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.     

Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries

Total DNA from sediment samples was isolated by a direct lysis technique. Purified DNA was used as template either undiluted or diluted 1 : 10 prior to polymerase chain reaction (PCR) amplification of 16S rRNA genes. Full-length inserts were analysed for restriction fragment length polymorphisms (RFLP) with the enzyme Cfo1, and the resulting distribution and abundance of RFLP patterns compared between the undiluted and diluted PCR reactions. Results indicate that for low PCR template concentrations, in the range from a few picograms to tens of picograms DNA, proportional representation of specific RFLP types was not reproducible upon template dilution, confirming that PCR amplification of 16S rDNA cannot be used directly to infer microbial abundance. In particular, only 15–24% of the RFLP types recovered from a sample were present in both the undiluted and diluted extracts. We propose that very low template concentrations in the PCR generate random fluctuations in priming efficiency, which led to the contrast in the RFLP types observed in the libraries from the undiluted and diluted extracts.     

Identification of active bacterial communities in a model drinking water biofilm system using 16S rRNA-based clone libraries

Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rRNA gene clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, an annular reactor was used to generate model drinking water biofilms grown on polycarbonate slides. High-quality RNA was extracted from 2-month-old biofilms and used to generate 16S rRNA-based clones. Sequencing analyses of 16S rRNA-based clones suggested that the active bacterial fraction consisted of a few dominant bacterial groups related to Nevskia ramosa and to uncultured bacteria. Several of these bacterial groups were closely related to clones characterized in a DNA-based clone library also generated in this study. Altogether, these results suggest that some of the predominant drinking water bacteria identified using DNA-based techniques are indeed active.     

PCR指纹图谱技术分析人体口腔内微生物菌群结构

目的 应用PCR-DGGE指纹图谱技术对人体口腔微生物菌群结构进行系统性研究.方法 对1例健康人唾液周期性采集的样品和8例健康人个体的唾液与牙菌斑采集的样品,进行微生物群落总DNA的抽提.以此为模板扩增16S rRNA V3可变区,产物经DGGE指纹图谱分析其组成结构,并运用UVIBAND/MAP等软件比较所得群落指纹图谱的相似性指数.结果 同一健康人个体不同采样时间的唾液菌群结构相似性系数＞74%,通过对不同健康个体口腔样本的研究,发现同一个体的唾液与牙菌斑菌群结构存在差异（84%～95%）.结论 同一健康个体其唾液微生物菌群在一定时间内基本稳定,仅有微小的变化;唾液与同个体牙菌斑的微生物组成虽然存在差异,但这种差异要明显小于个体间的差异.     

基于线粒体12S和16S rRNA基因部分序列探讨蚱科12种的系统发育关系

用ABI377自动测序仪测定了蚱科5属11个种的12s和16S rRNA基因部分序列,并从GenBank获得1属1种的同源序列;用Clustal X1．81比较其同源性,用Mega2．1计算序列变异性和遗传距离。在获得的736bp序列中,A T含量为71．2％～77．5％,平均为73．9％;G C含量为22．5％～28．8％,平均为26．1％。经Clustal X1．81软件比对,共得到755个位点,其中简约信息位点185个。以Cylindraustralia kochii为外群,构建NJ、MP和ML分子系统树,结果表明：(1)蚱属并非一个单系群,而是一个并系群;(2)环江柯蚱Coptltettix huanjiangensis和贡山柯蚱C.gongshanensis为同一个种,即贡山柯蚱,而环江柯蚱是贡山柯蚱的同物异名。     

Comparative analysis of bacterioplankton assemblages from maritime Antarctic freshwater lakes with contrasting trophic status

The bacterioplankton assemblages of eight maritime Antarctic lakes with a wide range of trophic status and geographic span (six lakes from Hope Bay, Antarctic Peninsula and two from Potter Peninsula, King George Island) were described using denaturing gradient gel electrophoresis and band sequencing during two consecutive austral summers (2003–2004). Analyses of the gels identified a total of 230 bands spread across 57 different positions. Among those bands, 14 were shared between lakes from Hope Bay and Potter Peninsula, 17 were observed only in particular lakes, and 17 were registered both years in the same lake. We successfully reamplified and sequenced 43 bands located in 36 different positions belonging to Bacteroidetes, Actinobacteria, Betaproteobacteria and Cyanobacteria. The closest matches for 63% of the sequenced bands were from Antarctic or from other cold environment clones and sequences already in the databases, suggesting the widespread dominance of microbial communities adapted to cold habitats. The results of the multivariate analyses (Cluster Analysis and CCA) indicated that the nutrient status of the lake influences the bacterioplankton assemblages. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

A comparison of the 16S ribosomal RNAs from mesophilic and thermophilic bacilli: Some modifications in the sanger method for RNA sequencing

Summary Two modifications in the Sanger two dimensional electrophoretic procedure for RNA analysis are reported. One increases resolution on the primary fingerprint to the point that digests of large RNAs, of the size 1500–3000 nucleotides yield well resolved fingerprint patterns. The other is a novel endonucleolytic procedure that proves useful in determining sequences of the large oligonucleotides produced by T1 ribonuclease.These modifications have been used in determining the catalogs of oligomers produced by T1 ribonuclease digestion of 16S rRNAs from three related organisms,Bacillus subtilis, B.pumilus andB.stearothermophilus. The possible effects of adaptation to a thermophilic niche on ribosomal RNA primary structure and the phylogenetic relatedness of the two mesophilic Bacilli are discussed.This is contribution No.6 in a series on procaryote phylogeny.     

The influence of external perturbations on the functional composition of phytoplankton in a Mediterranean reservoir

The Salton Sea is a large, shallow, endorheic, polymictic, saline lake in Southern California sustained by runoff from local agricultural and municipal wastewater. As a closed-basin lake in an arid region with declining water supply, it is becoming increasingly saline. Due to its eutrophic status and episodic wind-driven mixing, basin-wide anoxia events are becoming more common during summer. Sequencing of 16S rRNA gene clones obtained from the water column and sediments provided the first molecular assessment of bacterial community structure in the Salton Sea. Similar to other saline habitats, the water column community was dominated by Gamma- and Alphaproteobacteria, and Bacteroidetes, although there was significant seasonal variability in diversity, richness, and the specific genera recovered. Sediments showed high diversity and richness and were dominated by sequences from cultured and uncultured phyla typical of marine sediments, especially the Deltaproteobacteria. This study has provided baseline data about the bacterial communities found in this important California water body and may serve as important indicators of change as the lake becomes increasingly saline over time or as remediation efforts are enacted. Handling editor: Darren Bade     

Molecular phylogeny of Collembola inferred from ribosomal RNA genes

The classification of taxa within Collembola (Springtails, Hexapoda) has been controversial. In this study, we combined complete 18S rRNA gene with partial 28S rRNA gene (D7-D10) sequences to investigate the phylogeny of Collembola. About 2500 aligned sites of thirty species representing 29 genera from14 families of Collembola were analyzed, including one species of Neelipleona from which no sequence has been reported previously.The phylogenetic trees were obtained by different methods (maximum parsimony, maximum likelihood, and Bayesian analysis). Our results supported the monophyly of two of the four taxonomic groups of Collembola summarized by Deharveng [Deharveng, L., 2004. Recent advances in Collembola systematics. Pedobiologia 48, 415–433.], namely of Poduromorpha and of Symphypleona. Within Poduromorpha, Neanuridae was monophyletic with high support, but Hypogastruridae was not. Entomobryomorpha was paraphyletic, as the Tomoceroidea (Tomoceridae and Oncopoduridae) was found to be apart from the other entomobryomorphs. In the latter Isotomoidea and Entomobryoidea joined into a group with moderate support. Within Symphypleona, the phylogenetic relationship [(Sminthuridae + Bourletiellidae) + Sminthurididae] was consistent with traditional morphological studies. Neelipleona grouped with Symphypleona in all trees, with moderate support in the ML and Bayesian analyses.     

Defining seasonal marine microbial community dynamics

Here we describe, the longest microbial time-series analyzed to date using high-resolution 16S rRNA tag pyrosequencing of samples taken monthly over 6 years at a temperate marine coastal site off Plymouth, UK. Data treatment effected the estimation of community richness over a 6-year period, whereby 8794 operational taxonomic units (OTUs) were identified using single-linkage preclustering and 21 130 OTUs were identified by denoising the data. The Alphaproteobacteria were the most abundant Class, and the most frequently recorded OTUs were members of the Rickettsiales (SAR 11) and Rhodobacteriales. This near-surface ocean bacterial community showed strong repeatable seasonal patterns, which were defined by winter peaks in diversity across all years. Environmental variables explained far more variation in seasonally predictable bacteria than did data on protists or metazoan biomass. Change in day length alone explains >65% of the variance in community diversity. The results suggested that seasonal changes in environmental variables are more important than trophic interactions. Interestingly, microbial association network analysis showed that correlations in abundance were stronger within bacterial taxa rather than between bacteria and eukaryotes, or between bacteria and environmental variables.     

Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea

In this study, we report on first 16S rRNA gene sequences from highly saline brine sediments taken at a depth of 1,515 m in the Kebrit Deep, northern Red Sea. Microbial DNA extracted directly from the sediments was subjected to PCR amplification with primers specific for bacterial and archaeal 16S rRNA gene sequences. The PCR products were cloned, and a total of 11 (6 bacterial and 5 archaeal) clone types were determined by restriction endonuclease digestion. Phylogenetic analysis revealed that most of the cloned sequences were unique, showing no close association with sequences of cultivated organisms or sequences derived from environmental samples. The bacterial clone sequences form a novel phylogenetic lineage (KB1 group) that branches between the Aquificales and the Thermotogales. The archaeal clone sequences group within the Euryarchaeota. Some of the sequences cluster with the group II and group III uncultivated archaea sequence clones, while two clone groups form separate branches. Our results suggest that hitherto unknown archaea and bacteria may thrive in highly saline brines of the Red Sea under extreme environmental conditions. Received: 5 February 1999 / Accepted: 14 July 1999     

Actinobacterial diversity from marine sediments collected in Mexico

Seventeen different media known to support the growth and isolation of members of the class Actinobacteria were evaluated as selective isolation media for the recovery of this microbial group from marine sediments samples collected in the Gulf of California and the Gulf of Mexico. A general selective isolation procedure was employed for six sediments and nearly 300 actinomycetes were recovered from the selective isolation plates. Full 16S rRNA gene sequencing revealed that the isolates belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia, Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora, Saccharopolyspora, Salinispora, Streptomyces, “Solwaraspora” and Verrucosispora. Previous works on marine sediments have been restricted to the isolation of members of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides further evidence that Actinobacteria present in marine habitats are not restricted to the Micromonospora-Rhodococcus-Streptomyces grouping. Indeed, this first systematic study shows the extent of actinobacterial diversity that can be found in marine sediments collected in Mexico and probably, worldwide. The 16S rRNA gene sequences of marine isolates A1, AA2, AA6, AB1, AB2, AG1, AI2, AK1, AL2, AO1, AO3, AR1, AW1, B1, BB1, BC1, C5, R1, R2, R3, AV1, AE1, AI1, AN1 and AP1 determined in this study have been deposited under GenBank accession numbers EU714241–EU714258 and FJ462359–FJ462365, respectively.     

Analysis and comparison of the bacterial community in fermented grains during the fermentation for two different styles of Chinese liquor

Bacterial populations in fermented grains during fermentation may play important roles in Chinese liquor flavor. PCR-based denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene library analysis were performed to analyze the bacterial community structure of two styles of liquor. The results of DGGE profiles showed that bacterial diversity decreased with the fermentation process and Lactobacillus acetotolerans became the predominant species at the end of the fermentation. But the obvious differences of bacterial community appeared in the middle stage of two styles of liquor fermentation, in which the different upstream production techniques were used. Moreover, 16S rRNA gene libraries of two styles were constructed. A total of 125 and 107 clones, chosen from two libraries, were grouped into 46 and 49 operational taxonomic units (OTUs) by amplified ribosomal DNA restriction analysis. According to sequencing results of clones, the predominant bacteria in strong aroma style fermented grains were those from the class Bacilli, Bacteroidetes, and Clostridia, whereas the predominant bacteria in fermented grains of roasted sesame aroma style belonged to Bacilli, Flavobacteria, and Gammaproteobacteria. Molecular analysis of the bacterial diversity of the liquor fermentation will benefit the analysis of important microorganisms playing key roles in the formation of liquor flavor components.     

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO₂ by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO₂. Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed < 95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real‐time PCR (qRT PCR), was 9.38 ± 0.75 × 10⁷ and 5.43 ± 0.79 × 10⁸ (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO₂‐fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.     

耐热嗜盐菌的分离及16S rRNA基因序列分析

从位于西藏自治区澜沧江边一个47℃的盐井中分离筛选到一株耐热嗜盐菌菌株 YJ0238.对其进行了生理生化特性研究,采用PCR方法扩增其16S rRNA基因序列,并进行了测定.基于生理生化特性和16S rRNA基因序列的同源性比较,以及系统发育分析,发现菌株YJ0238是Idiomarina属中成员zobellii的一个亚种,其16S rRNA基因序列已被GenBank数据库收录,序列号为EF693953.迄今为止,国内极少有关高温、高盐环境中微生物研究的报道,本研究可为今后研究同类极端环境中新的物种资源以及微生物多样性提供素材和参考.     

耐热嗜盐菌的分离及16S rRNA基因序列分析

从位于西藏自治区澜沧江边一个47℃的盐井中分离筛选到一株耐热嗜盐菌菌株YJ0238, 对其进行了生理生化特性研究, 采用PCR方法扩增其16S rRNA基因序列, 并进行了测定。基于生理生化特性和16S rRNA基因序列的同源性比较, 以及系统发育分析, 发现菌株YJ0238是Idiomarina属中成员zobellii的一个亚种, 其16S rRNA基因序列已被GenBank数据库收录, 序列号为EF693953。迄今为止, 国内极少有关高温、高盐环境中微生物研究的报道, 本研究可为今后研究同类极端环境中新的物种资源以及微生物多样性提供素材和参考。