Raman-deuterium isotope probing to study metabolic activities of single bacterial cells in human intestinal microbiota.

Yi Wang,Hongjuan Wang,Jiabao Xu,Tang Liu,Lingchao Kong,Chunmiao Zheng,Wei E Huang,Lingbo Yi

doi:10.1111/1751-7915.13519

Abstract

SummaryHuman 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 (D2O), D2O 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.

Highlights

The human gut microbiota consists of up to 3.8 9 1013 microbes, which is closer to the number of human cells of 70 kg males (Sender et al, 2016) and contains ~100 times more genes than the human’s genome(Backhed et al, 2005; Gill et al, 2006; Ley et al, 2006; Thursby and Juge, 2017)
After anaerobically incubating the human faeces with heavy water (D2O), D2O with specific substrates 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
At 8-h post-cultivation, a broad Raman band appeared in the region between 2020 and 2300 cmÀ1, peaked at 2160 cmÀ1 (Fig. 1A), which is C–D stretching vibrations shifted from the C–H stretching vibrations at 2800– 3200 cmÀ1 (Berry et al, 2015). This shift was due to the incorporation of deuterium from D2O to bacterial biomass via NADPH-mediated H/D exchange reactions in the metabolically active bacteria

Summary

Introduction

The human gut microbiota consists of up to 3.8 9 1013 microbes, which is closer to the number of human cells of 70 kg males (Sender et al, 2016) and contains ~100 times more genes than the human’s genome(Backhed et al, 2005; Gill et al, 2006; Ley et al, 2006; Thursby and Juge, 2017). After anaerobically incubating the human faeces with heavy water (D2O), D2O 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. We used Raman–DIP to study the metabolic activities and reveal their pathways of the intestinal microbiota in response to different nutrient sources such as sugar (glucose), amino acids (tyrosine and tryptophan) and fatty acids (oleic acid).

Results

Conclusion