Microbial diversity and function in soil: from genes to ecosystems

Soils sustain an immense diversity of microbes, which, to a large extent, remains unexplored. A range of novel methods, most of which are based on rRNA and rDNA analyses, have uncovered part of the soil microbial diversity. The next step in the era of microbial ecology is to extract genomic, evolutionary and functional information from bacterial artificial chromosome libraries of the soil community genomes (the metagenome). Sophisticated analyses that apply molecular phylogenetics, DNA microarrays, functional genomics and in situ activity measurements will provide huge amounts of new data, potentially increasing our understanding of the structure and function of soil microbial ecosystems, and the interactions that occur within them. This review summarizes the recent progress in studies of soil microbial communities with focus on novel methods and approaches that provide new insight into the relationship between phylogenetic and functional diversity.     

Environmental paleomicrobiology: using DNA preserved in aquatic sediments to its full potential

In-depth knowledge about spatial and temporal variation in microbial diversity and function is needed for a better understanding of ecological and evolutionary responses to global change. In particular, the study of microbial ancient DNA preserved in sediment archives from lakes and oceans can help us to evaluate the responses of aquatic microbes in the past and make predictions about future biodiversity change in those ecosystems. Recent advances in molecular genetic methods applied to the analysis of historically deposited DNA in sediments have not only allowed the taxonomic identification of past aquatic microbial communities but also enabled tracing their evolution and adaptation to episodic disturbances and gradual environmental change. Nevertheless, some challenges remain for scientists to take full advantage of the rapidly developing field of paleo-genetics, including the limited ability to detect rare taxa and reconstruct complete genomes for evolutionary studies. Here, we provide a brief review of some of the recent advances in the field of environmental paleomicrobiology and discuss remaining challenges related to the application of molecular genetic methods to study microbial diversity, ecology, and evolution in sediment archives. We anticipate that, in the near future, environmental paleomicrobiology will shed new light on the processes of microbial genome evolution and microbial ecosystem responses to quaternary environmental changes at an unprecedented level of detail. This information can, for example, aid geological reconstructions of biogeochemical cycles and predict ecosystem responses to environmental perturbations, including in the context of human-induced global changes.     

土壤微生物群落多样性解析法:从培养到非培养

土壤微生物群落多样性是土壤微生物生态学和环境科学的重点研究内容之一.传统的土壤微生物群落多样性解析技术是指纯培养分离法(平板分离和形态分析法以及群落水平生理学指纹法).后来,研究者们建立了多样性评价较为客观的生物标记法(磷脂脂肪酸法和呼吸醌指纹法).随着土壤基因组提取技术和基因片段扩增(PCR)技术的发展,大量的现代分子生物学技术不断地涌现并极大地推动了土壤微生物群落多样性的研究进程.这些技术主要包括:G+C％含量、DNA复性动力学、核酸杂交法(FISH和DNA芯片技术)、土壤宏基因组学以及DNA指纹图谱技术等.综述了这些技术的基本原理、比较了各种技术的优缺点并且介绍了他们在土壤微生物群落多样性研究中的应用,展望了这些技术的发展方向.     

土壤微生物在法庭科学领域中的应用研究进展

土壤物证在法庭科学领域的应用由来已久,主要是基于其外观、颜色、元素组成、矿物学等理化特性及土壤夹杂物等的比对检验。近年来,随着高通量测序技术的发展,法医土壤微生物检验不再完全依赖于传统培养技术,而是直接挖掘分析土壤中的全部微生物DNA信息,并将这些信息应用于法庭科学领域内的样本比对、土壤物证区域环境推断和溯源研究等,从而凸显出土壤微生物物证在案件侦查和法庭诉讼方面的巨大价值和应用潜力。本文通过综述国内外有关法医土壤微生物研究的最新进展,指出了土壤微生物多样性检验在法庭科学领域的应用潜力,分析了法医土壤微生物群落多样性的影响因素,最后探讨了法医土壤微生物研究中存在的问题和未来的发展方向。     

海洋微生物宏基因组工程进展与展望

据初步统计,生活于海洋环境包括大洋深处的微生物有100万种以上,构成了一个动态的遗传基因库,其中绝大多数微生物或者从来没有经过实验室培养,或者至今无法培养,因而其分类地位及其生态学功能尚未为人类所认识。随着16S rRNA序列分析与系统分类学的广泛应用,海洋微生物多样性研究领域已经发生了很可观的改变,这些变化极大的丰富了人们对的微生物多样性及其生态功能的认识和理解。这里结合笔者近十年来的工作实践,讨论近年来在海洋微生物资源开发利用方面的研究进展,提出一个带有自动化特征的宏基因组功能表达平台,探讨海洋微生物资源利用的新途径。可以预见在不久的将来,海洋环境宏基因组工程研究将在一定程度上使得传统未培养海洋微生物基因资源及其功能产物能够为人类所开发和利用。     

Isolation of DNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that is suitable for a wide variety of GI tract samples, including biopsies with minute amounts of material. The protocol is set up in such a way that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The DNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction. In addition, it includes an alternative DNA isolation protocol that is based on a commercial kit. These protocols have all been successfully used in our laboratory, resulting in isolation of DNA of sufficient quality for microbial diversity studies. Depending on the number of samples and sample type, the whole procedure will take approximately 2.5-4 hours.     

Comparison of DNA extraction methods for the PCR-single-strand-conformation polymorphism analysis of kefir

Experiments were performed to determine the influence of three DNA extraction methods (i.e. lysozyme, sonication and CTAB methods) from kefir on the microbial diversity analysis by PCR-single strand conformation polymorphism (PCR-SSCP). The results showed that the band of DNA extracted using CTAB was clearer than that using other methods. In addition, the yield and purity of DNA extracted using CTAB were the highest and reached, respectively, 915 μg/ml and 1.694.The results from the experiments indicated that the CTAB-based DNA extraction method was the most efficient method for DNA extraction from kefir. The heterogeneity of PCR products, amplified from community DNA with universal primers spanning the V3 region of 16S rRNA genes, was analysed by using SSCP. The results showed that the SSCP profile based on the sonication method gave the highest microbial diversity of kefir. One conclusion from these results was that the DNA extraction method was an important factor affecting the SSCP-based microbial diversity analysis of kefir.     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

种植体周围炎生物膜的微生物群落多样性研究进展

种植体周围炎是发生在骨性结合种植体周围组织的炎症,是由微生物引发的感染性疾病,可引起种植体周围支持组织丧失而导致种植失败。阐明种植体周围炎生物膜的微生物学基础,可为制定相应防治策略提供理论依据。随着测序技术的发展,基于16S rRNA基因的测序分析技术逐渐应用于与口腔种植体相关的微生物学研究,使人们对种植体周围炎生物膜的微生物群落多样性有了更全面的了解,也进一步认识到种植体周围炎和牙周炎菌斑生物膜的微生物结构存在显著差别。本文根据基于16S rRN基因A序列分析技术的最新研究成果,对种植体周围炎菌斑生物膜的微生物学研究进展作一综述。     

Metagenomics

The total number of prokaryotic cells on earth has been estimated to be approximately 4–6 × 10³⁰, with the majority of these being uncharacterized. This diversity represents a vast genetic bounty that may be exploited for the discovery of novel genes, entire metabolic pathways and potentially valuable end‐products thereof. Metagenomics constitutes the functional and sequence‐based analysis of the collective microbial genomes (microbiome) in a particular environment or environmental niche. Herein, we review the most recent sequence‐based metagenomic analyses of some of the most microbiologically diverse locations on earth; including soil, marine water and the insect and human gut. Such studies have helped to uncover several previously unknown facts; from the true microbial diversity of extreme environments to the actual extent of symbiosis that exists in the insect and human gut. In this respect, metagenomics has and will continue to play an essential part in the new and evolving area of microbial systems biology.     

覆土栽培食用菌的微生物病害防控

微生物病害问题会造成覆土栽培食用菌的产量和质量严重下降。防控微生物病害一直是食用菌研究和产业发展关注的热点,但常规的防控措施均存在局限性,尚难以在生产实践中有效地防控病害。本文列举了近年来常规的生物和非生物方法在覆土栽培食用菌微生物病害防控中的应用,对其特点进行了总结,并综述了近年来覆土栽培食用菌土壤微生物群落多样性研究的进展。基于此,提出了应用合成土壤微生物组来防控食用菌微生物病害的新策略,对构建和应用合成土壤微生物组面临的挑战和前景进行展望。这将有助于有效地防控覆土栽培食用菌的微生物病害和维系土壤健康。     

不同煤阶生物成因煤层气微生物群落的功能及多样性研究进展

生物成因煤层气是煤层气形成的主要途径之一,在各种煤阶的煤层气田中均发现有生物成因煤层气。高通量测序技术、宏基因组学等高新技术的应用逐步揭示了生物成因煤层气微生物群落的组成及多样性,为揭示煤层气资源的微生物生成机理提供了理论依据。本文综述了近年来国内外针对不同煤阶条件下微生物群落结构及多样性方面的研究进展,总结了煤层气生物成因过程中主要微生物的功能及产气途径,并探讨了生物成因煤层气领域的研究前景。     

大庆盐碱地九种植物根际土壤微生物群落结构及功能多样性

盐碱土是陆地表面生态脆弱区域。它与荒漠化过程相伴而生,不但造成了资源的破坏、农业生产的巨大损失,而且还对生物圈和生态环境构成威胁。研究盐碱地植物根际土壤微生物群落的多样性,对于盐碱土壤的植被恢复和生态重建具有重要意义。运用PCR-DGGE技术和Biolog微平板法,对大庆盐碱地9种不同植物根际土壤微生物结构和功能的多样性进行了分析。结果表明,不同植物根际土壤微生物组成不同,同一科的植物具有相似的微生物组成。对11个克隆进行了序列测定,发现这一地区植物根际优势微生物菌群为变形菌门(Proteobacteria)和酸杆菌门(Acidobacteria)。利用Biolog微平板法分析了微生物群落功能多样性。结果表明,不同植物根际土壤细菌群落对底物碳源的代谢特征存在着一定的差异,其中豆科的野大豆根际土壤细菌对底物碳源的代谢能力最强。     

metaBIT,an integrative and automated metagenomic pipeline for analysing microbial profiles from high‐throughput sequencing shotgun data

Micro‐organisms account for most of the Earth's biodiversity and yet remain largely unknown. The complexity and diversity of microbial communities present in clinical and environmental samples can now be robustly investigated in record times and prices thanks to recent advances in high‐throughput DNA sequencing (HTS). Here, we develop metaBIT, an open‐source computational pipeline automatizing routine microbial profiling of shotgun HTS data. Customizable by the user at different stringency levels, it performs robust taxonomy‐based assignment and relative abundance calculation of microbial taxa, as well as cross‐sample statistical analyses of microbial diversity distributions. We demonstrate the versatility of metaBIT within a range of published HTS data sets sampled from the environment (soil and seawater) and the human body (skin and gut), but also from archaeological specimens. We present the diversity of outputs provided by the pipeline for the visualization of microbial profiles (barplots, heatmaps) and for their characterization and comparison (diversity indices, hierarchical clustering and principal coordinates analyses). We show that metaBIT allows an automatic, fast and user‐friendly profiling of the microbial DNA present in HTS shotgun data sets. The applications of metaBIT are vast, from monitoring of laboratory errors and contaminations, to the reconstruction of past and present microbiota, and the detection of candidate species, including pathogens.     

变性梯度凝胶电泳技术在微生物多样性研究中的应用

变性梯度凝胶电泳是不依赖于培养的、依据DNA分子的大小和所带电荷分析微生物多样性和动态变化的分子生物学技术,具有检测极限低、分析速度快及重复性好等优点。主要对变性梯度凝胶电泳原理、特点及其在微生物多样性应用方面进行综述。     

Accessing the soil metagenome for studies of microbial diversity

Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.     

一种从活性污泥中提取微生物总DNA的方法

对活性污泥的微生物群落进行研究的首要前提是获得大量的高纯度微生物基因组DNA。本文建立了一种高效、简便的提取活性污泥总DNA方法。从提取的核酸总量、纯度、基因组完整性等多方面对所得到的DNA质量进行了评价,结果表明,本法从单位活性污泥中提取的DNA得率为105-823μg/g,结构完整,纯度很高,无需进一步的纯化,可直接进行微生物群落分析及构建文库等后续分子生物学操作。现在实验室使用的提取活性污泥中DNA的方法,纯度普遍都无法达到PCR反应和建立文库的要求,本文建立的活性污泥DNA提取方法则可以克服这一难题。     

Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh,India to Explore Bacterial Diversity

Extraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.