Bacterial community composition of a shallow hypertrophic freshwater lake in China, revealed by 16S rRNA gene sequences

The phylogenetic composition of a bacterial community from a hypertrophic freshwater lake in China was investigated by sequencing cloned 16S rRNA genes. Three hundred and thirty-six bacterial clones from four clone libraries in different months (March, May, July and September in 2004) were classified into 142 operational taxonomic units, most of which were affiliated with bacterial divisions commonly found in freshwater ecosystem, e.g. Alpha-, Beta-, Gamma- and Deltaproteobacteria, Bacteriodetes and Actinobacteria. The results showed that the composition of bacterial community in the July library was the most diverse one. Actinobacteria was the most significant lineage in Lake Taihu, with dominant numbers of operational taxonomic units in the May, July and September libraries. Phylogenetic analysis suggested that 53 sequences were grouped into six novel clusters which may represent specific populations indigenous to the environment. Coverage analyses indicated that the clone libraries could provide a fine inventory of bacterial diversity in the lake.     

新疆顿巴斯他乌盐湖沉积物免培养古菌多样性

【目的】了解新疆顿巴斯他乌盐湖沉积物免培养古菌组成及多样性。【方法】利用免培养法直接从顿巴斯他乌盐湖沉积物样品中提取环境总DNA,采用古菌通用引物对16S rRNA基因进行扩增,构建基因克隆文库。对随机挑选的59个阳性克隆进行HaeⅢ限制性酶切分型并测序、BLAST比对及构建16S rRNA基因系统发育树。【结果】文库覆盖率为89%,Shannon-Wiener指数为2.69,共得到21个不同的可操作分类单元,分属于广古菌门(Euryarchaeota,92%)和泉古菌门(Crenarchaeota,8%),其中多数为盐杆菌科(Halobacteriaceae,88%)的盐杆菌属(Halobacterium,24%)、盐盒菌属(Haloarcula,18%)、盐碱红菌属(Natronorubrum,14%)、盐红菌属(Halorubrum,8%)等,与海盐环境(thalassohaline)获得的16S rRNA基因序列相似性最高(﹥95%);整个文库中约11%的克隆与可培养古菌多个属的相似性小于97%。【结论】顿巴斯他乌盐湖古菌多样性略低于同类高盐环境,组成较为一致,只是各类群所占百分比稍有不同,且可能存在一些潜在新物种或新类群。     

Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and cultivation

Fecal microbial diversity in a strictly vegetarian woman was determined by the 16S rDNA library method, terminal restriction fragment length polymorphism (T-RFLP) analysis and a culture-based method. The 16S rDNA library was generated from extracted fecal DNA, using bacteria-specific primers. Randomly selected clones were partially sequenced. T-RFLP analysis was performed using amplified 16S rDNA. The lengths of T-RF were analyzed after digestion by HhaI and MspI. The cultivated bacterial isolates were used for partial sequencing of 16S rDNA. Among 183 clones obtained, approximately 29% of the clones belonged to 13 known species. About 71% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). A total of 55 species or phylotypes were identified among the 16S rDNA library, while the cultivated isolates included 22 species or phylotypes. In addition, many new phylotypes were detected from the 16S rDNA library. The 16S rDNA library and isolates commonly included the Bacteroides group, Bifidobacterium group, and Clostridium rRNA clusters IV, XIVa, XVI and XVIII. T-RFLP analysis revealed the major composition of the vegetarian gut microbiota were Clostridium rRNA subcluster XIVa and Clostridium rRNA cluster XVIII. The dominant feature of this strictly vegetarian gut microbiota was the detection of many Clostridium rRNA subcluster XIVa and C. ramosum (Clostridium rRNA cluster XVIII).     

规模化牛场犊牛直肠细菌多样性分析

【目的】研究腹泻犊牛直肠细菌多样性,以及与健康犊牛直肠细菌多样性的差异。【方法】通过建立直肠菌群16S rRNA基因克隆文库,分别用限制性内切酶MspⅠ和HhaⅠ对阳性克隆的PCR产物进行限制性酶切片段长度多态性(RFLP)分析,通过测定16S rRNA基因序列,绘制系统发育树,确定犊牛直肠菌群的组成。【结果】腹泻组克隆阳性率达98.75%(474/480),优势菌群以乳杆菌属(14%)、肠球菌属(10%)和埃希菌属(8%)等需氧和兼性厌氧菌为主,健康组克隆阳性率达96.45%(488/506),优势菌群以梭菌属(13%)、双歧杆菌属(8%)和巨型球菌属(5%)等专性厌氧菌为主。【结论】2周龄犊牛直肠菌群复杂多样,并且具有自己的独特性菌群,且腹泻时乳杆菌属、肠球菌属、埃希氏菌属等显著增加。     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

Bacterial and archaeal assemblages in sediments of a large shallow freshwater lake, Lake Taihu, as revealed by denaturing gradient gel electrophoresis

Aims:  To explore the association of microbial community structure with the development of eutrophication in a large shallow freshwater lake, Lake Taihu.
Methods and Results:  The bacterial and archaeal assemblages in sediments of different lake areas were analysed using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. The bacterial DGGE profiles showed that eutrophied sites, grass-bottom areas and relatively clean sites with a eutrophic (albeit dredged) site are three respective clusters. Fifty-one dominant bacterial DGGE bands were detected and 92 corresponding clones were sequenced, most of which were affiliated with bacterial phylotypes commonly found in freshwater ecosystems. Actinobacteria were detected in the centre of the lake and not at eutrophied sites whereas the opposite was found with respect to Verrucomicrobiales . Twenty-five dominant archaeal DGGE bands were detected and 31 corresponding clones were sequenced, most of which were affiliated with freshwater archaeal phylotypes.
Conclusions:  The bacterial community structures in the sediments of different areas with similar water quality and situation tend to be similar in Taihu Lake.
Significance and Impact of the Study:  This study may expand our knowledge on the relationship between the overall microbial assemblages and the development of eutrophication in the shallow freshwater lake.     

新疆艾比湖和伊吾湖可培养嗜盐古菌多样性

新疆地区盐湖密布,蕴藏着丰富的微生物资源。为保护和利用微生物物种与基因资源,作者从新疆准噶尔盆地的艾比湖和天山山间盆地的伊吾湖分离纯化嗜盐微生物。采用PCR方法扩增其中65株嗜盐古菌16SrRNA基因序列。序列分析表明,分离的嗜盐古菌分属6个属,艾比湖以Haloterrigena和Natrinema属的菌株为主,伊吾湖由Haloarcula和Halorubrum两个属的菌株构成。通过多样性指数、丰富度指数、均匀度指数和物种相对多度模型对分离的菌株进行多样性分析和比较,结果表明,盐湖嗜盐古菌的多样性指数、丰富度指数和均匀度指数具有一定相关性,艾比湖可培养嗜盐古菌的多样性高于伊吾湖。研究发现了一些新的物种资源,表明新疆盐湖中孕育的特色微生物资源亟待保护与利用。     

Diversity of bacterial community in freshwater of Woopo wetland

Diversity of bacterial community in water layer of Woopo wetland was investigated. Cultivable bacterial strains were isolated by the standard dilution plating technique and culture-independent 16S rRNA gene clones were obtained directly from DNA extracts of a water sample. Amplified rDNA restriction analysis (ARDRA) was applied onto both of the isolates and 16S rRNA gene clones. Rarefaction curves, coverage rate and diversity indices of ARDRA patterns were calculated. Representative isolates and clones of all the single isolate/clone phylotype were partially sequenced and analyzed phylogenetically. Sixty-four and 125 phylotypes were obtained from 203 bacterial isolates and 235 culture-independent 16S rRNA gene clones, respectively. Bacterial isolates were composed of 4 phyla, of which Firmicutes (49.8%) and Actinobacteria (32.0%) were predominant. Isolates were affiliated with 58 species. Culture-independent 16S rRNA gene clones were composed of 8 phyla, of which Proteobacteria (62.2%), Actinobacteria (15.5%), and Bacteroidetes (13.7%) were predominant. Diversity of 16S rRNA gene clones originated from cultivation-independent DNA extracts was higher than that of isolated bacteria.     

A comparison of ammonia-oxidiser populations in eutrophic and oligotrophic basins of a large freshwater lake

A combination of PCR amplification and oligonucleotide probing was used to investigate the populations of ammonia-oxidisers of the -Proteobacteria in the eutrophic and oligotrophic basins of Lake Windermere, a large temperate lake in the English Lake District. Numbers of ammonia-oxidisers (MPN) in the Windermere lakewater were low (< 100 cells ml^–1) throughout the year with the exception of peaks in August, which coincided with stratification, and November in the South Basin where overturn may have introduced ammonia-oxidising bacteria into the water column. Sediment samples contained larger populations of ammonia oxidisers, usually ca. 10⁴ per g. dry weight, which remained relatively constant throughout the seasonal cycle in both Basins. DNA was recovered from lakewater and sediment samples and Nitrosospiraand N. europaea-eutrophalineage16S rRNA genes amplified in a nested PCR reaction, with confirmation of identity by oligonucleotide hybridisation. Nitrosospira 16S rDNA was readily detected in all samples and therefore found to be ubiquitous. In contrast, nitrosomonad DNA of the N. europaea-eutropha lineage could only be detected in the oligotrophic North Basin. Enrichment cultures of lakewater samples only exhibited nitrification at low (0.67 mM) and medium (5 mM) ammonium concentrations, whilst sediment enrichments nitrified at all concentrations tested including high (12.5 mM) ammonium medium. These data suggest that ammonia-oxidiser populations may be physiologically distinguished between lakewater and sediment, and that species distribution in a single lake is non-uniform.     

PCR-RFLP法探讨三个人工湖微生物群落多样性

目的：探讨牡丹江市区三个人工湖（月牙湖、牛角湖、南湖）的微生物群落多样性。方法：从三个水样中分别提取微生物DNA,选取通用引物扩增16S rDNA片段,对扩增产物进行RFLP分析。结果：不同的样品由于其中的微生物多样性的差异,酶切产物在凝胶上显示的相对位置和条带数目都有一定差异。结论：牛角湖样品的条带数目最多,表明牛角湖的微生物群落多样性最为丰富。     

健康藏族与汉族大学生肠道细菌多样性研究

【目的】探究生活在相同环境下,同年龄段健康藏族人群和汉族人群肠道细菌组成的多样性特征及其可能的成因。【方法】入选同专业的20–24岁男性健康藏族学生和汉族学生各20名,同一时间段收集新鲜粪便,提取新鲜粪便细菌总DNA,通过高通量测序和生物信息学分析,结合对饮食习惯的问卷调查,研究健康藏族学生与汉族学生肠道细菌多样性与饮食习惯之间的关系。【结果】从40份样本中,测序共得到2880个OTU,alpha和beta多样性分析表明,生活在相同环境下,不同民族男性学生的肠道细菌组成有明显差异。Prevotella 9、Bacteroides和Lachnoclostridium等18个属为两民族学生共有,同时,Prevotella 9、Faecalibacterium和Succinivibrio等10个属细菌为对两民族学生肠道细菌组成差异具有显著性影响的标志性菌属。进一步聚类分析表明,所有学生样本分为ET F型、ET P型、ET F和ET B混合型3种不同肠型,随访调查结果显示不同类型分别对应以脂肪为主和以淀粉为主的不同饮食习惯。【结论】生活在相同环境下,健康藏族和汉族学生的肠道细菌组成特征及其差异可能与遗传因素及饮食习惯密切相关。     

石油污染对土壤微生物群落多样性的影响

土壤中的微生物主要有细菌、放线菌、真菌三大类群,微生物在石油污染的土壤中发挥着维持生态平衡和生物降解的功能。文中以四川省遂宁市射洪县某废弃油井周围不同程度石油污染土壤为供试土壤,首先对各组供试土壤的基本理化性质进行测定分析;然后采用平板菌落计数法测定了供试土壤中三大类微生物数量的变化,结果表明:相比未被污染的对照土壤,石油污染的土壤中细菌、放线菌、真菌数量均减少,并且土壤中可培养微生物的数量与土壤含水量呈正相关;再采用454焦磷酸测序技术对土壤中的细菌群落多样性及变化进行16S rRNA基因分析。在所有供试的4个土壤样品中,共鉴定出不少于23 982个有效读取序列和6 123种微生物,相比于未被污染的对照土壤,石油污染土壤中细菌的种类更加丰富,主要优势门类为酸杆菌门、放线菌门、拟杆菌门、绿弯菌门、浮霉菌门和变形菌门。但不同土壤样品中优势菌群的群落结构有所差异,石油污染的土壤中,酸杆菌门、放线菌门和变形菌门的数量最多,未被石油污染的土壤中,放线菌门、拟杆菌门和变形菌门的数量最多。     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes (Xuanwu,Mochou) have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly differ-ent from these two lakes.Furthermore,the DGGE pro-files of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100% homology with known sequences (Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96% homology with known sequences (Acinetobacter,and Bacillus).The study shows that the PCR-DGGE tech-nique combined with sequence analysis is a feasible and efficient method for the determination of microbial com-munities in sediment samples.     

Phytoplankton diversity and cyanobacterial dominance in a hypereutrophic shallow lake with biologically produced alkaline pH

In this work, we have characterized the diversity of phytoplanktonic species in a highly alkaline and hypereutrophic shallow lake, Santa Olalla (southwestern Spain), the evolution of their relative abundances, and that of several physicochemical parameters over 2 years. In the absence of an external input of alkaline water, Santa Olallas stable high pH (average pH 9.52, with several maxima >10.5) is explained by an extremely high photosynthetic primary productivity. A variety of phytoplankton species was observed even during pH maxima. These included several species of green algae, diatoms, and euglenoids and several cyanobacteria from the orders Nostocales and Chroococcales. Quantitatively, cyanobacteria dominated. A blooming event due to Aphanothece clathrata was observed at one pH maximum, during which the diversity as measured by the Shannon-Weaver index was extremely low. Santa Olallas cyanobacteria are alkaliphilic and/or extremely alkalitolerant and appear to be responsible for the generation and maintenance of stable high-pH conditions in their environment.Communicated by W.D. Grant     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]