Transcriptomic and proteomic changes associated with cobalamin-dependent propionate production by the rumen bacterium...

Xylanibacter ruminicola is an abundant rumen bacterium that produces propionate in a cobalamin (vitamin B₁₂)-dependent manner via the succinate pathway. However, the extent to which this occurs across ruminal Xylanibacter and closely related bacteria, and the effect of cobalamin supplementation on the expression of propionate pathway genes and enzymes has yet to be investigated. To assess this, we screened 14 strains and found that almost all strains produced propionate when supplemented with cobalamin. X. ruminicola KHP1 was selected for further study, including complete genome sequencing, and comparative transcriptomics and proteomics of KHP1 cultures grown with and without supplemented cobalamin. The complete KHP1 genome was searched for cobalamin-binding riboswitches and four were predicted, though none were closely located to any of the succinate pathway genes, which were dispersed at numerous genomic loci. Cobalamin supplementation led to the differential expression of 17.5% of genes, including genes encoding the cobalamin-dependent methylmalonyl-CoA mutase and some methylmalonyl-CoA decarboxylase subunits, but most propionate biosynthesis pathway genes were not differentially expressed. The effect of cobalamin supplementation on the KHP1 proteome was much less pronounced, with the only differentially abundant propionate pathway enzyme being methylmalonyl-CoA mutase, which had greater abundance when supplemented with cobalamin. Our results demonstrate that cobalamin supplementation does not result in induction of the entire propionate biosynthesis pathway, but consistently increased expression of methylmalonyl-CoA mutase at transcriptome and proteome levels. The magnitude of the differential expression of propionate pathway genes observed was minor compared to that of genes proximate to predicted cobalamin riboswitches.

In ruminants, the rumen microbial community plays a critical role in nutrition through the fermentation of feed to provide vital energy substrates for the host animal. Propionate is a major rumen fermentation end-product and increasing its production is desirable given its importance in host glucose production and impact on greenhouse gas production. Vitamin B₁₂ (cobalamin) can induce propionate production in the prominent rumen bacterium Xylanibacter ruminicola, but it is not fully understood how cobalamin regulates propionate pathway activity. Contrary to expectation, we found that cobalamin supplementation had little effect on propionate pathway expression at transcriptome and proteome levels, with minor upregulation of genes encoding the cobalamin-dependent enzyme of the pathway. These findings provide new insights into factors that regulate propionate production and suggest that cobalamin-dependent propionate production by X. ruminicola is controlled post-translationally.