南海西沙海槽表层沉积物微生物多样性

利用非培养的分子技术研究了西沙海槽表层沉积物中的微生物群落.沉积物中扩增的古菌16S rDNA 序列分属两个大类:泉古生菌(Crenarchaeota)和广古生菌(Euryarchaeota).以Marine Crenarchaeotic GroupⅠ (古菌16S rDNA文库的49.2%)和Terrestrial Miscellaneous Euryarchaeotal Group (16.9%)为主要类群;其余为Marine Benthic Group B (9.7%)、 Marine Benthic Group A (4%)、 Marine Benthic Group D (1.6%)、Novel Euryarchaeotic Group (0.8%)和 C3(0.8%).细菌克隆子多样性明显高于古菌,16S rDNA序列分别来自变形杆菌(Proteobacteria)(细菌16S rDNA文库的30.5%)、浮霉菌(Planctomycetes)(20.3%)、放线菌(Actinobacteria)(14.4%)、厚壁菌(Firmicutes)(15.3%)、屈桡杆菌(Chloroflexi)(8.5%)、酸杆菌(Acidobacteria)(3.4%)、candidate division OP8 (2.5%)、拟杆菌/绿菌(Bacterioidetes/Chlorobi)(1.7%)和疣微菌(Verrucomicrobia)(1.7%).变形杆菌为优势类群(包括Alpha-和Delta-Proteobacteria亚群).多数克隆子为未培养细菌和古菌.结果表明南海表层沉积物中蕴含大量未知的微生物资源.     

南海北部陆坡神狐海域HS-PC500 岩心微生物多样性

[目的]本文研究南海北部陆坡神狐HS-PC500重力活塞岩心沉积物中微生物多样性.[方法]使用吖啶橙染色法计数沉积物中微生物丰度;提取沉积物微生物总DNA,使用特异性引物扩增古菌及细菌16SrRNA基因序列;对克隆文库进行系统发育分析.[结果]系统发育分析显示表层PC500-l(0-5 cm below sea floor,bsf)古菌以C3为主要类群,占该层总序列的25.6%;中层PC500-6(350-355 cm bsf)和底层PC500-11(790-795 cm bsf)古菌以Marine Benthic Group(MBG)-B为主要类群,分别占该层总序列的48.1%和38.9%.另有部分克隆序列属于MBG-A、Miscellaneous Crenarchaeotic Group(MCG)、Thermoprotei、NGC、Halobacteriales、MBG-E、South African Gold Mine Euryarchaeotic Group(SAGMEG).表层细菌以变形菌(Proteobacteria)为主要类群,占该层文库的38.3%.中层和底层细菌以绿弯菌(Choloflexi)和JS1为主要类群,分别占该层文库的28.1%、29.2%和39%、24.7%.另有部分克隆序列属于硝化螺旋菌(Nitrospirae)、放线菌(Actinobacteria)、酸杆菌(Acidobacteria)、OP8、螺旋体菌(Spirochaetes)、TM6、脱铁杆菌(Deferribacteres)、浮霉菌(Plantomycete).[结论]结果显示,HS-PC500岩心微生物丰度与甲烷浓度变化相吻合;微生物丰度较低可能与较低的总有机碳量有关;微生物多样性较高,并且随深度的增加群落结构变化明显;岩心中有关硫酸盐还原的微生物类群占优势,说明微生物的硫代谢在该海区沉积物的物质循环过程中占有重要地位.     

中国南海北部陆坡沉积物古菌多样性及丰度分析

【目的】海洋沉积物中的古菌在全球生物地球化学循环中充当重要的角色,深入了解沉积物中古菌群落的结构及功能特征是探究海洋沉积物中古菌参与生物地球化学循环和生态学功能的基础。【方法】采用高通量测序技术,分别对南海北部陆坡不同海域(东部,西部和神狐海域的7个站位)沉积物中古菌16SrRNA基因进行Illumina Mi Seq测序。【结果】中国南海北部陆坡沉积物中古菌的主要门类是Bathyarchaeota、Thermoplasmata、Woesearchaeota(DHVEG-6)、Thaumarchaeota(Marine Group I)、Lokiarchaeota和Marine Hydrothermal Vent Group(MHVG),还存在少量的AK8、Marine Benthic Group A和Terrestrial Hot Spring Crenarchaeota Group(THSCG)等。在潜在水合物区沉积物中还发现了甲烷代谢相关古菌(Anaerobic methanotrophic archaea,ANME)类群,主要为ANME-1、ANME-2ab和ANME-2c等。甲烷代谢古菌的分布特征也从甲烷代谢保守功能基因mcr A(Methyl coenzyme-Mreductase A)的扩增中得到了验证。利用定量PCR对南海沉积物中的细菌、古菌的16SrRNA基因和mcrA基因进行了定量,发现细菌16SrRNA基因拷贝数为10~5-10~7 copies/g(湿重),古菌16SrRNA基因拷贝数为10~5-10~6 copies/g(湿重),潜在水合物区mcrA基因拷贝数为10~3-10~5 copies/g(湿重)。【结论】揭示了中国南海北部陆坡沉积物中具有丰富的微生物资源,其中古菌种类多样且丰度较高,同时发现冷泉特征古菌群落,为深入认识和理解南海沉积物中微生物丰度和古菌多样性,以及解析古菌地球化学功能奠定基础。     

南海北部表层沉积物中原核微生物多样性

[目的]为研究南海北部沉积物原核微生物的多样性和群落结构.[方法]从南海北部XSCS13站位表层沉积物中扩增古菌和细菌的16S rDNA并构建文库,随机挑出阳性克隆子进行测序,选出所有的OTU构建系统进化树,进行系统发育学分析.[结果]多数克隆子来自于未培养原核微生物,沉积物中的古菌分属3大门类:泉古菌(Crenarchaeota)、奇古菌(Thaumarchaeota)和广古菌(Euryarchaeota),其中泉古菌(Crenarchaeota)为主要门类,占71％;广古菌(Euryarchaeota)最少,只有3个克隆子.泉古菌(Crenarchaeota)又以MG Ⅰ为主要类群,占61％.细菌多样性明显高于古菌,共9个门类:变形杆菌(Proteobacteria)(32.6％)、疣微菌(Verrucomicrobia)(3.0％)、拟杆菌门(Bacteroidete)(5.2％)、酸杆菌(Acidobacteria)(4.4％)、绿弯菌(Chloroflexi)(6.0％)、厚壁菌(Firmicute)(3.7％)、浮霉菌(Planctomycete)(5.2％)、芽单胞菌(Gemmatimonadete)(11.1％)、放线细菌(Actinobacteria)(4.4％).变形杆菌为优势类群(包括α-Proteobacteria、γy-Proteobacteria和δ-Proteobacteria 3个亚群),其中γ-Proteobacteria是Proteobacteria中的优势种群,占54.5％.另外所有原核微生物总共有超过50％的克隆子与硫酸盐的还原以及甲烷的形成相关.[结论]结果表明南海北部XSCS13站位表层沉积物中原核微生物的多样性非常丰富,其中蕴含大量未知的微生物资源;另外古菌和细菌群落结构表明该位点可能处于富含甲烷的冷泉活动区.     

海南东寨港红树林不同植被土壤微生物群落结构比较

【目的】比较不同植被下红树林土壤细菌和古菌的多样性及群落结构,认识红树林土壤微生物资源多样性。【方法】直接提取红树林土壤总DNA,采用细菌通用引物27F/1492R和古菌通用引物Arch21F/Arch958R进行PCR扩增,构建细菌和古菌16S rRNA基因文库,对海南东寨港自然保护区秋茄林、无瓣海桑林和无红树林裸滩土壤的细菌和古菌多样性和群落结构进行分析和比较。【结果】3种土壤样品的细菌类群包括变形细菌门(Proteobacteria)等16个类群,其中变形细菌门(Proteobacteria)与绿屈挠菌门(Chloroflexi)是优势类群;古菌包括6个嗜泉古菌界(Crenarchaeota)类群和7个广域古菌界(Euryarchaeota)类群,分别以Marine Benthic Group C、Marine Benthic Group D为优势类群。多样性指数(H’)和物种丰富度指数(Schao1)表明,本地种秋茄林下土壤细菌和古菌的多样性指数最高,外来种无瓣海桑显著低于秋茄林,甚至明显低于相邻无红树林裸滩沉积物;不同植被下土壤细菌和古菌群落结构存在显著差异,秋茄林土壤微生物群落结构和无红树林裸滩沉积物更相似。【结论】红树林土壤微生物类群丰富,不同植被下土壤细菌和古菌多样性和群落结构存在显著差异。     

西藏羊八井废弃地热热井的细菌多样性

【目的】旨在研究羊八井热田两废弃地热热井沉积物及热水中的细菌多样性。【方法】采用非培养分子分析技术分别构建了热井A沉积物A样、水样A以及热井B沉积物B样总DNA的16S rRNA基因文库,并对每个文库中随机挑选的克隆子进行扩增rDNA限制性酶切片段分析（amplified ribosomal DNA restriction analysis, ARDRA）,分别选取文库中不同分类单元的阳性克隆子进行测序,将所得到的数据与国际基因数据库GenBank 的序列进行相似性比较并构建系统发育树。【结果】热井A和热井B的细菌类群大多为典型的栖热细菌,均以变形菌（Proteobacteria）为优势类群（在热井A沉积物A中为41.08% 、在水样A中为38.00%,以及在热井B的沉积物B中为42.57%,下同）,以恐球菌－栖热菌（Deinococcus-Thermus）为亚优势类群（10.71%、20.00%、21.30%）;此外两热井沉积物中均发现热泉中比较少见的且为亚优势类群的酸杆菌（Acidobacteria）(在热井A沉积物A中为16.07%、在热井B的沉积物B中为19.15%),热井A沉积物中第三个亚优势类群为壁厚菌门(Firmicutes)的真杆菌（Eubacterium sp.）（14.28%）;水样A中则没有检测到酸杆菌,而以产水菌门（Aquificae）的产氢杆菌属（Hydrogenobacter）为另一亚优势类群(16.00%)。此外两热井中均检测到绿弯菌（Chloroflexi）、蓝藻（Cyanobacteria）以及噬纤维菌-屈挠杆菌-拟杆菌群 (Cytophaga-Flexibacter-Bacteroides,CFB group)等类群的细菌。【结论】通过与文献比较分析,发现羊八井热田中细菌类群大多为典型的栖热细菌,如恐球菌－栖热菌、产水菌、绿弯菌、蓝藻以及candidate division OP类群的微生物等;但是尚有一些不常见的细菌类群如弧菌（Vibrio sp.）、Bacteriovorax sp.、未培养的酸杆菌和Holophaga（uncultured Holophaga/Acidobacterium）和未确定的疣微菌（Unidentified Verrucomicrobium）等,羊八井热田地热的开发变迁和地下冷热水交融可能与细菌类群的复杂性有关。     

珠海市海-陆交错带水环境原核生物多样性

采用16S rRNA基因PCR-RFLP分析技术,对珠海市海-陆交错带的海水和地下水进行原核生物多样性分析,并将所得数据与国际基因数据库Genbank进行相似性比较及聚类分析.结果表明：海水细菌以变形菌居多,其次是古菌和芽单胞菌,以及Candidate division OP3、OP8和浮霉菌等;海 陆交错带地下水细菌则以古菌居多,其次是变形菌和鞘脂杆菌,以及Candidate division OP3、放线菌和假单胞菌等.海-陆交错带地下水中大部分优势类群与源于海洋的不可培养细菌具有很高的相似性.地下水样中存在大量能降解有机质和具有净化水体潜力的细菌,说明在长期演化下,海-陆交错带兼备了海洋生态系统和陆地生态系统的特征.     

福建省3座水库库心沉积物聚磷菌的群落特征

聚磷菌在水库沉积物磷的代谢循环中起着重要作用.采用T-RFLP技术和克隆测序,以多聚磷酸盐激酶基因(ppk1)作为标记基因,研究对比了福建省3座富营养化水库(九龙江西陂水库、平潭三十六脚湖水库和三明东牙溪水库)库心沉积物中聚磷菌的多样性及群落结构.结果表明:3座水库库心沉积物聚磷菌的多样性存在一定差异,但不显著,以三十六脚湖库心样品中聚磷菌多样性最高(Shannon指数H=2.89±0.03,Simpson指数D=0.06±0.01).克隆测序和比对结果分析表明,三十六脚湖水库沉积物样品中聚磷菌种类最多,结构最复杂,这与T-RFLP的结果一致.3座水库库心沉积物样品中聚磷菌优势菌属差异较为明显,但均以变形菌门、放线菌门和酸杆菌门为主,这3个门在3座水库库心沉积物样品中所占比重分别达74.5%、85.0%和75.0%.其中,变形菌门中的厌氧粘细菌属、酸杆菌门中的Solibacter属在3座水库库心沉积物样品中均为优势菌属.沉积物中Fe/Al-P与聚磷菌多样性指数的相关性最显著.Fe/Al-P对聚磷菌群落结构影响最为显著,3个水库共有优势菌属厌氧粘细菌与各形态磷呈正相关,且与OP、Ca-P等难溶解性磷呈显著相关,Solibacter与各形态磷均呈负相关.表明聚磷菌与富营养化水库沉积物磷代谢循环密切相关.     

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。     

洋山深水港海域表层海水中古菌多样性特点

利用DGGE、定量PCR以及16S rRNA基因克隆文库技术对洋山深水港海域表层海水中的古菌多样性组成进行了调查和分析。通过Quantity One软件分析不同样品的DGGE图谱,发现洋山深水港3个不同海域(小洋山港口区、大洋山港口区、中间航道)的古菌群落多样性组成不存在明显的区别;利用定量PCR的方法对3个不同样品中的古菌16S rRNA基因拷贝进行计数,发现3个不同海域的古菌16S rRNA基因拷贝数的数量关系为:大洋山海域((1.69±0.19)×10~6copies/mL)中间航道((2.17±0.20)×10~6copies/mL)小洋山港口区((2.42±0.22)×10~6copies/mL),这说明洋山深水港3个不同海域的古菌群落组成是一致的,只是在数量上略有差异,为对该海域古菌的更深入调查研究提供了便利和数据基础。洋山深水港海域表层海水的古菌16S rRNA基因文库分析表明,古菌由三大门类组成:广古菌门(Euryarchaeota)、泉古菌门(Crenarchaeota)和奇古菌门(Thaumarchaeota)。广古菌门所占比例约为26.8%,泉古菌门约为33.0%,奇古菌门约为40.2%。广古菌门含有2个类群,分别是Mathanobacteriales(10.3%)和Marine GroupⅡ(16.5%);泉古菌门含有1个类群,为Unidentified Crenarchaeotic Group(33.0%);奇古菌门含有1个类群,为Marine GroupⅠ(40.2%)。结果显示,Mathanobacteriales类群和Marine GroupⅡ类群的比例失调,这可能预示着洋山深水港海域存在油污污染及外来微生物物种入侵的风险。     

Microbial diversity in surface sediments of the Xisha Trough, the South China Sea

Microbial communities were obtained from the surface sediments of the Xisha Trough using the culture-independent technique. The characteristics of the 16S rDNA gene amplified from the sediments indicated that archaeal clones could be grouped into Euryarchaeota and Crenarchaeota, respectively. Two archaeal groups, Marine Crenarchaeotic GroupI and Terrestrial Miscellaneous Euryarchaeotal Group, were the most dominant archaeal 16S rDNA gene components in the sediments. The remaining components were related to the members of Marine Benthic Group B, Marine Benthic Group A, Marine Benthic Group D, Novel Euryarchaeotic Group and C3. The bacterial clones exhibited greater diversity than the archaeal clones with the 16S rDNA gene sequences from the members of Proteobacteria, Planctomycetes, Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, candidate division OP8, Bacterioidetes/Chlorobi and Verrucomicrobia. Most of these lineages represented uncultured microorganisms. The result suggests that a vast amount of microbial resource in the surface sediments of the South China Sea has not been known.     

The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L.

The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated. A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA. The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation. The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library. The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48%. Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring. The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated. The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes. The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community. The diversity within the archaeal domain was low. Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity). The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea. The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.     

Community Structure of Archaea from Deep-Sea Sediments of the South China Sea

Using the archaeal 16S rRNA gene, we determined the community structures of archaea of subseafloor sediments (～9-11 m below seafloor) from two geographically distant cores (MD05-2896, south, water depth 1,657 m; MD05-2902, north, water depth 3,697 m) in the South China Sea. Euryarchaeota accounted for 61.4% of total archaeal clone libraries at MD05-2896 and 56.2% at MD05-2902. At both locations, the Euryarchaeota-related sequences were dominated by Marine Benthic Group D, Terrestrial Miscellaneous Eryarchaeotal Group, and South African GoldMine Euryarchaeotal Group; the Crenarchaeota-related sequences were dominated by Marine Benthic Group B, Marine Group I, pSL12, and C3. The community structure showed no significant difference with depth at each location, suggesting the lack of stratification of archaeal populations in the deep-sea marine sediments in the South China Sea. On the other hand, the community structure is significantly different between the two sites, which may be related to geographical difference in the South China Sea.     

Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting

Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.     

Microbial Diversity in the Deep Marine Sediments from the Qiongdongnan Basin in South China Sea

The continental shelf and slope in the northern South China Sea is well known for its prospect of oil/gas/gas-hydrate resources. To study microbial communities and their roles in carbon cycling, a 4.9-m sediment core was collected from the Qiongdongnan Basin on the continental slope of the South China Sea during our cruise HY4-2005-5 in 2005. Geochemical, mineralogical, and molecular phylogenetic analyses were carried out. Sulfate concentration in pore water decreased with depth. Abundant authigenic carbonates and pyrite were observed in the sediments. The bacterial community was dominated by aerobic and facultative organisms. Bacterial clone sequences belonged to the Gamma-, Alpha-, Deltaproteobacteria and Firmicutes group, and they were related to Fe(III) and/or Mn(IV) reducers, sulfate reducers, aromatic hydrocarbon degraders, thiosulfate/sulfite oxidizers, and denitrifiers. Archaeal clone sequences exhibited greater overall diversity than the bacterial clones with most sequences related to Deep-Sea Archaeal Group (DSAG), Miscellaneous Crenarchaeotic Group (MCG), and Uncultured Euryarchaeotic Clusters (UECs). Archaeal sequences related to Methanosarcinales, South African Gold Mine Euryarchaeotic Group (SAGMEG), Marine Benthic Group-D (MBG-D) were also present. Most of these groups are commonly present in deep-sea sediments, particularly in methane/organic-rich or putative methane hydrate-bearing sediments.     

Characterization of Archaeal Community in Contaminated and Uncontaminated Surface Stream Sediments

Archaeal communities from mercury and uranium-contaminated freshwater stream sediments were characterized and compared to archaeal communities present in an uncontaminated stream located in the vicinity of Oak Ridge, TN, USA. The distribution of the Archaea was determined by pyrosequencing analysis of the V4 region of 16S rRNA amplified from 12 streambed surface sediments. Crenarchaeota comprised 76% of the 1,670 archaeal sequences and the remaining 24% were from Euryarchaeota. Phylogenetic analysis further classified the Crenarchaeota as a Freshwater Group, Miscellaneous Crenarchaeota group, Group I3, Rice Cluster VI and IV, Marine Group I and Marine Benthic Group B; and the Euryarchaeota into Methanomicrobiales, Methanosarcinales, Methanobacteriales, Rice Cluster III, Marine Benthic Group D, Deep Sea Hydrothermal Vent Euryarchaeota 1 and Eury 5. All groups were previously described. Both hydrogen- and acetate-dependent methanogens were found in all samples. Most of the groups (with 60% of the sequences) described in this study were not similar to any cultivated isolates, making it difficult to discern their function in the freshwater microbial community. A significant decrease in the number of sequences, as well as in the diversity of archaeal communities was found in the contaminated sites. The Marine Group I, including the ammonia oxidizer Nitrosopumilus maritimus, was the dominant group in both mercury and uranium/nitrate-contaminated sites. The uranium-contaminated site also contained a high concentration of nitrate, thus Marine Group I may play a role in nitrogen cycle.     

Bacterial Community Composition in Different Sediments from the Eastern Mediterranean Sea: a Comparison of Four 16S Ribosomal DNA Clone Libraries

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.     

Upstream-independent ribosomal RNA amplification analysis (URA): a new approach to characterizing the diversity of natural microbial communities

Here, we propose an advanced method for recently developed fingerprinting strategies to analyse microbial populations by direct detection of 16S rRNA sequences occurring in natural habitats. The differential display (DD) technique, which is widely used to analyse for eukaryotic gene expression, was optimized to assess bacterial rRNA diversity in environmental samples. Double-stranded cDNAs of rRNAs were synthesized without a forward primer digested with endonuclease and ligated with a double-stranded adapter. The fragments obtained were then amplified using an adapter-specific extended primer and a 16S rDNA universal reverse primer pair displayed by electrophoresis on a polyacrylamide gel. We validated this approach by characterization of a microbial community colonizing a geothermal (48°C) vent system located close to the eruption zone of the south-east crater of the Mount Etna volcano, Sicily. Analysis of the patterns of abundant 16S rRNA revealed a considerable diversity of metabolically active bacteria phylogenetically clustering within the Crenarchaeota , Cyanobacteria , Firmicutes , Planctomycetales and Thermus divisions. Two sequence phylotypes were affiliated with uncultivated representatives of the recently described candidate division OP10 from a Yellowstone hot spring.