Symbiotic bacteria inhabiting the human gut have evolved under intense pressure to utilize complex carbohydrates, primarily plant cell wall glycans in our diets. These polysaccharides are not ...digested by human enzymes, but are processed to absorbable short chain fatty acids by gut bacteria. The Bacteroidetes, one of two dominant bacterial phyla in the adult gut, possess broad glycan-degrading abilities. These species use a series of membrane protein complexes, termed Sus-like systems, for catabolism of many complex carbohydrates. However, the role of these systems in degrading the chemically diverse repertoire of plant cell wall glycans remains unknown. Here we show that two closely related human gut Bacteroides, B. thetaiotaomicron and B. ovatus, are capable of utilizing nearly all of the major plant and host glycans, including rhamnogalacturonan II, a highly complex polymer thought to be recalcitrant to microbial degradation. Transcriptional profiling and gene inactivation experiments revealed the identity and specificity of the polysaccharide utilization loci (PULs) that encode individual Sus-like systems that target various plant polysaccharides. Comparative genomic analysis indicated that B. ovatus possesses several unique PULs that enable degradation of hemicellulosic polysaccharides, a phenotype absent from B. thetaiotaomicron. In contrast, the B. thetaiotaomicron genome has been shaped by increased numbers of PULs involved in metabolism of host mucin O-glycans, a phenotype that is undetectable in B. ovatus. Binding studies of the purified sensor domains of PUL-associated hybrid two-component systems in conjunction with transcriptional analyses demonstrate that complex oligosaccharides provide the regulatory cues that induce PUL activation and that each PUL is highly specific for a defined cell wall polymer. These results provide a view of how these species have diverged into different carbohydrate niches by evolving genes that target unique suites of available polysaccharides, a theme that likely applies to disparate bacteria from the gut and other habitats.
Bacterial viruses (phages) are the most abundant biological group on Earth and are more genetically diverse than their bacterial prey/hosts. To characterize their role as agents shaping gut microbial ...community structure, adult germ-free mice were colonized with a consortium of 15 sequenced human bacterial symbionts, 13 of which harbored one or more predicted prophages. One member, Bacteroides cellulosilyticus WH2, was represented by a library of isogenic transposon mutants that covered 90% of its genes. Once assembled, the community was subjected to a staged phage attack with a pool of live or heat-killed virus-like particles (VLPs) purified from the fecal microbiota of five healthy humans. Shotgun sequencing of DNA from the input pooled VLP preparation plus shotgun sequencing of gut microbiota samples and purified fecal VLPs from the gnotobiotic mice revealed a reproducible nonsimultaneous pattern of attack extending over a 25-d period that involved five phages, none described previously. This system allowed us to (i) correlate increases in specific phages present in the pooled VLPs with reductions in the representation of particular bacterial taxa, (ii) provide evidence that phage resistance occurred because of ecological or epigenetic factors, (iii) track the origin of each of the five phages among the five human donors plus the extent of their genome variation between and within recipient mice, and (iv) establish the dramatic in vivo fitness advantage that a locus within a B. cellulosilyticus prophage confers upon its host. Together, these results provide a defined community-wide view of phage–bacterial host dynamics in the gut.
Knowledge of the spatial organization of the gut microbiota is important for understanding the physical and molecular interactions among its members. These interactions are thought to influence ...microbial succession, community stability, syntrophic relationships, and resiliency in the face of perturbations. The complexity and dynamism of the gut microbiota pose considerable challenges for quantitative analysis of its spatial organization. Here, we illustrate an approach for addressing this challenge, using (i) a model, defined 15-member consortium of phylogenetically diverse, sequenced human gut bacterial strains introduced into adult gnotobiotic mice fed a polysaccharide-rich diet, and (ii) in situ hybridization and spectral imaging analysis methods that allow simultaneous detection of multiple bacterial strains at multiple spatial scales. Differences in the binding affinities of strains for substrates such as mucus or food particles, combined with more rapid replication in a preferred microhabitat, could, in principle, lead to localized clonally expanded aggregates composed of one or a few taxa. However, our results reveal a colonic community that is mixed at micrometer scales, with distinct spatial distributions of some taxa relative to one another, notably at the border between the mucosa and the lumen. Our data suggest that lumen and mucosa in the proximal colon should be conceptualized not as stratified compartments but as components of an incompletely mixed bioreactor. Employing the experimental approaches described should allow direct tests of whether and how specified host and microbial factors influence the nature and functional contributions of “microscale” mixing to the dynamic operations of the microbiota in health and disease.
The interrelationships between our diets and the structure and operations of our gut microbial communities are poorly understood. A model community of 10 sequenced human gut bacteria was introduced ...into gnotobiotic mice, and changes in species abundance and microbial gene expression were measured in response to randomized perturbations of four defined ingredients in the host diet. From the responses, we developed a statistical model that predicted over 60% of the variation in species abundance evoked by diet perturbations, and we were able to identify which factors in the diet best explained changes seen for each community member. The approach is generally applicable, as shown by a follow-up study involving diets containing various mixtures of pureed human baby foods.
The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors ...such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities.
Feed efficiency is a crucial parameter in swine production, given both its economic and environmental impact. The gut microbiota plays an essential role in nutrient digestibility and is, therefore, ...likely to affect feed efficiency. This study aimed to characterize feed efficiency, fatness traits, and gut microbiome composition in three major breeds of domesticated swine and investigate a possible link between feed efficiency and gut microbiota composition.
Average daily feed intake (ADFI), average daily gain (ADG), feed conversion ratio (FCR), residual feed intake (RFI), backfat, loin depth, and intramuscular fat of 615 pigs belonging to the Duroc (DR), Landrace (LR), and Large White (LW) breeds were measured. Gut microbiota composition was characterized by 16S rRNA gene sequencing. Orthogonal contrasts between paternal line (DR) and maternal lines (LR+LW) and between the two maternal lines (LR versus LW) were performed. Average daily feed intake and ADG were statistically different with DR having lower ADFI and ADG compared to LR and LW. Landrace and LW had a similar ADG and RFI, with higher ADFI and FCR for LW. Alpha diversity was higher in the fecal microbial communities of LR pigs than in those of DR and LW pigs for all time points considered. Duroc communities had significantly higher proportional representation of the Catenibacterium and Clostridium genera compared to LR and LW, while LR pigs had significantly higher proportions of Bacteroides than LW for all time points considered. Amplicon sequence variants from multiple genera (including Anaerovibrio, Bacteroides, Blautia, Clostridium, Dorea, Eubacterium, Faecalibacterium, Lactobacillus, Oscillibacter, and Ruminococcus) were found to be significantly associated with feed efficiency, regardless of the time point considered.
In this study, we characterized differences in the composition of the fecal microbiota of three commercially relevant breeds of swine, both over time and between breeds. Correlations between different microbiome compositions and feed efficiency were established. This suggests that the microbial community may contribute to shaping host productive parameters. Moreover, our study provides important insights into how the intestinal microbial community might influence host energy harvesting capacity. A deeper understanding of this process may allow us to modulate the gut microbiome in order to raise more efficient animals. Video Abstract.
Colonization of germ-free mice with a normal gut microbiota elicits bacteria-specific IgA antibody responses. The effects of these responses on microbial and host biology remain poorly defined. ...Therefore, we developed a gnotobiotic mouse model where the microbiota is reduced to one bacterial species, and the antibody repertoire to a single, monoclonal IgA against the bacterium's capsular polysaccharide. Bacteroides thetaiotaomicron was introduced into germ-free wild-type, immunodeficient Rag1(-/-), or Rag1(-/-) mice harboring IgA-producing hybridoma cells. Without IgA, B. thetaiotaomicron elicits a more robust innate immune response and reacts to this response by inducing genes that metabolize host oxidative products. IgA reduces intestinal proinflammatory signaling and bacterial epitope expression, thereby balancing suppression of the oxidative burst with the antibody's negative impact on bacterial fitness. These results underscore the adaptive immune system's critical role in establishing a sustainable host-microbial relationship. Immunoselection of bacterial epitope expression may contribute to the remarkable strain-level diversity in this ecosystem.
Understanding how the human gut microbiota and host are affected by probiotic bacterial strains requires carefully controlled studies in humans and in mouse models of the gut ecosystem where ...potentially confounding variables that are difficult to control in humans can be constrained. Therefore, we characterized the fecal microbiomes and metatranscriptomes of adult female monozygotic twin pairs through repeated sampling 4 weeks before, 7 weeks during, and 4 weeks after consumption of a commercially available fermented milk product (FMP) containing a consortium of Bifidobacterium animalis subsp. lactis, two strains of Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. cremoris, and Streptococcus thermophilus. In addition, gnotobiotic mice harboring a 15-species model human gut microbiota whose genomes contain 58,399 known or predicted protein-coding genes were studied before and after gavage with all five sequenced FMP strains. No significant changes in bacterial species composition or in the proportional representation of genes encoding known enzymes were observed in the feces of humans consuming the FMP. Only minimal changes in microbiota configuration were noted in mice after single or repeated gavage with the FMP consortium. However, RNA-Seq analysis of fecal samples and follow-up mass spectrometry of urinary metabolites disclosed that introducing the FMP strains into mice results in significant changes in expression of microbiome-encoded enzymes involved in numerous metabolic pathways, most prominently those related to carbohydrate metabolism. B. animalis subsp. lactis, the dominant persistent member of the FMP consortium in gnotobiotic mice, up-regulates a locus in vivo that is involved in the catabolism of xylooligosaccharides, a class of glycans widely distributed in fruits, vegetables, and other foods, underscoring the importance of these sugars to this bacterial species. The human fecal metatranscriptome exhibited significant changes, confined to the period of FMP consumption, that mirror changes in gnotobiotic mice, including those related to plant polysaccharide metabolism. These experiments illustrate a translational research pipeline for characterizing the effects of FMPs on the human gut microbiome.
In pigs, gut bacteria have been shown to play important roles in nutritional, physiological, and immunological processes in the host. However, the contribution of their metagenomes or part of them, ...which are normally reflected by fragments of 16S rRNA-encoding genes, has yet to be fully investigated.
Fecal samples, collected from a population of crossbred pigs at three time points, including weaning, week 15 post weaning (hereafter "week 15"), and end-of-feeding test (hereafter "off-test"), were used to evaluate changes in the composition of the fecal microbiome of each animal over time. This study used 1205, 1295, and 1283 samples collected at weaning, week 15, and off-test, respectively. There were 1039 animals that had samples collected at all three time points and also had phenotypic records on back fat thickness (BF) and average daily body weight gain (ADG). Firmicutes and Bacteroidetes were the most abundant phyla at all three time points. The most abundant genera at all three time points included Clostridium, Escherichia, Bacteroides, Prevotella, Ruminococcus, Fusobacterium, Campylobacter, Eubacterium, and Lactobacillus. Two enterotypes were identified at each time point. However, only enterotypes at week 15 and off-test were significantly associated with BF. We report herein two novel findings: (i) alpha diversity and operational taxonomic unit (OTU) richness were moderately heritable at week 15, h
of 0.15 ± 0.06 to 0.16 ± 0.07 and 0.23 ± 0.09 to 0.26 ± 0.08, respectively, as well as at off-test, h
of 0.20 ± 0.09 to 0.33 ± 0.10 and 0.17 ± 0.08 to 0.24 ± 0.08, respectively, whereas very low heritability estimates for both measures were detected at weaning; and (ii) alpha diversity at week 15 had strong and negative genetic correlations with BF, - 0.53 ± 0.23 to - 0.45 ± 0.25, as well as with ADG, - 0.53 ± 0.32 to - 0.53 ± 0.29.
These results are important for efforts to genetically improve the domesticated pig because they suggest fecal microbiota diversity can be used as an indicator trait to improve traits that are expensive to measure.
Despite recent efforts to characterize longitudinal variation in the swine gut microbiome, the extent to which a host's genome impacts the composition of its gut microbiome is not yet well understood ...in pigs. The objectives of this study were: i) to identify pig gut microbiome features associated with growth and fatness, ii) to estimate the heritability of those features, and, iii) to conduct a genome-wide association study exploring the relationship between those features and single nucleotide polymorphisms (SNP) in the pig genome. A total of 1,028 pigs were characterized. Animals were genotyped with the Illumina PorcineSNP60 Beadchip. Microbiome samples from fecal swabs were obtained at weaning (Wean), at mid-test during the growth trial (MidTest), and at the end of the growth trial (OffTest). Average daily gain was calculated from birth to week 14 of the growth trial, from weaning to week 14, from week 14 to week 22, and from week 14 to harvest. Backfat and loin depth were also measured at weeks 14 and 22. Heritability estimates (±SE) of Operational Taxonomic Units ranged from 0.025 (±0.0002) to 0.139 (±0.003), from 0.029 (±0.003) to 0.289 (±0.004), and from 0.025 (±0.003) to 0.545 (±0.034) at Wean, MidTest, and OffTest, respectively. Several SNP were significantly associated with taxa at the three time points. These SNP were located in genomic regions containing a total of 68 genes. This study provides new evidence linking gut microbiome composition with growth and carcass traits in swine, while also identifying putative host genetic markers associated with significant differences in the abundance of several prevalent microbiome features.
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