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Observational Study
. 2017 Aug 25;7:383.
doi: 10.3389/fcimb.2017.00383. eCollection 2017.

The Glycolytic Versatility of Bacteroides uniformis CECT 7771 and Its Genome Response to Oligo and Polysaccharides

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Free PMC article
Observational Study

The Glycolytic Versatility of Bacteroides uniformis CECT 7771 and Its Genome Response to Oligo and Polysaccharides

Alfonso Benítez-Páez et al. Front Cell Infect Microbiol. .
Free PMC article

Abstract

Bacteroides spp. are dominant components of the phylum Bacteroidetes in the gut microbiota and prosper in glycan enriched environments. However, knowledge of the machinery of specific species isolated from humans (like Bacteroides uniformis) contributing to the utilization of dietary and endogenous sources of glycans and their byproducts is limited. We have used the cutting-edge nanopore-based technology to sequence the genome of B. uniformis CECT 7771, a human symbiont with a proven pre-clinical efficacy on metabolic and immune dysfunctions in obesity animal models. We have also used massive sequencing approaches to distinguish the genome expression patterns in response to carbon sources of different complexity during growth. At genome-wide level, our analyses globally demonstrate that B. uniformis strains exhibit an expanded glycolytic capability when compared with other Bacteroides species. Moreover, by studying the growth and whole-genome expression of B. uniformis CECT 7771 in response to different carbon sources, we detected a differential growth fitness and expression patterns across the genome depending on the carbon source of the culture media. The dietary fibers used exerted different effects on B. uniformis CECT 7771 activating different molecular pathways and, therefore, allowing the production of different metabolite types with potential impact on gut health. The genome and transcriptome analysis of B. uniformis CECT 7771, in response to different carbon sources, shows its high versatility to utilize both dietary and endogenous glycans along with the production of potentially beneficial end products for both the bacterium and the host, pointing to a mechanistic basis of a mutualistic relationship.

Keywords: Bacteroides uniformis; GABA; butyrate; genome; mucin-degrader; polysaccharides; transcriptome.

Figures

Figure 1
Figure 1
Phylogenetic relationships among Bacteroides species. The genome information of all species and strains presented in the figure was used for comparative analysis using algorithms implemented in the Jspecies tool (Richter and Rossello-Mora, 2009). Genetic distance based on the Average Nucleotide Identity is presented in the left UPGMA dendrogram. The relationships based on tetranucleotide distribution is presented in the UPGMA right dendrogram. Branch lengths are based on the RSMD distance coefficient.
Figure 2
Figure 2
Comparative analysis of Bacteroides uniformis strains. Circular representation of B. uniformis CECT 7771 (black inner line), B. unformis CL03T00C23, (red), B. unformis ATCC 8495 (purple), B. unformis 3978-T3i (blue), and B. unformis dnLKV2 (green) genomes. They are compared using whole-genome and blast-based alignment. Genomic regions exclusively found in B. uniformis CECT 7771 are highlighted with dashed rectangles.
Figure 3
Figure 3
B. uniformis CECT 7771 and carbohydrate metabolism. (A) Venn diagram showing the strain-specific and shared CAZy families among five B. uniformis strains. (B) Venn diagram showing the species-specific and shared CAZy families among six Bacteroides species. The number of non-redundant CAZy families present in respective genomes is showed below respective Venn diagrams following the color nomenclature.
Figure 4
Figure 4
Growth fitness of B. uniformis CECTC 7771 in different carbon sources. (A) Growth curve comparison between B. uniformis CECT 7771 cultures using glucose (blue line), gum arabic (purple), WBE (red), inulin (green), pectin (baby blue), or mucin (blue-green) as the carbon source. Growth was monitored measuring OD600 at 60 min intervals. The OD600 values are presented as a mean of three independent replicates (±SEM). (B) Doubling times calculated during the exponential growth phase by linear regression. The values are present as a mean of three independent replicates (±SEM). The asterisks indicate differential growth rates when compared to glucose used as the reference (p < 0.022 for gum arabic and p < 0.057 for WBE) supported on pairwise t-test comparison with Welch's correction.
Figure 5
Figure 5
Expression patterns in the B. uniformis CECT 7771 genome. (A) Venn diagram showing the number of genes differentially expressed in B. uniformis CECT 7771 cultures using gum arabic, WBE, inulin, pectin, and mucin as the carbon source. The expression pattern in glucose media was used as normalizer. The total number of genes showing differential expression in different conditions are depicted below the Venn diagram. (B) Heatmap with expression values obtained from the exploratory transcriptome analysis of the 633 genes showing differential expression in all condition tested. Hierarchical clustering was assessed for the treatments and genes using Euclidean distance and average linkage methods implemented in ClustVistweb server. (C) Detailed view of the gene expression profile for 15 genes associated with GABA, butyrate, and long-chain fatty acid biosynthesis. Both the treatment labels and gene expression score correspond with those observed in (B).
Figure 6
Figure 6
Quantification of GABA production in culture supernatants. Culture supernatants from conditions where over expression of glutamate decarboxylase (BUNIF7771_0544–K01580) and glutamate:GABA antiporter (BUNIF7771_0548–K20265) genes were inferred from the exploratory RNAseq and corroborated by qPCR were subject to LC-MS analysis in order to determine the GABA concentration at log phase of respective cultures (OD600 ~ 0.4). The comparisons were made using three independent replicates per condition and statistical analysis was assessed through pairwise t-test with Bonferroni correction.

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