Background: The gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel single-cell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. Results: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as highand medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin-responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. Conclusions: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota and could be exploited to improve human and animal health.

A diet rich in vegetables and fruit is generally considered healthy because of a high content of phytochemicals, vitamins, and fiber. The phytochemical indole-3-carbinol (I3C), a derivative of glucobrassicin, is sold as a dietary supplement promising diverse health benefits. I3C metabolites act as ligands of the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals. Here, we investigated how dietary AhR ligand supplementation influences AhR target gene expression and intestinal microbiota composition. For this, we used AhR repressor (AhRR)-reporter mice as a tool to study AhR activation in the intestine following dietary I3C-supplementation in comparison with AhR ligand-deprived diets, including a high fat diet. AhRR expression in intestinal immune cells was mainly driven by dietary AhR ligands and was independent of microbial metabolites. A lack of dietary AhR ligands caused enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis and correlated with the expansion of Enterobacteriaceae, whereas Clostridiales, Muribaculaceae, and Rikenellaceae were strongly reduced. I3C supplementation largely reverted this effect. Comparison of I3C-induced changes in microbiota composition using wild-type (WT), AhRR-deficient, and AhR-deficient mice revealed both AhR-dependent and -independent alterations in the microbiome. Overall, our study demonstrates that dietary AhR ligand supplementation has a profound influence on Ahrr expression in intestinal immune cells as well as microbiota composition.

BackgroundThe gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel singlecell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. ResultsInulin feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., and increased microbial metabolic function resulted in increased succinate concentrations in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning two phyla, four classes, seven orders, and 14 families, and 31 different strains of SAGs were graded as high-and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. ConclusionsOur single-cell genomics approach generated a massive amount of SAGs, enabling functional analysis of uncultured bacteria in the intestinal microbiome and consideration of host responses. This enabled us to estimate metabolic lineages involved in the bacterial 2 fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota, and could be exploited to improve human and animal health. BackgroundThe gut microbiota plays a crucial role in the control of host physiology and metabolism through the release and transformation of metabolites like short-chain fatty acids (SCFAs) [1,2]. Many studies have demonstrated that dietary fiber supplementation can modulate gut microbiota composition and promote SCFA production [3][4][5]. This dietary fiber metabolism is particularly important for non-digestible inulin-type fructans classified as prebiotics, which are substrates that promote the growth of beneficial microorganisms in the gut [6][7][8][9][10][11][12]. Thus, to enhance the benefits of dietary fiber to the host, it is important to identify inulin responders in the complex microbiome community of the host intestine, and characterize their mechanisms of inulin fermentation and potential for SCFA production.Recently developed metagenomic approaches can provide comprehensive microbial profiling and putative functions using 16S rRNA gene amplicon sequencing and shotgun metagenome sequencing. These methods have enabled the ...