采用未培养技术对荷斯坦奶牛瘤胃细菌多样性进行初步分析

采用未培养(Culture independent)技术直接从荷斯坦奶牛瘤胃液中提取瘤胃细菌微生物混合DNA(也叫元基因组DNA),利用细菌16SrDNA通用引物27F与1492R,扩增瘤胃混合微生物的16SrDNA,根据16SrDNA序列对瘤胃细菌多样性进行初步分析。通过16SrDNA序列同源性分析,发现有多于一半以上的序列与可培养的菌株的同源性小于90%,属于不可培养的菌株。选用45条测得序列与已知序列构建系统发育树,分析结果表明,它们分属于两大类LGCGPB(the lowG CGram positivebac teria)和CFB(Cytophaga_Flexibacter $CBacteroides group),剩下的克隆尚难确定其分类地位,可能是代表新属和种的序列,这些序列已向GenBank提交并得到序列号(AY986777_AY986791)。     

厚荚相思树根瘤菌HJ06菌株的16S rDNA全序列和nifA基因片段分析

对厚荚相思(Acaciacrassicarpa)根瘤菌HJ06菌株的16SrDNA全序列和nifA基因片段进行了测定。结果表明,HJ06菌株在以16SrDNA序列构建的系统发育树状图中位于根瘤菌属(Rhizobium)分支中,与根瘤菌属各个种的相似性达95%以上;从HJ06菌株克隆出的585bpnifA基因片段与Klebsiellapneumoniae的同源性达到99.3%,与Klebsiellaoxytoca的NifF,NifL,NifA,NifB蛋白基因的同源性为97.8%。     

柠檬酸废水IC反应器厌氧颗粒污泥真细菌结构分析

目的:分析柠檬酸工业废水IC厌氧反应器处理时产生的厌氧颗粒污泥中真细菌的菌群结构.方法:构建细菌的16S rDNA克隆文库,对文库中的16S rDNA基因序列进行测序,然后Blast比对,并进行分类、建系统发育树.结果:对获得的77个16S rDNA序列进行测序,按序列相似性≥97％的分类标准,这些序列可分为22个OTU,其中4个优势OTU分别与棒杆菌属(Corynebacterium)、梭菌属(Clostridium)、消化球菌属(Peptococcus)、疣微菌属(Verrucomicrobia)最为相近,其余OTU的克隆数较少.颗粒污泥中的真细菌主要为放线菌纲(Actinobacteria)、梭菌纲(Clostridia)、拟杆菌纲(Bacteroidetes)以及δ-变形菌纲(Deltaproteobacteria)的细菌,分别占克隆总数的34/77、31/77、6/77、6/77.结论:该文研究了柠檬酸废水处理过程中产生的厌氧颗粒污泥中细菌的菌群组成和结构,为深入了解柠檬酸废水的厌氧处理过程提供了一定的理论借鉴作用.     

南美白对虾肠道微生物群落的分子分析

采用分子生物学手段16S rDNA克隆文库方法对实验室养殖条件下的南美白对虾肠道细菌进行了多样性研究。用限制性片段长度多态性(RFLP)方法从文库中筛选出可能不同细菌来源的克隆子12个,测定其16S rDNA片段核甘酸序列,将所获得的序列与GenBank数据库进行BLAST比对,结果表明:南美白对虾肠道的16S rDNA克隆文库中126个克隆子分属2个不同的细菌类群:变形细菌(Proteobacteria)和厚壁细菌(Firmicutes),其中厚壁细菌为优势菌群占到75.4%,且与最相似序列同源性均低于94%;变形细菌占到24.6%,与最相似序列同源性均高于98%,分别为希瓦氏菌属(Shewanella),泛菌属(Pantoea),Aranicola属,假单胞菌属(Pseudomonas)和弧菌属(Vibrio)。     

海南温泉嗜热菌的16S rDNA分析

目的:确定24株海南温泉嗜热菌菌株的分类地位。方法:Blastn分析菌株16S rDNA序列同源性;邻接法构建菌株16SrDNA序列系统发育进化树并分析菌株的进化位置;Clustax比对分析菌株的相似度和进化距离。结果:菌株LY5和LY4的16SrDNA序列与Geobacillus pallidus strain B1,partial sequence(GenBank:HM030740.1)的16S rDNA序列同源性分别为98%和97%,其他菌株的16S rDNA序列与Geobacillus subterraneus,strain R-35641(GenBank:FN428689.1)的16S rDNA序列的同源性均大于96%。Clustax比对分析表明26株菌16S rDNA序列前段(1～70bp)、中段(70bp～1 420bp)、后段(1 420～1 484bp)的相似度分别为40%、100%和60%,进化距离分析表明菌株GT7、LY4和LY5与其他菌株进化距离较远,其余菌株之间进化距离差异不明显。综上所述,初步将24株温泉嗜热菌鉴定为土芽孢杆菌属(Geobacillus sp.)。结论:16S rDNA序列分析可用于温泉嗜热菌的鉴定。     

新疆玛纳斯热气泉免培养土壤细菌多样性分析

【目的】了解新疆玛纳斯热气泉土壤免培养细菌群落组成及多样性。【方法】采用免培养法直接从土壤样品中提取总DNA,利用细菌通用引物对土壤总DNA进行16S rDNA扩增,构建细菌16SrDNA文库。使用HaeⅢ限制性内切酶对阳性克隆进行限制性片段长度多态性分析(restriction fragment length polymorphism,RFLP),挑选具有不同酶切图谱的克隆进行测序、比对并构建16SrDNA系统发育树。【结果】从土壤细菌16S rDNA文库中随机挑选了170个阳性克隆,共得到29个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析归为6个门:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes)。其中厚壁菌门(Firmicutes)为绝对优势类群,占整个细菌文库的71%。29个OTUs中有14条序列与GenBank中相关序列的相似性低于97%(序列长度约1.5kb),占序列总数的48%。【结论】热气泉土壤细菌种群多样性较低,但存在大量潜在细菌新种。     

醉马草免培养内生细菌的多样性

为了解醉马草内生细菌的组成和多样性,采用液氮研磨法提取醉马草总DNA,利用内生细菌16S rRNA基因特异性引物对醉马草总DNA 进行16S rRNA 基因扩增,构建醉马草内生细菌16S rRNA基因文库。对筛选到249个克隆进行Hae Ш酶切分析,得到57个操作分类单元(OTUs)。系统发育分析将其归为4个门:变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)以及1个未知类群。其中74%的克隆与可培养细菌中的37个属具有高的16S rRNA基因序列相似性 (97％-100％),其中芽孢杆菌属(Bacillus spp.)、红球菌属(Rhodococcus spp.)、黄杆菌属(Flavobacterium spp.)、黄单胞杆菌属(Xanthomonas spp.)最为丰富。另外26%的克隆序列与GenBank中已存细菌16S rRNA基因序列相似性小于96%。研究结果表明,醉马草中可培养内生细菌丰富,多样并且可能存在一些潜在新物种或新类群。     

人参细胞器核糖体小亚单位DNA的PCR扩增与序列测定

采用CTAB法提取了人参(Panax ginseng)根基因组DNA,根据植物叶绿体16S rDNA和线粒体18SrDNA与细菌16S rDNA序列具有高度同源性,用扩增细菌16S rDNA的一对通用引物(8f,1492r)扩增了人参细胞器核糖体小亚单位DNA.对扩增产物进行了克隆与测序,经多序列比对,扩增片段分别与已知植物叶绿体16S rDNA和线粒体18S rDNA具高度同源性,表明该对引物可以用来扩增绝大多数植物细胞器核糖体小亚单位DNA,可以作为鉴定植物叶绿体16S rDNA和线粒体18S rDNA的一种基本实验技术.     

高效产氢细菌新种--Ethanologenbacterium sp.X-1的分离鉴定及其产氢效能

为获得高效产氢发酵细菌 ,采用改进的厌氧Hungate培养技术 ,从生物制氢反应器CSTR中分离一株产氢细菌X 1。对该株细菌进行了形态学特征、生理生化指标、16SrDNA和 16S 2 3SrDNA间隔区序列分析等研究。结果表明与最相近的种属Clostridiumcellulosi和Acetanaerobacteriumelongatum等的 16SrRNA基因序列同源性为 94 %以下。16S 2 3SrRNA间隔区基因序列比对分析显示保守区域仅为tRNAAla和tRNAIle序列 ,其它可变部位没有同源性区域 ,鉴定为新属Ethanologenbacteriumsp .。该株细菌为专性厌氧杆菌 ,代谢特征为乙醇发酵 ,葡萄糖发酵产物主要为乙醇、乙酸、H2 和CO2 。在pH4 0和 36℃条件下最大产氢速率是 2 8 3mmolH2 (gdrycell·h)。经鉴定和产氢效能分析表明该菌株是一新属的高效产氢细菌     

一株海洋细菌的初步鉴定及其产黑色素相关新基因(簇)的分离

对分离自近海沼泽地大米草根际的一株供试海洋细菌MWYL1进行了形态观察、生理特性检测以及16SrDNA序列分析。实验结果表明:该菌株属于海洋单胞菌属(Marinomonas),革兰氏染色呈阴性,直杆状,好氧生长,适于28℃生长。基于16SrDNA序列的Blast分析表明,菌株MWYL1与Marinomonas pontica和Marinomonas dokdonensis的序列相似性分别为97%和95%。通过基因组fosmid文库的构建,直接分离到一个产生黑色素的克隆,进一步亚克隆和测序后获得与黑色素产生相关的功能新基因(簇),并且对其进行了生物信息学的初步分析。     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

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。     

16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica

The prokaryotic diversity of aerobic and anaerobic bacterial isolates and of bacterial and archaeal 16S rDNA clones was determined for a microbial mat sample from the moated region of Lake Fryxell, McMurdo Dry Valleys, Antarctica. Among the anaerobic bacteria, members of Clostridium estertheticum and some other psychrotolerant strains dominated whereas methanogens and other Archaea were lacking. Isolates highly related to Flavobacterium hibernum, Janthiniobacterium lividum, and Arthrobacter flavus were among the aerobic bacteria most frequently isolated. Assessment of more than 350 partial 16S rDNA clone sequences of libraries generated by Bacteria- and Archaea-specific PCR primers revealed a rich spectrum of bacterial diversity but only two different archaeal clone sequences. Among the Bacteria, representative sequences belonged to the class Proteobacteria, order Verrucomicrobiales, class Actinobacteria, Clostridium/Bacillus subphylum of Gram-positives, and the Cytophaga-Flavobacterium-Bacteroides phylum. The clones formed about 70 higher taxonomy groups (<98% sequence similarity) and 133 potential species, i.e., groups of clones sharing greater than 98% similarity. Only rarely were clone sequences found to be highly related to Lake Fryxell isolates and to strains of described species. Subsequent analysis of ten sequencing batches of 36 individual clones indicated that the diversity might be still higher than had been assessed.     

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).     

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.     

High bacterial diversity in permanently cold marine sediments.

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones.     

Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869^T in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.

The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.     

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

Culture-independent phylogenetic analysis of the faecal flora of the rat

The dominant faecal flora of the rat was determined using randomly cloned 16S rDNA comparative sequence analysis. A total of 109 near full-length 16S rDNA clones were sequenced, representing 69 unique 16S rRNA phylotypes or operational taxonomic units (OTUs). Estimates of species richness indicated that approximately 338 species were present in the faeces, suggesting that only 20% of species were identified. Only two of 39 Gram-negative clones aligned with previously cultured species, the remainder fell into a separate lineage within the Bacteroides-Cytophaga phylum. Several clones within this new group were related to 16S rDNA sequences previously identified from mouse faeces. Lactobacilli were the most abundant Gram-positive species, representing 23% of the total clones but only 7% of OTUs. The remaining Gram-positive clones were distributed among the Clostridium coccoides group (9%), the Clostridium leptum subgroup (18%), and throughout the low GC Gram-positive bacteria (13%). The majority of OTUs (63/69 or 91%) were less than 97% homologous to previously cultured bacteria. Faecal samples were also cultured using a variety of anaerobic media. With the exception of the lactobacilli, the cultured isolates demonstrated low species diversity and poorly reflected the population, as defined through comparative sequence analysis.