Abstract
RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that “cross-feeding” occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system.
Highlights
It is well documented that symbiotic gut inhabitants are required for maintaining host health and well-being [1, 2], as they greatly influence several host functions, those in the intestinal system [3,4,5,6,7,8]
The average density of the centrifugation gradients ranged from 1.743 g mL−1 to 1.84 g mL−1, which is in line with previous RNA-based stable isotope probing (RNA-stable isotope probing (SIP)) studies and indicated an adequate density spectrum for efficient separation of isotope-labeled RNA [21,22,23, 25, 26]
While a noticeable amount of unlabeled control RNA (0 h) could be measured at averaged buoyant density (BD) between 1.796 g mL−1 and 1.761 g mL−1, the bulk of it accumulated in fraction 13, showing a BD of 1.767 g mL−1 (Figure 1(b))
Summary
It is well documented that symbiotic gut inhabitants are required for maintaining host health and well-being [1, 2], as they greatly influence several host functions, those in the intestinal system [3,4,5,6,7,8]. The differences in the mean relative abundance of bacterial taxa found in “heavy” and “medium” RNA-SIP fractions were assessed using one-way ANOVA.
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