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.     

Functional Analysis of Type 1 Isopentenyl Diphosphate Isomerase from Halobacterium sp. NRC-1

Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 °C at pH 6.0. This type-1 enzyme appears to be the first for which the Co²⁺ ion is required for activity.     

Haloarchaeal proteases and proteolytic systems

Proteases play key roles in many biological processes and have numerous applications in biotechnology and industry. Recent advances in the genetics, genomics and biochemistry of the halophilic Archaea provide a tremendous opportunity for understanding proteases and their function in the context of an archaeal cell. This review summarizes our current knowledge of haloarchaeal proteases and provides a reference for future research.     

基于元基因组学数据集的海洋微生物多样性分析

通过对海洋13个样品元基因组数据的BLAsT搜索,筛选到了16s rRNA基因序列1600条,18s RNA基因序列61条。分类结果显示,细菌在海岸、公开海域深层海水和表层海水3种海洋环境类型中都占优势,其相对百分比分别为98%、59%和91%。相比于海岸和公开海域表层海水,公开海域深层海水中古生菌和Deltaproteobacteria所占的相对含量较高,各31%和27%。海岸检测到的古生菌主要为Euryarcheata,公开海域深层海水检测到的古生菌主要为Crenarchaaeota(93%为与氨氧化相关的MGI纲)。结果表明,氨氧化相关古生菌在深海生态系统中的作用可能较大。     

嗜盐菌耐盐机制相关基因的研究进展

嗜盐微生物能够在高盐环境中生存,其耐盐机制一直是微生物学家研究的热点。目前嗜盐微生物耐盐机制的研究主要集中在细胞吸K+排Na+作用、胞内积累小分子相容性溶质及嗜盐酶的氨基酸组成特性三个方面。文章从基因水平综述了嗜盐菌的耐盐机制,并对其在高盐废水处理上的应用进行讨论与展望。     

不同风化时间碳酸岩表面古菌群落结构与功能特征

【背景】古菌群落是碳酸岩表面微生物群落的重要成员,也是碳酸岩表面生物演替的先锋生物,能够促进碳酸岩风化和加快土壤形成,在生物地球化学循环中起重要作用。【目的】揭示在不同风化时间碳酸岩表面风化残积物中的古菌群落结构及生态功能。方法】采集19-213年风化时间段废弃碳酸岩墓碑表面风化残积物样品(n=18),基于宏基因组测序技术分析其古菌群落结构与功能特征。【结果】门水平上,优势门有广古菌门(Euryarchaeota),随后为奇古菌门(Thaumarchaeota)、未鉴定古菌门(unclassified Archaea)、深古菌门(Bathyarchaeota)和泉古菌门(Crenarchaeota);属水平上,优势属主要由甲烷八叠球菌属(Methanosarcina)、甲烷丝状菌属(Methanothrix)、Methanoperedens、氨氧化古菌属(Nitrosocosmicus)、亚硝化球菌属(Nitrososphaera)及其他未鉴定属组成;C/N、C/P、N/P是显著影响碳酸岩表面古菌群落的主要环境因子。进一步分析发现,碳酸岩表面古菌群落功能丰富,其中新陈代谢(metabo...     

Stabilization of an archaeal DNA-sliding clamp protein, PCNA, by proteasome-activating nucleotidase gene knockout in Haloferax volcanii

Many details of structure, function and substrate specificity of eukaryotic proteasomal systems have been elucidated. This information far-exceeds that available for the archaeal and bacterial counterparts. While structural and functional studies have provided some insight into the workings of prokaryotic proteasomes, the question of substrate targeting and global cellular influence remain largely unaddressed. In this communication, we report an over 720-fold increase in the half-life of the DNA-sliding clamp protein proliferating cell nuclear antigen after knockout of the panA gene, encoding a proteasome-activating nucleotidase A, on the chromosome of the halophilic archaeon Haloferax volcanii . This discovery marks the first identification of a protein stabilized by an archaeal proteasome mutation and provides a starting point for investigations into substrate recognition mechanisms. The findings also begin to address the functional role of proteasomal systems within the scope of the archaeal cell.     

Spatial Distribution and Molecular Diversity of Archaeal Communities in the Extreme Hypersaline Meromictic Brâncoveanu Lake (Transylvanian Basin,Romania)

Dating from the Middle Miocene, the massive halite deposits lying beneath the Transylvanian Basin (Central Romania) have been valuable mineral resources quarried for millennia. Among the numerous hypersaline pit lakes that resulted from this mining, Brâncoveanu Lake is unique by its extreme salinity. Assessment of physicochemical variables, water chemistry and trophic status indicated that Brâncoveanu Lake is a permanently stratified, pH-neutral, NaCl-rich and eutrophied system. We investigated the abundance, molecular diversity and vertical distribution of archaeal community by culture-independent approaches. Additionally, the most relevant environmental parameters shaping the archaeal community composition were evaluated by statistical methods. Archaea appeared to largely outnumber Bacteria; altogether the great prevalence of Halobacteriaceae-related sequences could imply a major contribution of this group to the biogeochemical carbon turnover. The fairly distinct composition of archaeal communities reflects the lake's physicochemical stratification. Among the limnological factors, salinity and oxygen showed a significant impact on determining the composition and structure of archaeal assemblages. Furthermore, Brâncoveanu Lake might harbor novel microorganisms such as members of the recently described phylum Nanohaloarchaea. Overall, this study reported the occurrence of halophilic Archaea in a little explored hydrogeochemical system and provided a better insight into geomicrobiology of meromictic hypersaline pit lakes.     

从宏基因组推测扎龙湿地未培养甲烷古菌Rice Cluster II的代谢途径与可能的盐碱适应性

【背景】湿地是重要的甲烷排放源,因为其中栖息着各种产甲烷古菌。已知未培养甲烷古菌Rice Cluster Ⅱ (RCⅡ)类群广泛分布于低温酸性和北方泥炭藓湿地、淡水湿地及草本沼泽等环境,但它们在低温盐碱湿地中的分布及代谢途径尚未知。【目的】分析扎龙盐碱湿地未培养甲烷古菌RCⅡ类群的多样性、推测产甲烷代谢途径及其潜在的盐碱适应机制。【方法】16S rRNA基因扩增子测序分析扎龙湿地土壤中甲烷古菌群组成;构建16S rRNA基因克隆文库分析扎龙湿地土壤RCⅡ的多样性;宏基因组分析推测RCⅡ古菌编码的产甲烷途径及与耐盐碱相关物质的合成基因。【结果】16SrRNA基因高通量测序发现未培养甲烷古菌的RCⅡ类群占扎龙盐碱湿地总甲烷古菌的13.280%±0.019%;系统发育学分析表明该湿地的RCⅡ由3个分支组成;宏基因组分析组装了2个优势的未培养RCⅡ的基因组,均含完整的氢还原二氧化碳产甲烷途径的基因,并编码海藻糖的转运与合成基因。【结论】扎龙盐碱湿地土壤富含未培养RCⅡ甲烷古菌,推测它们通过氢还原二氧化碳产甲烷,利用细胞内高的海藻糖适应盐碱环境。     

西黑冠长臂猿粪便微生物宏基因组来源的高比活α-l-阿拉伯呋喃糖苷酶的重组表达及酶学性质

【背景】α-l-阿拉伯呋喃糖苷酶是一类重要的半纤维素酶,能协同其他半纤维素酶降解木聚糖,在食品、医药、生物质能转化中具有应用价值。【目的】挖掘新型α-l-阿拉伯呋喃糖苷酶基因,对其进行异源表达、纯化并研究其酶学性质。【方法】从西黑冠长臂猿粪便微生物宏基因组中扩增α-l-阿拉伯呋喃糖苷酶基因,在大肠杆菌BL21(DE3)中进行异源表达,并进行酶学性质研究。【结果】从粪便微生物宏基因组中扩增得到α-l-阿拉伯呋喃糖苷酶基因AbfNC2b_38,并获得重组α-l-阿拉伯呋喃糖苷酶AbfNC2b_38,其分子量为57.04 kDa。AbfNC2b_38的最适作用条件为55 ℃、pH 6.0,K_m和V_max分别为(6.48±0.73) mmol/L和(1 248.0±114.6) U/mg,与其他宏基因组来源的α-l-阿拉伯呋喃糖苷酶相比具有最高比活300.81 U/mg。AbfNC2b_38具有较好的乙醇和NaCl耐受性,30%乙醇下耐受1 h保持68%的活性;25% NaCl中耐受1 h,相对酶活仍保持在约70%。与木聚糖酶协同降解山毛榉木聚糖时,协同率最高为1.21。【结论】从西黑冠长臂猿粪便微生物宏基因组中获得新型α-l-阿拉伯呋喃糖苷酶基因AbfNC2b_38并成功异源表达。AbfNC2b_38具有较好的乙醇和NaCl耐受性,能与木聚糖酶协同作用提高木聚糖的降解效率,在饲料、食品加工等领域具有潜在的应用价值。     

Heterotrophic dinitrogen fixation (acetylene reduction) in phosphate-fertilised Microcoleus chthonoplastes microbial mat from the hypersaline inland lake ‘la Salada de Chiprana’ (NE Spain)

Microcoleus chthonoplastes dominated microbial mats are conspicuous along the shallow littoral zone in Lake Chiprana, a hypersaline lake located in the Ebro river basin in north-eastern Spain. Pigment data show that these mats included diatom species and anoxygenic phototrophs, Chloroflexus-type bacteria and purple bacteria. In situ, these mats showed low rates of dinitrogen fixation (acetylene reduction). Acetylene reduction was stimulated about 30-fold in excised mats after moderate phosphate fertilisation during 2 weeks incubation in a mesocosm. Pigment analyses showed that this treatment had little impact on the phototrophic community structure, except that it induced a decrease of Chloroflexus-type bacteria. The use of metabolic inhibitors indicated that methanogenic archaea and aerobic heterotrophic bacteria were the major dinitrogen fixers in this system. This is in agreement with the fact that the mat-building cyanobacterium M. chthonoplastes lacks the dinitrogenase reductase nifH gene and with the fact that acetylene reduction rates were strongly stimulated by additions of H₂/CO₂, methanol, fructose and sucrose, but not by lactate, acetate, formate and glucose. No significant differences where found for acetylene reduction rates when comparing light and dark incubations of these microbial mats. However, acetylene reduction rates were enhanced in the light when the near infrared (NIR) light was filtered out, which arrested anoxygenic photosynthesis. We suggest, therefore, that the chemoheterotrophic dinitrogen fixing bacteria were in competition with anoxygenic phototrophic bacteria for organic substrates, while the latter did not contribute to dinitrogen fixation in the mat.     

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.     

Spatial and temporal variability in a stratified hypersaline microbial mat community

Hypersaline microbial mat communities have recently been shown to be more diverse than once thought. The variability in community composition of hypersaline mats, both in terms of spatial and temporal dimensions, is still poorly understood. Because this information is essential to understanding the complex biotic and abiotic interactions within these communities, terminal restriction fragment analysis and 16S rRNA gene sequencing were used to characterize the near-surface community of a hypersaline microbial mat in Guerrero Negro, Mexico. Core samples were analyzed to assay community variability over large regional scales (centimeter to kilometer) and to track depth-related changes in population distribution at 250-μm intervals over a diel period. Significant changes in total species diversity were observed at increasing distances across the mat surface; however, key species (e.g. Microcoleus sp.) were identified throughout the mat. The vertical position and abundance of >50% of the 60 peaks detected varied dramatically over a diel cycle, including Beggiatoa sp., cyanobacteria, Chloroflexus sp., Halochromatium sp., Bacteroidetes sp. and several as-yet-identified bacteria. Many of these migrations correlated strongly with diel changes in redox conditions within the mat, contributing to strong day–night community structure differences.