16S rDNA clone library-based bacterial phylogenetic diversity associated with three South China Sea sponges

Culture-independent molecular techniques, 16S rDNA clone library alongside RFLP and phylogenetic analysis, were applied to investigate the bacterial diversity associated with three South China Sea sponges, Stelletta tenui, Halichondria rugosa and Dysidea avara. A wide bacterial diversity was detected according to total genomic DNA-based 16S rDNA clone library, abundant clones with low identify with sequences retrieved from database were found as well as uncultured sponge symbionts. The phylogenetic analysis shows that the bacterial community structure of Stelletta tenui is similar to that of Halichondria rugosa comprising gamma-Proteobacteria and Firmicutes. Whereas, alpha-Proteobacteria, gamma-Protebacteria, Bacteroidetes and uncultured sponge symbionts were found in sponge Dysidea avara, suggesting that Dysidea avara has the highest bacteria diversity among these sponges. A specific sponge–microbe association is suggested based on the difference of bacterial diversity among these three sponges from the same geography location and the observed sponge species-specific bacteria.     

基于PCR-DGGE指纹的南海海绵共附生细菌优势种群的揭示与系统发育分析

采用PCR-DGGE指纹、克隆测序和系统发育分析技术较系统地对我国南海贪婪倔海绵(Dysidea avara)和澳大利亚厚皮海绵(Craniella australiensis)共附生的优势细菌进行了研究。研究发现变形菌门(Proteobacteria)细菌是这两种海绵中的主要优势细菌,贪婪倔海绵中的变形菌包含了α、β、γ三种类型,而澳大利亚厚皮海绵中仅有γ一种类型。两种海绵都有拟杆菌(Bacteroidetes),但是具体的种类不同。这些细菌都是第一次在海绵中被发现。澳大利亚厚皮海绵共附生的优势细菌还包括放线菌属(Actinobacterium)及厚壁菌门(Firmicutes)细菌,菌群多样性要比贪婪倔海绵的丰富。两种海绵尽管来自于同一海域但其共附生优势细菌的组成明显不同,这说明海绵共附生微生物具有宿主特异性。     

Bacterial diversity based on 16S rRNA and gyrB genes at Yinshan mine, China

The diversity of bacterial communities at three sites impacted by acid mine drainage (AMD) from the Yinshan Mine in China was studied using comparative sequence analysis of two molecular markers, the 16S rRNA and gyrB genes. The phylogenetic analyses retrieved sequences from six classes of bacteria, Nitrospira, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Acidobacteria, and Actinobacteria, as well as sequences related to the plastid of the cyanobacterium Cyanidium acidocaldarium and also some unknown bacteria. The results of phylogenetic analyses based on gyrB and 16S rRNA were compared. This confirmed that gyrB gene analysis may be a useful tool, in addition to the comparative sequence analysis of the 16S rRNA gene, for the analysis of microbial community compositions. Moreover, the Mantel test showed that the geochemical characteristics, especially the pH value and the concentration of iron, strongly influenced the composition of the microbial communities.     

16S rDNA-RFLP分析六氯苯好氧降解菌群的结构及其多样性

从某化工厂排水沟底泥中取样,经2个月的富集驯化得到六氯苯好氧降解菌群。通过测定该微生物菌群在降解六氯苯过程中累积耗氧量、微生物生长曲线及Cl-浓度的变化,证明在好氧条件下该微生物菌群能够以六氯苯为唯一碳源和能源生长。当培养温度为30℃,pH为7.0时,该菌群能在18d内将无机盐培养基中浓度为4.5mg/L的六氯苯降解55%以上,降解速率达到137.5μg/(L.d)。对降解菌群提取总DNA,选择性扩增细菌16S rDNA片段,建立克隆文库。通过限制性内切酶(限制性内切酶HaeⅢ和RsaⅠ)分析,得到9种不同的谱型,其中3种谱型是主要谱型。对主要谱型的克隆子测序,结果表明,它们分别与Alcaligenes和Azospirillum菌属相似性最高。该菌群在去除环境中难降解的有机氯污染物方面具有应用前景。     

南海硇洲岛潮汐带栉江珧可培养细菌的系统发育多样性

【目的】研究南海硇洲岛潮汐带栉江珧(Atrina pectinata)样品相关可培养细菌的多样性。【方法】采用纯培养法和基于16S r RNA基因序列的系统发育分析,法对样品中可培养细菌(含放线菌)的类群多样性、物种多样性和遗传多样性进行研究。【结果】用补充0–25%(质量体积比)Na Cl的MA、MH和NA培养基从栉江珧样品中分离到125株细菌。在形态观察和部分生理生化实验结果的基础上去冗余,选取90个代表性菌株进行基于16S r RNA基因序列的系统发育多样性分析。结果表明,这90个分离菌株分属于3个大的系统发育类群(Gamma-proteobacteria、Firmicutes、Actinobacteria)、6个科、10个属,可分为33个物种。优势类群为厚壁菌门(Firmicutes)(56株,62.2%)和γ-变形杆菌亚门(Gamma-proteobacteria)(31株,34.5%)。大多数菌株与其系统发育关系最密切的已知物种的典型菌株之间存在一定的遗传差异(16S r RNA基因序列相似性为95.7%–99.9%),其中有5株可能代表新的分类单元(Potential new taxa)。分析表明,菌株JSM 112024可能代表了盐单胞菌科(Halomonadaceae)的一个属一级新分类单元;菌株JSM 112019、JSM 114045、JSM 114058和JSM 114083可能分别代表盐弧菌属(Salinivibrio)、芽孢杆菌属(Bacillus)、盐单胞菌属(Halomonas)和枝芽孢菌属(Virgibacillus)的新物种。【结论】湛江硇洲岛潮汐带栉江珧中存在较为丰富的可培养细菌物种多样性和系统发育多样性,并潜藏着较多的新微生物类群(物种)。     

海绵共附生细菌种群组成的PCR-DGGE基因指纹分析

采用不依赖于分离培养的16S rDNA的PCR-DGGE基因指纹技术对我国南海的细薄星芒海绵、皱皮软海绵、贪婪倔海绵、澳大利亚厚皮海绵体内的优势细菌的种群组成进行了比较分析。结果显示:每种海绵体内都含有丰富多样的细菌;通过DGGE指纹图的聚类分析发现来自同一海域的不同海绵的共附生细菌的种群组成具有明显不同,即共附生细菌具海绵宿主特异性;同时也发现有相同的细菌存在于不同的海绵体内。     

Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses

Bacterial diversity associated with Baer Soda Lake in Inner Mongolia of China was investigated using a culture-independent method. Bacterial 16S rRNA gene libraries were generated using bacterial oligonucleotide primers, and 16S rRNA gene sequences of 58 clones were analyzed phylogenetically. The library was dominated by 16S rDNAs of Gram-negative bacteria (24% -Proteobacteria, 31% -Proteobacteria, 33% -Proteobacteria, and 2% -Proteobacteria), with a lower percentage of clones corresponding to Gram-positive bacteria. Forty cloned sequences were similar to that of known bacterial isolates (>97% sequence similarity), represented by the species of the genera Brevundimonas, Comamonas, Alcaligenes, Stenotrophomonas, and Klebsiella. Eighteen cloned sequences showed less affiliation with known taxa (<97% sequence similarity) and may represent novel taxa.Communicated by K. Horikoshi     

中国南海海域海洋深层水细菌多样性

中国南海与作为海洋生物多样性中心的印太珊瑚大三角区具有一定的环境与生物连通性,其生境多样,为各种生物类群提供了有利的热带和亚热带海洋环境栖息地,具有极其丰富的生物多样性,是中国海洋生物多样性热点地区,也是重要的海洋生物多样性资源宝库。海洋细菌在生态系统循环中发挥重要作用,通过对南海海洋深层水细菌的探测,可以更加清晰地了解西太平洋和印太交汇区的生物多样性。研究基于参加开放项目获得的南海的深海站点水样,采用高通量测序进行了16S rRNA基因序列测序,分析了南海深海海水细菌群落多样性、菌群构成和多样性特征。该站点南海海洋深层水测定的细菌分属于15门、20纲、24目、86科、140属、150种,表明该区海洋深层水细菌群落丰富。测得优势菌属包括类芽胞杆菌属(Paenibacillus)、芽孢杆菌属(Bacillus)、海旋菌属(Thalassospira)、噬甲基菌属(Methylophaga)、拟杆菌属(Bacteroides)、纤细芽胞杆菌属(Gracilibacillus)、海洋芽胞杆菌属(Oceanobacillus)、海源菌属(Idiomarina)、嗜油菌属(Oleiphilus)、食碱菌属(Alcanivorax),这些属的细菌在生物材料或药物、生物燃料、水处理、降解石油烃等方面均有开发利用的潜力。属内的解淀粉芽孢杆菌(Lentibacillus amyloliquefaciens)、施氏假单胞菌(Pseudomonas stutzeri)、柴油食烷菌(Alcanivorax dieselolei)、三浦半岛盐乳杆菌(Halolactibacillus miurensis)、海迪茨氏菌(Dietzia maris)可以具体鉴定到种,这些深海细菌目前已在产抗生素、生物防治、石油降解、反硝化等方面被开发利用。基于高通量测序显示出采样点南海海洋深层水细菌具有独特菌群特征和多样性,有其极为丰富的生物学开发利用价值。     

南海南部陆坡表层沉积物细菌和古菌多样性

从南海南部陆坡表层沉积物中扩增了细菌和古菌16S rDNA序列,并对克隆子文库进行系统发育分析.细菌序列以变形杆菌(Proteobacteria)居多,其次是浮霉菌(Planctomycete)、酸杆菌(Acidobacteria)和candidate division OP10,另外还有少量铁还原杆菌(Deferrobacteres)、candidate division OP3、OP11、OP8、TM6、疣微菌(Verrucomicrobia)和螺旋体(Spirochaetes).古菌序列分别来自泉古生菌(Crenarchaeota)和广古生菌(Euryarchaeota),以Marine Benthic Group B(MBGB)、MarineCrenarchaeotic Group Ⅰ(MGⅠ)、Marine Benthic Group D(MBGD)和South African Gold Mine Euryarchaeotic Group(SAGMEG)为主.少量序列为C3、甲烷杆菌(Methanobacteriales)和Novel Euryarchaeotic Group(NEG).结果表明海底表层沉积物中有丰富多样的微生物群落.     

Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences

The 16S rDNA sequences of 11 strains, nine type strains of validated Pseudonocardia species and Actinobispora yunnanensis, and two strains of unnamed Pseudonocardia species, were determined and compared with those of representatives of the family Pseudonocardiaceae. The phylogenetic analysis indicated that all of the validated species of the genera Pseudonocardia and Actinobispora consistently formed a monophyletic unit and separated well from the other genera of the family Pseudonocardiaceae. One unnamed Pseudonocardia strain was related to members of the genus Pseudonocardia, whereas the other unnamed Pseudonocardia strain formed a distinct clade within the radiation of the genus Amycolatopsis.     

硇洲岛潮汐带牡蛎相关可培养细菌多样性

[目的]了解南海硇洲岛潮汐带香港巨牡蛎(Crassostrea hongkongensis)相关可培养细菌的多样性.[方法]应用纯培养法分离样品中的细菌(含放线菌),采用基于16S rRNA基因序列的系统发育分析方法研究这些分离菌株的生物多样性.[结果]用补充0-25％(W/V) NaC1的MA、MH和NA培养基从样品中分离到102株细菌,在形态观察和部分生理生化实验结果的基础上去冗余,选取74个代表性菌株进行基于16S rRNA基因序列的系统发育分析.结果表明,这些菌株归为38个物种,属于4个大的系统发育类群(Gamma-Proteobacteria,Alpha-Proteobacteria,Bacteroidetes,Firmicutes)的16个科、18个属.大多数菌株属于Gamma-Proteobacteria亚门(45株,60.8％),其余依次是Firmicutes门(12株,16.2％)、Bacteroidetes门(11株,14.9％)和Alpha-Proteobacteria亚门(6株,8.1％).大多数菌株与其系统发育关系最密切的有效发表种典型菌株之间(16S rRNA基因序列相似性为94.3％-99.8％)存在一定的遗传差异,其中JSM 111069可能代表Carnobacteriaceae科的潜在新属;菌株JSM 111039和JSM 111085可能分别代表Bacillus属的两个新物种,菌株JSM 111020、JSM 111072和JSM 111090可能分别代表Pseudoalteromonas、Proteus和Idiomarina属的新物种.[结论]南海硇洲岛潮汐带牡蛎中存在较为丰富的原核生物多样性,并潜藏着一定数量的微生物新类群(物种).     

Microbial community dynamics based on 16S rRNA gene profiles in a Pacific Northwest estuary and its tributaries

We analyzed bacterioplankton community structure in Tillamook Bay, Oregon and its tributaries to evaluate phylogenetic variability and its relation to changes in environmental conditions along an estuarine gradient. Using eubacterial primers, we amplified 16S rRNA genes from environmental DNA and analyzed the PCR products by length heterogeneity polymerase chain reaction (LH-PCR), which discriminates products based on naturally occurring length differences. Analysis of LH-PCR profiles by multivariate ordination methods revealed differences in community composition along the estuarine gradient that were correlated with changes in environmental variables. Microbial community differences were also detected among different rivers. Using partial 16S rRNA sequences, we identified members of dominant or unique gene fragment size classes distributed along the estuarine gradient. Gammaproteobacteria and Betaproteobacteria and members of the Bacteroidetes dominated in freshwater samples, while Alphaproteobacteria, Cyanobacteria and chloroplast genes dominated in marine samples. Changes in the microbial communities correlated most strongly with salinity and dissolved silicon, but were also strongly correlated with precipitation. We also identified specific gene fragments that were correlated with inorganic nutrients. Our data suggest that there is a significant and predictable change in microbial species composition along an estuarine gradient, shifting from a more complex community structure in freshwater habitats to a community more typical of open ocean samples in the marine-influenced sites. We also demonstrate the resolution and power of LH-PCR and multivariate analyses to provide a rapid assessment of major community shifts, and show how these shifts correlate with environmental variables.     

Bacterial community composition of a shallow hypertrophic freshwater lake in China, revealed by 16S rRNA gene sequences

The phylogenetic composition of a bacterial community from a hypertrophic freshwater lake in China was investigated by sequencing cloned 16S rRNA genes. Three hundred and thirty-six bacterial clones from four clone libraries in different months (March, May, July and September in 2004) were classified into 142 operational taxonomic units, most of which were affiliated with bacterial divisions commonly found in freshwater ecosystem, e.g. Alpha-, Beta-, Gamma- and Deltaproteobacteria, Bacteriodetes and Actinobacteria. The results showed that the composition of bacterial community in the July library was the most diverse one. Actinobacteria was the most significant lineage in Lake Taihu, with dominant numbers of operational taxonomic units in the May, July and September libraries. Phylogenetic analysis suggested that 53 sequences were grouped into six novel clusters which may represent specific populations indigenous to the environment. Coverage analyses indicated that the clone libraries could provide a fine inventory of bacterial diversity in the lake.     

具有多重酶活性的澳大利亚厚皮海绵共附生放线菌的研究

从我国南海澳大利亚厚皮海绵分离得到23株放线菌,并对其蛋白酶、琼胶酶、纤维素酶、几丁质酶和酯酶活性进行了筛选,同时基于16S rDNA序列信息对放线菌进行了分子鉴定。研究发现23株放线菌全部具有较强的生物酶活性,其中21株放线菌具有至少3种以上的酶活性,17株具有除酯酶活性以外的4种酶活性。通过序列比对与系统发育分析证实12株放线菌属于链霉菌属(Streptomyces)。这些分离自海绵具有多重生物酶活性的放线菌具有较大的潜在应用价值。     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

基于PCR-DGGE基因指纹的对虾体内优势细菌组成分析

采用不依赖分离培养的16S rDNA的PCR-DGGE基因指纹技术对刀额新对虾与中国对虾的鳃部与肠道优势细菌种群组成进行比较分析。研究发现:对虾鳃部与肠道存在着丰富多样的细菌;根据DGGE指纹图的聚类分析发现不同对虾及同一种对虾的鳃部与肠道内的细菌组成差异性非常大;同时也发现不同对虾体内有相同的细菌存在。首次尝试建立基于16S rDNA的PCR-DGGE基因指纹的对虾体内细菌组成揭示方法,对于今后建立对虾与养殖水体微生物和相关疾病的关系具有重要意义。     

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis

Abstract: Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.