海绵Pacnychalina sp.体内古菌多样性非培养技术分析

采用非分离培养分析方法,即16S rDNA限制性酶切片段长度多态性(ARDRA)和测序方法对南海湛江海域海绵Pachychalina sp.体内的古菌多样性进行了研究.从海绵体内直接提取古菌总DNA.以样品总DNA为模板,用古菌16S rDNA通用引物进行PCR扩增获得16S rDNA,回收、纯化16S rDNA产物并克隆到T-Vector.进行第二次PCR扩增反应,且对扩增产物进行ARDRA.在古菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱上存在差异;随机挑选8个克隆子进行测序,获得古菌16S rDNA的部分序列,并对16S rDNA序列进行聚类分析构建了系统进化树,结果发现海绵体内的古菌主要属于Methanogenium organophilum、Methanoplanus petrolearius等古菌类.但它们与目前数据库中收录的古细菌间的相似性均不超过90%,它们极有可能是一些新的古菌.     

海绵Pachychalina sp.体内古菌多样性非培养技术分析

采用非分离培养分析方法,即16S rDNA限制性酶切片段长度多态性（ARDRA）和测序方法对南海湛江海域海绵Pachychalina sp.体内的古菌多样性进行了研究。从海绵体内直接提取古菌总DNA。以样品总DNA为模板,用古菌16S rDNA通用引物进行PCR扩增获得16S rDNA,回收、纯化16S rDNA产物并克隆到TVector。进行第二次PCR扩增反应,且对扩增产物进行ARDRA。在古菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱上存在差异;随机挑选8个克隆子进行测序,获得古菌16S rDNA的部分序列,并对16S rDNA序列进行聚类分析构建了系统进化树,结果发现海绵体内的古菌主要属于Methanogenium organophilum、Methanoplanus petrolearius等古菌类。但它们与目前数据库中收录的古细菌间的相似性均不超过90%,它们极有可能是一些新的古菌。     

海绵Pachychalina sp.体内细菌多样性的研究

通过非分离培养分析方法,直接从海绵体内提取细菌总DNA。以样品总DNA为模板进行PCR扩增获得细菌16S rDNA。用16S rDNA限制性酶切片段长度多态性(ARDRA)和测序方法对南海湛江海域海绵Pachychalina sp．体内的细菌多样性进行了研究。在细菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱间存在差异;随机挑选22个克隆进行测序得到它们的16S rDNA部分序列,大部分序列属于γ-proteobacterium和α-proteobacterium,但有少数克隆序列与RDP数据库中收录的16S rDNA序列间的相似性极小,不参与系统发育树的构建。研究结果表明海绵Pachychalina sp.体内细菌组成具有丰富的多样性。     

海绵Pachychalina sp.体内细菌多样性的研究

通过非分离培养分析方法,直接从海绵体内提取细菌总DNA。以样品总DNA为模板进行PCR扩增获得细菌16S rDNA。用16S rDNA限制性酶切片段长度多态性（ARDRA）和测序方法对南海湛江海域海绵Pachychalina sp.体内的细菌多样性进行了研究。在细菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱间存在差异;随机挑选22个克隆进行测序得到它们的16S rDNA部分序列,大部分序列属于γproteobacterium和αproteobacterium,但有少数克隆序列与RDP数据库中收录的16S rDNA序列间的相似性极小,不参与系统发育树的构建。研究结果表明海绵Pachychalina sp.体内细菌组成具有丰富的多样性。     

西藏扎布耶茶卡盐碱湖古菌多样性的非培养技术分析

采用非培养技术,直接从西藏扎布耶茶卡盐碱湖样品中提取微生物总DNA。以样品总DNA为模板,PCR扩增湖中古菌的16S rDNA序列。扩增产物经过克隆并随机挑选60个克隆进行测序得到它们的16S rDNA部分序列,大部分序列与嗜盐碱古菌的16S rDNA相近。在系统发育树上,部分克隆与已知古菌属归于同一分支,主要分布在嗜盐菌科的Natronococcus、Natronorubrum、Natronobacterium、Natronomonas、Natrinema、Halorubrum、Haloterrigena和Halorhabdus等8个嗜盐古菌属中, 也有一些克隆形成了独立的分支。它们共同显示出扎布耶茶卡湖中的古菌具有丰富的多样性。     

温度对厚指海绵Pachychalina sp.体内真细菌的影响

摘要：温度是影响微生物多样性的重要因素之一。【方法】本研究采用16S rDNAs扩增产物酶切片段多态性（amplified rDNA restriction analysis , ARDRA）和16S rDNAs序列分析方法,对生长在16℃和30℃条件下厚指海绵Pachychalina sp.体内真细菌（eubacteria）的多样性进行了研究,【目的】探讨温度对海绵体内细菌的影响。【结果】根据ARDRA 聚类分析,16℃条件下海绵体内100个真细菌的16S rDNAs克隆片断被分成34个类群,而30℃条件下,海绵体内100个细菌的16S rDNAs克隆片断则被分为32个类群,说明在不同温度条件下,海绵体内真细菌的组成与群落结构有所变化,但研究发现温度没有明显改变海绵体内真细菌总的群落结构。【结果】根据厚指海绵Pachychalina sp.体内真细菌的16S rDNAs序列分析结果发现：在16℃和30℃条件下,海绵体内的真细菌均属于α、γ、δ-变形菌、硫细菌、硫还原菌和烃类分解菌,此外还有少数放线菌。16℃条件下海绵体内的优势菌为γ-变形菌,而且体内的硫细菌和硫还原菌主要是耐寒细菌,而30℃条件下海绵体内的优势菌为α-变形菌。该研究还发现：同一ARDRA类型的克隆序列分析表明在同一ARDRA群内各组分间彼此关系比较密切。     

新疆艾比湖湿地博乐河入湖口古菌多样性分析

旨为了解艾比湖湿地土壤中古菌的多样性及其群落结构组成。以艾比湖湿地博乐河入口土壤为样本,首次利用古菌特异性16S r DNA引物构建克隆文库。使用限制性内切酶AfaⅠ和MspⅠ对目的片段进行酶切分型(amplified r DNA restraction analysis,ARDRA),每种谱型随机选取若干阳性克隆子进行测序,序列比对构建古菌16S r DNA系统进化树。结果显示,测序得到的117条序列划分为61个OTUs,通过序列比对及系统发育分析归类为广古菌门(Euryarchaeota)和奇古菌门(Thaumarchaeota)两大门类。广古菌门的盐杆菌科(Halobacteriaceae)为第一优势菌群,包括盐微菌属(Halomicrobium)、盐碱球菌属(Natronococcus)、盐碱团菌属(Halalkalicoccus)等10个属,共48个OTU(78.7%),阳性克隆子77个(63.6%)。艾比湖湿地博乐河入口土壤古菌多样性丰富并存在大量的潜在新种。     

口虾蛄肠道细菌种群多样性的分子分析

目的探讨口虾蛄肠道细菌种群的多样性。方法通过不依赖于分离培养的分子生物学分析方法,以直接提取虾蛄肠道细菌的总DNA为模板经过PCR扩增16S DNA,然后经与T载体连接建立质粒文库。用限制性内切酶(BsuRⅠ和Hin6Ⅰ)对阳性克隆的PCR扩增产物进行限制性酶切片段长度多态性(RFLP)分析,选取有代表性的克隆进行测序。结果16S DNA序列通过CLUSTALX进行多序列比对及NCBI数据库中的BLAST分析后发现口虾蛄肠道细菌主要分成4类:未培养细菌,未培养支原体科细菌,未培养δ变形菌和马特斯支原体。结论口虾蛄肠道细菌种类较为简单,且多为未培养的细菌。     

太湖梅梁湾冬季浮游细菌的多样性

运用分子生物学方法对太湖梅梁湾2005年3月冬季水体中的浮游细菌多样性进行了研究。提取水样中细菌基因组总DNA,以细菌的16SrDNA通用引物进行PCR扩增,扩增产物经分子克隆、酶切归类分析、测序和序列分析,对水样中的细菌群落建立了针对16SrDNA的克隆文库和系统发生树。基于细菌16SrDNA克隆文库和系统发生树的分析,结果表明,该水域的优势细菌类群为拟杆菌(Cy-tophaga-Flavobacterium-Bacteroides,44.3%)和β、γ变形杆菌(Proteobacteria;β-Proteobacteria,25.3%和γ-Proteobacteria,27.8%),其中大部分细菌与黄杆菌属(Flavobacterium)、假单胞菌属(Pseudomonas)和食酸菌属(Acidovorax)细菌的关系密切。与国外富营养化湖泊的研究报道相比较,γ-Proteobacteria类群为水体中的优势类群,是严重富营养化的太湖在冬季所具有的独特种群结构特征。     

健康人肠道中溶血菌的调查与分子鉴定

目的 调查无临床症状健康人肠道内有无溶血菌的存在,并利用分子方法对溶血菌进行鉴定。方法 通过血平板分离培养,对17例无临床症状个体肠道内溶血菌的存在情况做了3周的动态调查,对分离到的溶血菌的16SrDNA进行ARDRA（Amplified Ribosomal DNA Restriction Analysis）酶切分型,将溶血菌分为不同类型,最后利用ERIC—PCR指纹图谱技术对不同类型的溶血菌进行菌株水平的鉴定。结果17例个体中发现5例个体能检测到溶血菌,其中4例个体（A～D）只有1次样品检测到溶血菌,而个体E在3周的5次采样中均能检测到溶血菌。根据ARDRA酶切图谱将溶血菌分为2种类型,Ⅰ型16SrDNA全长测序后与蜡状芽胞杆菌（Bacillus cereus ATCC 10987）序列同源性达99％,Ⅱ型与大肠埃希菌菌（Escherichia coli CFT 073）的16SrDNA序列同源性达99％。结论 在健康个体肠道内也有溶血菌的存在,且溶血菌的存在可能与机体的疲劳程度和饮食情况有关。     

垃圾填埋场渗滤液中古细菌群落16S rRNA基因的ARDRA分析

利用特异性的引物对,选择性扩增垃圾填埋场渗滤液中古细菌群落的18S rRNA基因片断,在此基础上建立16S rDNA克隆文库,经古细菌通用寡核苷酸探针的原位杂交筛选后,克隆文库内古细菌16S rDNA扩增片断的多样性通过ARDRA分析（amplified rDNA restriction analysis)而获得,利用PCR将各组重克隆子内的16S rDNA外源片断再扩增出来后,两种限制性内切酶－Hha I和HaeⅢ－被分别用于16S rDNA克隆片断的限制酶切分析,结果表明,随机选出的70个古细菌16S rDNA克隆片断被妥为21个不同的ARDRA型（组）,其中的两个优势型总共占了所有被分析克隆子的60％,而其余19个型的相对丰度均处于较低的水平,当中的14个型更仅含有1个克隆子,通过对16S rRNA基因的PCR扩增,克隆及其ARDRA分析,能快速地获得有关填埋场渗滤液中古细菌群落的结构及其多样性的初步信息。     

Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes.

The diversity of microorganisms associated with the leaves of the seagrass Halophila stipulacea in the northern Gulf of Elat was examined by culture-independent analysis. Microorganisms were harvested by a sonication treatment for total-community genomic DNA isolation. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used for PCR amplification. The 16S rDNA PCR products were subcloned and further characterized by a restriction fragment length analysis termed ARDRA (amplified rDNA restriction analysis). These analyses were carried out after reamplifying the cloned fragments with two primers binding symmetrically to the plasmid immediately on both sides of the cloned insert. Computer-aided clustering was performed after separate restriction analysis with enzymes HinfI and HpaII. By this method, 103 cloned 16S rDNA fragments were clustered into a total of 58 different groups. Sequence analysis of clones with an identical ARDRA pattern confirmed that members of an ARDRA group were closely related to each other. The sequenced clones were found to be affiliated with a marine snow-associated plastid-like rRNA clone and with a marine Hyphomonas strain, respectively. The method applied in this study could be useful for the routine study of other microbial communities of interest.     

Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval

The diversity and structure of the archaeal community in the effluent leachate from a full-scale recirculating landfill was characterized by direct 16S rRNA gene (16S rDNA) retrieval. Total-community DNA was extracted from the microbial assemblages in the landfill leachate, and archaeal 16S rDNAs were amplified with a universally conserved primer and an Archaea-specific primer. The amplification product was then used to construct a 16S rDNA clone library, and 70 randomly selected archaeal clones in the library were grouped by restriction fragment length polymorphism (RFLP) analysis. Sequencing and phylogenetic analysis of representatives from each unique RFLP type showed that the archaeal library was dominated by methanogen-like rDNAs. Represented in the kingdom of Euryarchaeota were phylotypes highly similar to the methanogenic genera Methanoculleus, Methanosarcina, Methanocorpusculum, Methanospirillum and Methanogenium, where the clone distribution was 48, 11, 3, 1 and 1, respectively. No sequences related to known Methanosaeta spp. were retrieved. Four rDNA clones were not affiliated with the known methanogenic Archaea, but instead, they were clustered with the uncultured archaeal sequences recently recovered from anaerobic habitats. Two chimeric sequences were identified among the clones analyzed.     

昆明盐矿古老岩盐沉积中的原核生物多样性

应用PCR-DGGE和rRNA分析法研究了昆明盐矿古老岩盐沉积中的原核生物多样性。样品的细菌DGGE分析得到27条带,古菌得到18条带。样品与纯培养得到的19个属菌株的DGGE图谱对比分析发现,细菌18个属菌株,只有1个属菌株与样品中的1条带迁移位置都不一致;古菌1个属的菌株不与样品中任何条带迁移位置一致。表明纯培养所得菌株并非该环境中的优势类群。同时,建立了样品细菌和古菌的16S rDNA克隆文库,从中分别挑取36个细菌克隆和20个古菌克隆进行ARDRA分析。细菌可分为10个OTUs,其中3个OTUs是优势类群,分别占38.9%,25.0%,16.7%,其余7个OTUs各含有1个克隆。古菌分为8个OTUs,没有明显的优势类群。每个OTU的代表克隆16S rDNA序列分析表明,细菌分属3大类群:α-Proteobacteria,γ-Proteobacteria和Actinobacteria,以Pseudomonas属菌为优势,含有其它岩盐沉积中没有发现的Actinobacteria。古菌主要是Halorubrum属、Haloterrigena属菌和未培养古菌。本研究表明,昆明盐矿古老岩盐沉积具有较丰富的原核生物多样性,含有大量未知的、未培养或不可培养的原核生物,但在原核生物物种组成和丰度上,免培养与此前的纯培养研究结果存在一定差异。因此,结合使用两类方法才能较全面地认识高盐极端环境微生物的多样性。     

Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea

In this study, we report on first 16S rRNA gene sequences from highly saline brine sediments taken at a depth of 1,515 m in the Kebrit Deep, northern Red Sea. Microbial DNA extracted directly from the sediments was subjected to PCR amplification with primers specific for bacterial and archaeal 16S rRNA gene sequences. The PCR products were cloned, and a total of 11 (6 bacterial and 5 archaeal) clone types were determined by restriction endonuclease digestion. Phylogenetic analysis revealed that most of the cloned sequences were unique, showing no close association with sequences of cultivated organisms or sequences derived from environmental samples. The bacterial clone sequences form a novel phylogenetic lineage (KB1 group) that branches between the Aquificales and the Thermotogales. The archaeal clone sequences group within the Euryarchaeota. Some of the sequences cluster with the group II and group III uncultivated archaea sequence clones, while two clone groups form separate branches. Our results suggest that hitherto unknown archaea and bacteria may thrive in highly saline brines of the Red Sea under extreme environmental conditions. Received: 5 February 1999 / Accepted: 14 July 1999     

Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.     

Microbial Diversity in Sediments Collected from the Deepest Cold-Seep Area, the Japan Trench

The Japan Trench land slope at a depth of 6,400 m is the deepest cold-seep environment with Calyptogena communities. Sediment samples from inside and beside the Calyptogena communities were collected, and the microbial diversity in the sediment samples was studied by molecular phylogenetic techniques. From DNA extracted directly from the sediment samples, 16S rDNAs were amplified by the polymerase chain reaction method. The sequences of the amplified 16S rDNAs selected by restriction fragment length polymorphism analysis were determined and compared with sequences in DNA databases. The results showed that 33 different bacterial 16S rDNA sequences from the two samples analyzed fell into similar phylogenetic categories, the α-, γ-, δ-, and ɛ-subdivisions of Proteobacteria, Cytophaga, and gram-positive bacteria; some of the 16S rDNA sequences were common to both samples. δ- and ɛ-Proteobacteria-related sequences were abundant in both sediments. These sequences are mostly related to sulfate-reducing or sulfur-reducing bacteria and epibionts, respectively. Eight different archaeal 16S rDNA sequences were cloned from the sediments. The majority of the archaeal 16S rDNA sequences clustered in Crenarchaeota and showed high similarities to marine group I archaeal rDNA. A Methanococcoides burtonii–related sequence obtained from the sediment clustered in the Euryarchaeota indicating that M. burtonii–related strains in the area of Calyptogena communities may contribute to production of methane in this environment. From these results, we propose a possible model of sulfur circulation within the microbial community and that of Calyptogena clams in the cold-seep environment. Received June 15, 1998; accepted November 10, 1998.