Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.     

The microbial loop concept as used in terrestrial soil ecology studies

Components and processes in the aquatic microbial loop are compared with the composition and functioning of the soil microbial loop. Relative to their bacterial and/or fungal food sources, many of the soil water-film fauna (e.g., protozoa, nematodes) are conspicuous by low biomasses and high turnover rates of carbon and mineral nutrients. Comparisons with production and turnover rates of aerial (pore-inhabiting) fauna are made, and the highly patchy nature of soil microhabitats is shown to be similar to that of aquatic (marine) habitats.     

分子生物学方法在环境微生物生态学中的应用研究进展

随着分子生物学方法的不断发展和改进,微生物在生态系统中的作用被更好的挖掘出来。目前快速发展的先进的分子生物学技术,已经开始应用于分析环境微生物的多样性、微生物的生物地理学及微生物对气候变化的响应等。一般环境微生物的研究目标主要有3个,即确定微生物的种类和多样性、微生物的功能或潜在作用及在特定时间点活跃的微生物等。然而,现有微生物的研究方法复杂多样,容易给研究者在方法的选择上带来困惑。将从微生物的多样性和功能研究两个方面介绍和分析相应的分子生物学方法,尤其是近年来快速发展的高通量测序、宏组学和单细胞水平研究方法(如纳米二次离子质谱与荧光原位杂交相结合的方法)等新技术及其应用情况,以期为研究者选择合适的研究方法进行环境微生物的研究提供依据。     

The maturing of microbial ecology.

A.J. Kluyver and C.B. van Niel introduced many scientists to the exceptional metabolic capacity of microbes and their remarkable ability to adapt to changing environments in The Microbe's Contribution to Biology. Beyond providing an overview of the physiology and adaptability of microbes, the book outlined many of the basic principles for the emerging discipline of microbial ecology. While the study of pure cultures was highlighted, provided a unifying framework for understanding the vast metabolic potential of microbes and their roles in the global cycling of elements, extrapolation from pure cultures to natural environments has often been overshadowed by microbiologists inability to culture many of the microbes seen in natural environments. A combination of genomic approaches is now providing a culture-independent view of the microbial world, revealing a more diverse and dynamic community of microbes than originally anticipated. As methods for determining the diversity of microbial communities become increasingly accessible, a major challenge to microbial ecologists is to link the structure of natural microbial communities with their functions. This article presents several examples from studies of aquatic and terrestrial microbial communities in which culture and culture-independent methods are providing an enhanced appreciation for the microbe's contribution to the evolution and maintenance of life on Earth, and offers some thoughts about the graduate-level educational programs needed to enhance the maturing field of microbial ecology.     

非培养方法在土壤微生物生态学研究中的应用

由于有相当数量的土壤微生物是目前不可培养的,因此利用传统培养技术来研究土壤微生物,不仅费时费力,所得到的结果可能和真实的情况相差甚远。近年来发展了三类不需培养的方法来研究土壤微生物的种类和数量,这些方法大体上分为生物化学、生理学和分子生物学三类。生物化学方法主要根据细胞膜磷脂酸(PLFA)的种类和数量来判定微生物的多样性;BIOLOG微量板分析系统是生理学方法的代表,它主要是根据土样细胞悬液对95种单一碳源的利用模式来说明群落结构的变化;分子生物学方法是发展应用最广的方法。基本步骤是提取土壤的总DNA,然后用通用引物或选择性高的引物来扩增16SrRNA基因。由于对扩增产物分析方法的不同,该方法又可分为PCR-DGGE,PCR-RFLP等。最近在PCR-RFLP基础上发展起来的T—RFLP分析方法,将微生物的多样性分析工作同RDP(ribosomal database project)数据库结合,充分利用了Internet的数据资源共享的优势,具有分辨率高,可实现自动化等优点。是未来土壤微生物生态学研究的有力工具。     

Phosphoglycerides of Trichophyton terrestre and One Phenotype Selected from the Apollo 16 Microbial Ecology Evaluation Device

Total lipid extracted from wild-type Trichophyton terrestre CDC-X285 was found to be 2.0 percent of the dry cell weight. The total lipid contained the following phospholipid components identified by silicic acid-impregnated thin-layer and paper chromatography: phosphatidyl inositol, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The total lipid extracted from the phenotype T. terrestre 7048-1 isolated from the Apollo 16 Microbial Ecology Evaluation Device (MEED) was found to vary according to the time at which the phospholipids were extracted. The Trichophyton phenotype was selected from a cuvette housed in the MEED exposed to specific space parameters including ultraviolet light of known wavelengths and energy levels in deep space. The phospholipid components, identified in the phenotype were phosphatidyl ethanolamine and cardiolipin. The major lipid fraction was composed of digalactosyl diglyceride and monogalactosyl diglyceride. An unusual lipid was detected in the phenotype, which appeared to be sterol glycoside.     

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。     

Direct extraction of DNA from soils for studies in microbial ecology

Molecular analyses for the study of soil microbial communities often depend on the extraction of DNA directly from soils. These extractions are by no means trivial, being complicated by humic substances that are inhibitory to PCR and restriction enzymes or being too highly colored for blot hybridization protocols. Many different published protocols exist, but none have been found to be suitable enough to be generally accepted as a standard. Most direct extraction protocols start with relatively harsh cell breakage steps such as bead-beating and freeze-thaw cycles, followed by the addition of detergents and high salt buffers and/or enzymic digestion with lysozyme and proteases. After typical organic extraction and alcohol precipitation, further purification is usually needed to remove inhibitory substances from the extract. The purification steps include size-exclusion chromatography, ion-exchange chromatography, silica gel spin columns, and cesium chloride gradients, among others. A direct DNA extraction protocol is described that has been shown to be effective in a wide variety of soil types. This protocol is experimentally compared to several published protocols.     

The microbial ecology of the Great Salt Lake

The Great Salt Lake is actually two lakes. A highly saline (330-gml^–1) northern arm and a moderately saline (120-gml^–1) southern arm separated by a semipermeable rock causeway. The lake, particularly the northern arm, has a massive accumulation of organic matter resulting from more than 100,000 years of productivity, cycling from a freshwater to a saline lake, plus the influence of human industry and agriculture in more recent times. The north arm planktonic and attached community consists principally of, in order of biomass: bacteria of at least two genera,Halobacterium andHalococcus; two algae,Dunaliella salina andD. viridis; the brine shrimp,Anemia salina; and, two species of brine fly,Ephydra gracilis andE. hians and possibly one more species. The algae and the bacteria appear to depend on each other for nutrients. The bacteria use organic matter produced by the algae and the algae use ammonia produced by the bacteria and possibly the brine shrimp. The production of ammonia appears to be the rate-limiting step although there is no shortage of other forms of nitrogen in the north arm. Based on aquarium studies, the potential for biomass production of algae and bacteria is much higher than actually observed in the north arm, leading to the postulation of two additional factors controlling population; the grazing of the algae by invertebrates with the excretion of compounds rich in nitrogen, and the effect of a low habitat temperature and winter cold on the bacteria, reducing their metabolic activities to nearly zero. Some aspects of the various organisms and their metabolism are discussed. A comparison is made with recent work on the Dead Sea.     

The role of ecological theory in microbial ecology

Microbial ecology is currently undergoing a revolution, with repercussions spreading throughout microbiology, ecology and ecosystem science. The rapid accumulation of molecular data is uncovering vast diversity, abundant uncultivated microbial groups and novel microbial functions. This accumulation of data requires the application of theory to provide organization, structure, mechanistic insight and, ultimately, predictive power that is of practical value, but the application of theory in microbial ecology is currently very limited. Here we argue that the full potential of the ongoing revolution will not be realized if research is not directed and driven by theory, and that the generality of established ecological theory must be tested using microbial systems.     

空间分析方法在微生物生态学研究中的应用

微生物生态正在受到越来越多的关注,对其研究也渐趋深入。然而由于微生物个体微小的特点及研究手段的限制,多数研究还停留在探索阶段,研究方法也在不断完善当中。近年来,较多的研究开始探讨空间因素在微生物多样性和分布中的影响,对空间分布的探讨有助于更好地认识生态过程,是一种有力的研究手段。微生物空间分析方法已经成为微生物生态学领域中重要的研究方向之一,我国空间方法在微生物生态研究中的应用还没有得到普遍的重视。从不同研究角度出发,结合空间统计的作用,对空间统计方法在微生物生态研究中的应用的必要性及现状做了评述。介绍了空间自相关性的检验,方差图,Mantel检验,Kriging插值等方法在微生物生态研究中的应用,并论述了微生物研究中的尺度问题。这一梳理,对丰富微生物生态学研究中的新方法、新手段具有一定价值。