Fig. 4: Genome-centric metaproteomic analysis of colon samples collected from pigs fed either the control of 4% AcGGM diet.

a Principle Component Analysis of metaproteomes generated from randomly selected colon samples collected from pigs fed either the control (C1-4) or 4% AcGGM (M1-4) diet. A clear separation was observed between the two diets, highlighting that the detected proteins in the colon microbiomes from piglets fed the control, and 4% AcGGM diets were distinct. b Hierarchal clustering and heatmap of detected protein group abundance profiles. Rows are scaled so that red represents the highest abundance for that protein group and blue the lowest. Five different clusters were observed, with protein groups differentially detected in AcGGM-fed pigs (M1-4: red), control pigs (C1-4: blue), all pigs (M1-4 + C1-4: purple), AcGGM-fed pigs plus one control (M1-4 + C4: brown) and only in individual pigs (Individual: green). c Phylogeny and metaproteomic detection of 355 MAGs sampled from the colon of weaned piglets. This maximum likelihood tree is based on an alignment of 22 concatenated ribosomal proteins from the 355 MAGs reconstructed in this study from 24 colon metagenomes (12 control pigs, 12 fed 4% AcGGM diet), in addition to 239 reference genomes closely related to the MAGs. Branches are shaded with color to highlight phylum-level affiliations (see legend). Colored bars on the outside of the tree depict the average number of unique and shared protein groups detected for each MAG in four randomly selected samples that were analyzed from pigs fed either the control- (gray) or 4% AcGGM diet (green). Total number of proteins for each MAG in each pig is detailed in Supplementary Data 5. Purple circles on the inside of the tree represent nodes with bootstrap support ≥70%, relative to size. MAG041 and MAG243 were found to encode CE2/CE17-containing mannan PULs (Fig. 5) are indicated by *. All MAGs depicted in Fig. 6 (blue circles) and Fig. 7 (red circles) are listed, with average detected protein counts in both diets, which metaproteomic expression cluster they are enriched in (part b, adjusted p-values are indicated: * denotes p < 0.05, ** denotes p < 0.001, exact values listed in Supplementary Data 7), and their MAG taxonomic affiliation determined via GTDB-Tk. MAG enrichment analysis was performed using the hypergeometric distribution function phyper in R with the false discovery rate controlled at 5% using the function p.adjust with method = ‘BH’. Recently reclassified Roseburia species are denoted as Agathobacter. The full tree in Newick format is provided in Supplementary Data 4.