印度尼西亚Padang Cermin热泉古菌多样性分析

通过印度尼西亚苏门答腊岛Padang Cermin热泉环境基因组DNA构建古菌16S rRNA基因文库,并利用PCR-RFLP技术对其古菌多样性和系统发育分析进行了研究.根据限制性内切酶AluⅠ和MspⅠ的特征性酶切图谱,将62个阳性克隆归类为14个分类操作单位(operational taxonomic unit,OTU),文库的覆盖度达90.32％.克隆文库的优势类群为OTU2和OTU1,分别占克隆文库的27.42％和20.96％.从每个OUT中选取一个代表性克隆进行16S rRNA基因的序列测定与系统发育分析,结果表明,测定的Padang Cermin热泉中的古菌均属于泉古菌门,包括热变形菌目(Thermoproteales)、硫还原球菌目(Desulfurococcales)、杂色泉古菌(miscellaneous crenarchaeotic group,MCG)和未培养泉古菌(uncultured Crenarchaeota,UC),该热泉代表性克隆与GenBank数据库已有16S rRNA序列的相似性为91.9％～97.8％,而且与其相似性最高的序列均来自未培养的古菌克隆,由结果可见,Padang Cermin热泉古菌群落与已报道的其他热泉古菌群落的相似性较低,表明该热泉可能具有某些独特的特征,存在着特殊的古菌生态类群.     

甘油二醚膜类脂化合物的生物地球化学研究进展

甘油二醚膜类脂化合物相对其他类脂生物标志物而言,通常具有较特定的微生物来源及环境指示意义,目前国内外还未有相关的综述性报道。综述了该类型化合物的测试方法,古菌和细菌二醚的组成特征,重点评述了如产甲烷菌、甲烷氧化菌、嗜盐菌等古菌,以及硫酸盐还原菌、Aquificales及一些嗜热细菌,在极端环境(如冷泉、热泉、热液系统)研究中甘油二醚膜类脂化合物应用的进展和可能经历的生物地球化学过程。简要介绍了环境对甘油二醚膜类脂分布的影响,并对其及其他类脂生物标志物的应用前景和发展趋势进行了展望。     

东太平洋中国多金属结核区锰结核样品中微生物群落结构的研究

通过非培养手段研究了东太平洋中国多金属结核区ES0303站点锰结核样品中的微生物群落结构.细菌16S rRNA基因克隆文库的研究结果表明：结核内细菌种群结构复杂,微生物种类丰富且各种群丰度不一（61个OTUs）,其中变形杆菌类群为优势种群,占所有细菌克隆子比例的64%,且主要分布于β/γ-、α-和δ-等3类变形杆菌（Proteobacteria）亚群之中,占比分别为34%、18%和12%.此外还存在包括酸杆菌（Acidobacteria）,放线菌纲（Actinobacteria）,绿弯菌门（Chloroflexi）,厚壁菌门（Firmicutes）,浮霉菌门（Planctomycetes）等在内的细菌类群的分布,克隆子比例依次为9%、7%、8%、2%和5%.古菌16S rRNA基因克隆文库的研究结果表明：古菌的群落结构单一（仅12个OTUs）,全部是由泉古菌海洋类群I（crenarchaeote marine group I,MGI）组成;其中MGI-η类群最为丰富,达到44%,而MGI-α、MGI-ζ和MGI-ε类群的克隆子比例分别为25%、18%和9%,另外还发现2个新的MGI分类类群.相关克隆子的数据库比对和系统发育树分析表明,并未发现已报道的直接参与铁锰氧化还原相关类群的存在,但它们大多数与来自多金属结核来源或深海来源的不可培养微生物具有较高的同源性.进一步的分析表明,锰结核内存在相当数量的氨氧化菌、硫酸盐还原菌、酸杆菌等能够改变pH值的细菌和古菌类群的存在,意味着它们可能在锰结核的形成过程中起到重要的作用.     

潮间带污泥中厌氧发酵产氢混合菌群组成与 Fe-氢酶基因多样性分析

以采自天津海水浴场潮间带的污泥为研究材料,将污泥热休克处理后富集混合菌群进行厌氧发酵产氢,测定混合菌群在发酵过程中累积产氢量和相关产氢指标(吸光度、酸碱度和氧化还原电位)的变化。结果表明,热休克处理后富集的产氢混合菌群产氢量为0.41mol H2/mol葡萄糖。产氢结束后,利用变性梯度凝胶电泳(DGGE)分析混合菌群组成和Fe-氢酶基因的多样性。混合菌群基因组DNA的16S rRNAV3区扩增产物经过DGGE电泳分离,结果得到6条丰度比较高的条带,测序结果表明优势菌为Clostridium sp.。利用梭菌氢酶基因保守性引物克隆混合产氢菌群中梭菌Fe-氢酶基因,扩增产物经DGGE电泳分离,电泳结果表明混合菌群中Fe-氢酶基因分为3个分类单元,NCBI-BLAST比对结果与Clostridium roseum和Clostridium perfringens的Fe-氢酶基因相似度分别为79%和98%。分析产氢混合菌群组成和Fe-氢酶基因多样性,可以为扩大产氢微生物种质资源奠定基础。     

超嗜热古菌Palaeococcus pacificus PolB DNA聚合酶和dUTPase在PCR反应中的应用

为了获取具有广泛适用性的DNA聚合酶,将深海热液区获得的超嗜热古菌Palaeococcus pacificus DY20341的Pol B DNA聚合酶和d UTPase进行原核表达和纯化,将得到的Pol B DNA聚合酶及d UTPase混合后用于扩增古菌、细菌和真核生物的基因.通过PCR反应成功地扩增了商业化Pfu DNA聚合酶所不能扩增的Palaeococcus pacificus DY2034的琼脂酶基因.由此可知,尽管商业化的DNA聚合酶多种多样,针对不同扩增目的片段所需的特异DNA聚合酶体系仍然是必要的,这是又一嗜热古菌B家族DNA聚合酶应用的报道.     

基于高通量测序方法分析荣成月湖潮间带趋磁细菌多样性

荣成月湖是一个典型天然潟湖,潮间带沉积物中存在多种形态趋磁细菌。通过Roche454高通量测序平台,对沉积物样品(B_S)和磁收样品(B_M)进行16SrRNA基因高通量测序,认识荣成潮间带沉积物的细菌群落结构,并了解趋磁细菌多样性及系统进化地位。研究结果表明沉积物样品中主要的细菌类群为δ-变形菌纲,占总细菌数的26.4%,其次是γ-变形菌纲和α-变形菌纲;而磁收样品中细菌多样性和种类明显降低,以α-变形菌纲占绝对优势,相对比例达72.6%。在磁收样品和沉积物样品分别发现了1 612条和186条reads与趋磁细菌相关,分别占细菌总数的5.76%和0.85%,磁收样品中趋磁细菌数是沉积物样品的6.8倍。对两个样品中获得的趋磁细菌序列进行系统进化分析,发现这些序列多数属于变形菌门的α-变形菌纲,以趋磁球菌占绝对优势,少数属于δ-变形菌纲,与多细胞趋磁原核生物亲缘关系最近。海洋趋磁螺菌属仅在B_M样品中检出,趋磁弧菌属在B_M样品优势度高于B_S样品,而多细胞趋磁原核生物和趋磁螺菌属在B_S样品中优势度更高。通过分析样品间的差异OTUs,认为荣成潮间带沉积物中可能存在大量未知的趋磁细菌新类群。研究结果为下一步培养和开发趋磁细菌这一功能菌群,发现趋磁细菌新类群及趋磁细菌生态功能提供了基础资料。     

雅浦海沟海参(Synallactidae)肠道微生物多样性及系统进化分析

为研究超深渊海参肠道微生物多样性,了解其所在沉积环境的物质循环特征,利用MiSeq高通量测序技术,对雅浦海沟海参肠道微生物16S rDNA的V3-V4和V4-V5可变区基因序列进行了扩增,并与近岸养殖刺参(Apostichopus japonicus)肠道微生物的多样性和组成进行比较。实验结果显示,与养殖刺参比雅浦海沟海参肠道细菌的多样性(辛普森指数和Chao1指数)和丰富度(ACE指数和香农指数)均较低;雅浦海沟海参肠道细菌主要包括γ-变形菌(Gammaproteobacteria,34.1%)、α-变形菌(Alphaproteobacteria,16.0%)和拟杆菌(Bacteroidetes,24.0%),古菌主要由奇古菌门(Thaumarchaeota,98.8%)组成;雅浦海沟海参肠道中,化能自养的氨氧化古菌亚硝化侏儒菌属(Nitrosopumilus)相对丰度达95.6%,甲基营养的硝酸盐还原菌(Methylotenera)相对丰度为10.6%;而养殖海参肠道中硫酸盐还原菌(28.1%)和硫氧化菌硫卵形菌属(Sulfurovum,13.7%)的相对丰度较高。16S rDNA基因序列的系统进化分析结果表明,亚硝化侏儒菌与超深渊沉积环境中奇古菌门亲缘关系最近,且遵循氨氧化古菌从陆地到深海进化的理论;雅浦海沟海参和养殖海参的肠道微生物组成差异可能反映了超深渊和近海沉积环境中氮循环、硫循环特征。本研究可以为揭示超深渊物质循环和能量流动及生命演化提供参考。     

北黄海微表层细菌丰度与可培养细菌群落结构分析

为分析海洋微表层这一特殊生境中的细菌类群,于2010—2011年4个航次对北黄海微表层和次表层海水中的总菌丰度、可培养细菌丰度和群落结构进行了分析。采用流式细胞仪测定总菌丰度,平板计数法测定可培养细菌丰度,PCR-16SrDNA分析可培养细菌的群落结构。结果表明,调查海域微表层海水总菌均值为1.76×106 cell/mL,次表层海水总菌均值为1.07×106 cell/mL。可培养细菌丰度范围是1.00×102~1.70×106 CFU/mL,微表层和次表层可培养细菌所占总菌数量的百分比分别为13.05%和0.45%。微表层对总菌的富集因子(EF)均值为2.02,可培养细菌的EF均值为74.16。PCR-16SrDNA序列分析结果表明,该海域可培养细菌分属变形菌门(Proteobacteria)(94.34%)、厚壁菌门(Firmicutes)(1.89%)、拟杆菌门(Bacteroidetes)(1.89%)和放线菌门(Actinobacteria)(1.89%)4个类群。本研究初步发现,微表层对细菌具有较强的聚集作用,尤其对可培养细菌聚集作用更为明显。微表层中可培养细菌的群落结构与次表层有所不同,其种类丰富,来源和功能多样。由此可见,微表层独特的生境成就了其独特的微生物类群,其生态功能有待于进一步研究。     

西南印度洋龙旂热液区神盾螺共附生微生物多样性分析

深海热液区的极端环境促使热液区生物形成了独特的生存机制,即与微生物的共附生关系。本文利用高通量测序技术分析了与西南印度洋龙旂热液区神盾螺（Gigantopelta aegis）共附生微生物的种类及功能。基于16S rRNA基因 V3-V4区扩增子测序结果发现：①神盾螺共附生菌中99.99%都属于细菌域,丰度最高的是变形菌门γ-,ε-,和α-变形菌纲;②神盾螺个体Ga1和Ga3的共附生菌群落中以γ-变形菌为主,个体Ga2 中以ε-变形菌为主,表明在同一热液区生活的不同个体间其共附生菌种类也存在较大差异;③宏转录组测序结果与扩增子测序结果基本一致,即神盾螺共附生细菌中变形菌门相对丰度最高,同时发现了高丰度的硫代谢、氢代谢、甲烷代谢以及环境胁迫应答基因。神盾螺共附生微生物的研究将有助于探究深海热液喷口生物的生存和适应策略,为进一步研究奠定基础。     

劳盆地热液喷口沉积物微生物多样性初步研究

采用聚合酶链式反应限制性片段长度多态性分析(PCR-RFLP)技术对劳盆地热液区TVG9站位沉积物中细菌、古菌多样性进行了调查,并构建其细菌、古菌种群的16S rRNA基因文库.研究结果表明,该站位细菌包含变形杆菌(Proteobacteria)、硝化螺旋菌(Nitrospira),疣微菌(Verrucomicrobia)等3个类群,其中变形杆菌占据优势地位,由Alphaproteobactria,Gamaproteobactria,Deltaproteobactria等3个亚群组成.古菌包含泉古菌(Crenarchaeota)和广古菌(Euryarchaeota),其中泉古菌占优势,由MG Ⅰ (Marine Group Ⅰ)和MCG(Miscellaneous Crenarchaeotal Group)两类群组成,而广古菌主要由MBGE(Marine Benthic Group E)组成.旨在揭示深海热液区的微生物多样性,为生态环境的研究提供理论支持.     

中国近海区域古菌群落结构研究概述

古菌作为海洋微生物的重要组成部分,是海洋生态系统中物质与能量传递的重要贡献者,在全球生物地球化学循环中扮演着不可或缺的角色。虽然目前绝大多数的古菌尚未获得纯培养,但近年来分子生物学技术的发展使人们认识到古菌参与甚至主导了海洋生源要素循环的关键过程。本文介绍了目前研究海洋古菌多样性的主要手段,包括:荧光定量PCR、16S rRNA基因扩增子高通量测序、宏组学以及单细胞测序等技术,系统地阐述了中国近海不同区域(包括辽河口、黄河口、渤海、南北黄海、长江口、东海北部、珠江口以及南海区域)海水及沉积物中的古菌群落结构和多样性特征。研究表明我国近海地区的主要古菌类群为广古菌门(Euryarchaeota)、奇古菌门(Thaumarchaeota)、乌斯古菌门(Woesearchaeota)和深古菌门(Bathyarchaeota)等,其生物地理学分布主要受到环境因子和距离因素的影响,且在河口和近海海域呈现出完全不同的分布特点。在此基础上,对未来的研究方向进行展望,为全面深入了解我国边缘海生态系统中古菌群落及其参与的生物地球化学循环奠定了基础。     

非培养手段分析珠江口淇澳岛海岸带沉积物中的古菌多样性

利用古菌16SrDNA特异引物对珠江口淇澳岛海岸带沉积物中古菌的多样性及垂直分布特征进行研究。结果表明珠江口淇澳岛海岸带沉积物中古菌多样性丰富,大部分为新的不可培养古菌;泉古菌在整个沉积物柱中是优势菌群,约占81%;古菌多样性随沉积物深度增加而增加,区系结构也随深度变化而呈现出明显的不同,在表层沉积物中,88%的序列属于Ⅰ型海洋泉古菌(MGⅠ),而在中层和底层检测到的古菌序列大部分与不可培养的富含甲烷的环境序列有最高的同源性,并且有15%的克隆子序列属于甲烷八叠球菌目(Methanosarcinales)和甲烷微菌目(Methanomicrobiales)。QC-PCR结果表明珠江口淇澳岛海岸带沉积物中古菌含量丰富[(1.93±0.60)×106~6.45±0.25×10716S rDNA拷贝/g],呈现随深度增加含量增加的趋势。     

A culture-dependent survey of thermophilic bacteria from hot springs in Xiamen area in China

Microbes are believed to play important roles in ecosystem function in many environments. The hot springs of Xiamen Island are close to the Xiamen Sea, and may have some characteristics different from those of inland hot springs. Microbes living in the hot springs of Xiamen may have new characteristics. However, little is known about microbial communities of hot springs close to the Xiamen Sea. A culturedependent survey of microbial population in the Xiamen hot springs was performed by using an approach combining total cellular protein profile identification and 16S rRNA gene sequencing. A total of 328 isolates of bacteria were obtained from liquid and sediment samples from the Xiamen hot springs, including neutrophilic thermophilic bacteria and moderately thermophilic acidophiles. Neutrophilic thermophilic bacteria, which grow at a temperature range of 55—90℃ including Rhodothermus marinus (Strain 1), Thermus thermophilus (Strain 2), Thermus thiopara (Strain 3), Geobacillus stearothermophilus (Strain 4), Geobacillus thermoleovorans (Strain 5), and Pseudomonas pseudoalcaligenes (Strain 6), were recovered by 2216E plates. Moderately thermophilic acidophiles, which can grow at temperatures above 50℃ and a pH range of 1.8—3.5 such as Alicyclobacillus acidoterrestris (Strain 8), Sulfobacillus acidophilus (Strain 9), and Sulfobacillus thermosulfidooxidans (Strain 10), were isolated on selective solid medium containing sulfur and Fe2+. Among these strains, Rhodothermus marinus, Thermus thermophilus and Geobacillus stearothermophilus are not only thermophiles, but also halophiles. One bacterium strain (Strain 6) shared 99% nucleotide sequence homology with Pseudomonas pseudoalcaligenes on the 16S rRNA gene sequence, but was quite different from Pseudomonas pseudoalcaligenes in biological characteristics, suggesting that it may represent a novel thermophilic species. Results indicated that various species of neutrophilic thermophiles and moderately thermophilic acidophiles were widely distributed in the Xiamen hot springs and that Rhodothermus marinus and Thermus thermophilus dominated the cultivable microbial community.     

地芽孢杆菌属DYth03 DNA聚合酶基因的克隆、表达及性质分析

从东太平洋热液区E53站位的深海沉积物样品中分离出1株能在65℃生长的嗜热菌（DYth03）.该菌的16S rDNA序列与地芽孢杆菌属（Geobacillus）内各种之间的同源性为98%以上.克隆得到DYth03的DNA聚合酶基因（DYth-pol）,序列分析表明该基因全长为2 631 bp,G＋C含量为55.5%,推测编码为876个氨基酸,与BstDNApolI的同源性最高（达98%）.将该聚合酶基因克隆到pTTQ-h表达载体上,并在大肠杆菌DH1中进行表达.对纯化到的表达产物进行酶活性测定,结果表明该酶具有聚合酶活性和5’-3’外切酶活性.     

深海冷泉古菌厌氧甲烷氧化研究进展

在富含甲烷水合物的海相冷泉沉积物中, 古菌厌氧甲烷氧化作用(anaerobic oxidation of methane, AOM)越来越受到人们的重视。目前普遍认为, AOM是由嗜甲烷古菌和硫酸盐还原菌共同调节的生物地球化学过程。16S rRNA基因分析表明, 包括AEME-1、AEME-2和AEME-3在内的多种甲烷古菌参与了AOM的过程, 它们广泛分布于全球大洋海底缺氧带。AOM过程与全球环境变化密切相关, 从深海底部冷泉区向上渗漏的甲烷气体, 绝大部分在穿透缺氧带沉积层过程中被甲烷氧化古菌所消耗, 有效减少了具有强烈温室效应的甲烷气体向大气的释放。对AOM生物地球化学过程的研究, 在认识冷泉系统碳酸盐的形成机理、控制强温室气体甲烷从海底的渗漏和开发可燃冰新能源等方面具有重要意义。     

A culture-dependent survey of thermophilic bacteria from hot springs in Xiamen area in China

Microbes are believed to play important roles in ecosystem function in many environments. The hot springs of Xiamen Island are close to the Xiamen Sea, and may have some characteristics different from those of inland hot springs. Microbes living in the hot springs of Xiamen may have new characteristics. However, little is known about microbial communities of hot springs close to the Xiamen Sea. A cuhure-dependent survey of microbial population in the Xiamen hot springs was pcrformed by using an approach combining total cellular protein profile identification and 16S rRNA gene sequencing. A total of 328 isolates of bacteria were obtained from liquid and sediment samples from the Xiamen hot springs, including neutrophilie thermophilic bacteria and moderately thermophilic acidophiles. Neutrophilic thermophilic bacteria, which grow at a temperature range of 55-90℃ including Rhodothermus marinus (Strain 1) , Thermus thermophilus (Strain 2), Thermus thiopara (Strain 3) , Geobacillus stearothermophilus(Strain 4) , Geobacillus thermoleovorans (Strain 5) , and Pseudomonas pseudoal-caligenes (Strain 6), were recovered by 2216E plates. Moderately thermophilic acidophiles, which can grow at temperatures above 50℃ and a pH range of 1. 8-3.5 such as Alicyclobacillus acidoterrestris (Strain 8) , Sul-fobacillus acidophilus (Strain 9), and Sulfobacillus thermosulfidooxidans (Strain 10), were isolated on selective solid medium containing sulfur and Fe2+. Among these strains, Rhodothermus marinus, Thermus thermophilus and Geobacillus stearothermophilus are not only thermophilcs, but also halophiles. One bacterium strain (Strain 6) shared 99% nucleotide sequence homology with Pseudomonas pseudoalcaligenes on the 16S rRNA gene se-quence, but was quite different from Pseudomonas pseudoalcaligenes in biological characteristics, suggesting that it may represent a novel thermophilic species. Results indicated that various species of neutrophilic thermophiles and moderately thermophilic acidophiles were widely distributed in the Xiamen hot springs and that Rhodother-mus marinus and Thermus thermophilus dominated the cultivable microbial community.     

俄罗斯库页岛潮间带沉积物可培养细菌的系统发育多样性

采用2216E、R2A、M1培养基,首次从俄罗斯库页岛潮间带沉积物样品中分离到82株可培养海洋细菌,经查重并菌后选取其中45株具有代表性的菌株进行了16S r RNA基因序列分析和多样性研究。结果发现,它们共分布于变形细菌门(Proteobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)4个类群(分别占45株代表菌株的42.2%、20.0%、20.0%和17.8%),代表着6个纲、27个属、44个种;同时发现其中有8个潜在新种和1个潜在新属。本研究结果表明,俄罗斯库页岛潮间带沉积物可培养细菌的系统发育多样性非常高,大部分菌株与分离自日本和韩国近海的细菌物种在系统发育关系上高度同源,但也存在不少潜在的新物种。     

Fish assemblage structure of Koycegiz Lagoon–Estuary, Turkey: Spatial and temporal distribution patterns in relation to environmental variation

Spatial and temporal variation in fish assemblage structure of Koycegiz Lagoon–Estuarine System (KLES), located on the northwestern Turkish coast of Mediterranean, was investigated along an estuarine gradient where salinity ranged from 5 in upper reaches to 40 in lower reaches during October 1993–September 1994. Throughout the study, 42 species, consisting of marine (25), marine–estuarine-dependent (12), freshwater (3), catadromous (1), and estuarine resident (1) forms, were collected in trammel nets. Although species richness of marine species was greater than that of other groups, numerical contribution by marine species to the total catch was only 16%. Tilapia spp., the most abundant species mostly during summer and early spring at upper reaches, contributed 17% of the total samples. Among the seven species of Mugilidae, which contributed 42% of the total catch, Mugil cephalus, Liza aurata, and Liza salines contributed 10, 13, and 10% of the total catch, respectively. Consistent with findings from other studies, species richness and abundance were highest during late spring and summer and the lowest during winter and early spring. Samples from sites at or near the sea had more marine species. Samples from upper reaches had more freshwater and marine–estuarine-dependent species. Canonical correspondence analysis (CCA) indicated that salinity and turbidity were the most important environmental parameters affecting fishes. Sites near the sea were associated with high salinity and low turbidity, and sites in upper reaches had low salinity and high turbidity. Thus, the pattern observed in fish assemblage structure appears to be strongly influenced by species' responses to dominant salinity and turbidity gradients.     

Characterizing lipid biomarkers in methanotrophic communities of gas hydrate-bearing sediments in the Sea of Okhotsk

We studied specific lipid biomarkers of archaea and bacteria, that are associated with the anaerobic oxidation of methane (AOM) in a cold seep environment as well as the origin of sedimentary organic matter on the continental slope off NE Sakhalin in the Sea of Okhotsk. The organic geochemical parameters demonstrated that most of the sedimentary organic matter containing hydrate layers could be derived from marine phytoplankton and bacteria, except for a station (LV39-29H) which was remarkably affected by terrestrial vascular plant. Specific methanotrophic archaea biomarkers was vertically detected in hydrate-bearing cores (LV39-40H), coinciding with the negative excursion of the δ¹³C_org at core depths of 90–100 cm below the seafloor. These results suggest that methane provided from gas hydrates are already available substrates for microbes thriving in this sediment depth. In addition, the stable isotope mass balance method revealed that approximately 2.77–3.41% of the total organic carbon (or 0.036–0.044% dry weight sediment) was generated by the activity of the AOM consortium in the corresponding depth of core LV39-40H. On the other hand, the heavier δ¹³C values of archaeol in the gas hydrate stability zone may allow ongoing methanogenesis in deeper sediment depth.