters (5.1, 5.2, and 5.3). Three novel clades as part of Synechococcus cluster were detected only in SB and one novel clade in BL. The majority of OTUs were found to be exclusive to each site, whereas some were shared by two or more sites as revealed by beta‐diversity analysis.     

16s rRNA基因克隆文库分析比较牙周炎患者和健康人口腔唾液微生物多样性

【目的】通过对同一地区、同一民族牙周炎患者和健康人的唾液微生物群落结构的分析,探寻牙周炎患者口腔微生物的多样性。【方法】采集甘肃东乡族自治县的东乡族牙周炎患者和健康人唾液各5例,分别记作DP(东乡牙周)和DH(东乡健康),提取细菌总DNA,构建16S r RNA基因克隆文库,测序后利用MOTHUR、MEGA 4.0、Clustal X 3.0等软件对测序结果进行分析。【结果】所有样本共检测出115个OTUs(DP 60,DH 75),归属于6个门,27个属。TM7是DP组特有的优势菌门。仅在DP组中检测到的优势菌属是梭菌属(Fusobacterium)、卟啉单胞菌属(Porphyromonas)、消化链球菌属(Peptostreptococcus)和TM7_genera。【结论】发现牙周炎患者与健康人口腔唾液微生物存在一定差异。其中,TM7、梭菌属和消化链球菌属在牙周病中的作用值得进一步研究。     

Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture-based method. Direct counts ranged from 1.9 X 10" to 4.0 X 10" cells/g (wet weight), and plate counts totaled 6.6 X 10(10) to 1.2 X 10(11) CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.     

Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries

The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries. Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected. The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles. The majority of clones obtained in this study belonged to the Clostridium coccoides (C. coccoides) group, the Bacteroides group or the Lactobacillus group. Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C. coccoides and Bacteroides groups. The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries. T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data. T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.     

不同粪便DNA提取方法比较分析北大核心CSCD

肠道微生物群落结构和多样性与人体疾病密切相关。然而,相关群落结构分析结果可能受到DNA提取质量等实验因素影响。因此,评估不同DNA提取方法对肠道特定种属的提取效果,对于全面、准确获取人体肠道微生物谱,深入探究肠道微生物群落结构具有指导意义。本研究旨在借助实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT qPCR)技术,以DNA提取纯度、浓度,以及对肠道中特定种属微生物基因组DNA的提取丰度为指标,对5种DNA提取方法进行比较分析。结果表明,试剂盒Q的提取效果最佳,特别是对乳杆菌属和双歧杆菌属等革兰氏阳性菌的提取效果较好。N试剂盒的平均DNA提取浓度较Q试剂盒低,但在纯度方面,二者无显著性差异。与其他3种商用试剂盒(M、PSP、TG)相比,N方法对肠道内指定微生物基因组的提取效果仅次于Q试剂盒,位居第二。相比之下,M试剂盒提取所得DNA,质量较高,但浓度偏低,对于肠道内革兰氏阳性菌的提取效果不很理想。TG试剂盒和PSP试剂盒提取所得DNA在浓度、质量以及细菌丰度方面均不及其他验证的试剂盒。综上,Q试剂盒可作为肠道微生态研究相关实验中获取高质量基因组DNA的提取方法。本研究结果为肠道微生态研究相关实验中基因组DNA提取方法的选择提供参考依据。     

Barcoding marine nematodes: an improved set of nematode 18S rRNA primers to overcome eukaryotic co-interference

Nematodes form an important component of many benthic marine ecosystems and DNA barcoding approaches could provide an insight into nematode community composition from different environments globally. We have amplified nematode 18S rRNA sequences using standard nematode18S rRNA primers from environmental DNA extracted from intertidal sediment collected from New Jersey coast, USA to test whether the published marine nematode 18S rRNA sequences from GenBank and EMBL databases can effectively assign unknown nematode sequences into genus or species level. Most of the sequenced clones showed some degree of identities with published marine nematode 18S rRNA sequences. However, relatively very few of the sequences could be assigned even to genus level based on sequence assignment rule. In addition, other eukaryotic 18S rRNA sequences were found to be co-amplified with commonly used nematode 18S rRNA primers. We found that the majority of the current nematode 18S rRNA primers will co-amplify other eukaryotes if environmental DNA is the target template. We therefore designed a new set of nematode 18S rRNA primers and evaluated them using environmental DNA in intertidal sediment from the New Jersey coast. In total, 40 clones were screened and subsequently sequenced and all the sequences showed varying degree of identities with published nematode 18S rRNA sequences from GenBank and EMBL databases, and no obvious eukaryotic co-amplicons were detected with new primers. Only 13 out of 40 clones amplified with the new primer set showed 100% identity to published Daptonema and Metachromadora 18S rRNA sequences. The current molecular databases for nematodes are dominated by sequences from NW Europe and need to be more extensively populated with new full length 18S rRNA nematode sequences collected from different biogeographic locations. The new primers developed in this study, in combination with an updated nematode 18S rRNA sequence database, would help us to better investigate and understand the diversity and community composition of free-living marine nematodes based on DNA barcoding approaches during biodiversity or biomonitoring surveys on a global-scale.     

Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples

To determine the significance of differences between clonal libraries of environmental rRNA gene sequences, differences between homologous coverage curves, CX(D), and heterologous coverage curves, CXY(D), were calculated by a Cramér-von Mises-type statistic and compared by a Monte Carlo test procedure. This method successfully distinguished rRNA gene sequence libraries from soil and bioreactors and correctly failed to find differences between libraries of the same composition.     

Comparison of eukaryotic phytobenthic community composition in a polluted river by partial 18S rRNA gene cloning and sequencing

We compared the species composition in phytobenthic communities at different sampling sites in a small French river presenting polluted and unpolluted areas. For each sampling point, the total DNA was extracted and used to construct an 18S rRNA gene clone library after PCR amplification of a ca 400 bp fragment. Phytobenthic community composition was estimated by random sequencing of several clones per library. Most of the sequences corresponded to the Bacillariophyceae and Chlorophyceae groups. By combining phylogenetic and correspondence analyses, we showed that our molecular approach is able to estimate and compare the species composition at different sampling sites in order to assess the environmental impact of xenobiotics on phytobenthic communities. Changes in species composition of these communities were found, but no evident decrease in the diversity. We discuss the significance of these changes with regard to the existing level of pollution and their impact on the functionality of the ecosystem. Our findings suggest that it is now possible to use faster molecular methods (DGGE, ARISA...) to test large numbers of samples in the context of ecotoxicological studies, and thus to assess the impact of pollution in an aquatic ecosystem.     

基于16S rRNA基因测序分析微生物群落多样性

微生物群落多样性的研究对于挖掘微生物资源,探索微生物群落功能,阐明微生物群落与生境间的关系具有重要意义。随着宏基因组概念的提出以及测序技术的快速发展,16S rRNA基因测序在微生物群落多样性的研究中已被广泛应用。文中系统地介绍了16S rRNA基因测序分析流程中的四个重要环节,包括测序平台与扩增区的选择、测序数据预处理以及多样性分析方法,就其面临的问题与挑战进行了探讨并对未来的研究方向进行了展望,以期为微生物群落多样性相关研究提供参考。     

Spatial and temporal dynamics of a freshwater eukaryotic plankton community revealed via 18S rRNA gene metabarcoding

The Brazilian Environmental Ministry (MMA) released a report in 2016 listing 163 non-native species into Brazilian inland waters. Reservoirs are among the freshwater habitats most frequently associated with the release of non-native species. Therefore, the aim of the present study was to evaluate the occurrence and distribution of the non-native species listed by the MMA, in large Brazilian reservoirs (≥ 30 km²). Further, we have tested the hypothesis that beta diversity of non-native species within reservoirs from the same basin is lower than those among reservoirs from distinct basins. A literature search was performed for 70 Brazilian reservoirs, resulting in the records of 91 non-native species. Reservoirs from the Paraná basin showed the highest number of occurrences, with 33 non-native species recorded only in the Itaipu reservoir. Beta diversity of non-native species showed higher variability among reservoirs from different basins than those within the same basin. Some basins were also distinguished by their composition of non-native species, as supported by the IndVal index. Non-native species were widespread along Brazilian reservoirs, and their distribution can be even higher than reported.     

Bacterial diversity at different depths in lead-zinc mine tailings as revealed by 16S rRNA gene libraries

Bacterial communities at 10 cm, 100 cm, and 200 cm depths in a 100-year-old lead-zinc tailing heap were evaluated by constructing 16S rRNA gene libraries. In total, 98 operational taxonomic units (OTUs) were identified from 193 clones at a 3% sequence difference level. The OTU number and species richness decreased with the depth. Species composition was significantly different between the three libraries. Fifty-seven percent of the examined clones were Acidobacteria and 27% belonged to Proteobacteria. Other sequences included Chloroflexi, Firmicutes, Chlamydiae, Actinobacteria, Gemmatimonadetes, Nitrospira, and three unclassified OTUs. Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were mainly distributed in the rhizosphere of naturally colonizing plants; however, Deltaproteobacteria, Acidobacteria, and Chloroflexi tended to inhabit the deeper tailings (below the 100 cm-depth).