Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection

Abstract

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B₁₂, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ∼75% of the genus-level bacterial and archaeal taxa present in the rumen.

Figure 1. Microbial community composition data from the Global Rumen Census overlaid with the 16S rRNA gene sequences (yellow dots) from the 501 Hungate catalog genomes.

Two groups of abundant but currently unclassified bacteria are indicated by blue (Bacteroidales, RC-9 gut group) and orange (Clostridiales, R-7 group) dots. The colored rings around the trees represent the taxonomic classifications of each OTU from the Ribosomal Database Project database (from the innermost to the outermost): genus, family, order, class and phylum. The strength of the color is indicative of the percentage similarity of the OTU to a sequence in the RDP database of that taxonomic level.

Figure 2. Functions of the rumen microbiome.

(a) Number of degradative CAZymes (GH, glycoside hydrolases and PL, polysaccharide lyases) in distinct families in each of the 501 Hungate catalog genomes. Genomes are colored by phylum. (b) Simplified illustration showing the degradation and metabolism of plant structural carbohydrates by the dominant bacterial and archaeal groups identified in the Global Rumen Census project using information from metabolic studies and analysis of the reference genomes. The abundance and prevalence data shown in the table are taken from the Global Rumen Census project. Abundance represents the mean relative abundance (%) for that genus-level group in samples that contain that group, while prevalence represents the prevalence of that genus-level group in all samples (n = 684).* The conversion of choline to trimethylamine, and propanediol to propionate generate toxic intermediates that are contained within bacterial microcompartments (BMC). Cultures from the reference genome set that encode the genes required to produce the structural proteins required for BMC formation are shown in Supplementary Table 5. (c) Number of polysaccharide-degrading CAZymes encoded in the genomes of representatives from the eight most abundant bacterial groups. Cellulose: GH5, GH9, GH44, GH45, GH48; pectin: GH28, PL1, PL9, PL10, PL11, CE8, CE12; xylan: GH8, GH10, GH11, GH43, GH51, GH67, GH115, GH120, GH127, CE1, CE2.

Figure 3. Survey of enolase genes in Butyrivibrio strains.

Maximum likelihood tree based on concatenated alignment of 56 conserved marker proteins from genomes of all Butyrivibrio strains in the Hungate Collection. Strains lacking a detectable enolase gene are indicated by pale pink shading while those with a truncated enolase are indicated by lavender shading. Strains without shading possess an intact enolase.

Figure 4. Recruitment of metagenomic proteins by Hungate catalog genomes.

Maximum likelihood tree based on 16S rDNA gene alignment of rumen strains. The tree clades are color coded according to phylum. Multi-bar-chart depicting the average % coverage of total CDS of an isolate by metagenome samples from each ecosystem category was drawn using iTOL. Dashed boxes highlight interesting examples of recruitment such as isolates detected in both rumen and human samples (maroon boxes) or detected in human but not rumen samples (red boxes), and others. Number key is as follows (average % coverage is given in parentheses): 1. Sharpea azabuensis str. (∼88%), Kandleria vitulina str. (∼87%); 2. Staphylococcus epidermidis str. (∼40%), Lactobacillus ruminis str. (∼51%); 3. Streptococcus equinus str. (∼38% by rumen, ∼35% by human); 4. Prevotella bryantii str. (∼38% by rumen, ∼9% by human); 5. Bacteroides spp.(∼38%); 6. Bifidobacterium spp. (∼24%), Propionibacterium acnes (∼39%); 7. Shigella sonnei (∼30% by human), E. coli PA3 (∼31% by human), Citrobacter sp. NLAE-zl-C269 (20% by human); 8. Clostridium beijerinckii HUN142 (87% by plant); 9. Methanobrevibacter spp. (∼32%⋆Figure 4 Maximum likelihood tree based on 16S rDNA gene alignment of rumen strains. The tree clades are color coded according to phylum. Multi-bar-chart depicting the average % coverage of total CDS of an isolate by metagenome samples from each ecosystem category was drawn using iTOL55. Dashed boxes highlight interesting examples of recruitment such as isolates detected in both rumen and human samples (maroon boxes) or detected in human but not rumen samples (red boxes), and others. Number key is as follows (average % coverage is given in parentheses): 1. Sharpea azabuensis str. (~88%), Kandleria vitulina str. (~87%); 2. Staphylococcus epidermidis str. (~40%), Lactobacillus ruminis str. (~51%); 3. Streptococcus equinus str. (~38% by rumen, ~35% by human); 4. Prevotella bryantii str. (~38% by rumen, ~9% by human); 5. Bacteroides spp.(~38%); 6. Bifidobacterium spp. (~24%), Propionibacterium acnes (~39%); 7. Shigella sonnei (~30% by human), E. coli PA3 (~31% by human), Citrobacter sp. NLAE-zl-C269 (20% by human); 8. Clostridium beijerinckii HUN142 (87% by plant); 9. Methanobrevibacter spp. (~32%). The innermost circle identifies Hungate isolates of fecal (★) or salivary (♦) origin. Please refer to Supplementary Table 9 for data and other specifics.

Figure 5. Differentially abundant Pfams between rumen and human intestinal isolates.

X axis is individual Pfams detected by Metastats to be differentially abundant with a Q-value < 0.001. Y axis is the log2-fold difference of mean counts for each population (rumen or intestinal). Select Pfams are highlighted as discussed in the text. OPPP, oxidative pentose phosphate pathway.