De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities

This study describes reconstruction of two highly unusual archaeal genomes by de novo metagenomic assembly of multiple, deeply sequenced libraries from surface waters of Lake Tyrrell (LT), a hypersaline lake in NW Victoria, Australia. Lineage-specific probes were designed using the assembled genomes to visualize these novel archaea, which were highly abundant in the 0.1–0.8 μm size fraction of lake water samples. Gene content and inferred metabolic capabilities were highly dissimilar to all previously identified hypersaline microbial species. Distinctive characteristics included unique amino acid composition, absence of Gvp gas vesicle proteins, atypical archaeal metabolic pathways and unusually small cell size (approximately 0.6 μm diameter). Multi-locus phylogenetic analyses demonstrated that these organisms belong to a new major euryarchaeal lineage, distantly related to halophilic archaea of class Halobacteria. Consistent with these findings, we propose creation of a new archaeal class, provisionally named ‘Nanohaloarchaea''. In addition to their high abundance in LT surface waters, we report the prevalence of Nanohaloarchaea in other hypersaline environments worldwide. The simultaneous discovery and genome sequencing of a novel yet ubiquitous lineage of uncultivated microorganisms demonstrates that even historically well-characterized environments can reveal unexpected diversity when analyzed by metagenomics, and advances our understanding of the ecology of hypersaline environments and the evolutionary history of the archaea.     

水牛瘤胃宏基因组的一个新的b-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性

以木质纤维素为原料、应用同步糖化共发酵工艺发酵生产酒精时需要酸性中低温高活力纤维素酶包括b-葡萄糖苷酶。本工作分6次构建了水牛瘤胃未培养微生物宏基因组文库, 获得1.26×105个克隆, 文库含外源DNA的总长度 约为4.8×106 kb。从文库中筛选到118个表达b-葡萄糖苷酶活性的独立克隆。发现其中8个克隆表达的b-葡萄糖苷酶在pH5.0、37oC条件下活性较强。对其中一个克隆进行了亚克隆, 序列分析发现一个2223 bp的潜在的编码b-葡萄糖苷酶基因(umcel3G)的开放阅读框(ORF), 其编码产物的氨基酸序列与来自于 Bacillus sp.的一个b-葡萄糖苷酶同源性最高, 具有60%的一致性和73%的相似性。该ORF在E.coli中的表达产物Umcel3G的分子量与预测大小相似, 酶谱分析表明该表达产物具有b-葡萄糖苷酶活性, 证实该基因为一个b-葡萄糖苷酶基因。测定了用Ni-NTA纯化的Umcel3G的酶学特性, 其最适pH和最适温度分别为6.0~6.5和45oC。一些金属离子如Ca2+、Zn2+能显著提高该酶的酶活, 而另外一些金属离子如Fe3+、Cu2+能抑制Umcel3G的活性。在pH4.5、35oC和5 mmol/L的 Ca2+存在的条件下, 用Ni-NTA纯化的重组酶的比活为22.8 IU/mg, 说明该酶在用SSCF工艺发酵生产酒精中有潜在的应用价值。     

Decreased plant productivity resulting from plant group removal experiment constrains soil microbial functional diversity

Anthropogenic environmental changes are accelerating the rate of biodiversity loss on Earth. Plant diversity loss is predicted to reduce soil microbial diversity primarily due to the decreased variety of carbon/energy resources. However, this intuitive hypothesis is supported by sparse empirical evidence, and most underlying mechanisms remain underexplored or obscure altogether. We constructed four diversity gradients (0–3) in a five‐year plant functional group removal experiment in a steppe ecosystem in Inner Mongolia, China, and quantified microbial taxonomic and functional diversity with shotgun metagenome sequencing. The treatments had little effect on microbial taxonomic diversity, but were found to decrease functional gene diversity. However, the observed decrease in functional gene diversity was more attributable to a loss in plant productivity, rather than to the loss of any individual plant functional group per se. Reduced productivity limited fresh plant resources supplied to microorganisms, and thus, intensified the pressure of ecological filtering, favoring genes responsible for energy production/conversion, material transport/metabolism and amino acid recycling, and accordingly disfavored many genes with other functions. Furthermore, microbial respiration was correlated with the variation in functional composition but not taxonomic composition. Overall, the amount of carbon/energy resources driving microbial gene diversity was identified to be the critical linkage between above‐ and belowground communities, contrary to the traditional framework of linking plant clade/taxonomic diversity to microbial taxonomic diversity.     

生物元件的挖掘、改造与标准化

生物元件是合成生物学中的三大基本要素之一,是合成生物学的基石。现阶段,生物元件的挖掘、鉴定和改造仍然是合成生物学领域的重要研究方向之一。合成生物学与基因工程和代谢工程最显著的差别在于能够将大量的生物元件进行快速、随意的组装,而实现这一目标的前提是将生物元件标准化。目前,已经有大量基因组被解析,通过这些基因组数据库的注释与功能验证,并借助于各种生物信息学软件预测启动子、终止子、操纵了、转录因子和转录因子结合位点、核糖体结合位点以及蛋白质编码区等部件,为合成生物学提供丰富的生物元件信息资源。随着元基因组技术的兴起,大量未培养微生物中的基因和基因簇信息被解析,使得我们可以从占自然界中实际存在微生物总数99％的未知微生物中挖掘更多的生物元件。另外,生物元件可以从自然界分离出来,也可以对天然生物元件进行修饰、重组和改造后得到新的元件。酵母是异源蛋白表达的通用宿主和生物基产品生产的细胞工厂,但其本身可用的启动子非常有限,近年来各国学者在酵母启动子改造和文库构建方面做了很多工作,该文也将概述酵母启动子改造和在合成生物生物学研究领域中的应用方面的研究进展。     

Viral metagenome analysis to guide human pathogen monitoring in environmental samples

Aims: The aim of this study was to develop and demonstrate an approach for describing the diversity of human pathogenic viruses in an environmentally isolated viral metagenome. Methods and Results: In silico bioinformatic experiments were used to select an optimum annotation strategy for discovering human viruses in virome data sets and applied to annotate a class B biosolid virome. Results from the in silico study indicated that <1% errors in virus identification could be achieved when nucleotide‐based search programs (BLASTn or tBLASTx), viral genome only databases and sequence reads >200 nt were considered. Within the 51 925 annotated sequences, 94 DNA and 19 RNA sequences were identified as human viruses. Virus diversity included environmentally transmitted agents such as parechovirus, coronavirus, adenovirus and aichi virus, as well as viruses associated with chronic human infections such as human herpes and hepatitis C viruses. Conclusions: This study provided a bioinformatic approach for identifying pathogens in a virome data set and demonstrated the human virus diversity in a relevant environmental sample. Significance and Impact of the Study: As the costs of next‐generation sequencing decrease, the pathogen diversity described by virus metagenomes will provide an unbiased guide for subsequent cell culture and quantitative pathogen analyses and ensures that highly enriched and relevant pathogens are not neglected in exposure and risk assessments.     

宏基因组与宏基因组学

宏基因组学诞生于上世纪90年代,是指不经过微生物培养阶段,采用直接提取环境中总DNA的方法,对微生物基因总和进行研究的一门新学科.宏基因组技术的出现,使得人们对占微生物总体99%以上不可培养微生物的研究成为现实,微生物基因的可探测空间显著增大.总的来说,目前宏基因组技术的应用主要分为两个方面:一方面是筛选功能基因,开发具有所需功能的蛋白;另一方面是通过对宏基因组文库进行分析,探讨在各种环境下微生物间相互作用和微生物与周围环境间相互影响的规律,以便我们能更加客观、全面地认识微生物世界.在宏基因组技术的应用范围被不断扩展的同时,围绕着宏基因组文库的构建和筛选、测序和分析等方面的研究已成为宏基因组学发展的主要推动力,宏基因组技术的进步将不断提升其应用价值.     

肉牛剩余采食量相关瘤胃及粪便微生物特征比较分析

本试验旨在研究不同剩余采食量（residual feed intake, RFI）相关肉牛胃肠道微生物物种组成及相对丰度、微生物基因功能注释与富集特征。选取30头牛进行81 d饲喂试验,试验结束后选取极端RFI个体各5头屠宰并采集瘤胃液及肠道末端粪便样用于宏基因组测序。结果表明：共获得259 045.47 Mb有效数据,组装得到 4 318 393条Scaftigs,基因预测得到7 008 053个开放阅读框（ORFs）。进行物种注释后发现粪便样中Top10物种相对丰度在高剩余采食量（high residual feed intake, HRFI）、低剩余采食量（low residual feed intake, LRFI）组间无差异（P>0.05）,瘤胃液中的Top10微生物在LRFI组的相对丰度均低于HRFI组;粪便中、瘤胃液中优势菌门均为拟杆菌门和厚壁菌门;粪便中的优势属为拟杆菌属,瘤胃液中的优势属为普雷沃菌属。LefSe分析显示,在粪便中LRFI组的丹毒丝菌纲（Erysipelotrichia）显著富集（P<0.05）,瘤胃液中差异最显著的是甲烷杆菌纲（Methanobacteria）,且该菌在HRFI组的相对丰度显著高于LRFI组（P<0.05）。使用KEGG、eggNOG和CAZy数据库进行功能注释分析表明,在胃肠道中微生物的一些功能基因的丰度与RFI的分组有关。不同RFI肉牛瘤胃液、粪便中的微生物结构存在显著差异,丹毒丝菌纲、甲烷杆菌纲可能是区分肉牛饲料效率的潜在生物标志物之一。     

Life in Hot Spring Microbial Mats Located in the Trans-Mexican Volcanic Belt: A 16S/18S rRNA Gene and Metagenomic Analysis

The geothermal system of the Araró region, located in the Trans-Mexican Volcanic Belt of México, hosts various hot springs with unique physicochemical characteristics, including temperatures ranging from 45°C to 78°C. The microbial diversity in these hot springs has been explored only by culture-dependent surveys. In this study, we performed metagenomic Illumina MiSeq, and 16S and 18S rRNA pyrosequencing analysis of the microbial life are residing in the microbial mats of the springs called “Tina–Bonita”. Our results show the presence of 186 operational taxonomic units, 99.7% of which belong to bacteria, 0.27% to eukaryotes, and 0.03% to archaea. The most abundant bacterial divisions are the Proteobacteria, Chloroflexi, and Cyanobacteria, which include 105 genera. The ecological indexes indicate that the microbial mats have moderate microbial diversity. An abundant group of genes that participate in photosynthesis, including photosynthetic electron transport, as well as photosystems I and II, were detected. Another cluster of genes was found that participates in sulfur, nitrogen, and methane metabolism. Finally, this phylogenetic and metagenomic analysis revealed an unexpected taxonomic and genetic diversity, expanding our knowledge of microbial life under specific extreme conditions.     

Next‐Generation Sequencing Assessment of Eukaryotic Diversity in Oil Sands Tailings Ponds Sediments and Surface Water

Tailings ponds in the Athabasca oil sands (Canada) contain fluid wastes, generated by the extraction of bitumen from oil sands ores. Although the autochthonous prokaryotic communities have been relatively well characterized, almost nothing is known about microbial eukaryotes living in the anoxic soft sediments of tailings ponds or in the thin oxic layer of water that covers them. We carried out the first next‐generation sequencing study of microbial eukaryotic diversity in oil sands tailings ponds. In metagenomes prepared from tailings sediment and surface water, we detected very low numbers of sequences encoding eukaryotic small subunit ribosomal RNA representing seven major taxonomic groups of protists. We also produced and analysed three amplicon‐based 18S rRNA libraries prepared from sediment samples. These revealed a more diverse set of taxa, 169 different OTUs encompassing up to eleven higher order groups of eukaryotes, according to detailed classification using homology searching and phylogenetic methods. The 10 most abundant OTUs accounted for > 90% of the total of reads, vs. large numbers of rare OTUs (< 1% abundance). Despite the anoxic and hydrocarbon‐enriched nature of the environment, the tailings ponds harbour complex communities of microbial eukaryotes indicating that these organisms should be taken into account when studying the microbiology of the oil sands.