黄海繁茂膜海绵中微生物多样性的研究

构建了中国黄海繁茂膜海绵中细菌16S rDNA克隆,对其遗传多样性进行了分析,发现海绵中相关细菌16S rDNA基因主要归类于紫硫细菌门(Proteobacteria)中的α-亚门、γ-亚门,和放线菌门(Actinobacteria)等类群。所获得的16S rDNA序列与GenBank中的已知序列差异较大,反映出该海绵存在尚未发现的微生物新信息。     

16S rDNA_RFLP分析繁茂膜海绵可培养放线菌的多样性

海绵是迄今为止已知海洋天然产物的最大来源。由于海绵的底栖过滤性摄食和消化选择性等生理特性,其体内蕴藏了丰富的微生物种群。近几年来,发现越来越多的海绵微生物产生很强的生物活性物质,有些并被证明是海绵天然产物的真正生产者。对大连海域繁茂膜海绵中的放线菌进行分离,对于得到的菌株进行16SrDNA扩增和RFLP及测序分析。研究表明海绵中蕴含着丰富的放线菌资源。其中包含链霉菌(Streptomycetes),拟诺卡氏菌(Nocardiopsis),假诺卡氏菌(Pseudonocardia),诺卡氏菌(Nocardia),小单孢菌(Micromonospora),红球菌(Rhodococcus),异壁放线菌(Actinoalloteichus)等属。利用限制性内切酶HhaⅠ对16SrDNA进行的RFLP分析能够对海绵中放线菌的16SrDNA多样性进行有效的分析,结果达到属,部分到种的级别。揭示了繁茂膜海绵中蕴藏着丰富的放线菌资源。     

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。     

青藏铁路沿线唐古拉山口土壤微生物的ARDRA分析

通过构建16S rDNA文库及文库的限制性片段长度多态性分析（ARDRA）,对青藏铁路沿线唐古拉山口的土壤微生物多样性进行了研究。采用限制性内切酶HaeIII和RsaI对克隆文库中的90个克隆子进行了酶切分型,根据ARDRA酶切图谱的不同,可将其分为23个OTUs。16SrDNA序列分析结果表明,该克隆文库中主要包括变形菌门（Proteobacteria）的alpha、beta、detla亚类、厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）、拟杆菌门（Bacteroidetes）、酸杆菌门（Acidobacteria）及浮霉菌门（Planctomycetes）等8类细菌及未培养细菌。Alpha变形细菌为该文库中的主要菌群,占克隆总数的33.3%;其次为未培养细菌,占克隆总数的22.2%,Bradyrhizobium为优势菌属。研究结果揭示,青藏铁路唐古拉山口的土壤微生物种群不仅具有丰富的多样性,还存在丰富的潜在新菌种。     

繁茂膜海绵硅聚合酶抗体制备及其在造骨细胞鉴别中的应用

为准确鉴别海绵造骨细胞,分别提取了繁茂膜海绵、多皱软海绵和澳大利亚厚皮海绵的硅聚合酶,以繁茂膜海绵硅聚合酶为抗原制备抗体,效价为1∶9600。SDSPAGE显示三种海绵硅聚合酶的亚基分布在28kD左右;建立竞争抑制性检测方法并结合WesternBlotting检测,显示该抗体可与繁茂膜海绵硅聚合酶特异性结合,且与另外两种海绵硅聚合酶几乎无交叉反应。利用该抗体对繁茂膜海绵组织和体外培养细胞进行免疫组织化学染色,均可显示造骨细胞的分布。结果提示硅聚合酶抗体可以特异性与繁茂膜海绵造骨细胞内的硅聚合酶结合,因此,该抗体可以用于造骨细胞的鉴别。     

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 16SrDNA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Pachychalinasp .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 16SrDNA通用引物进行PCR扩增获得 16SrDNA ,回收、纯化 16SrDNA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 16SrDNA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 16SrDNA的部分序列 ,并对 16SrDNA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Methanogeniumorganophilum、Methanoplanuspetrolearius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     

溴代阻燃剂污染底泥与未污染底泥中细菌群落结构之比较分析

【目的】通过免培养的分子生物学方法,比较受溴代阻燃剂污染严重的河流底泥和与未污染水库底泥中细菌多样性差异,解析二者间细菌群落结构,为污染河流的治理与生物修复提供相关的理论依据。【方法】从中国贵屿溴代阻燃剂污染区练江底泥样品和未污染水库底泥样品中分别提取微生物总DNA,用细菌通用引物27F和1500R扩增16S rRNA基因,构建16S rRNA基因文库。用HhaⅠ和HinfⅠ2种限制性内切酶对克隆子进行扩增产物rDNA的限制性酶切分析(ARDRA),挑取不同的操作分类单元OUT中的克隆进行测序并构建系统发育树,比较代表克隆子的基本分类类群和生物多样性构成。【结果】未污染水库底泥中细菌群落组成主要包括变形细菌门(Proteobacteria)的α、β、γ和δ4个亚门、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)、疣微菌门(Verrucomicrobia)和厚壁菌门(Firmicutes)。优势菌是酸杆菌,占文库克隆的30.2%。污染河流底泥中细菌群落组成包括变形细菌门(Proteobacteria)的α、β、δ和ε4个亚门、浮霉菌门(Planctomycetes)、绿菌门或拟杆菌门(Chlorobi orBacteroidetes)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)、待定菌群candidatedivision OP01、candidate division OP08和candidate division WS3的相似菌。优势菌是ε-变形细菌和绿弯菌,占文库克隆的44.9%。【结论】受溴代阻燃剂污染河流底泥中的细菌群落具有较高水平的多样性,与未污染底泥有显著区别,主要体现在ε-变形细菌和绿弯菌在细菌群落中具有优势地位。这种优势种群的改变可能与污染物的过度富集具有一定的相关性,对于我们进一步探索和了解溴代阻燃剂的微生物修复具有一定的指导意义。     

一株海绵放线菌的分离鉴定及活性研究*

从大连潮间带繁茂膜海绵中分离到一株具有很强抑制革兰氏阳性菌活性和较强抗肿瘤活性的链霉菌。通过其形态观察,生理生化测定和16SrDNA序列的测定,初步判定属于假浅灰链霉菌(Streptomyces pseudogriseolus)。并对其活性物质的生产条件进行了初步探索和优化,确定了用天然海水配制的TSB培养基为最佳发酵培养基,3d为最适发酵时间。     

黑龙江大豆胞囊线虫胞囊可培养细菌多样性

【目的】了解黑龙江省大豆田大豆胞囊线虫胞囊可培养细菌的多样性。【方法】运用稀释平板法和16SrDNA基因序列的系统发育分析对胞囊可培养细菌多样性进行研究。【结果】用NA培养基从胞囊上分离90株具有不同菌落形态的细菌。16S rDNA序列分析结果表明:90株菌株分属于7个属22个种。46株属于变形菌门γ亚群(Gammaproteobacteria),32株属于厚壁菌门(Firmicutes),10株属于变形菌门β亚群(Betaproteobacteria),2株属于变形菌门ɑ亚群(Alphaproteobacteria)。假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)为优势菌属。【结果】黑龙江省大豆胞囊线虫胞囊中存在丰富的细菌物种多样性,这些细菌对大豆胞囊线虫可能具有一定的生理生态作用。     

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)。     

Isolation and diversity of planctomycetes from the sponge Niphates sp., seawater, and sediment of Moreton Bay, Australia

Planctomycetes are ubiquitous in marine environment and were reported to occur in association with multicellular eukaryotic organisms such as marine macroalgae and invertebrates. Here, we investigate planctomycetes associated with the marine sponge Niphates sp. from the sub-tropical Australian coast by assessing their diversity using culture-dependent and -independent approaches based on the 16S rRNA gene. The culture-dependent approach resulted in the isolation of a large collection of diverse planctomycetes including some novel lineages of Planctomycetes from the sponge as well as sediment and seawater of Moreton Bay where this sponge occurs. The characterization of these novel planctomycetes revealed that cells of one unique strain do not possess condensed nucleoids, a phenotype distinct from other planctomycetes. In addition, a culture-independent clone library approach identified unique planctomycete 16S rRNA gene sequences closely related to other sponge-derived sequences. The analysis of tissue of the sponge Niphates sp. showed that the mesohyl of the sponge is almost devoid of microbial cells, indicating this species is in the group of ‘low microbial abundant’ (LMA) sponges. The unique planctomycete 16S rRNA gene sequences identified in this study were phylogenetically closely related to sequences from LMA sponges in other published studies. This study has revealed new insights into the diversity of planctomycetes in the marine environment and the association of planctomycetes with marine sponges.     

Sponge-specific Bacterial Associations of the Mediterranean Sponge <Emphasis Type="Italic">Chondrilla nucula</Emphasis> (Demospongiae,Tetractinomorpha)

A stable and specific bacterial community was shown to be associated with the Mediterranean sponge Chondrilla nucula. The associated bacterial communities were demonstrated to be highly similar for all studied specimens regardless of sampling time and geographical region. In addition, analysis of 16S rDNA clone libraries revealed three constantly C. nucula-associated bacterial phylotypes belonging to the Acidobacteria, the Gamma- and Deltaproteobacteria present in sponge specimens from two Mediterranean regions with distinct water masses (Ligurian Sea and Adriatic Sea). For the first time, candidate division TM7 bacteria were found in marine sponges. A major part (79%) of the C. nucula-derived 16S rDNA sequences were closely related to other sponge-associated bacteria. Phylogenetic analysis identified 14 16S rRNA gene sequence clusters, seven of which consisted of exclusively sponge-derived sequences, whereas the other seven clusters contained additional environmental sequences. This study adds to a growing database on the stability and variability of microbial consortia associated with marine sponges.     

Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Microbial Diversity and Community Structure on Corroding Concretes

The objective of this study was to analyze bacterial diversity in two different concrete samples to understand the dominant types of bacteria that may contribute to concrete corrosion. Two concrete samples, HN-1 from the sunny side and HN-2 from dark and damp side, were collected from Zijin Mountain in Nanjing and genomic DNA was extracted. The partial bacterial 16S rRNA gene fragment was PCR amplified and two clone libraries were constructed. Amplified ribosomal DNA restriction analysis (ARDRA) was performed by digestion of the 16S rRNA gene and each unique restriction fragment polymorphism pattern was designated as an operational taxonomic unit (OTU). Phylogenetic trees of bacterial 16S rDNA nucleotide sequences were constructed. Sample HN-1 and HN-2 contained 21 OTUs and 26 OTUs, respectively. Proteobacteria and Planctomycetes were the predominant bacteria in both samples, and they are distributed among Herbaspirillum, Archangium, Phyllobacteriaceae and Planctomycetaceae. Cyanobacteria and Rubrobacter sp. are dominant in HN-1; while Acidobacteriaceae, Adhaeribacter sp. and Nitrospira sp. are predominant in HN-2. This distribution pattern was consistent with local environmental conditions of these two samples. The inferred physiological characteristics of these bacteria, based on relatedness of the DNA clone sequences to cultivated species, revealed different mechanisms of concrete corrosion depending on the local environmental conditions.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge <Emphasis Type="Italic">Gelliodes carnosa</Emphasis> Collected from the Hainan Island Coastal Waters of the South China Sea

Several molecular techniques were employed to document the bacterial diversity associated with the marine sponge Gelliodes carnosa. Cultivation-dependent and cultivation-independent methods were used to obtain the 16S rRNA gene sequences of the bacteria. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the bacterial community structure was highly diverse with representatives of the high G + C Gram-positive bacteria, cyanobacteria, low G + C Gram-positive bacteria, and proteobacteria (α-, β-, and γ-), most of which were also found in other marine environments, including in association with other sponges. Overall, 300 bacterial isolates were cultivated, and a total of 62 operational taxonomic units (OTUs) were identified from these isolates by restriction fragment length polymorphism (RFLP) analysis and DNA sequencing of the 16S rRNA genes. Approximately 1,000 16S rRNA gene clones were obtained by the cultivation-independent method. A total of 310 clones were randomly selected for RFLP analysis, from which 33 OTUs were acquired by further DNA sequencing and chimera checking. A total of 12 cultured OTUs (19.4% of the total cultured OTUs) and 13 uncultured OTUs (39.4% of the total uncultured OTUs) had low sequence identity (≤97%) with their closest matches in GenBank and were probably new species. Our data provide strong evidence for the presence of a diverse variety of unidentified bacteria in the marine sponge G. carnosa. A relatively high proportion of the isolates exhibited antimicrobial activity, and the deferred antagonism assay showed that over half of the active isolates exhibited a much stronger bioactivity when grown on medium containing seawater. In addition to demonstrating that the sponge-associated bacteria could be a rich source of new biologically active natural products, the results may have ecological implications. This study expands our knowledge of the diversity of sponge-associated bacteria and contributes to the growing database of the bacterial communities within sponges.     

A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones

The anaerobic oxidation of ammonium (anammox) contributes significantly to the global loss of fixed nitrogen and is carried out by a deep branching monophyletic group of bacteria within the phylum Planctomycetes. Various studies have implicated anammox to be the most important process responsible for the nitrogen loss in the marine oxygen minimum zones (OMZs) with a low diversity of marine anammox bacteria. This comprehensive study investigated the anammox bacteria in the suboxic zone of the Black Sea and in three major OMZs (off Namibia, Peru and in the Arabian Sea). The diversity and population composition of anammox bacteria were investigated by both, the 16S rRNA gene sequences and the 16S-23S rRNA internal transcribed spacer (ITS). Our results showed that the anammox bacterial sequences of the investigated samples were all closely related to the Candidatus Scalindua genus. However, a greater microdiversity of marine anammox bacteria than previously assumed was observed. Both phylogenetic markers supported the classification of all sequences in two distinct anammox bacterial phylotypes: Candidatus Scalindua clades 1 and 2. Scalindua 1 could be further divided into four distinct clusters, all comprised of sequences from either the Namibian or the Peruvian OMZ. Scalindua 2 consisted of sequences from the Arabian Sea and the Peruvian OMZ and included one previously published 16S rRNA gene sequence from Lake Tanganyika and one from South China Sea sediment (97.9-99.4% sequence identity). This cluster showed only 相似文献   

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp.

The microbial community associated with a marine sponge (Haliclona sp.) collected from Tateyama city, Japan was studied using 16S rRNA gene clone libraries. Two DNA templates were prepared using methods recommended for Gram-positive and Gram-negative bacteria in the Qiagen kit manual. From each DNA template, two 16S rRNA genes were PCR amplified, using the combination of universal bacterial primer 27f and primers 1385r and 1492r, respectively. A total of 347 clones were sequenced and compared with those available in DNA data banks. These sequences were members of ten bacterial phyla. Interestingly, more than 30 % of the clones represent novel sequences. A comparison of these sequences with sequences in a library prepared from DNA extracted from the surrounding water shows minimum DNA contamination. Taxonomically, the highest diversity was detected in the clone library prepared using a combination of primers 27f and 1492r and DNA isolated using the Gram-positive bacteria protocol. The potential of Haliclona sp.-associated bacteria to produce secondary metabolites was studied by cloning and sequencing the polyketide synthase (PKS, type 1) gene using the same DNA samples. Analysis of partial sequences derived from the sponge metagenome revealed 27 unique ketosynthase domains of PKS type I. This study suggests strongly that this Haliclona sp. plays host to diverse novel bacteria with a potential to produce novel polyketides.     

Endophytic Bacterial Diversity in Rice (Oryza sativa L.) Roots Estimated by 16S rDNA Sequence Analysis

The endophytic bacterial diversity in the roots of rice (Oryza sativa L.) growing in the agricultural experimental station in Hebei Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of rice, a pair of bacterial PCR primers (799f–1492r) was selected to specifically amplify bacterial 16S rDNA sequences directly from rice root tissues. Among 192 positive clones in the 16S rDNA library of endophytes, 52 OTUs (Operational Taxonomic Units) were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Cytophaga/Flexibacter/Bacteroides (CFB) phylum, low G+C gram-positive bacteria, Deinococcus-Thermus, Acidobacteria, and archaea. The dominant group was Betaproteobacteria (27.08% of the total clones), and the most dominant genus was Stenotrophomonas. More than 14.58% of the total clones showed high similarity to uncultured bacteria, suggesting that nonculturable bacteria were detected in rice endophytic bacterial community. To our knowledge, this is the first report that archaea has been identified as endophytes associated with rice by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in rice roots.