Diversity of the bacterial community in the rice rhizosphere managed under conventional and no-tillage practices

Bacterial diversity in the rice rhizosphere at different rice growth stages, managed under conventional and no-tillage practices, was explored using a culture-based approach. Actinobacteria are among the bacterial phyla abundant in the rice rhizosphere. Their diversity was further examined by constructing metagenomic libraries based on the 16S rRNA gene, using actinobacterial- and streptomycete-specific polymerase chain reaction (PCR) primers. The study included 132 culturable strains and 125 clones from the 16S rRNA gene libraries. In conventional tillage, there were 38% Proteobacteria, 22% Actinobacteria, 33% Firmicutes, 5% Bacteroidetes, and 2% Acidobacteria, whereas with no-tillage management there were 63% Proteobacteria, 24% Actinobacteria, 6% Firmicutes, and 8% Bacteroidetes as estimated using the culture-dependent method during the four stages of rice cultivation. Principal coordinates analysis was used to cluster the bacterial communities along axes of maximal variance. The different growth stages of rice appeared to influence the rhizosphere bacterial profile for both cultivation practices. Novel clones with low similarities (89–97%) to Actinobacteria and Streptomyces were retrieved from both rice fields by screening the 16S rRNA gene libraries using actinobacterial- and streptomycete-specific primers. By comparing the actinobacterial community retrieved by culture-dependent and molecular methods, it was clear that a more comprehensive assessment of microbial diversity in the rice rhizosphere can be obtained using a combination of both techniques than by using either method alone. We also succeeded in culturing a number of bacteria that were previously described as unculturable. These were in a phylogenetically deep lineage when compared with related cultivable genera.     

云南江城和黑井盐矿沉积物未培养放线菌多样性比较

类群特异性引物的应用使得研究者可以对感兴趣的微生物类群进行针对性研究.围绕云南江城和黑井两个地区的3个盐矿样点沉积物中放线菌的多样性和群落组成,我们通过放线菌特异性引物对总DNA进行16S rRNA基因扩增,经过克隆文库构建,利用酶切并选择其中不同带型的133个克隆的16S rRNA基因插入片段进行测序.系统发育分析和统计学结果表明,两地放线菌16S rRNA基因克隆广泛分布于整个放线菌门,同时发现部分序列可能属于放线菌的新类群.分析结果还预示,江城和黑井两地盐矿虽处云南不同地域含盐区,但两地未培养放线菌物种多样性和系统发育关系均较为相似.     

Intra- and inter-lake variability of free-living and particle-associated Actinobacteria communities

We have analysed the inter- and intra-lake variability of free-living and particle-associated freshwater Actinobacteria communities in four limnological different lakes of the Mecklenburg Lake District, Northeastern Germany. Denaturing gradient gel electrophoresis (DGGE) specific for Actinobacteria was used to investigate phylogenetic diversity and seasonal dynamics of actinobacterial communities in the epilimnion of all lakes (inter-lake variability) and to assess differences between Actinobacteria communities of the epi-, meta- and hypolimnion of a single lake (intra-lake variability) respectively. DGGE analyses showed significant inter- and intra-lake differences between Actinobacteria communities of all lakes and water layers as well as between free-living and particle-associated Actinobacteria. Phylogenetic inferences of 16S rRNA gene sequences suggest that particular members of particle-associated Actinobacteria were exclusively affiliated to certain actinobacterial lineages. The phylogenetic comparison of 16S rRNA gene sequences of all lakes and water layer, however, indicated the occurrence of almost similar phylogenetic lineages in all studied habitats and suggest high intracluster diversity within already known actinobacterial lineages. Non-metric multidimensional scaling (NMS) ordination analyses and Pearson's product moment correlations revealed several strong correlations between the investigated Actinobacteria communities and various limnological parameters, such as conductivity, total phosphorous, alkalinity or primary production. However, no uniform correlation patterns were found between lakes, water layers and bacterial fractions. These heterogeneous correlation patterns together with the phylogenetic similarities of Actinobacteria communities from different lakes indicate that particular Actinobacteria represent various ecotypes or exhibit a pronounced ecophysiological plasticity.     

Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil

Bacterial diversity in 16S ribosomal DNA and reverse-transcribed 16S rRNA clone libraries originating from the heavy metal-contaminated rhizosphere of the metal-hyperaccumulating plant Thlaspi caerulescens was analysed and compared with that of contaminated bulk soil. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Acidobacteria and the Planctomycetales. Only 14.7% of all phylotypes (sequences with similarities> 97%), but 45% of all clones, were common in the rhizosphere and the bulk soil clone libraries. The combined use of rDNA and rRNA libraries indicated which taxa might be metabolically active in this soil. All dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries compared with the rDNA libraries. Clones belonging to the Verrucomicrobiales, Firmicutes, Cytophaga-Flavobacterium-Bacteroides and OP10 were found only in rDNA clone libraries, indicating that they might not represent active constituents in our samples. The most remarkable result was that sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA (50% and 60% of all phylotypes respectively). Seventy per cent of these clone sequences were related to the Rubrobacteria subgroups 2 and 3, thus providing for the first time evidence that this group of bacteria is probably metabolically active in heavy metal-contaminated soil.     

Effects of Transgenic Hybrid Aspen Overexpressing Polyphenol Oxidase on Rhizosphere Diversity

This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.     

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.     

芒草种植对土壤细菌群落结构和功能的影响

芒草作为第二代能源植物的代表,其生长过程中根际土壤细菌群落的结构与功能尚不清楚.本研究以种植5年的芒草(湘杂芒1号)为研究对象,选取裸地作为对照,采用16S rRNA基因Miseq测序技术研究其细菌群落组成,同时采用PICRUSt功能预测分析其功能.结果表明: 芒草根际细菌由变形菌门、酸杆菌门、放线菌门、绿弯菌门、拟杆菌门和芽单胞菌门等23个门、231个属的细菌组成,表现出群落组成的丰富性.细菌群落分析表明,种植湘杂芒1号改变了根际细菌群落结构,其细菌群落多样性低于裸地对照.PICRUSt功能预测分析表明,湘杂芒1号根际细菌主要涉及氨基酸运输和代谢、细胞壁/细胞膜/膜结构的生物合成、信号转导机制等24个基因功能家族,表现出功能上的丰富性,并有22个基因功能家族预测基因相对丰度高于裸地,表明种植湘杂芒1号提高了根际细菌功能.对氮、磷循环相关基因进行分析表明,种植湘杂芒1号改变了土壤氮、磷代谢能力.     

Molecular profiling of rhizosphere microbial communities associated with healthy and diseased black spruce (Picea mariana) seedlings grown in a nursery

Bacterial and fungal populations associated with the rhizosphere of healthy black spruce (Picea mariana) seedlings and seedlings with symptoms of root rot were characterized by cloned rRNA gene sequence analysis. Triplicate bacterial and fungal rRNA gene libraries were constructed, and 600 clones were analyzed by amplified ribosomal DNA restriction analysis and grouped into operational taxonomical units (OTUs). A total of 84 different bacterial and 31 different fungal OTUs were obtained and sequenced. Phylogenetic analyses indicated that the different OTUs belonged to a wide range of bacterial and fungal taxa. For both groups, pairwise comparisons revealed that there was greater similarity between replicate libraries from each treatment than between libraries from different treatments. Significant differences between pooled triplicate samples from libraries of genes from healthy seedlings and pooled triplicate samples from libraries of genes from diseased seedlings were also obtained for both bacteria and fungi, clearly indicating that the rhizosphere-associated bacterial and fungal communities of healthy and diseased P. mariana seedlings were different. The communities associated with healthy and diseased seedlings also showed distinct ecological parameters as indicated by the calculated diversity, dominance, and evenness indices. Among the main differences observed at the community level, there was a higher proportion of Acidobacteria, Gammaproteobacteria, and Homobasidiomycetes clones associated with healthy seedlings, while the diseased-seedling rhizosphere harbored a higher proportion of Actinobacteria, Sordariomycetes, and environmental clones. The methodological approach described in this study appears promising for targeting potential rhizosphere-competent biological control agents against root rot diseases occurring in conifer nurseries.     

秦皇岛新开河河口邻近海域细菌多样性及其影响因子

【目的】分析秦皇岛新开河河口及其邻近海域3个站位的细菌多样性,了解入海口污染对微生物多样性的影响,为该海域微生物的生态功能研究提供理论基础。【方法】于2014年8月选取秦皇岛新开河入海河口(XKH)及其邻近海域(W1,W2)共3个站位采集水样,采用荧光显微镜计数以及构建16S rRNA基因克隆文库的方法,分析细菌群落结构多样性与海水环境条件的关系。【结果】邻近海域W1站位的细菌总数(2.62×10~6 cells/m L)和多样性均高于新开河河口XKH站位(细菌总数:6.62×10~5 cells/m L)和W2站位(细菌总数:2.02×10~6 cells/m L);从XKH、W1、W2 3个站位的16S r RNA基因克隆文库中分别获得57、89、87条有效序列,按97%的序列相似性分别划分为46、51、56个OTU,分别属于Proteobacteria、Cyanobacteria、Bacteroidetes、Firmicute、Actinobacteria、Planctomycetes和Verrucomicrobia七个门。其中在XKH和W2站位中,Proteobacteria门的克隆子分别占总克隆数的50.9%和75.9%,是最优势的类群,分属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria、Deltaproteobacteria和Epsilonproteobacteria纲;在W1站位中,Cyanobacteria门的克隆子占总克隆数的38.2%,是该站位的最优势类群,这些优势类群可通过利用水体中的氮等营养来调节水体生态环境。影响细菌群落分布的环境因子主要为溶解氧、p H和氮营养盐。【结论】秦皇岛新开河河口及其邻近海域的细菌具有丰富的多样性,处于河口海域过渡带的水样具有更高的多样性,细菌群落多样性的分布受氮营养盐的影响更为显著。     

Diversity analyses of microbial communities in petroleum samples from Brazilian oil fields

Recent studies of oil fields have shown that the microbial diversity is represented by bacteria and archaea of wide distribution, and that many of these organisms have potential to metabolize organic and inorganic compounds. Biodegradation processes in oil industry are of great relevance, since it may be related with the loss of petroleum quality and can bring problems during production. The aim of this study was to compare the microbial communities present in biodegraded (GMR75) and non-biodegraded (PTS1) terrestrial oils from the Potiguar Basin (RN, Brazil) by using cultivation (microbial enrichments and isolation) and molecular approaches (16S rRNA gene libraries). The cultivated microorganisms recovered were affiliated with the phyla Actinobacteria, Firmicutes and Proteobacteria. Both bacterial 16S rRNA gene libraries revealed a great diversity, encompassing representatives from 8 different phyla (Actinobacteria, Bacteroidetes, Deferribacteres, Spirochaetes, Firmicutes, Proteobacteria, Thermotogae and Synergistetes) for the GMR75 sample, and from 5 different phyla (Actinobacteria, Chloroflexi, Firmicutes, Proteobacteria and Thermotoga) for the PTS1 sample. The archaeal 16S rRNA gene library was obtained only for GMR75 oil and all phylotypes were affiliated with the family Methanomicrobiaceae. Diversity results suggest that methanogenesis is the dominant terminal process for hydrocarbon degradation in GMR oil field, driven by anaerobic biodegradation.     

Phylogenetic diversity of winter bacterioplankton of eutrophic siberian reservoirs as revealed by 16S rRNA gene sequence

Using 16S rRNA gene sequence analyses we investigated the bacterial diversity of winter bacterioplankton of two eutrophic Siberian reservoirs. These reservoirs show similarity in phytoplankton community composition in spring and autumn but tend to differ in summer in exhibiting cyanobacterial bloom. Forty-eight unique partial 16S RNA gene sequences retrieved from two libraries were mostly affiliated with the class Actinobacteria, b subdivision of the class Proteobacteria, and the phylum Cytophaga-Flavobacterium-Bacteroides. The clone library of the pond exhibiting summer cyanobacterial bloom showed more diversity in sequence composition. A significant number of bacterial 16S rRNA gene clones were closely related to freshwater bacteria previously found in different aquatic ecosystems. This finding confirms the assumption that some bacterial clades are globally distributed.     

Effects of transgenic fructan-producing potatoes on the community structure of rhizosphere and phyllosphere bacteria

The rhizosphere and phyllosphere microbial communities of transgenic potatoes producing fructan were studied in comparison with isogenic controls and conventional varieties in a field release experiment over a period of 3 years. Population densities and 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analysis of the rhizosphere bacterial community only displayed the influence of annual and seasonal effects and the influence of field heterogeneity. In contrast, the T-RFLP analysis of the phyllosphere bacteria revealed in two of the 3 years significant differences in the community structure between the transgenic lines producing inulin and the other variants. This effect was studied in more detail through the analysis of bacterial isolates and a 16S rRNA gene clone library obtained from a transgenic line and the control. Both methods revealed a lower genetic diversity in the transgenic line and changes in the abundance of several bacterial groups. The isolates of the transgenic line were dominated by Bacilli, whereas most of the control isolates represented Actinobacteria. The clones were dominated by Proteobacteria, with main differences between both variants in Deltaproteobacteria, Bacilli and Bacteroidetes. However, all in all, the impact of the transgenic lines did not exceed the natural variability of the phyllosphere community structure on potato plants.     

Ecotypes of planktonic actinobacteria with identical 16S rRNA genes adapted to thermal niches in temperate, subtropical, and tropical freshwater habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6 degrees C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

Comparison of Soil Bacterial Communities of Pinus patula of Nilgiris, Western Ghats with Other Biogeographically Distant Pine Forest Clone Libraries

The bacterial community structure of the rhizosphere and non-rhizosphere soil of Pinus patula, found in the Nilgiris region of Western Ghats, was studied by constructing 16S rRNA gene clone libraries. In the rhizosphere and non-rhizosphere soil clone libraries constructed, 13 and 15 bacterial phyla were identified, respectively. The clone libraries showed the predominance of members of culturally underrepresented phyla like Acidobacteria and Verrucomicrobia. The Alphaproteobacteria and Acidobacteria clones were predominant in rhizosphere and non-rhizosphere soil samples, respectively. In rhizosphere, amongst Alphaproteobacteria members, Bradyrhizobium formed the significant proportion, whereas in non-rhizosphere, members of subdivision-6 of phylum Acidobacteria were abundant. The diversity analysis of P. patula soil libraries showed that the phylotypes (16S rRNA gene similarity cutoff, ≥97 %) of Acidobacteria and Bacteroidetes were relatively predominant and diverse followed by Alphaproteobacteria and Verrucomicrobia. The diversity indices estimated higher richness and abundance of bacteria in P. patula soil clone libraries than the pine forest clone libraries retrieved from previous studies. The tools like principal co-ordinate analysis and Jackknife cluster analysis, which were under UniFrac analysis indicated that variations in soil bacterial communities were attributed to their respective geographical locations due to the phylogenetic divergence amongst the clone libraries. Overall, the P. patula rhizosphere and non-rhizosphere clone libraries were found significantly unique in composition, evenly distributed and highly rich in phylotypes, amongst the biogeographically distant clone libraries. It was finally hypothesised that the phylogenetic divergence amongst the bacterial phylotypes and natural selection plays a pivotal role in the variations of bacterial communities across the geographical distance.     

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).     

Investigation of total bacterial and ammonia-oxidizing bacterial community composition in a full-scale aerated submerged biofilm reactor for drinking water pretreatment in China

The community composition of total bacteria and ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment was characterized by analysis of 16S rRNA gene and the functional gene amoA, respectively. Sampling was performed in February and in July. 16S rRNA gene clone libraries revealed 13 bacterial divisions. At both sampling dates, the majority of clone sequences were related to the Alpha- and Betaproteobacteria. A minor proportion belonged to the following groups: Gammaproteobacteria, Deltaproteobacteria, Nitrospira, Firmicutes, Acidobacteria, Verrucomicrobia, Actinobacteria, Planctomycetes, Chloroflexi, Gemmatimonadetes and the Cytophaga-Flavobacterium-Bacteroides group. Some sequences related to bacteria owning high potential metabolic capacities were detected in both samples, such as Rhodobacter-like rRNA gene sequences. Surveys of cloned amoA genes from the two biofilm samples revealed ammonia-oxidizing bacterial sequences affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage. An unknown Nitrosomonas group of amoA gene sequences was also detected.     

Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes

The diversity of freshwater bacterioplankton communities has not been extensively studied despite their key role in foodwebs and the cycling of carbon and associated major elements. In order to explore and characterize the composition of bacterioplankton associated with cyanobacterial blooms, large 16S rRNA clone libraries from four lakes experiencing such blooms were analysed. The four libraries contained 1461 clones, of which 559 were prokaryotic sequences of non-cyanobacterial origin. These clones were classified into 158 operational taxonomic units affiliated mainly with bacterial divisions commonly found in freshwater systems, e.g. Proteobacteria, Bacteriodetes, Actinobacteria, Verrucomicrobia and Planctomycetes. Richness and evenness of non-cyanobacterial clones were similar to other clone libraries obtained for freshwater bacterioplankton, suggesting that bacterial communities accompanying cyanobacterial blooms are as diverse as non-bloom communities. Many of the identified operational taxonomic units grouped with known freshwater clusters but the libraries also contained novel clusters of bacterial sequences that may be characteristic for cyanobacterial blooms. About 25% of the operational taxonomic units were detected in more than one lake. Even so, 16S rRNA heterogeneity analysis demonstrated large differences in community composition between lakes regardless of their similar characteristics and close proximity. Hence even the similar environmental conditions created by different cyanobacterial blooms may foster very dissimilar bacterial communities, which could indicate that the genetic diversity in lake bacteria have been underestimated in the past.     

Genetic diversity of rhizobia associated with Desmodium species grown in China

AIMS: Desmodia are leguminous plants used as important forage and herbal medicine in China. Little information is available about the nodule bacteria of Desmodium species. To understand the genetic diversity of rhizobia associated with Desmodium species grown in China, isolates from temperate and subtropical regions were obtained and analysed. METHODS AND RESULTS: A total of 39 rhizobial strains isolated from 9 Desmodium species grown in China were characterized by PCR-based 16S rDNA gene and 16S-23S rDNA intergenic spacer gene restriction fragment length polymorphism (RFLP) and 16S rRNA gene sequencing. The results showed high diversity among rhizobia symbiotic with Desmodium species. Most microsymbionts of Desmodium species belonged to Bradyrhizobium closely related to Bradyrhizobium elkanii, Bradyrhizobium japonicum and Bradyrhizobium yuanmingense. Several small groups or single strain were related to Rhizobium, Sinorhizobium or Mesorhizobium. CONCLUSIONS: Desmodium species formed nodules with diverse rhizobia in Chinese soils. SIGNIFICANCE AND IMPACT OF THE STUDY: These results offered the first systematic information about the microsymbionts of desmodia grown in the temperate and subtropical regions of China.     

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.     

Bacterial biodiversity from Roopkund Glacier, Himalayan mountain ranges, India

The bacterial diversity of two soil samples collected from the periphery of the Roopkund glacial lake and one soil sample from the surface of the Roopkund Glacier in the Himalayan ranges was determined by constructing three 16S rRNA gene clone libraries. The three clone libraries yielded a total of 798 clones belonging to 25 classes. Actinobacteria was the most predominant class (>10% of the clones) in the three libraries. In the library from the glacial soil, class Betaproteobacteria (24.2%) was the most predominant. The rarefaction analysis indicated coverage of 43.4 and 41.2% in the samples collected from the periphery of the lake thus indicating a limited bacterial diversity covered; at the same time, the coverage of 98.4% in the glacier sample indicated most of the diversity was covered. Further, the bacterial diversity in the Roopkund glacier soil was low, but was comparable with the bacterial diversity of a few other glaciers. The results of principal component analysis based on the 16S rRNA gene clone library data, percentages of OTUs and biogeochemical data revealed that the lake soil samples were different from the glacier soil sample and the biogeochemical properties affected the diversity of microbial communities in the soil samples.