School of Environment, Harbin Institute of Technology, Harbin, 150090, China.
Guangdong Provincial Key Laboratory of Soil and Groundwater Pollution Control, School of Environmental Science and Engineering, Southern University of Science and Technology, Shenzhen, 518055, China.
Microb Biotechnol. 2020 Mar;13(2):572-583. doi: 10.1111/1751-7915.13519. Epub 2019 Dec 10.
Human intestinal microbiota is important to host health and is associated with various diseases. It is a challenge to identify the functions and metabolic activity of microorganisms at the single-cell level in gut microbial community. In this study, we applied Raman microspectroscopy and deuterium isotope probing (Raman-DIP) to quantitatively measure the metabolic activities of intestinal bacteria from two individuals and analysed lipids and phenylalanine metabolic pathways of functional microorganisms in situ. After anaerobically incubating the human faeces with heavy water (D O), D O with specific substrates (glucose, tyrosine, tryptophan and oleic acid) and deuterated glucose, the C-D band in single-cell Raman spectra appeared in some bacteria in faeces, due to the Raman shift from the C-H band. Such Raman shift was used to indicate the general metabolic activity and the activities in response to the specific substrates. In the two individuals' intestinal microbiota, the structures of the microbial communities were different and the general metabolic activities were 76 ± 1.0% and 30 ± 2.0%. We found that glucose, but not tyrosine, tryptophan and oleic acid, significantly stimulated metabolic activity of the intestinal bacteria. We also demonstrated that the bacteria within microbiota preferably used glucose to synthesize fatty acids in faeces environment, whilst they used glucose to synthesize phenylalanine in laboratory growth environment (e.g. LB medium). Our work provides a useful approach for investigating the metabolic activity in situ and revealing different pathways of human intestinal microbiota at the single-cell level.
人类肠道微生物群对宿主健康很重要，与各种疾病有关。在肠道微生物群落中，识别单个细胞水平上微生物的功能和代谢活性是一个挑战。在这项研究中，我们应用拉曼微光谱和氘同位素探测（Raman-DIP）定量测量了来自两个人的肠道细菌的代谢活性，并原位分析了功能微生物的脂质和苯丙氨酸代谢途径。将人的粪便在无氧条件下与重水（D2O）、含有特定底物（葡萄糖、酪氨酸、色氨酸和油酸）的 D2O 和氘代葡萄糖孵育后，粪便中一些细菌的单细胞拉曼光谱中出现了 C-D 带，这是由于 C-H 带的拉曼位移。这种拉曼位移用于表示一般代谢活性和对特定底物的活性。在两个人的肠道微生物群中，微生物群落的结构不同，一般代谢活性分别为 76±1.0%和 30±2.0%。我们发现，葡萄糖而不是酪氨酸、色氨酸和油酸显著刺激了肠道细菌的代谢活性。我们还证明，在粪便环境中，微生物群内的细菌优先利用葡萄糖来合成脂肪酸，而在实验室生长环境（如 LB 培养基）中，它们利用葡萄糖来合成苯丙氨酸。我们的工作为研究原位代谢活性和揭示人类肠道微生物群在单细胞水平上的不同途径提供了一种有用的方法。
