Novel major archaebacterial group from marine plankton.

Marine bacteria often dominate the plankton biomass and are responsible for much of the cycling of organic matter, but bacterial diversity is poorly understood because conventional identification methods (requiring culturing) miss about 99% of the organisms. Recent advances permit characterization of microbial communities by analysis of 16S ribosomal RNA gene sequences directly from biomass without the need to culture the organisms; such studies from surface ocean samples have found only eubacteria, not archaebacteria (or Archaea), which are profoundly different. Here we report 16S rRNA sequences obtained from Pacific Ocean bacterioplankton samples collected from depths of 100 m and 500 m. Among these we found sequences only distantly related to those of any organisms previously characterized by 16S rRNA sequences, with similarities to the nearest such relatives (extreme thermophiles) approximately the same as those between animals and plants. We suggest that these sequences are from a previously undescribed archaebacterial group that may have diverged from the ancestors of characterized organisms very early in evolution.     

Cultivation of the ubiquitous SAR11 marine bacterioplankton clade

The alpha-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation-independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems. This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods. The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems, which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study. Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities into very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 micro m(3), places them among the smallest free-living cells in culture.     

A newly discovered Roseobacter cluster in temperate and polar oceans

Bacterioplankton phylotypes of alpha-Proteobacteria have been detected in various marine regions, but systematic biogeographical studies of their global distribution are missing. Alpha-Proteobacteria comprise one of the largest fractions of heterotrophic marine bacteria and include two clades, SAR11 and Roseobacter, which account for 26 and 16% of 16S ribosomal RNA gene clones retrieved from marine bacterioplankton. The SAR11 clade attracted much interest because related 16S rRNA gene clones were among the first groups of marine bacteria to be identified by cultivation-independent approaches and appear to dominate subtropical surface bacterioplankton communities. Here we report on the global distribution of a newly discovered cluster affiliated to the Roseobacter clade, comprising only as-yet-uncultured phylotypes. Bacteria of this cluster occur from temperate to polar regions with highest abundance in the Southern Ocean, but not in tropical and subtropical regions. Between the south Atlantic subtropical front and Antarctica, we detected two distinct phylotypes, one north and one south of the polar front, indicating that two adjacent but different oceanic provinces allow the persistence of distinct but closely related phylotypes. These results suggest that the global distribution of major marine bacterioplankton components is related to oceanic water masses and controlled by their environmental and biogeochemical properties.     

武进港浮游微生物群落研究

通过 T-RFLP 分析和构建 16S rRNA小型基因文库相结合的方法, 研究了江苏省常州市武进港塘桥段微生物群落结构特点。T-RFLP 分析结果表明, 该群落的多样性较高,但均匀度较低。基于 Ribosomal Database Project Ⅱ的分析结果表明, 该处水样的浮游微生物群落种类较多, 还含有尚未分出门的微生物种群, 而变形菌门为最优势的类群。通过与 GenBank 中已知序列对比(BLAST)发现, 具有较高相似性的克隆数较多, 但也存在很多未知种类的新细菌。此外,还探讨了一些已知属的微生物的环境意义。     

Fine-scale phylogenetic architecture of a complex bacterial community

Although molecular data have revealed the vast scope of microbial diversity, two fundamental questions remain unanswered even for well-defined natural microbial communities: how many bacterial types co-exist, and are such types naturally organized into phylogenetically discrete units of potential ecological significance? It has been argued that without such information, the environmental function, population biology and biogeography of microorganisms cannot be rigorously explored. Here we address these questions by comprehensive sampling of two large 16S ribosomal RNA clone libraries from a coastal bacterioplankton community. We show that compensation for artefacts generated by common library construction techniques reveals fine-scale patterns of community composition. At least 516 ribotypes (unique rRNA sequences) were detected in the sample and, by statistical extrapolation, at least 1,633 co-existing ribotypes in the sampled population. More than 50% of the ribotypes fall into discrete clusters containing less than 1% sequence divergence. This pattern cannot be accounted for by interoperon variation, indicating a large predominance of closely related taxa in this community. We propose that such microdiverse clusters arise by selective sweeps and persist because competitive mechanisms are too weak to purge diversity from within them.     

土壤细菌和真菌群落的物种续存模式对扰动的响应

通过研究物种存活概率和局域多度间的关系, 对微生物群落动态是否符合中性假说进行检验. 使用野外采集的土壤进行土壤微宇宙实验,微宇宙经受了稀释扰动,剔除一些非常稀有的物种. 微宇宙经过恢复培养后, 用454高通量焦磷酸测序方法分别对土壤细菌16SrRNA 基因和真菌18SrRNA 基因进行测序, 检测多度≥1的物种, 从而探究细菌和真菌物种组成相对于源土壤发生的变化. 结果发现: 恢复生长过程中细菌和真菌物种丰富度都显著降低(>50%); 受到扰动的微宇宙恢复生长以后, 土壤细菌和真菌物种存活概率与其在源土壤中的初始相对多度之间呈正相关, 这个结果符合中性假说的预测,即最初多度较高的物种平均灭绝概率较低.      

Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation.

The taxonomic group Prochlorales (Lewin 1977) Burger-Wiersma, Stal and Mur 1989 was established to accommodate a set of prokaryotic oxygenic phototrophs which, like plant, green algal and euglenoid chloroplasts, contain chlorophyll b instead of phycobiliproteins. Prochlorophytes were originally proposed (with concomitant scepticism) to be a monophyletic group sharing a common ancestry with these 'green' chloroplasts. Results from molecular sequence phylogenies, however, have suggested that Prochlorothrix hollandica is not on a lineage that leads to plastids. Our results from 16S ribosomal RNA sequence comparisons, which include new sequences from the marine picoplankter Prochlorococcus marinus and the Lissoclinum patella symbiont Prochloron sp., indicate that prochlorophytes are polyphyletic within the cyanobacterial radiation, and suggest that none of the known species is specifically related to chloroplasts. This implies that the three prochlorophytes and the green chloroplast ancestor acquired chlorophyll b and its associated structural proteins in convergent evolutionary events. We report further that the 16S rRNA gene sequence from Prochlorococcus is very similar to those of open ocean Synechococcus strains (marine cluster A), and to a family of 16S rRNA genes shotgun-cloned from plankton in the north Atlantic and Pacific Oceans.     

玉龙雪山冰川雪坑中细菌多样性群落结构及其与气候环境的关系

通过DAPI荧光计数、恢复培养和变性梯度凝胶电泳,分析了玉龙雪山不同深度积雪中的细菌数量、多样性及其群落结构.结果显示:玉龙雪山雪坑样品中细菌数量和多样性指数与气候环境替代指标W(Ca~(2+)),W(Mg~(2+)),W(Cl~-)等具有相关性.可培养细菌隶属于三个不同系统发育群:Actinobactema(37%),Firmicutes(11%)和Proteobacteria(α-Proteobacteria)(52%),其中α-Proteobacteria类为优势类群.DGGE测序结果表明细菌属β-Proteobacteria,Actinobacteria和Bacteroidetes类群,以β-Proteobacteria为优势类群,并且不同深度雪坑中细菌多样性和群落结构没有明显变化.结果表明:海洋型冰川雪中细菌数量和多样性、群落结构与大陆型冰川雪相比,冰雪微生物具有其特殊性.     

Unsuspected diversity among marine aerobic anoxygenic phototrophs

Aerobic, anoxygenic, phototrophic bacteria containing bacteriochlorophyll a (Bchla) require oxygen for both growth and Bchla synthesis. Recent reports suggest that these bacteria are widely distributed in marine plankton, and that they may account for up to 5% of surface ocean photosynthetic electron transport and 11% of the total microbial community. Known planktonic anoxygenic phototrophs belong to only a few restricted groups within the Proteobacteria alpha-subclass. Here we report genomic analyses of the photosynthetic gene content and operon organization in naturally occurring marine bacteria. These photosynthetic gene clusters included some that most closely resembled those of Proteobacteria from the beta-subclass, which have never before been observed in marine environments. Furthermore, these photosynthetic genes were broadly distributed in marine plankton, and actively expressed in neritic bacterioplankton assemblages, indicating that the newly identified phototrophs were photosynthetically competent. Our data demonstrate that planktonic bacterial assemblages are not simply composed of one uniform, widespread class of anoxygenic phototrophs, as previously proposed; rather, these assemblages contain multiple, distantly related, photosynthetically active bacterial groups, including some unrelated to known and cultivated types.     

Light stimulates growth of proteorhodopsin-containing marine Flavobacteria

Proteorhodopsins are bacterial light-dependent proton pumps. Their discovery within genomic material from uncultivated marine bacterioplankton caused considerable excitement because it indicated a potential phototrophic function within these organisms, which had previously been considered strictly chemotrophic. Subsequent studies established that sequences encoding proteorhodopsin are broadly distributed throughout the world's oceans. Nevertheless, the role of proteorhodopsins in native marine bacteria is still unknown. Here we show, from an analysis of the complete genomes of three marine Flavobacteria, that cultivated bacteria in the phylum Bacteroidetes, one of the principal components of marine bacterioplankton, contain proteorhodopsin. Moreover, growth experiments in both natural and artificial seawater (low in labile organic matter, which is typical of the world's oceans) establish that exposure to light results in a marked increase in the cell yield of one such bacterium (Dokdonia sp. strain MED134) when compared with cells grown in darkness. Thus, our results show that the phototrophy conferred by proteorhodopsin can provide critical amounts of energy, not only for respiration and maintenance but also for active growth of marine bacterioplankton in their natural environment.     

古矿井区域酸性矿坑水微生物群落的多样性

通过生态群落16S rRNA基因库的重建并采用限制性酶切长度多态性(RFLP)分析的方法,对铜绿山铜矿酸性矿水中微生物群落的多样性进行研究。通过RFLP对扩增出的327个16S rRNA PCR产物进行分析,对44个16S rRNA的克隆子进行测序。每个样点各有2～6个主要操作分类单元,这些操作分类单元所对应的克隆子数分别占各样点克隆子总数的56.3%～69.6%。93.8%的克隆子序列与现在的数据库中序列相似性大于90%。样点酸性矿坑水主成分分析与系统发育分析结果显示:在低pH值、高离子浓度的样点tls1与tls2中多数克隆子属于gamma-Proteobacteria(主要为Acidithiobacillus ferrooxidans)和Nitrospira科(主要为Leptospirillum);而在较高pH值、低离子浓度的样点tls3的克隆子多属于gamma-Proteobacteria(没有A.ferrooxidans)和alpha-Proteobacteria。在古矿井区域酸性矿坑水中微生物群落的多样性很低,生物地球化学特性相近的酸性矿坑水由具有相近的微生物群落组成。     

青岛海域固氮细菌的系统发育多样性研究

采用严格人工无氮海水培养基富集、分离培养方法及用3对固氮基因(nifH)引物进行PCR检测,从青岛近海沉积物和海水中分离到16株异养型可培养海洋固氮细菌,经16S rDNA全序列测定分析了它们的系统发育多样性。结果显示,16株菌分布于Alphaproteobacteria、Gammaproteobacteria、Flavobacteria的3个纲中,分别代表着6个属、10个种,其中有50%归于Pseudoalteromonas属;海底沉积物中固氮菌的多样性要远高于海水,并在其中发现2个潜在新种。研究表明,青岛近海海域的固氮菌具有一定程度的多样性,它们大部分在系统发育关系上与分离自韩国、南极洲、新喀里多尼岛等海水的标准菌高度同源,但也存在少量潜在新物种。结果可为其他不同海域异养固氮菌的多样性及其生态功能研究提供有益参考。     

胜利油田纯梁采油厂某区块油藏中微生物菌群分析

构建细菌16S rDNA基因文库以及硫酸盐还原菌的dsrAB基因文库,研究了胜利油田纯梁采油厂35区块某油井采出水中微生物的多样性以及SRB的菌群结构组成.结果表明,样品中微生物的多样性指数不高,样品中的微生物主要来自β变形菌纲以及γ变形菌纲,SRB主要由Thermodes-ulfobacterium、Desulfomicrobium、Thermodesulforhabdus以及Archaeoglobus fulgidus等属的微生物组成.将序列相似性≥97%作为一个OTU的划分原则,16S rDNA文库中有2个优势OTU,分别与Arco-bacter以及Pseudomonas stutzeri相近,分别占总克隆数的44%和56%;dsrAB基因文库有3个优势OTU,分别与Archaeoglobus fulgidus、Archaeoglobus infectus、Desulfotomaculum geothermicum相近,分别占总克隆数的46.6%、16.6%以及13.3%.16S rDNA基因序列与已知序列相似性较高,优势菌种在国内油田中较为常见,但不同油藏之间菌群结构存在较大差异.dsrAB序列与已知序列的相似性较小,可能存在新的SRB.     

盐度影响珠江口浮游细菌形成特殊生态位

通过对珠江口水域进行水样的高通量测序, 获得 16S rRNA 基因序列, 分析结果表明, 盐度是影响珠江口浮游细菌Alpha和Beta多样性的主要环境参数之一, 并且低盐度样品中浮游细菌具有相对高的遗传多样性。将盐度分为低、中、高3个组, 通过群落组成分析验证表明, 该研究区域内总样本与盐度分组样本浮游细菌的优势菌和核心菌的分类学信息差异较大。总样本中优势菌是变形杆菌门, 核心菌多为酸杆菌门。低盐度组样本中核心菌主要为C111科, 中盐度和高盐度组均为OCS111科, 来自酸杆菌门。低盐度浮游细菌与中高盐度浮游细菌的核心菌物种完全不重合。对盐度进行交叉细分, 并对浮游细菌进行共发生网络分析, 结果表明, 细分的盐度分组下, 珠江口浮游细菌群落形成紧密联系且彼此独立的模块, 且模块内部物种组成关系复杂。     

云台山白云岩表层土可培养细菌多样性

运用纯培养法和基于16S rRNA基因序列的系统发育分析贵州云台山白云岩表层土可培养细菌的多样性.稀释涂布平板分离共获得131株细菌,限制性内切酶HhaⅠ和HaeⅢ对扩增的16S rRNA基因片段进行酶切分型,根据ARDRA酶切图谱划分为40个操作分类单元.16S rRNA基因序列测定和系统发育分析结果显示,这些菌株隶属于包括厚壁菌门(Firmicutes,64.89%)、β-变形菌纲(β-Proterbacteria,3.82%)、γ-变形菌纲(γ-Proterbacteria,17.56%)、放线细菌门(Actinobacteria,11.45%)和拟杆菌门(Bacteroidetes,3.82%)等5大类群,共13个属.优势菌群为芽胞杆菌属(Bacillus,53.44%),亚优势菌群为寡养单胞菌属(Stenotrophomonas,9.16%)、假单胞菌属(Pseudomonas,8.40%)和微小杆菌属(Exiguobacterium,8.40%).15.00%的有效序列与GeneBank已知序列相似性小于等于97%,可能为潜在新类群.研究表明,云台山白云岩喀斯特地区不仅含有较为丰富的细菌物种多样性,且潜藏着许多新的微生物资源.     

16S rRNA sequences reveal numerous uncultured microorganisms in a natural community.

Microbiologists have been constrained in their efforts to describe the compositions of natural microbial communities using traditional methods. Few microorganisms have sufficiently distinctive morphology to be recognized by microscopy. Culture-dependent methods are biased, as a microorganism can be cultivated only after its physiological niche is perceived and duplicated experimentally. It is therefore widely believed that fewer than 20% of the extant microorganisms have been discovered, and that culture methods are inadequate for studying microbial community composition. In view of the physiological and phylogenetic diversity among microorganisms, speculation that 80% or more of microbes remain undiscovered raises the question of how well we know the Earth's biota and its biochemical potential. We have performed a culture-independent analysis of the composition of a well-studied hot spring microbial community, using a common but distinctive cellular component, 16S ribosomal RNA. Our results confirm speculations about the diversity of uncultured microorganisms it contains.     

青海茶卡盐湖嗜盐碱芽胞杆菌资源分析

采用3种不同的培养基,通过可培养法,从青海茶卡盐湖样品中共分离获得110株嗜盐碱芽胞杆菌.基于16S rRNA基因序列相似性鉴定及系统发育分析,得出这些菌株为芽胞杆菌10个属的41个种,它们与最近匹配模式菌株的16S rRNA基因序列相似性在96%~100%之间,以芽胞杆菌属为优势菌,共计25种83株;存在6个潜在新种.3种酶筛实验结果表明,87%的嗜盐碱芽胞杆菌菌株具有产酶能力,产蛋白酶70株,产纤维素酶81株,产木聚糖酶50株.青海茶卡盐湖中的芽胞杆菌种类丰富多样且能产多种酶,并且潜藏着新的微生物物种,研究结果可为嗜盐碱芽胞杆菌资源开发提供理论基础.     

Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton

Phylogenetic information from ribosomal RNA genes directly amplified from the environment changed our view of the biosphere, revealing an extraordinary diversity of previously undetected prokaryotic lineages. Using ribosomal RNA genes from marine picoplankton, several new groups of bacteria and archaea have been identified, some of which are abundant. Little is known, however, about the diversity of the smallest planktonic eukaryotes, and available information in general concerns the phytoplankton of the euphotic region. Here we recover eukaryotes in the size fraction 0.2-5 microm from the aphotic zone (250-3,000 m deep) in the Antarctic polar front. The most diverse and relatively abundant were two new groups of alveolate sequences, related to dinoflagellates that are found at all studied depths. These may be important components of the microbial community in the deep ocean. Their phylogenetic position suggests a radiation early in the evolution of alveolates.     

3种色型仿刺参线粒体和核糖体序列片段遗传结构比较研究

仿刺参作为我国重要的海水养殖物种,良种选育是目前研究的重点,其中体色是重要的考虑因素之一。为了比较3种不同体色仿刺参的遗传结构,采用PCR扩增与测序技术对我国3种体色(青色、白色和紫色)仿刺参线粒体16S rRNA和COⅠ与核糖体18S rRNA-ITS-28S rRNA序列片段进行分析,揭示其遗传结构差异及系统发育关系。结果表明, 16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列长度分别为812~830 bp、877~915 bp、1 536~1 572 bp。16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列在青色、紫色和白色仿刺参中分别检测到单倍型个数分别为4/5/5、5/3/4和4/4/3, COⅠ序列多态性位点数目所比例最大, 16S rRNA序列最小。3种色型仿刺参16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列遗传距离数值均比较小,范围分别为0~0.014 7、0~0.021 2和0~0.010 3,没有达到种的分化水平。以紫海胆为外群,基于COⅠ序列构建系统发育树,结果表明白刺参单独聚为一支,但是紫色和青色仿刺参不同个体间相互交叉聚集,无法通过发育树区分。     

海洋沉积物中特有细菌类群的初步探讨

通过构建16S rRNA基因文库对中国南沙南海区沉积物中细菌多样性进行分析,并将获得16S rRNA基因序列与国际基因数据库GenBank进行相似性比较及聚类分析,结果发现来自海洋沉积物的未能培养的微生物往往组成一簇,并与现已获培养并鉴定的微生物自然分开,这一表象表明,海洋沉积物中存在特有的微生物属种。