Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.     

Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity

This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the α-subunit of particulate methane monooxygenase ( pmoA ) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum . Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.     

实时荧光定量PCR(TaqMan)法测定外源基因的拷贝数

实时荧光定量PCR是近年新兴的一项技术,因其快速、方便、便宜,需要DNA样品量少,无需放射性检测等优点被广泛应用于基因的定量分析。该文就实时荧光定量PCR(TaqMan)技术的发展、基本原理及测定外源基因拷贝数的技术流程做一介绍。     

基于TaqMan实时荧光定量PCR技术的西花蓟马快速检测

西花蓟马Frankliniella occidentalis（Pargande）是世界性害虫,2003年在我国首次发生危害。针对西花蓟马与其他种类蓟马形态相似、难以快速区分的问题,本文在SCAR标记基础上,采用TaqMan实时荧光定量PCR技术,设计1对特异性引物和1条MGB探针,扩增出大小为138bp的特异片段。以质粒DNA为标准品建立了标准曲线（R2=0.9965）,种特异性检验结果显示,该引物和探针只能检测到西花蓟马的荧光信号,而对其他种类的蓟马不具有检测能力。并且可以定量检测西花蓟马不同虫态靶标DNA片段的拷贝数。该检测体系重复性强、稳定性高,在口岸检疫以及植物种苗及其产品调运中的有害生物检测和监测中具有重要意义。     

莫克兰冷泉区沉积物原核微生物群落组成及其环境响应

【目的】冷泉系统广泛存在于大陆边缘地区,其典型特征是在海底渗漏出大量富含以甲烷为主的碳氢化合物和硫化氢等成分的低温流体。冷泉也因其独特的地球化学条件孕育着独特的原核微生物群落结构,然而,原核微生物组成与冷泉环境之间的响应关系却并不清楚。【方法】本文以莫克兰大陆边缘活跃冷泉区沉积物柱状样为研究对象,沿深度剖面分析了沉积物中的CH4以及孔隙水SO42–、H2S浓度等关键地球化学参数,并基于16S rRNA基因高通量测序对冷泉沉积物原核微生物的群落结构及其空间变化进行了系统分析。【结果】根据其硫酸盐-甲烷浓度剖面特征,从上向下,将沉积物垂向剖面划分为硫酸盐还原区(SZ)、硫酸盐-甲烷转换区(SMTZ)和产甲烷区(MZ)。通过原核微生物α多样性与基因定量研究发现,随着深度增加微生物多样性与丰度呈逐渐降低的趋势。16SrRNA基因高通量测序结果表明,SZ中以硫氧化细菌γ-变形菌纲、α-变形菌纲和埃普西隆杆菌门为主,且以硫酸盐为电子受体的与有机质降解相关的原核微生物JS1、绿弯菌门、洛基古菌纲、深古菌纲及底栖古菌纲的相对含量也较高;SMTZ存在较高含量的ANME-1a、ANME-1b与SEEP-S...     

基于mcrA基因的沁水盆地煤层气田产甲烷菌群与途径分析

【目的】分析沁水盆地煤层气田不同煤层气井产出水样中产甲烷菌群和生物成因气的生成途径。【方法】以甲基辅酶M还原酶基因(mcr A)作为目标基因,采用454焦磷酸高通量测序方法,同时比对NCBI功能基因文库中的mcr A序列,分析不同煤层气井产出水中的产甲烷菌群。【结果】高通量测序表明,5个出水样产甲烷菌群OTUs(Operational taxonomic units)数为64–157个,共有的为22个,各占样品总数14%-34%;样品共检测到4种已知菌属,即甲烷杆菌属(Methanobacterium)、甲烷微菌属(Methanomicrobium)、甲烷叶菌属(Methanolobus)和甲烷螺菌属(Methanospirillum),优势菌属均为Methanobacterium。系统发育分析表明,未明确地位的菌属主要与Methanobacterium、Methanomicrobium、产甲烷球菌属(Methanococcus)和甲烷囊菌属(Methanoculleus)有较近的亲缘关系。5个样品中菌属所占比例不同,检测到的菌属类别大致相同。所有检测样品生物成因煤层气(Coalbed methane,CBM)的生成途径主要为氢营养型产甲烷途径。【结论】沁水盆地不同煤层气田产甲烷菌群菌种差异比较大,但生物成因气生成途径基本相似,与地理位置和煤藏条件没有相关性。     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

荧光定量PCR检测人巨细胞病毒的方法学建立

目的建立人巨细胞病毒（HCMV）的TaqMan MGB探针荧光定量PCR（FQ—PCR）检测方法。方法选取HCMV MIE exon4为PCR扩增靶序列,经TA克隆构建重组质粒作为定量标准品,经FQ—PCR反应条件的优化及方法学评价,再将其应用于临床检测。结果FQ—PCR最适循环参数为：95℃ 5 min;95℃ 20 s,60℃ 60 s（40 cycles）,20μl最适反应体系为：2．0mmol／L Mg^2＋、0．5μmol／L引物、1．5μmol／L探针、200μmol／L dNTP、2110×buffer、1．0 U Taq酶、2．0μl DNA模板。检测批内CV（变异系数）值为1．32％,批间CV值为1．96％;特异性较好;线性范围为10^2-10^8copies／μl。结论成功地建立了检测HCMV的FQ—PCR法,完全适用于临床检测。     

基于Taqman-MGB探针的亚稀褶红菇实时荧光定量PCR检测方法

本研究建立了一种基于Taqman-MGB探针的亚稀褶红菇Russula subnigricans实时荧光定量PCR检测方法。根据亚稀褶红菇与其近似种的内转录间隔区（internal transcribed spacers,ITS）序列差异,设计合成1对引物和1条特异性Taqman-MGB探针,并用常见有毒红菇种类进行验证。结果显示,引物特异性良好,仅亚稀褶红菇出现荧光信号,完成整个检测过程只需2h。该法能够为毒蘑菇中毒的快速检测提供技术支持。     

甲烷氧化菌研究进展

甲烷氧化菌以甲烷为其唯一的碳源和能源 ,在全球大气甲烷平衡中起着重要的作用 ,它还可以降解卤代化合物 ,在污染治理方面具有潜在价值。本文从甲烷氧化菌的分类出发 ,对甲烷氧化菌氧化甲烷的机理及影响因素、甲烷氧化菌的生理、生态分布及检测方法、甲烷氧化菌降解有机污染物的潜在应用等作一综述 ,分析目前研究中存在的问题 ,并指出今后应加强研究的方面。     

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.     

High rates of denitrification and nitrate removal in cold seep sediments

We measured denitrification and nitrate removal rates in cold seep sediments from the Gulf of Mexico. Heterotrophic potential denitrification rates were assayed in time-series incubations. Surficial sediments inhabited by Beggiatoa exhibited higher heterotrophic potential denitrification rates (32 μ N reduced day⁻¹) than did deeper sediments (11 μ N reduced day⁻¹). Nitrate removal rates were high in both sediment horizons. These nitrate removal rates translate into rapid turnover times (<1 day) for the nitrate pool, resulting in a faster turnover for the nitrate pool than for the sulfate pool. Together, these data underscore the rigorous nature of internal nitrogen cycling at cold seeps and the requirement for novel mechanisms to provide nitrate to the sediment microbial community.     

基于三步荧光定量PCR技术揭示不同产区白酒酿造系统中Lactobacillus sp.的分布特征

[背景]Lactobacillus sp.是一株存在于白酒酿造系统中的功能微生物,但是针对Lactobacillus sp.的定量方法及其在中国白酒酿造系统中的分布尚未被研究。[目的]建立一种基于特异性引物的荧光定量PCR定量方法,并应用于实际生产检测,揭示Lactobacillus sp.在中国白酒酿造系统中的分布特征。[方法]基于16S rRNA基因序列设计特异性引物,并通过PCR验证引物特异性;优化荧光定量PCR反应程序,提高引物扩增效率;定量分析所采集样本中Lactobacillus sp.的含量,揭示Lactobacillus sp.在中国白酒酿造系统中的分布特征。[结果]设计了一对扩增产物大小为445 bp的特异性引物。通过优化扩增条件,构建含有变性、退火、延伸过程的三步荧光定量PCR方法,该方法扩增线性较强,R²>0.99;灵敏度高,定量限为17.9 copies/μL;重复性好,Ct值的变异系数小于1%。跟踪检测全国10个产区代表产地的白酒酿造系统,其中8个产区均检测到Lactobacillus sp.,在相同产地不同酿造工艺的酿造系统中均检...     

Methane as an Organic Matter Source and the Trophic Basis of a Laptev Sea Cold Seep Microbial Community

An area of cold methane seeps at the bottom of the Laptev Sea was investigated. High rates of methane oxidation were revealed in the sediments and in the water column. Anaerobic methane oxidation carried out by the ANME-2 a/b consortium was coupled to sulfate reduction. Bacteria of the genera Sulfurovum and Arcobacter were the agents of the sulfur cycle. Methane unconsumed in the sediments diffused into the near-bottom water, where it was oxidized by methanotrophic bacteria. Methanotrophic activity was essential for development of symbiotrophic tubeworms of the upper sediment layers, which were responsible for the process of bioturbation.     

Novel archaeal macrocyclic diether core membrane lipids in a methane-derived carbonate crust from a mud volcano in the Sorokin Trough, NE Black Sea

A methane-derived carbonate crust was collected from the recently discovered NIOZ mud volcano in the Sorokin Trough, NE Black Sea during the 11th Training-through-Research cruise of the R/V Professor Logachev. Among several specific bacterial and archaeal membrane lipids present in this crust, two novel macrocyclic diphytanyl glycerol diethers, containing one or two cyclopentane rings, were detected. Their structures were tentatively identified based on the interpretation of mass spectra, comparison with previously reported mass spectral data, and a hydrogenation experiment. This macrocyclic type of archaeal core membrane diether lipid has so far been identified only in the deep-sea hydrothermal vent methanogen Methanococcus jannaschii. Here, we provide the first evidence that these macrocyclic diethers can also contain internal cyclopentane rings. The molecular structure of the novel diethers resembles that of dibiphytanyl tetraethers in which biphytane chains, containing one and two pentacyclic rings, also occur. Such tetraethers were abundant in the crust. Compound-specific isotope measurements revealed delta13C values of -104 to -111/1000 for these new archaeal lipids, indicating that they are derived from methanotrophic archaea acting within anaerobic methane-oxidizing consortia, which subsequently induce authigenic carbonate formation.     

微生物介导的甲烷厌氧氧化过程及其影响因子研究进展

温室气体甲烷减排是全球变化领域的研究热点,甲烷厌氧氧化(anaerobic methane oxidation,AOM)过程是一个以前被忽视的甲烷汇,在调控全球甲烷收支平衡及减缓温室效应等方面扮演着十分重要的角色。AOM微生物以甲烷为唯一电子供体,与硫酸盐(SO42-)、亚硝酸盐(NO2-)/硝酸盐(NO3-)、金属离子(Fe3+、Mn4+、Cr6+)等结合完成氧化还原过程,该过程是耦合碳、氮、硫循环的关键环节。本文系统整理分析了不同AOM类型、发生机理、相关功能微生物类群(ANME-1、ANME-2、ANME-3、NC10、MBG-D)及影响AOM过程的关键调控因子的最新研究进展。结果发现,目前80%以上研究都集中在对最常见电子受体类型(SO42-/NO3-/NO2-/Fe3+/Mn4+)的AOM相关过程,而忽视了潜在的新型电子受体(AQDS/HAs O42-/Cr6+/ClO4-等)的耦合作用过程和相对应的微生物类型及作用机理。对未来AOM研究方向提出展望,以期为研究甲烷厌氧氧化菌在不同生态系统中的生态分布及减缓全球温室气体排放提供新的思路。     

Effect of tea saponin on methanogenesis,microbial community structure and expression of mcrA gene,in cultures of rumen micro‐organisms

Aims: To determine the in‐vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. Methods and Results: Saponin extracted from tea seeds was added to (1) an in‐vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme‐M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real‐time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. Conclusions: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal‐associated methanogens. Significance and Impact of the Study: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.