Department of Molecular Systems Biology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Germany; Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
J Proteomics. 2020 Jun 30;222:103791. doi: 10.1016/j.jprot.2020.103791. Epub 2020 Apr 23.
Stable isotope probing (SIP) approaches are a suitable tool to identify active organisms in bacterial communities, but adding isotopically labeled substrate can alter both the structure and the functionality of the community. Here, we validated and demonstrated a substrate-independent protein-SIP protocol using isotopically labeled water that captures the entire microbial activity of a community. We found that O yielded a higher incorporation rate into peptides and thus comprised a higher sensitivity. We then applied the method to an in vitro model of a human distal gut microbial ecosystem grown in two medium formulations, to evaluate changes in microbial activity between a high-fiber and high-protein diet. We showed that only little changes are seen in the community structure but the functionality varied between the diets. In conclusion, our approach can detect species-specific metabolic activity in complex bacterial communities and more specifically to quantify the amount of amino acid synthesis. Heavy water makes possible to analyze the activity of bacterial communities for which adding an isotopically labeled energy and nutrient sources is not easily feasible. SIGNIFICANCE: Heavy stable isotopes allow for the detection of active key players in complex ecosystems where many organisms are thought to be dormant. Opposed to the labelling with energy or nutrient sources, heavy water could be a suitable replacement to trace activity, which has been shown for DNA and RNA. Here we validate, quantify and compare the incorporation of heavy water either labeled with deuterium or 18‑oxygen into proteins of Escherichia coli K12 and of an in vitro model of a human gut microbial ecosystem. The significance of our research is in providing a freely available pipeline to analyze the incorporation of deuterium and 18‑oxygen into proteins together with the validation of the applicability of tracing heavy water as a proxy for activity. Our approach unveils the relative functional contribution of microbiota in complex ecosystems, which will improve our understanding of both animal- and environment-associated microbiomes and in vitro models.
稳定同位素探测（SIP）方法是一种识别细菌群落中活性生物的合适工具，但添加同位素标记的底物会改变群落的结构和功能。在这里，我们使用同位素标记水验证并展示了一种不依赖于底物的蛋白质 SIP 方案，该方案可捕获群落中整个微生物的活性。我们发现 18O 掺入肽中的速率更高，因此具有更高的灵敏度。然后，我们将该方法应用于在两种培养基配方中生长的人类远端肠道微生物生态系统的体外模型中，以评估高纤维和高蛋白饮食之间微生物活性的变化。我们表明，群落结构变化不大，但饮食之间的功能不同。总之，我们的方法可以检测复杂细菌群落中的种特异性代谢活性，更具体地说，可以定量测定氨基酸合成的量。重水可以分析添加同位素标记的能量和营养源不易实现的细菌群落的活性。意义：重稳定同位素允许检测复杂生态系统中的活性关键参与者，其中许多生物被认为处于休眠状态。与标记能量或营养源相反，重水可以作为一种合适的替代方法来追踪活性，这已经在 DNA 和 RNA 中得到了证明。在这里，我们验证、量化和比较了重水（标记有氘或 18 氧）掺入大肠杆菌 K12 蛋白质和人类肠道微生物生态系统体外模型中的蛋白质的情况。我们的研究意义在于提供了一种免费的分析方法，用于分析氘和 18 氧掺入蛋白质的情况，并验证了将重水作为活性示踪剂的适用性。我们的方法揭示了复杂生态系统中微生物的相对功能贡献，这将提高我们对动物和环境相关微生物组和体外模型的理解。
