Phylogenetic identification and in situ detection of individual microbial cells without cultivation
Phylogenetic identification and in situ detection of individual microbial cells without cultivation
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DOI：
10.1128/mr.59.1.143-169.1995
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发表时间：
1995-03
期刊：
影响因子：
--
通讯作者：
R. Amann;W. Ludwig;K. Schleifer
中科院分区：
文献类型：
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作者：
R. Amann;W. Ludwig;K. Schleifer
直接显微镜计数与环境样本中可培养细菌数量之间的频繁差异，只是我们目前只知道自然界微生物多样性的一小部分的几个迹象之一。RRNA序列的直接检索和全细胞寡核苷酸探针的组合可用于检测自然样品中未培养细菌的特定rRNA序列，并在显微镜下鉴定单个细胞。已经对各种复杂的微生物组合进行了研究，从简单的两组分细菌内共生组合到含有趋磁细菌的多物种丰富，再到高度复杂的海洋和土壤群落。对检索到的未培养微生物的rRNA序列进行的系统发育分析揭示了其最接近的可培养亲缘关系，并可能结合有关其自然栖息地的物理化学条件的信息，促进更有针对性的培养尝试。对于多物种生物膜和活性污泥絮体等复杂群落的分析，另一种方法已被证明是有利的。连续地应用针对不同分类级别的探针组，从更一般的开始，到更具体的结束(分层的自上而下的方法)，从而生成关于社区结构的越来越精确的信息。以rRNA为靶点的全细胞杂交不仅产生关于细胞形态、特定细胞计数和确定的系统发育组的原位分布的数据，而且杂交信号的强度也反映了单个细胞的细胞rRNA含量。根据特定探针发出的信号强度，可以估计已知物种的单个细胞的原位生长速度和活动。在许多生态系统中，低细胞rRNA含量和/或有限的细胞渗透性，加上背景荧光，阻碍了对原地种群的原位识别。详细讨论了规避这些问题的方法。
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.