Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing.

Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.     

A few cosmopolitan phylotypes dominate planktonic archaeal assemblages in widely different oceanic provinces

We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces. Samples from eight different environments were collected at two depths (surface and aphotic zone), and 16 genetic libraries of PCR-amplified archaeal 16S rRNA genes were constructed. The libraries were analyzed by using a three-step hierarchical approach. Membrane hybridization experiments revealed that most of the archaeal clones were affiliated with one of the two groups of marine archaea described previously, crenarchaeotal group I and euryarchaeotal group II. One of the 2,328 ribosomal DNA clones analyzed was related to a different euryarchaeal lineage, which was recently recovered from deep-water marine plankton. In temperate regions (Pacific Ocean, Atlantic Ocean, and Mediterranean Sea) both major groups were found at the two depths investigated; group II predominated at the surface, and group I predominated at depth. In Antarctic and subantarctic waters group II was practically absent. The clonal compositions of archaeal libraries were investigated by performing a restriction fragment length polymorphism (RFLP) analysis with two tetrameric restriction enzymes, which defined discrete operational taxonomic units (OTUs). The OTUs defined in this way were phylogenetically consistent; clones belonging to the same OTU were closely related. The clonal diversity as determined by the RFLP analysis was low, and most libraries were dominated by only one or two OTUs. Some OTUs were found in samples obtained from very distant places, indicating that some phylotypes were ubiquitous. A tree containing one example of each OTU detected was constructed, and this tree revealed that there were several clusters within archaeal group I and group II. The members of some of these clusters had different depth distributions.     

A Few Cosmopolitan Phylotypes Dominate Planktonic Archaeal Assemblages in Widely Different Oceanic Provinces

We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces. Samples from eight different environments were collected at two depths (surface and aphotic zone), and 16 genetic libraries of PCR-amplified archaeal 16S rRNA genes were constructed. The libraries were analyzed by using a three-step hierarchical approach. Membrane hybridization experiments revealed that most of the archaeal clones were affiliated with one of the two groups of marine archaea described previously, crenarchaeotal group I and euryarchaeotal group II. One of the 2,328 ribosomal DNA clones analyzed was related to a different euryarchaeal lineage, which was recently recovered from deep-water marine plankton. In temperate regions (Pacific Ocean, Atlantic Ocean, and Mediterranean Sea) both major groups were found at the two depths investigated; group II predominated at the surface, and group I predominated at depth. In Antarctic and subantarctic waters group II was practically absent. The clonal compositions of archaeal libraries were investigated by performing a restriction fragment length polymorphism (RFLP) analysis with two tetrameric restriction enzymes, which defined discrete operational taxonomic units (OTUs). The OTUs defined in this way were phylogenetically consistent; clones belonging to the same OTU were closely related. The clonal diversity as determined by the RFLP analysis was low, and most libraries were dominated by only one or two OTUs. Some OTUs were found in samples obtained from very distant places, indicating that some phylotypes were ubiquitous. A tree containing one example of each OTU detected was constructed, and this tree revealed that there were several clusters within archaeal group I and group II. The members of some of these clusters had different depth distributions.     

Diverse microbial communities inhabit Antarctic sponges

Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus. The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales. Of the 150 archaeal clones from L. apicalis, K. varialosa and M. acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota. Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones. Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity. Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group. Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species. Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives. In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K. varialosa.     

新疆沙湾冷泉沉积物中免培养古菌多样性初步研究

【目的】了解新疆沙湾冷泉沉积物的古菌组成及多样性。【方法】采用免培养法,液氮研磨提取冷泉沉积物总DNA,使用古菌通用引物进行16S rRNA基因扩增,构建16S rRNA基因文库。对阳性克隆进行HhaI限制性酶切分型,选出具有不同酶切图谱的序列进行测序,将所得序列与GenBank数据库中序列比对并构建16S rRNA基因系统发育树。【结果】从冷泉沉积物古菌16S rRNA基因文库中随机挑选了121个阳性克隆,共得到22个不同的可操作分类单元,BLAST结果表明全部克隆子归属于泉古菌门(Crenarchaeote)中免培养类群。系统发育分析归类为Soil-Freshwater-subsurface group和MarinegroupI,2个亚群并且各占整个文库的50%。其中40%左右的克隆子与具有无机碳和硝酸盐同化能力的泉古菌有高的相似性。此外还发现40%的克隆子与低温泉古菌类群具有很高的相似性。【结论】新疆沙湾冷泉沉积物中古菌类群多样性较低,但存有大量高度适应此低温、贫营养环境的泉古菌类群。     

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.     

新疆沙湾冷泉沉积物的细菌系统发育多样性

为了解新疆沙湾冷泉沉积物的细菌群落组成与类群多样性,利用免培养方法直接从沙湾冷泉沉积物中提取环境总DNA,构建细菌16S rRNA基因文库。对随机挑选的241个细菌阳性克隆子进行HaeIII酶切分型得到86个可操作分类单元(OTUs),系统发育分析将其归为11个门:放线菌门(Actinobacteria),酸杆菌门(Acidobacteria),拟杆菌门(Bacteroidetes),绿菌门(Chlorobi),蓝细菌门(Cyanobacteria),厚壁菌门(Firmicutes),芽单胞菌门(Gemmatimonadetes),硝化螺旋菌门(Nitrospirae),变形菌门(Proteobacteria),浮霉菌门(Planctomycetes),疣微菌门(Verrucomicrobia)。其中酸杆菌门和变型菌门为优势类群,分别占细菌克隆文库的48%和25%。超过1/3的OTUs序列与GenBank中已存序列具有较低相似性(相似性小于95%)。此外20%左右的克隆子与固氮细菌和硝酸盐氧化细菌相关。研究结果表明,新疆沙湾冷泉沉积物中细菌种类丰富,代谢类型多样而且存在大量未知类群。     

Microbial community analysis of a coastal hot spring in Kagoshima, Japan, using molecular- and culture-based approaches

Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared <92% sequence similarity with their closest sequences in a public database, suggesting that the Ibusuki hot spring may harbor a unique and novel bacterial community. By contrast, 77 clones from the Site 2 archaeal library contained only three OTUs, most of which were affiliated with Thaumarchaeota.     

晋南牛瘤胃中古菌分子多样性的研究

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立克隆库来研究晋南牛瘤胃古菌的多样性.每个克隆库随机挑选100个克隆.引物Arch f364/1386建立的克隆库中,克隆分为四类,分别与四种甲烷短杆菌1Y(61%)、SM9(23%)、NT7(14%)和AK-87(2%)相似.引物1Af/1100Ar建立的克隆库中,克隆分为两类,分别与Methanobacterium aarhusense(72%)和Methanosphaera stadtmanae DSM 3091(28%)相似.引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面,除以上4种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外,还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列,还有7%的未匹配序列.系统进化分析表明,这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium,和未知广域古菌等5个分支.有25类属于广域古菌的未知序列,提示瘤胃中存在大量的未知产甲烷菌.     

Nitrous oxide reductase (nosZ) gene fragments differ between native and cultivated Michigan soils

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mean) = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.     

大庆油田油藏采出水的细菌群落结构

采用ARDRA (扩增性rDNA限制性酶切片段多态性分析)技术对大庆油田聚驱、水驱和过渡带3种油藏采出水中的细菌群落的基因组总DNA的16S rDNA克隆文库进行分析,研究了细菌群落结构.结果表明：随机挑取的596个阳性克隆可分为85个操作分类单元 (OTUs),其中聚驱、水驱和过渡带文库分别含有28、41和33个.通过对优势OTUs测序,并与GenBank进行序列比对,发现油藏采出水中的优势菌群为不动杆菌属、弓形杆菌属、厚壁菌门、假单胞菌属和硫磺单胞菌属.聚驱样品中细菌群落组成最简单,优势菌群为不动杆菌属,占库容的85%,假单胞菌属占7%;水驱样品中的优势菌群也是不动杆菌属,占库容的62%,假单胞菌属和硫磺单胞菌属各占20%和6%;过渡带文库的细菌群落的优势菌群为弓形杆菌属,占库容的50%,不动杆菌属和厚壁菌门各占19%和18%.     

Characterization of the methanogen community in a household anaerobic digester fed with swine manure in China

Household anaerobic digesters have been installed across rural China for biogas production, but information on methanogen community structure in these small biogas units is sparsely available. By creating clone libraries for 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes, we investigated the methanogenic consortia in a household biogas digester treating swine manure. Operational taxonomic units (OTUs) were defined by comparative sequence analysis, seven OTUs were identified in the 16S rRNA gene library, and ten OTUs were identified in the mcrA gene library. Both libraries were dominated by clones highly related to the type strain Methanocorpusculum labreanum Z, 64.0 % for 16S rRNA gene clones and 64.3 % for mcrA gene clones. Additionally, gas chromatography assays showed that formic acid was 84.54 % of the total volatile fatty acids and methane was 57.20 % of the biogas composition. Our results may help further isolation and characterization of methanogenic starter strains for industrial biogas production.     

Picoeukaryotic Plankton Diversity at the Helgoland Time Series Site as Assessed by Three Molecular Methods

We analyzed picoeukaryote assemblages in the German Bight at the Helgoland time series site by sequencing cloned eukaryotic 18S rRNA genes in six genetic libraries plus one library from the Orkney Islands from a cruise of opportunity. The libraries were constructed from environmental samples collected at different periods of the year. The same samples were also analyzed using a fingerprinting technique, single-strand conformational polymorphism (SSCP), and DNA microarrays with class-level oligonucleotide probes. One hundred unique clones were analyzed from each library, thus insuring over 85% coverage of the library. The V4 region of the 18S rRNA gene was sequenced from each of these clones, thus providing the most discrimination among the clones. The nonphotosynthetic picoeukaryotic component dominated over the photosynthetic one and was represented by the ciliates at 45% and group II alveolates at 42%. Prasinophytes dominated the photosynthetic group at 40%, but other picoplankton groups, such as bolidomonads and chrysophytes, were also present. Totally novel groups were found in the cryptomonads and in the dinoflagellates. A new algal group sister to the cryptophyte nuclear gene and the glaucocystophytes was also found. These three groups have been found in other picoeukaryotic planktonic clone libraries. SSCP analyses at closer time intervals suggest that clone libraries should be made at weekly intervals if succession in the picoeukaryotic plankton community is to be monitored accurately. A comparison of annual samples suggests thatthere appears to be an annual cycle with regard to species composition. Microarray analysis supported the clone library data and offered a faster means of community analysis, which can be performed with similar accuracy and with higher throughput for a more in-depth analysis.     

Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.

YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.     

Analysis of Rumen Methanogen Diversity in Water Buffaloes (Bubalus bubalis) Under Three Different Diets

The water buffalo (Bubalus bubalis) is a prominent livestock species for the production of milk and meat in many countries. We investigated the diversity of rumen methanogens in Mediterranean water buffaloes maintained in Brazil under different diets: corn silage, grazing pasture, or sugar cane. A total of 467 clones were isolated from three methanogen 16S rRNA gene clone libraries that each represented a distinct feed type. The 467 clones were assigned to 19 species-level operational taxonomic units (OTUs). Four OTUs were represented in all three libraries, eight OTUs were library-specific, six OTUs were found in only the corn silage and pasture grazing libraries, and one OTU was shared only between pasture grazing and sugar cane libraries. We found that Methanobrevibacter-related sequences were the most abundant in the water buffaloes sampled for our analysis, in contrast to previously reported studies showing that Methanomicrobium mobile-like methanogens were the most abundant methanogens in water buffaloes of Murrah and Surti breeds sampled in India. Considering the worldwide distribution of water buffaloes and the likely wide variety of diets provided, our results combined with studies from other groups support that larger scope analyses of microbiomes for this livestock species would provide great insight into the contribution of geographical location, breed, and diet in determining the population structure of rumen microorganisms.     

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.     

Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass

The two-phase leach-bed system is a biogas reactor system optimized for the utilization of energy crop silages at maximized loading rates under maintenance of an optimal microbial activity. In this study, a characterization of the methanogenic microbial community within this reactor system was conducted for the first time. Accordingly, effluent samples from the anaerobic filter and the silage digesting leach-bed reactors of both a laboratory-scale two-phase biogas reactor system and a scaled-up commercial on-farm pilot plant were investigated. In total, five Archaea-specific 16S rDNA libraries were constructed and analyzed by amplified rDNA restriction analysis (ARDRA), with subsequent phylogenetic analysis of nucleotide sequences for individual ARDRA patterns. A quantification of major methanogenic Archaea groups was conducted by real-time PCR. A total of 663 clones were analyzed and 45 operational taxonomic units (OTUs) related to methanogenic Archaea were detected. These OTUs were related to the orders Methanosarcinales, Methanomicrobiales and Methanobacteriales, as well as the hitherto uncultured CA-11 and ARC-I groups, and most of them occurred throughout all the compartments of both two-phase biogas reactors. The proportion of acetotrophic to hydrogenotrophic methanogens differed between the laboratory and the pilot scale system. A total of 56% of the clones from the 16S rDNA library derived from the laboratory biogas system were assigned to presumably acetotrophic members of Methanosarcinales. In contrast, these OTUs were less abundant in the 16S rDNA library derived from samples of the pilot plant. Therein, the most dominant OTUs were Methanoculleus-related OTUs, which presumably indicated the predominant presence of hydrogenotrophic methanogens. These findings were confirmed by group-specific quantitative real-time PCR assays. The results indicated that the fraction of acetotrophic and hydrogenotrophic methanogens within a biogas reactor caused certain variations, which may reflect varying substrate utilization during methanogenesis.     

共存于厌氧真菌分离培养液中瘤胃甲烷菌的检测及其多样性分析

对分离自山羊瘤胃的真菌分离培养液中甲烷菌进行16SrDNA扩增、DGGE分析、RFLP及测序分析,研究共存于真菌分离培养液中甲烷菌的种类及其多样性。DGGE结果显示:从厌氧真菌分离至第45代,甲烷菌多样性指数由1·32降至0·99,相似性最低为34·7%;第45代至62代,多样性指数由0·99升至1·15,相似性最低为89·2%。RFLP多态性分析69个克隆共得到5个操作分类单元,选择其中6个具有代表性的序列进行测序。序列及系统进化分析表明,属于其中3个操作分类单元的克隆最相似菌都是UnculturedarchaealsymbiontPA202,相似性均为95%,没有与这些克隆相似性较高的已培养甲烷菌;属于另外2个操作分类单元的克隆最相似菌都是Unculturedrumenmethanogen956,相似性均为97%,最相似已知菌为Methanobrevibactersp.NT7,相似性为97%。结果表明,真菌培养液中存在目前尚未分离培养的瘤胃甲烷菌。     

How stable is stable? Function versus community composition.

The microbial community dynamics of a functionally stable, well-mixed, methanogenic reactor fed with glucose were analyzed over a 605-day period. The reactor maintained constant pH and chemical oxygen demand removal during this period. Thirty-six rrn clones from each of seven sampling events were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) for the Bacteria and Archaea domains and by sequence analysis of dominant members of the community. Operational taxonomic units (OTUs), distinguished as unique ARDRA patterns, showed reproducible distribution for three sample replicates. The highest diversity was observed in the Bacteria domain. The 16S ribosomal DNA Bacteria clone library contained 75 OTUs, with the dominant OTU accounting for 13% of the total clones, but just 21 Archaea OTUs were found, and the most prominent OTU represented 50% of the clones from the respective library. Succession in methanogenic populations was observed, and two periods were distinguished: in the first, Methanobacterium formicicum was dominant, and in the second, Methanosarcina mazei and a Methanobacterium bryantii-related organism were dominant. Higher variability in Bacteria populations was detected, and the temporal OTU distribution suggested a chaotic pattern. Although dominant OTUs were constantly replaced from one sampling point to the next, phylogenetic analysis indicated that inferred physiologic changes in the community were not as dramatic as were genetic changes. Seven of eight dominant OTUs during the first period clustered with the spirochete group, although a cyclic pattern of substitution occurred among members within this order. A more flexible community structure characterized the second period, since a sequential replacement of a Eubacterium-related organism by an unrelated deep-branched organism and finally by a Propionibacterium-like species was observed. Metabolic differences among the dominant fermenters detected suggest that changes in carbon and electron flow occurred during the stable performance and indicate that an extremely dynamic community can maintain a stable ecosystem function.     

Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study

We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36-53 OTUs using the DOTUR program when sequence similarities of 95%-100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon-Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.