Fig 1.
Phylogenetic analysis of taxa enriched in the presence of quercetin.
The phylogenetic tree shows six ASVs (black dots) whose abundance increased in the presence of quercetin; distances in the tree were inferred using the UPGMA method [27]. The optimal tree with the sum of branch length = 2.18101115 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches [29]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The scale bar refers to evolutionary distances in substitutions per site. The evolutionary distances were computed using the Maximum Composite Likelihood method [26] and are in the units of the number of base substitutions per site. The analysis involved 44 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 225 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [25].
Fig 2.
Bar plots for the relative abundances of the 6 Amplicon Sequence Variants (ASV) enriched in quercetin.
Human subjects are labeled as Subject #1, #2, #3, #4, #5, #6, #7, #8, #9 in bar plots, for better visualization intercalated libraries are highlighted in gray. Each library has 6 bars, 3 corresponds to replicates from first incubation and 3 from second incubation (Subject #3 had only 2 replicates for second incubation).
Fig 3.
Relative abundances of ASV_65f4 and ASV_a45d are negatively correlated.
Relative abundance of ASV_65f4 and ASV_a45d in human (A) and human microbiota-associated mice fecal samples (B). ASV_65f4 is represented in gray and ASV_a45d in black. In the bottom panel, libraries from HMAM mice fed a diet high in fiber are shown in solid color and mice fed a diet low in fiber are shown with a line pattern. Error bars correspond to 3 incubations done with fecal matter from the same donor individually sampled. For human samples (n = 9), relative abundances obtained for the second incubation are shown and for HMAM mice (n = 6), relative abundances obtained after 7 days of incubation with quercetin are shown.
Fig 4.
Principal Component Analysis (PCA) plot of the libraries from in vitro incubations with fecal samples from Human Microbiota-Associated Mice (HMAM).
(A) PCA plot of the HMAM samples at 0 days of incubation. Libraries from HMAM mice fed a high fiber diet are shown in gray and libraries from HMAM mice fed a low fiber diet in black. (B) PCA plot of the HMAM samples at 7 days of incubation. Libraries that were enriched in ASV_65f4 are shown in gray and libraries enriched in ASV_a45d are shown in black. Each symbol represents a library (n = 6, 3 replicates), different shapes represent libraries from a different subject: #10, star (*); #11, diamond (♦); #12, dot (●); #13, inv. triangle (▼); #14, triangle (▲); and #15, square (■).
Fig 5.
Relative abundance of ASV_65f4 and ASV_a45d in in vitro incubations with fecal sample combinations.
Fecal samples from subjects #3 and #4 were combined (50:50) or not (100:0 and 0:100). In vitro incubations with the fecal samples from subject #3 (enriched in ASV_65f4) are shown in gray (right) and from subject #4 (enriched in ASV_a45d) are shown in black (left).
Fig 6.
Representative BMC Loci for Flavonifractor spp..
The ethanolamine utilization operons (Eut operon 1 and 2) and 1,2-propanediol utilization operon (Pdu operon). Genes are drawn on the F. plautii YL31 genome using Benchling [Biology Software] (2019). Eut operon 1 is 12,247 bp, Pdu operon is 22,527 bp, and Eut operon 2 is 18,769 bp. Abbreviations are as follows: AlcDH, Alcohol dehydrogenase; AldDH, Aldehyde dehydrogenase; PTAC, phosphotransacetylase; BMC, bacterial microcompartment; PdtaS, two-component system, sensor histidine kinase; PdtaR, two-component system, response regulator. Genes are color-coded according to their annotation: light blue, BMC-containing proteins; red, aldehyde dehydrogenase; green, alcohol dehydrogenase; solid pink, PduL-type phosphotransacylase; light purple, re-activating proteins; dark blue, signature enzymes (ethanolamine ammonia lyase subunits and propanediol dehydratase subunits); brown, regulatory element including two-component signaling elements; yellow, transporter; gray, other Eut or Pdu proteins. Circles show the presence (filled circle) or absence (white circle) of proteins in the strains depicted in the phylogenetic tree. The UPGMA phylogenetic tree involved 8 nucleotide sequences: F. plautii 2789STDY5834932 (STDY5834932), F. plautii An248 (An248), F. plautii ATCC 29863 (ATCC29863), F. plautii YL31 (YL31), Flavonifractor sp. An306 (An306), Flavonifractor sp. An82 (An82), Flavonifractor sp. An4 (An4), and Flavonifractor sp. An10 (An10). All positions containing gaps and missing data were eliminated. There were a total of 1008 positions in the final dataset. S8 Table contains all accession numbers for each protein in each strain. Pdu operon was reconstructed by its similarity with the one in Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (S9 Table).
Table 1.
Gene clusters enriched in the genomes more closely related to ASV_65f4.
ASV_65f4-related group includes F. plautii YL31, F. plautii 2789STDY5834932, F. plautii ATCC 29863, and F. plautii An248.
Table 2.
Gene clusters enriched in the genomes more closely related to ASV_a45d.
ASV_a45d-related group includes Flavonifractor sp. An4, Flavonifractor sp. An10, Flavonifractor sp. An82, Flavonifractor sp. An306.
Table 3.
Blast results for Phloretin Hydrolase (Phy) for Flavonifractor spp.