Phenotypic and Genomic Diversification in Complex Carbohydrate-Degrading Human Gut Bacteria.

Symbiotic bacteria are responsible for the majority of complex carbohydrate digestion in the human colon. Since the identities and amounts of dietary polysaccharides directly impact the gut microbiota, determining which microorganisms consume specific nutrients is central for defining the relationship between diet and gut microbial ecology. Using a custom phenotyping array, we determined carbohydrate utilization profiles for 354 members of the Bacteroidetes, a dominant saccharolytic phylum. There was wide variation in the numbers and types of substrates degraded by individual bacteria, but phenotype-based clustering grouped members of the same species indicating that each species performs characteristic roles. The ability to utilize dietary polysaccharides and endogenous mucin glycans was negatively correlated, suggesting exclusion between these niches. By analyzing related Bacteroides ovatus/Bacteroides xylanisolvens strains that vary in their ability to utilize mucin glycans, we addressed whether gene clusters that confer this complex, multilocus trait are being gained or lost in individual strains. Pangenome reconstruction of these strains revealed a remarkably mosaic architecture in which genes involved in polysaccharide metabolism are highly variable and bioinformatics data provide evidence of interspecies gene transfer that might explain this genomic heterogeneity. Global transcriptomic analyses suggest that the ability to utilize mucin has been lost in some lineages of B. ovatus and B. xylanisolvens, which harbor residual gene clusters that are involved in mucin utilization by strains that still actively express this phenotype. Our data provide insight into the breadth and complexity of carbohydrate metabolism in the microbiome and the underlying genomic events that shape these behaviors. IMPORTANCE Nonharmful bacteria are the primary microbial symbionts that inhabit the human gastrointestinal tract. These bacteria play many beneficial roles and in some cases can modify disease states, making it important to understand which nutrients sustain specific lineages. This knowledge will in turn lead to strategies to intentionally manipulate the gut microbial ecosystem. We designed a scalable, high-throughput platform for measuring the ability of gut bacteria to utilize polysaccharides, of which many are derived from dietary fiber sources that can be manipulated easily. Our results provide paths to expand phenotypic surveys of more diverse gut bacteria to understand their functions and also to leverage dietary fibers to alter the physiology of the gut microbial community.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Improved Hybrid Genome Assemblies of Two Strains of Bacteroides xylanisolvens, SD_CC_1b and SD_CC_2a, Obtained Using Illumina and 454 Sequencing Technologies.

Bacteroides xlyanisolvens strains (SD_CC_1b, SD_CC_2a) isolated from human feces were grown on crystalline cellulose. Cellulolytic properties are not common in Bacteroides species. Here, we report improved genome sequences of both of the B. xlyanisolvens strains.

Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns.

One-hundred-and-three isolates of Bacteroides ovatus, B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade.

Newly cultured bacteria with broad diversity isolated from eight-week continuous culture enrichments of cow feces on complex polysaccharides.

One of the functions of the mammalian large intestinal microbiota is the fermentation of plant cell wall components. In ruminant animals, the majority of their nutrients are obtained via pregastric fermentation; however, up to 20% can be recovered from microbial fermentation in the large intestine. Eight-week continuous culture enrichments of cattle feces with cellulose and xylan-pectin were used to isolate bacteria from this community. A total of 459 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented: Firmicutes (51.9%), Bacteroidetes (30.9%), Proteobacteria (11.1%), Actinobacteria (3.5%), Synergistetes (1.5%), and Fusobacteria (1.1%). The majority of bacterial isolates had <98.5% identity to cultured bacteria with sequences in the Ribosomal Database Project and thus represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, B. ovatus, and B. xylanisolvens and members of the Porphyromonadaceae family. Many of the Firmicutes and Bacteroidetes isolates were related to species demonstrated to possess enzymes which ferment plant cell wall components; the others were hypothesized to cross-feed these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 98% of the isolates not represented as previously cultured, there are new opportunities to study the genomic and metabolic capacities of these members of the complex intestinal microbiota.

Broad diversity and newly cultured bacterial isolates from enrichment of pig feces on complex polysaccharides.

One of the fascinating functions of mammalian intestinal microbiota is fermentation of plant cell wall components. Eight-week continuous culture enrichments of pig feces with cellulose and xylan/pectin were used to isolate bacteria from this community. A total of 575 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented in the bacterial isolates: Firmicutes (242), Bacteroidetes (185), Proteobacteria (65), Fusobacteria (55), Actinobacteria (23), and Synergistetes (5). The majority of the bacterial isolates had ≥ 97 % similarity to cultured bacteria with sequences in the RDP, but 179 isolates represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families of Lachnospiraceae, Enterococcaceae, Staphylococcaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, Bacteroides ovatus, and B. xylanisolvens. Many of the Firmicutes and Bacteroidetes isolates were identified as species that possess enzymes that ferment plant cell wall components, and the rest likely support these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 30 % of the isolates not represented in culture, there are new opportunities to study genomic and metabolic capacities of these members of the complex intestinal microbiota.

Swine odor analyzed by odor panels and chemical techniques.

The National Research Council identified odors as a significant animal emission and highlighted the need to develop standardized protocols for sampling and analysis. The purpose of our study was to compare different odor sampling techniques for monitoring odors emitted from stored swine manure. In our study, odorous headspace air from swine manure holding tanks were analyzed by human panels and analytical techniques. Odorous air was analyzed by human panels using dynamic dilution olfactometry (DDO). Chemical analysis used acid traps for ammonia (NH3), fluorescence for hydrogen sulfide (H2S), and thermal desorption gas chromatography-mass spectrometry for volatile organic compounds (VOCs). Chemical analysis included the use of gas chromatography-olfactometry (GC-O) for determining key odorants. Chemical odorant concentrations were converted to odor activity values (OAVs) based on literature odor thresholds. The GC-O technique used was GC-SNIF. Dilution thresholds measured by different odor panels were significantly different by almost an order of magnitude even though the main odorous compound concentrations had not changed significantly. Only 5% of the key odorous VOCs total OAVs was recovered from the Tedlar bags used in DDO analysis. Ammonia was the only chemical odorant significantly correlated with DDO analysis in the fresh (1 wk) and aged manure. Chemical analysis showed that odor concentration stabilized after 5 to 7 wk and that HS was the most dominant odorant. In aged manure, neither volatile fatty acids (VFAs) nor HS was correlated with any other chemical odorant, but NH, phenols, and indoles were correlated, and phenols and indoles were highly correlated. Correlation of odorant concentration was closely associated with the origin of the odorant in the diet. Key odorants determined by chemical and GC-O included indoles, phenols, NH3, and several VFAs (butanoic, 3-methylbutanoic, and pentanoic acids).

Dietary protein and cellulose effects on chemical and microbial characteristics of Swine feces and stored manure.

The objectives of this study were to investigate the effects of dietary crude protein (14.5 or 12.0%) and cellulose (8.7 or 2.5%) levels on composition of feces and manure after 8 wk of diet feeding and storage. Pigs were fed twice daily; after each feeding, urine and feces were collected and added to manure storage containers. On weeks 2 and 8 after initiation of the experiment, fresh fecal and manure samples were obtained. On Week 8, increased dietary cellulose resulted in significantly higher levels of volatile fatty acids (VFA) and phenols in feces compare to other diets. In contrast, dietary protein had the greatest effect on manure chemical composition; lower protein decreased sulfur content, ammonia, and phenolic compound concentrations. High levels of either dietary cellulose or protein tended to increase microbial community similarity in fecal samples, but only high protein increased similarity among manure sample microbial communities. Fecal and manure samples from Week 8 differed from samples taken in Week 2 both in chemical and microbiological composition. Week 2 samples had lower concentrations of many of chemical compounds and microbial diversity than samples from Week 8. The fecal results indicate that after 2 wk of feeding experimental diets the animals were not fully adapted to the diets. More importantly, after only 2 wk of urine and fecal collection, manure was not representative of stored manure, limiting its usefulness in developing standards and recommendations for on-farm management practices.

CP/MAS 13C NMR analysis of cellulase treated bleached softwood kraft pulp.

Fully bleached softwood kraft pulps were hydrolyzed with cellulase (1,4-(1,3:1,4)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) from Trichoderma reesei. Supra-molecular structural features of cellulose during enzymatic hydrolysis were examined by using CP/MAS 13C NMR spectra in combination with line-fitting analysis. Different types of cellulose allomorphs (cellulose I(alpha), cellulose I(beta), para-crystalline) and amorphous regions were hydrolyzed to a different extent by the enzyme used. Also observed was a rapid initial phase for hydrolysis of regions followed by a slow hydrolysis phase. Cellulose I(alpha), para-crystalline, and non-crystalline regions of cellulose are more susceptible to enzymatic hydrolysis than cellulose I(beta) during the initial phase. After the initial phase, all the regions are then similarly susceptible to enzymatic hydrolysis.