School of Life Sciences, University of Warwick, Coventry, UK.
Aquatic Geochemistry, Institute of Biodiversity, Friedrich Schiller University, Jena, Germany.
Methods Mol Biol. 2023;2555:261-282. doi: 10.1007/978-1-0716-2795-2_17.
Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of C, O, or N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses.
稳定同位素探测（SIP）使研究人员能够针对复杂微生物群落中的活跃种群，这是通过提供富含重同位素的生长基质来实现的，通常是以 C、O 或 N 的形式。在基质上生长并随后提取微生物生物标志物后，通常是核酸或蛋白质，SIP 技术用于从活跃微生物种群中回收和分析同位素标记的生物标志物。在最初开发基于 DNA 和 RNA 的 SIP 后的几年中，通过靶向基因分析来表征标记种群是一种常见的做法。此类方法通常涉及基于指纹的分析或测序包含 16S rRNA 基因或功能标记基因扩增子的克隆文库。尽管分子指纹分析仍然是快速确认同位素标记的有价值的方法，但测序技术的最新进展意味着可以获得负担得起且全面的扩增子图谱，甚至可以从 SIP 实验中获得宏基因组和宏转录组。不仅可以从宏基因组推断微生物组的丰度，而且研究人员可以进行分类、组装和探索单个基因组，以建立关于标记微生物代谢能力的假设。标记 mRNA 的分析是最近的一项进展，可以提供基于活跃微生物的独立宏转录组分析。宏转录组学的强大之处在于 mRNA 丰度通常与相应酶的活性密切相关，因此可以深入了解采样时的微生物代谢。这些进展共同提高了 SIP 方法的灵敏度，并允许在环境相关浓度下使用标记底物。特别是随着方法的改进和成本的持续下降，我们预计 SIP 与多种基于组学的方法的整合将成为微生物生态学研究的常见组成部分，从而在我们对新的微生物种群的理解和阐明复杂微生物群落的代谢功能方面取得进一步的突破。在本章中，我们提供了用于获得可用于下游基于组学分析的标记 DNA、RNA 和蛋白质的方案。
