Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA.
mBio. 2011 Apr 5;2(2):e00012-11. doi: 10.1128/mBio.00012-11. Print 2011.
The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNA-seq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo verbana and for utilizing this information to design a medium for cultivating members of the microbiome. Expression data suggested that a Rikenella-like bacterium, the most abundant but uncultured symbiont, forages on sulfated- and sialated-mucin glycans that are fermented, leading to the secretion of acetate. Histological stains were consistent with the presence of sulfated and sialated mucins along the crop epithelium. The second dominant symbiont, Aeromonas veronii, grows in two different microenvironments and is predicted to utilize either acetate or carbohydrates. Based on the metatranscriptome, a medium containing mucin was designed, which enabled the cultivation of the Rikenella-like bacterium. Metatranscriptomes shed light on microbial metabolism in situ and provide critical clues for directing the culturing of uncultured microorganisms. By choosing a condition under which the desired organism is rapidly proliferating and focusing on highly expressed genes encoding hydrolytic enzymes, binding proteins, and transporters, one can identify an organism's nutritional preferences and design a culture medium.
The number of prokaryotes on the planet has been estimated to exceed 10(30) cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNA-seq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes.
绝大多数细菌物种仍未被培养，这严重限制了对其生理学、代谢能力和在环境中作用的研究。高通量 RNA 转录物测序（RNA-seq）允许在其自然栖息地中对未培养微生物的多样化生理学进行研究。在这里，我们报告了使用 RNA-seq 来描述药用蛭 Hirudo verbana 简单肠道微生物组的元转录组，并利用这些信息来设计一种培养微生物组成员的培养基。表达数据表明，一种类似于 Rikenella 的细菌，作为最丰富但未培养的共生体，以硫酸盐和唾液酸化粘蛋白糖为食，这些糖被发酵，导致乙酸盐的分泌。组织学染色与沿作物上皮存在硫酸盐和唾液酸化粘蛋白一致。第二大优势共生体 Aeromonas veronii 生长在两种不同的微环境中，预计可利用乙酸盐或碳水化合物。基于元转录组，设计了一种含有粘蛋白的培养基，使类似于 Rikenella 的细菌能够培养。元转录组揭示了原位微生物代谢，并为指导未培养微生物的培养提供了关键线索。通过选择一种期望的生物体快速增殖的条件，并专注于高度表达的编码水解酶、结合蛋白和转运蛋白的基因，可以确定生物体的营养偏好并设计培养基。
据估计，地球上的原核生物数量超过 10^30 个细胞，其中绝大多数都无法培养，这使得很难研究它们的生态、医学和工业相关性。基于高通量 RNA 转录物测序的转录组学（RNA-seq）在自然环境中应用于微生物，可以为研究人员提供有关最佳生长条件下其生理学的深入了解。我们利用 RNA-seq 更多地了解构成药用蛭相对简单的肠道微生物组的未培养和培养共生体。表达数据显示高度表达的水解酶和转运蛋白，为设计一种能够分离以前未培养的类似于 Rikenella 的共生体的培养基提供了关键线索。这种定向培养方法将极大地帮助人们理解未培养的微生物，包括有益共生体、病原体和具有生态相关性的微生物，通过促进以前未培养微生物的基因组测序、生理特征描述和遗传操作。
