Occurrence and expression of genes encoding methyl-compound production in rumen bacteria

Abstract

Background: Digestive processes in the rumen lead to the release of methyl-compounds, mainly methanol and methylamines, which are used by methyltrophic methanogens to form methane, an important agricultural greenhouse gas. Methylamines are produced from plant phosphatidylcholine degradation, by choline trimethylamine lyase, while methanol comes from demethoxylation of dietary pectins via pectin methylesterase activity. We have screened rumen metagenomic and metatranscriptomic datasets, metagenome assembled genomes, and the Hungate1000 genomes to identify organisms capable of producing methyl-compounds. We also describe the enrichment of pectin-degrading and methane-forming microbes from sheep rumen contents and the analysis of their genomes via metagenomic assembly.

Results: Screens of metagenomic data using the protein domains of choline trimethylamine lyase (CutC), and activator protein (CutD) found good matches only to Olsenella umbonata and to Caecibacter, while the Hungate1000 genomes and metagenome assembled genomes from the cattle rumen found bacteria within the phyla Actinobacteria, Firmicutes and Proteobacteria. The cutC and cutD genes clustered with genes that encode structural components of bacterial microcompartment proteins. Prevotella was the dominant genus encoding pectin methyl esterases, with smaller numbers of sequences identified from other fibre-degrading rumen bacteria. Some large pectin methyl esterases (> 2100 aa) were found to be encoded in Butyrivibrio genomes. The pectin-utilising, methane-producing consortium was composed of (i) a putative pectin-degrading bacterium (phylum Tenericutes, class Mollicutes), (ii) a galacturonate-using Sphaerochaeta sp. predicted to produce acetate, lactate, and ethanol, and (iii) a methylotrophic methanogen, Methanosphaera sp., with the ability to form methane via a primary ethanol-dependent, hydrogen-independent, methanogenesis pathway.

Conclusions: The main bacteria that produce methyl-compounds have been identified in ruminants. Their enzymatic activities can now be targeted with the aim of finding ways to reduce the supply of methyl-compound substrates to methanogens, and thereby limit methylotrophic methanogenesis in the rumen.

Keywords: Bacterial; Methanol; Methyl-compound; Methylamines; Rumen.

Fig. 1

Choline TMA lyase (cutC) gene abundance and diversity in a combined rumen metagenome and metatranscriptome dataset, metagenome-assembled genomes and Hungate1000 bacterial genomes (a), and a phylogenetic tree showing the relationships of CutC protein sequences from all these sources (b)

Fig. 2

Abundance (%) and diversity of genes encoding pectin methyl esterase (PME; PF01095)-domain containing proteins in a combined rumen metagenome and metatranscriptome dataset (outer circle; n = 2414), metagenome-assembled genomes (middle circle; n = 505) and Hungate1000 bacterial genomes (inner circle; n = 315)

Fig. 3

a: Tetranucleotide plot of the 24 scaffolds defining the 3 organisms recovered from a pectin-utilising, methane-producing enrichment culture. b: Predicted metabolic relationship between the three organisms enabling pectin conversion to methane

Fig. 4

Phylogenetic analysis of alcohol dehydrogenase (a) and aldehyde dehydrogenase (b) genes from rumen methanogen genomes and rumen MAGs. Both trees were constructed with the Jones-Taylor Thornton (JTT) model. Saccharomyces cerevisiae ATCC 204508 was used as the outgroup. Numbers represent the relative frequency of branch clustering based on 1000 bootstrap runs, bootstrap values < 50% are removed. Rumen MAGs; MEC1, Organism 3 (Methanosphaera sp.) from the pectin enrichment culture in this study; TAG1265, metagenome assembled Methanosphaera sp. sequences from low methane yield sheep datasets [35]; RUG761, metagenome assembled Methanosphaera sp. sequences from cattle [27]