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Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing

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Author: Kovatcheva-Datchary, P. · Egert, M. · Maathuis, A. · Rajilić-Stojanović, M. · Graaf, A.A.de · Smidt, H. · Vos, W.M.de · Venema, K.
Type:article
Date:2009
Institution: TNO Kwaliteit van Leven
Source:Environmental Microbiology, 4, 11, 914-926
Identifier: 241464
doi: doi:10.1111/j.1462-2920.2008.01815.x
Keywords: Biology · biomedical research · acetic acid derivative · bacterial DNA · bacterial RNA · butyric acid derivative · isotope · propionic acid derivative · ribosome DNA · RNA 16S · starch · article · bacterium · chemistry · classification · DNA fingerprinting · DNA sequence · fermentation · genetics · growth, development and aging · human · in vitro study · large intestine · metabolism · microbiology · molecular genetics · nucleotide sequence · phylogeny · restriction fragment length polymorphism · RNA gene · sequence homology · staining · Acetates · Bacteria · Butyrates · DNA Fingerprinting · DNA, Bacterial · DNA, Ribosomal · fermentation · genes, rRNA · humans · large intestine · isotopes · molecular sequence data · phylogeny · polymorphism · restriction fragment length · propionates · bacterial RNA · ribosomal RNA 16S · sequence analysis DNA · sequence homology · nucleic acid · staining and labeling · starch · bacteria (microorganisms) · bifidobacterium adolescentis · eubacterium rectale · prevotella · ruminococcus · ruminococcus bromii

Abstract

Summary Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP), we identified starch-fermenting bacteria under human colon-like conditions. To the microbiota of the TIM-2 in vitro model of the human colon 7.4 g l-1 of [U-13C]-starch was added. RNA extracted from lumen samples after 0 (control), 2, 4 and 8 h was subjected to density-gradient ultracentrifugation. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting and phylogenetic analyses of the labelled and unlabelled 16S rRNA suggested populations related to Ruminococcus bromii, Prevotella spp. and Eubacterium rectale to be involved in starch metabolism. Additionally, 16S rRNA related to that of Bifidobacterium adolescentis was abundant in all analysed fractions. While this might be due to the enrichment of high-GC RNA in high-density fractions, it could also indicate an active role in starch fermentation. Comparison of the T-RFLP fingerprints of experiments performed with labelled and unlabelled starch revealed Ruminococcus bromii as the primary degrader in starch fermentation in the studied model, as it was found to solely predominate in the labelled fractions. LC-MS analyses of the lumen and dialysate samples showed that, for both experiments, starch fermentation primarily yielded acetate, butyrate and propionate. Integration of molecular and metabolite data suggests metabolic cross-feeding in the system, where populations related to Ruminococcus bromii are the primary starch degrader, while those related to Prevotella spp., Bifidobacterium adolescentis and Eubacterium rectale might be further involved in the trophic chain. © 2008 The Authors. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd.