五氯酚厌氧生物降解体系中细菌的多样性分析

为了解五氯酚(PCP)降解过程中参与PCP降解的微生物多样性,本文应用16S rRNA基因克隆文库方法对PCP厌氧生物降解体系中细菌群落的组成和相对丰度进行了研究.结果表明,TM7类群的微生物在整个细菌群落中占有最大丰度(48.6%),检测到的序列与在三氯乙烯污染的地下水中检测的克隆子有一定的序列相似性(93.6%).丰度位居第二的微生物类群为β-变形菌纲(Betaproteobacteria)细菌,其中的一些克隆子(10.8%)与脱氯微生物Dechlorosoma suillum具有极高的序列同缘性(99.7%).此外,也检测到少数Clostridium属[厚壁菌门(Firmicutes)类群]的微生物.克隆文库中发现许多序列(占整个克隆文库的51.3%)与GenBank中已报道的序列具有较远的同源性(小于93.4%),它们可能代表新的微生物.本研究进一步拓宽了对PCP降解微生物多样性的认识.     

应用16S rDNA克隆文库解析人工快速渗滤系统细菌种群多样性

本文出不穷通过构建16S rDNA克隆文库开展了人工快速渗滤(CRI)系统表层(10cm深)细菌种群多样性研究,结果表明,在CRI系统表层填料中细菌多样性十分丰富,存在7个主要类群,其中不可培养的酸杆菌纲(uncultured Acidobacteria)和其它不可培养细菌(uncultured bacteria)在整个克隆文库中比例最大,比例为53.72%,其次是浮霉菌纲(uncultured planctomycete)和β-变形菌纲(β-proteobacterium),分别占文库比例的13.89%和8.33%;克隆文库中反硝化菌的比例高于亚硝化单胞菌属细菌(O.925%),没有出现Nitrospira属的克隆子.本文通过16S rDNA克隆文库揭示了在生物膜上存在的优势茵为不可培养菌,而假单胞菌(Pseudomonas aeruginosa)和紫色色杆菌(Chromobacterium sp.)等在平板分离培养中出现的频率较高,两种方法之间的结果存在差异.本研究采用16S rDNA克隆文库方法揭示的结果,将为CRI系统生物降解的进一步提高提供依据.     

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。     

西南印度洋中脊深海沉积物砷抗性菌的富集分离与多样性分析

[目的]为了筛选深海砷抗性菌,了解印度洋中脊深海沉积物砷抗性菌的多样性情况.[方法]通过砷富集培养,筛选出砷抗性菌;通过变性梯度凝胶电泳(DGGE)分析与构建16S rDNA克隆文库法两种手段分析了富集物中砷抗性菌的多样性.利用兼并引物PCR,从抗性菌株中克隆与砷抗性相关的基因.[结果]共筛选到8株砷抗性菌,分别属于y-proteobacteria的5个不同的属.其中,菌株AS-I1-3的抗性最高,可以在26×10-3 mol/L三价砷存在的情况下生长,该菌株的16S rDNA序列与菌株Pseudoalteromonas tetraodoni IAM同源性最高(100%).DGGE分析显示,该菌是富集物中的最优势菌,其次是盐单胞菌(Halomonas)和海杆菌(Marinobacter).16S rDNA克隆文库分析结果进一步证明,与菌株AS-I1-3序列相同的克隆子占整个克隆文库的72.5%;而与菌株AS-I1-5(Halomonas meridiana,100%)和菌株Mn-I1-6(Marinobacter vinifirmus,99%)序相同的克隆子分别占10%和7.5%.然而,利用兼并引物PCR,仅能从2株非优势菌中克隆到了与砷抗性相关的基因,表明菌群中的优势抗性菌可能有其他的抗性机制.[结论]在深海环境中存在着多种砷抗性菌,其中Pseudoalteromonas属的菌株是该富集条件下的砷抗性优势菌.这些砷抗性菌在大洋环境砷元素的生物地球化学循环中的作用有待进一步研究.     

新疆泥火山细菌遗传多样性

为了解新疆乌苏泥火山细菌多样性,从泥火山泥浆样品中直接提取总DNA,构建了含150个有效转化子的泥火山细菌16S rDNA基因文库,转化子经菌液PCR及HaeⅢ酶切后获得16个不同带型,克隆测序结果表明,其分属于16个不同的分类单元.一部分序列与已知细菌类群的16S rDNA序列相似性较高,归属变形菌门(Proteobacteria),厚壁菌门(Firmicutes),梭杆菌门(Fusobacteria),放线菌门(Actinobacteria);另外一部分序列与已知细菌类群的16S rDNA序列同源性较低,可能代表新的分类单位.研究结果显示,泥火山环境中微生物种群丰富,值得进一步研究.     

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。     

应用16S rDNA克隆文库解析人工快速渗滤系统细菌种群多样性

本文通过构建16S rDNA克隆文库开展了人工快速渗滤(CRI)系统表层(10 cm深)细菌种群多样性研究, 结果表明, 在CRI系统表层填料中细菌多样性十分丰富, 存在7个主要类群, 其中不可培养的酸杆菌纲(uncultured Acidobacteria)和其它不可培养细菌(uncultured bacteria)在整个克隆文库中比例最大, 比例为53.72%, 其次是浮霉菌纲(uncultured planctomycete)和β-变形菌纲(β-proteobacterium), 分别占文库比例的13.89%和8.33%; 克隆文库中反硝化菌的比例高于亚硝化单胞菌属细菌(0.925%), 没有出现Nitrospira属的克隆子。本文通过16S rDNA克隆文库揭示了在生物膜上存在的优势菌为不可培养菌, 而假单胞菌(Pseudomonas aeruginosa)和紫色色杆菌(Chromobacterium sp.)等在平板分离培养中出现的频率较高, 两种方法之间的结果存在差异。本研究采用16S rDNA克隆文库方法揭示的结果, 将为CRI系统生物降解的进一步提高提供依据。     

红豆杉免培养内生细菌多样性的初步研究

【目的】了解红豆杉(Taxus chinensis)内生细菌的组成及多样性。【方法】提取红豆杉组织总DNA,选用细菌通用引物799F和1492R对总DNA进行16S rDNA特异性扩增,构建红豆杉内生细菌16S rDNA克隆文库,对阳性克隆进行PCR-RFLP(限制性内切酶片段长度多态性)分析,并对酶切带谱不同的菌液进行测序,构建系统发育树。【结果】根据酶切带谱分析和测序结果的不同,将随机挑取的158个阳性克隆归为26个不同的可操作分类单元(OUTs),系统发育分析表明这些克隆序列分别属于变形菌门(Proteobacteria,包含Alpha、Beta、Gamma、Delta亚群)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)4个门。其中,变形菌门(Proteobacteria,占克隆总数的58.86%)为最优势类群。序列比对结果表明这些克隆序列分别与已报道的20个属具有较高的相似性。此外,还有一个OUTs在系统发育树上形成独立分支且未能确定其分类。【结论】红豆杉内生细菌多样性丰富,并且可能存在新的分类单元。     

细菌性阴道病患者阴道分泌物16SrDNA序列分析

目的分析健康妇女及细菌性阴道病(Bacterial vaginosis,BV)患者阴道分泌物16S rDNA序列。方法提取20例健康妇女及40例BV患者阴道分泌物标本中的总DNA,针对细菌16S rDNA保守区设计通用引物进行PCR扩增、克隆、测序,将获得的16S rDNA序列与美国国立生物技术信息中心(NCBI)数据库中的发表序列进行比对,分析克隆群中细菌种类和比例。结果通过阴道分泌物16S rDNA序列分析,发现健康妇女阴道分泌物中以卷曲乳酸杆菌(Lactobacillus crispatus),惰性乳酸杆菌(Lactobacillus iners),加氏乳酸杆菌(Lactobacillus gasseri)为优势菌种,而BV患者阴道菌群种类繁多,以加德纳菌属(Gardnerella)和奇异菌属(Atopobium vaginae)克隆子占较大比例,仅4例患者可见卷曲乳酸杆菌(Lactobacillus crispatus),其他患者均未见有乳酸杆菌克隆子且奇异菌属阴道病患者甲硝唑治疗疗效较差。结论健康妇女和BV患者阴道分泌物菌群种类有较大区别,BV患者在治疗前进行16S rDNA序列分析检测有较大的临床意义。     

16S rDNA克隆文库方法分析太岁样品中细菌的多样性

通过构建16S rDNA克隆文库的方法,分析太岁样品中细菌的群落结构及多样性。太岁样品中的细菌归属于4个门9个目,优势类群依次是芽胞杆菌目(Bacillales,33.01%)、柄杆菌目(Caulobacterales,32.04%)和伯克霍尔德氏菌目(Burkholderiales,12.62%);优势属为短波单胞菌属(Brevundimonas,30.10%)、葡萄球菌属(Staphylococcus,29.13%)和食酸菌属(Acidovorax,7.77%)。并且其中的5个目中含有未培养的细菌,红杆菌目(Rhodobacterales)、伯克霍尔德氏菌目和红环菌目(Rhodocyclales)的11个克隆子的细菌16S rDNA序列同源性低于97%。研究表明太岁样品中细菌多样性较丰富,且蕴藏着许多未知的微生物资源。     

杂交水稻种子固有细菌群落多样性探究

采用传统的分离培养方法和分子生物学技术对我国高产杂交水稻(OryzasativaL.)金优611种子固有细菌进行研究,从而了解其中可培养细菌群落的多样性。对分离得到的91株细菌进行16SrDNA扩增、ARDRA分型和16SrDNA系统发育分析,结果表明,分离得到的91株细菌分属于10个属16个种。其中γ-变形杆菌(Gammaproteobacteria)(53.85%)占据优势地位,其次为α-变形杆菌(Alphaproteobacteria)(20.88%),其它分属放线菌门Actinobacteria(15.39%)及厚壁菌门Firmicutes(9.88%)。其中的泛菌属(Pantoea sp.)和鞘氨醇单胞菌属(Sphingomonas sp.)、假单胞菌属(Pseudomonas sp.)、微杆菌属(Microbacterium sp.)为分离到的优势种群,且在种子这一特殊的生存空间中有4株潜在的新种存在。首次报道了杂交水稻金优611种子具有丰富的微生物群落多样性,为进一步探索植物种子际微生态环境中微生物群落的形成和生态功能提供了基础信息。     

Phylogenetic Analysis of Microbial Diversity in the Rhizoplane of Oilseed Rape (Brassica napus cv. Westar) Employing Cultivation-Dependent and Cultivation-Independent Approaches

The structure of the microbial rhizoplane community of the important crop plant oilseed rape was studied by using a culture-dependent as well as a culture-independent approach based on 16S rDNA amplification. After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica napus cv. Westar), the collected suspension was divided into two parts. One part was used for cultivation of bacteria onto three different growth media to establish a culture collection. From the other part of the rhizoplane suspension, genomic DNA was isolated and purified. Thereafter, 16S rDNA was amplified by PCR and cloned to obtain a library of 16S rDNA genes representative for the bacterial communities of this habitat. Phylogenetic 16S rDNA sequence analysis of 103 clones of this library revealed considerable differences from the corresponding nucleotide sequences of 111 cultured bacteria. Whereas the 16S rDNA clone library was dominated by a-Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups. More than 64% of the cultivated isolates were allocated to the b- and g-subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the a-Proteobacteria of the library showed highest similarity to Bradyrhizobium sp. No such bacteria were found in the culture collection. Similarly, the second dominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate. The phylogenetic analysis of isolates of the culture collection clearly emphasized the need to use different growth media for recovery of rhizoplane bacteria. Whereas most of the a-Proteobacteria were recovered on complex medium, most of the b-Proteobacteria were isolated onto minimal media. Our results demonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.     

新疆艾比湖湿地博乐河入口处土壤细菌多样性分析

【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。     

四川冬菜中细菌群落组成及多样性

【目的】了解腌制4年的四川南充冬菜中细菌群落组成及多样性。【方法】通过16S rDNA多样性分析样品细菌落组成;采用16S rDNA-RFLP方法分析从样品中分离出的纯培养细菌。【结果】16S rDNA多样性分析结果表明,样品中细菌主要属于变形杆菌门(Proteobacteria)和厚壁菌门(Firmicutes),分别占克隆文库的87.9%、7.1%,其中包括Virgibacillus kekensis,Marinococcus albus,Salinicoccus sp.,Lactobacillus halophilus和Halomonas等中度嗜盐菌,仅有5%属于放线菌门(Actinobacteria)。通过纯培养方法从冬菜中分离到35株菌,16S rDNA-RFLP分析结果表明,34株属于厚壁菌门(Firmicutes),包括Virgibacillus,Bacillus megaterium和Gracilibacillus saliphilus等中度嗜盐菌,1株属于放线菌门(Actinobacteria)。【结论】冬菜中细菌群落多样性较低,以中度嗜盐菌为主。     

Phylogenetic analysis of the succession of bacterial communities in the Great South Bay (Long Island)

Bacterial community composition and succession were examined over the course of the summer season in the Great South Bay, Long Island, NY, USA, using a 16S rDNA clone library approach. There was a progression of changes in dominant species in the libraries during the summer of 1997. The July library had several groups dominant, the SAR407 relatives of the alpha-Proteobacteria (24%) and the SAR86 (18%), sulfur-oxidizing symbiont relatives (8%) of the gamma-Proteobacteria, and unidentified Cytophaga-Flexibacter representatives (22%). In August, the Cytophaga-Flexibacter (Gelidibacter sp. and unidentified Cytophaga-Flexibacter representative) and Cyanobacteria (Synechococcus sp.) increased to 28% and 14%, respectively. High GC Gram-positives appeared at 18%, and beta-Proteobacteria (Ralstonia sp.) at 10%. By September these groups had either declined or were absent, while the SAR86 cluster, Pseudoalteromonas and Alteromonas of the gamma-Proteobacteria were dominant in the community (61%). The dominance of open ocean bacteria along with the presence of Aureococcus anophagefferens (Pelagophyceae) in July suggests possible open ocean coupling to bloom events. Many clones in this study were related to previously described clones from a wide distribution of marine environments, substantiating the cosmopolitan nature of pelagic bacteria. Only one isolated bacterium was closely related to 16S rDNA found in the August library.     

PCR-RFLP分析桉树人工林土壤微生物的群落结构

本研究利用限制性内切酶片段长度多态性(RFLP)和16S rDNA序列分析相结合的方法研究了广西南宁高峰林场人工桉树林中土壤微生物的群落结构.通过采用直接法提取桉树人工林土壤中微生物总DNA,构建细菌的16S rDNA克隆文库,从中随机挑取了192个阳性克隆进行检测,分析显示188个阳性克隆子确定含有16S rDNA并分归为52个不同的类群OTUs,随机挑取其中35个克隆进行测序,并构建了系统发育进化树.研究结果表明:桉树人工林土壤中细菌种类较为丰富,含有大量的未培养微生物,进一步的序列分析表明桉树人工林土壤中占优势的类群分别为Acidobacteria(40%)和Proteobacteria(27%).本研究对桉树人工林土壤微生物群落结构进行了研究,为进一步研究桉树人工林环境质量变化提供基础资料.     

Phylogenetic analysis of the fecal flora of the wild pygmy loris

The bacterial diversity in fecal samples from the wild pygmy loris was examined with a 16S rDNA clone library and restriction fragment length polymorphism analysis. The clones were classified as Firmicutes (43.1%), Proteobacteria (34.5%), Actinobacteria (5.2%), and Bacteroidetes (17.2%). The 58 different kinds of 16S rDNA sequences were classified into 16 genera and 20 uncultured bacteria. According to phylogenetic analysis, the major genera within the Proteobacteria was Pseudomonas, comprising 13.79% of the analyzed clone sequences. Many of the isolated rDNA sequences did not correspond to known microorganisms, but had high homology to uncultured clones found in human feces. Am. J. Primatol. 72:699–706, 2010. © 2010 Wiley‐Liss, Inc.     

Cloning and identification of cellulase genes from uncultured microorganisms in pulp sediments from paper mill effluent]

The metagenomic DNA of pulp sediments from paper mill effluent was extracted and purified. The 16S rDNA was amplified using the purified metagenomic DNA as template and a 16S rDNA library was prepared. Sequence analysis of 16S rDNA clones showed that diverse of uncultured bacteria inhabit in this environment, which can be classified into 4 clusters as Spirochaetes, Proteobacteria, Bacteroidetes and Firmicutes. A metagenomic library containing 10000 clones was constructed into cosmid vector, and the capacity of inserted DNA of which was 3.53 x 10(8) bp. Functional screening of the library resulted in isolation of two independent clones expressing endoglucanase activity, three independent clones expressing exoglucanase activity and two independent clones expressing beta-glucosidase activity. One clone expressing strongest enzyme activity from each activity category was chosen to be further analyzed. Three novel cellulase genes designated as umcel5L, umcel5M and umbgl3D were identified by subcloning, sequencing and expression. The umcel5L encodes an endoglucanase belonging to glycosyl hydrolase family 5, which is most related to an endoglucanase from Bradyrhizobium japonicum at 43% identity and 59% similarity. The umcel5M encodes a cellodextrinase belonging to glycosyl hydrolase family 5, which is most similar to a cellodextrinase from Fibrobacter succinogenes at 48% identity and 69% similarity. The umbgl3D encodes a putative beta-glucosidase belonging to glycosyl hydrolase family 3, which shares highest homology with a beta-glucosidase from Thermotoga maritima at 46% identity and 61% similarity. It is the first time to reveal the bacterial diversity of pulp sediments from paper mill effluent and clone novel cellulase genes from the bacteria by culture-independent method.     

太湖地区典型菜地土壤微生物16S rDNA的PCR-RFLP分析

土壤微生物多样性是土壤生态功能的基础,但长期以来缺乏对高强度土地利用条件下的土壤微生物多样性的认识.作者采用间接法提取了江苏省太湖地区典型菜地土壤微生物的总DNA,以细菌的通用引物27F和1492R扩增16S rDNA片段,将扩增产物与T-载体酶连,转化大肠杆菌,建立土壤微生物16S rDNA克隆文库.PCR扩增基因文库中插入的16S rDNA外源片段,用两种限制性内切酶Hha I和Rsa I分别酶切,获得该土壤173个克隆的酶切指纹图谱.结果表明,Hha I和Rsa I联合酶切产生了63个基因分型,文库的覆盖度达76.30%,单一酶切产生的基因分型少,但文库的覆盖度高;克隆文库中存在两种优势类群,分别占总克隆的16%和12%.16S rDNA测序结果表明,太湖地区菜地土壤细菌在分类方面主要属于α-和γ-变形杆菌亚门.以上结果为进一步研究太湖地区菜地土壤微生物生态功能提供了基础资料.