马里亚纳海沟不同深度水样微生物分离与多样性分析

【背景】马里亚纳海沟挑战者深渊是地球表面最深点,认识其微生物群落结构和分离培养的微生物对挖掘新的微生物资源具有重要意义。【目的】对马里亚纳海沟不同深度水样进行细菌分离培养,并与这些样品的微生物群落结构进行比较,认识进一步要分离培养的微生物类型。【方法】采用不同培养基对马里亚纳海沟两个站位不同深度水样进行细菌分离培养,并通过Illumina MiSeq高通量测序分析各个水样的细菌和古菌的群落结构。【结果】从来自两个站位不同深度的6个水样样品中分离获得783株细菌,属于4个门6个纲28个属。其中,变形菌门占主导地位,67.8%的菌株属于γ-变形菌纲。分离获得的菌株主要属于亚硫杆菌属、假单胞菌属和交替假单胞菌属,它们在这些样品中广泛分布,且在高通量测序结果中也能检测到。高通量测序结果表明除浅层样品优势微生物为蓝细菌外,其他样品以变形菌门占主导;不同深度样品的微生物群落结构存在较大差异。【结论】马里亚纳海沟不同深度水样中不仅分离培养出了相对丰度较高的一些细菌属,也分离得到一些相对丰度较低的微生物类型。从马里亚纳海沟水样中分离培养获得的细菌菌株资源将用于功能微生物和功能酶挖掘等相关研究,这有利于深渊微生物资源挖掘。     

基于宏基因组学分析湘西腊肉的细菌多样性

目的 解析湘西成熟腊肉制品中微生物群落结构和种群丰度.方法 采集湖南慈利县、辰溪县和古丈县3个样地成熟腊肉样品,提取样品细菌总DNA,利用454焦磷酸高通量测序法进行测序,并进行生物信息学分析.结果 从慈利腊肉获得10449条优质序列,聚类得到132个OTUs,注释为8个菌门,鉴定出82个属;其中变形菌门为主要细菌类群...     

驯化对生物阴极微生物燃料电池中阴极微生物群落多样性的影响

【背景】生物阴极微生物燃料电池因其构造成本低和阴极可持续性发展的优点而成为一种很有前途的废水处理系统,但阴极微生物的氧化还原性能限制了其在实际应用中的推广。【目的】为了提高生物阴极的性能,需要深入了解影响阴极氧化还原性能的微生物群落。【方法】利用16S rRNA基因高通量测序技术分析对比原始接种污泥样品和驯化后阴极电极上生物膜样品多样性及结构变化。【结果】测序结果表明,原始接种污泥样品与驯化后阴极电极生物膜样品中微生物群落种类和结构存在显著差异,驯化后阴极电极生物膜样品中变形菌门(Proteobacteria)、γ-变形菌纲(Gammaproteobacteria)和特吕珀菌属(Trueperaceae)相对丰度比例高于原始污泥样品,成为优势菌群。【结论】驯化对系统阴极电极生物膜群落影响显著,随着产电量的输出,优势菌群不断富集,最终形成一个适应该实验环境下的新的微生物群落。对优势菌群结构和变化进行探讨,为生物阴极的研究补充更多生物学方面的理论基础。     

敦煌莫高窟第98窟壁画表面菌斑的群落结构分析

【目的】确定第98窟壁画表面白色污染物内微生物微观特征, 分析其群落组成、结构特点及诱发壁画病害微生物产生的因素, 为石窟寺保护和旅游管理提供建议。【方法】利用无菌手术刀收集壁画表面白色污染物样品; 利用扫描电子显微镜(SEM)分析样品中微生物体微观形貌; 通过提取样品总DNA、扩增细菌16S rDNA、构建克隆文库、测序和系统发生关系分析等技术研究壁画微生物群落组成与结构特点。【结果】壁画白色污染物中存在大量具有微生物特征的结构体, 形态多呈短杆状和卵圆形, 大小在 (3.0?5.5)?μm×(1.5?2.5) μm之间。共得到克隆文库序列111条, 主要为变形菌门γ-变形菌亚门肠杆菌科(Enterobacteriaceae)与假单孢菌科(Pseudomonadaceae)成员。群落组成和结构分析表明所得序列主要隶属于肠杆菌属(Enterobacter)、埃希菌属(Escherichia)、固氮菌属(Azotobacter)、沙雷氏菌属(Serratia)和克雷伯菌属(Klebsiella); 埃希菌属和肠杆菌属为优势属, 分别占克隆文库中总序列的46.8%和35.1%, 二者在自然界分布广泛, 大多属于人类致病菌。【结论】莫高窟第98窟壁画表面白色污染物主要为病害细菌生长所形成的菌斑群落集成。变形菌门在壁画细菌克隆文库中占绝对优势, 壁画病害微生物的出现和蔓延可能与该洞窟之前长期旅游开放存在一定关联。     

西北印度洋卡尔斯伯格脊卧蚕热液羽流影响区细菌群落结构特征及其演化

【目的】热液羽流影响区包括热液羽流流经区域和羽流中性浮力层下方受热液颗粒物影响的区域。随着热液羽流的演化,热液羽流影响区内微生物群落的结构组成也会发生相应的变化,但是,由于观测和取样困难等原因,迄今热液羽流影响区不同空间位置微生物的群落结构特征及其在月际尺度上的演化尚不清楚。【方法】中国大洋49航次在卧蚕1号热液喷口东南侧300 m处投放了沉积物捕获器锚系,在不同离底高度开展了为期18个月的观测和时序采水。本文采用Illumina MiSeq高通量测序技术对水样中的微生物类群进行测序分析,结合现场实时探测的浊度异常资料,研究卧蚕热液区附近中性浮力羽流和热液颗粒沉降区细菌群落结构的特征和演化及其影响因素。【结果】结果表明,样品中细菌群落以γ-变形菌纲(Gammaproteobacteria)、弯曲菌纲(Camplylobacteria)、α-变形菌纲(Alphaproteobacteria)、拟杆菌纲(Bacteroidia)、梭菌纲(Clostridia)和脱硫叶菌纲(Desulfobulbia)为主。在时间上,优势类群的相对丰度随浊度起伏发生变化,当浊度异常值升高时,弯曲菌纲相对丰度...     

基于高通量测序的辐射污染区细菌群落特征分析

【目的】为了更加全面地揭示辐射污染区细菌种群多样性,了解辐射污染对辐射区土壤中细菌群落结构的影响。【方法】运用高通量测序方法,分别进行了土样细菌16S r RNA基因的V3可变区测序,进而对无辐射污染对照和不同辐射污染程度的土样中细菌群落组成和多样性进行分析。【结果】研究共获得110 348条有效序列,17 604个OTUs,共涉及细菌域的19个门和6个潜在菌门和其它未分类菌群的726个属。多样性分析表明,辐射污染会引起土壤样品中微生物群落的分布显著差异化,显著提高细菌群落种群多样性和微生物丰度。微生物群落组成分析发现,在辐射污染胁迫下,辐射污染区样品中变形杆菌门分布比例显著下降;随着辐射污染程度的提高,放线菌门所占比例逐步提高,未分类菌门、厚壁菌门和酸杆菌门也有明显的提高。同时,研究发现辐射污染区中存在着大量未分类菌属。【结论】研究揭示了辐射污染区极为丰富的细菌多样性,大量微生物新物种资源有待发掘。     

基于高通量测序的4种硝化细菌富集培养物微生物群落结构分析

【目的】比较分析4种硝化细菌富集培养物(以铵盐为氮源的淡水富集物A、以亚硝酸盐为氮源的淡水富集物B、以铵盐为氮源的低温淡水富集物C和以亚硝酸盐为氮源的海水富集物D)的微生物群落结构与组成。【方法】分别提取样品的总DNA,采用高通量测序技术,分析微生物群落的组成、丰度和多样性。【结果】在不同微生物的分类水平,4个样品共检测到24门47纲129属。4个样品的优势菌门均为变形菌门;样品A、B、C的优势菌纲为β-变形菌纲和γ-变形菌纲,样品D的优势菌纲为γ-变形菌纲、δ-变形菌纲和芽孢杆菌纲;而优势菌属各不相同,其中样品A为亚硝化单胞菌属(24.56%),样品B为链霉菌属(7.15%),样品C为噬菌弧菌属(19.36%)和类诺卡氏菌属(19.35%),样品D为嗜酸菌属(13.6%)和柄杆菌属(11.5%)。共检测出7种具有硝化功能的细菌,其中样品A、B和D中主要是亚硝化单胞菌属,占比分别为24.56%、4.94%和0.63%,样品C主要为Nitrospirillum(0.69%)和硝化螺旋菌属(0.69%)。此外在样品中还检测到红灯食烷菌、羽扇豆根瘤菌等有益菌,以及弧菌属、伯克霍尔德菌等致病菌。【结论】阐述了4个样品微生物群落结构的多样性,确定了不同培养物中起主要作用的硝化细菌类群以及其它与环境物质循环相关或具有特殊生理特性的菌群,研究结果为硝化细菌富集培养物的实际应用奠定了基础。     

模拟增温对青海湖鸟岛土壤微生物的影响

【背景】土壤微生物对其生存的微环境变化极为敏感,鸟岛作为湖滨湿地,对气候变化具有敏感性,但目前关于青海湖鸟岛的土壤微生物鲜有研究。【目的】探究气候变暖后青海湖鸟岛土壤微生物群落特征的变化。【方法】利用开顶箱模拟增温,通过高通量测序方法了解温度升高后土壤细菌及真菌的群落结构以及多样性的变化情况。【结果】温度的升高并未改变青海湖鸟岛土壤微生物的优势菌群,细菌优势菌群为变形菌门和酸杆菌门;真菌优势菌门为子囊菌门,优势菌纲为座囊菌纲。但增温改变了土壤微生物的群落结构,显著升高了拟杆菌门、蓝细菌门、Patescibacteria及球囊菌纲的相对丰度,显著降低了锤舌菌纲的相对丰度。土壤微生物群落的多样性指数也发生了变化,温度上升后微生物的ACE指数及Chao1指数均降低,细菌的Simpson指数及真菌的Shannon指数降低。【结论】青海湖鸟岛土壤微生物对温度升高的响应明显,增温改变了土壤细菌拟杆菌门、蓝细菌门、Patescibacteria的相对丰度及真菌的球囊菌纲、锤舌菌纲的相对丰度,降低了土壤微生物的多样性。     

好氧-厌氧混合污泥启动微生物燃料电池产电性能及微生物群落动态特征

【目的】为探讨好氧-厌氧混合污泥启动微生物燃料电池(Microbial fuel cell,MFC)产电性能以及MFC对微生物群落的选择作用,【方法】以乳酸为底物,应用不依赖于培养的微生物分子生物学技术解析单室MFC启动过程中微生物群落的组成和结构动态学特征。【结果】结果表明,MFC经过3个周期启动成功,最高输出电压230 m V。当MFC外电阻为1656Ω时,最大功率密度11.15 W/m3,电池运行稳定。混合污泥启动MFC以后,阳极生物膜微生物群落结构同种泥差异较大,且多样性降低。生物膜中微生物类群按丰度依次为β-变形菌纲(Betaproteobacteria)24.90%、拟杆菌门(Bacteroidetes)21.30%、厚壁菌门(Firmicutes)9.70%、γ-变形菌纲(Gammaproteobacteria)8.50%、δ-变形菌纲(Deltaproteobacteria)7.90%、绿弯菌门(Chloroflexi)4.20%以及α-变形菌纲(Alphaproteobacteria)3.60%。有利于生物膜形成与稳定的动胶菌属(Zoogloea)和不动杆菌属(Acinetobacter)序列丰度分别占生物膜群落的5.00%和3.90%,与MFC产电能力直接相关的地杆菌属(Geobacter)序列由混合污泥中的0.60%上升至阳极生物膜中的2.60%。【结论】本研究表明,MFC阳极生物膜在驯化过程中对污泥中的微生物进行淘汰和选择,最终驯化形成了有利于生物膜形成与稳定、有机物厌氧发酵与产电的微生物菌群。     

云南洱源牛街热泉原核微生物多样性分析

【目的】通过分析富含高分子有机物的云南洱源牛街热泉原核微生物16SrRNA基因克隆文库,丰富对高温热泉原核微生物多样性的认识,为进一步开发和利用该热泉微生物资源奠定基础。【方法】构建洱源牛街高温热泉原核微生物16SrRNA基因克隆文库,通过测序和序列相似性比对以及聚类分析研究该热泉原核微生物的多样性。【结果】该热泉原核微生物以细菌为主,包括变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、梭杆菌门(Fusobacteria)等在内的约10个细菌类群,其中变形菌门中的β-变形菌纲(β-Proteobacteria)为优势菌群,其次为拟杆菌门(Bacteroidetes)、绿菌门(Chlorobi);古菌的生物量和丰度较细菌少,分属广古菌(Euryarchaeota)和泉古菌(Crenarchaeota)两个类群,以广古菌为优势类群。     

Molecular convergence of bacterial and eukaryotic surface order

The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.     

The Golgi protein p115 associates with gamma-tubulin and plays a role in Golgi structure and mitosis progression

The Golgi apparatus is a network of polarized cisternae localized to the perinuclear region in mammalian cells. It undergoes extensive vesiculation at the onset of mitosis and its reassembly requires factors that are in part segregated via the mitotic spindle. Here we show that unlike typical Golgi markers, the Golgi-protein p115 partitioned with the spindle poles throughout mitosis. An armadillo-fold in its N terminus mediated a novel interaction between p115 and γ-tubulin and functioned in its centrosomal targeting. Both the N- and C-terminal regions of p115 were required to maintain Golgi structure. Strikingly, p115 was essential for mitotic spindle function and the resolution of the cytokinetic bridge because its depletion resulted in spindle collapse, chromosome missegregation, and failed cytokinesis. We demonstrate that p115 plays a critical role in mitosis progression, implicating it as the only known golgin to regulate both mitosis and apoptosis.     

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

The Viroporin Activity of the Minor Structural Proteins VP2 and VP3 Is Required for SV40 Propagation

For nonenveloped viruses such as Simian Virus 40, the mechanism used to translocate viral components across membranes is poorly understood. Previous results indicated that the minor structural proteins, VP2 and VP3, might act as membrane proteins during infection. Here, purified VP2 and VP3 were found to form pores in host cell membranes. To identify possible membrane domains, individual hydrophobic domains from VP2 and VP3 were expressed in a model protein and tested for their ability to integrate into membranes. Several domains from the late proteins supported endoplasmic reticulum membrane insertion as transmembrane domains. Mutations in VP2 and VP3 were engineered that inhibited membrane insertion and pore formation. When these mutations were introduced into the viral genome, viral propagation was inhibited. This comprehensive approach revealed that the viroporin activity of VP2 and VP3 was inhibited by targeted disruptions of individual hydrophobic domains and the loss of membrane disruption activity impaired viral infection.     

The N- and C-terminal Domains of Tomosyn Play Distinct Roles in Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Binding and Fusion Regulation

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

膜生物反应器的研究进展

膜生物反应器是近年来发展的废水处理新技术,具有活性污泥浓度高、污泥龄长、占地面积小、投资省的特点。利用膜生物反应器进行污水处理不仅可以大大节约水资源,还可以大大节约能源,节省设备和运行费用,已成为二十一世纪研究热点。膜生物反应器是通过高效膜分离技术与活性污泥相结合,增大污泥中的特效菌来加快生化反应速率,提高废水处理效果。目前处理对象已从生活污水扩展到高浓度的有机废水和难降解的工业废水。本文综述了膜生物反应器在废水中的应用研究情况,并分析比较了各种膜材质的特点、适用范围以及膜的污染因素和清洗方法,展望了膜生物反应器的应用前景及进一步研究方向。     

Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.     

Recent advances in membrane technologies for biorefining and bioenergy production

The bioeconomy, and in particular, biorefining and bioenergy production, have received considerable attention in recent years as a shift to renewable bioresources to produce similar energy and chemicals derived from fossil energy sources, represents a more sustainable path. Membrane technologies have been shown to play a key role in process intensification and products recovery and purification in biorefining and bioenergy production processes. Among the various separation technologies used, membrane technologies provide excellent fractionation and separation capabilities, low chemical consumption, and reduced energy requirements. This article presents a state-of-the-art review on membrane technologies related to various processes of biorefining and bioenergy production, including: (i) separation and purification of individual molecules from biomass, (ii) removal of fermentation inhibitors, (iii) enzyme recovery from hydrolysis processes, (iv) membrane bioreactors for bioenergy and chemical production, such as bioethanol, biogas and acetic acid, (v) bioethanol dehydration, (vi) bio-oil and biodiesel production, and (vii) algae harvesting. The advantages and limitations of membrane technologies for these applications are discussed and new membrane-based integrated processes are proposed. Finally, challenges and opportunities of membrane technologies for biorefining and bioenergy production in the coming years are addressed.     

Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.