The enzyme Rnf completes the pathway for forming propionate during fermentation inPrevotella

Abstract

Propionate is a microbial metabolite that is formed in the gastrointestinal tract, and it affects host physiology as a source of energy and signaling molecule. Despite the importance of propionate, the biochemical pathways responsible for its formation are not clear in all microbes. For the succinate pathway used during fermentation, a key enzyme appears to be missing—one that can oxidize ferredoxin and reduce NAD. Here we show that Rnf [ferredoxin--NAD(+) oxidoreductase (Na(+)-transporting)] is this key enzyme in two abundant bacteria of the rumen (Prevotella brevisandPrevotella ruminicola). We found these bacteria form propionate, succinate, and acetate with the classic succinate pathway. At first, this pathway appears unbalanced, forming reduced ferredoxin and oxidized NAD in excess. If this continued unabated, fermentation would halt within 1.5 s. We found these bacteria solve this problem by oxidizing ferredoxin and reducing NAD with Rnf. This is demonstrated using growth experiments, genomics, proteomics, and enzyme assays. Genomic and phenotypic data suggest many bacteria use Rnf similarly. We cataloged fermentation products of >1,400 species of prokaryotes, and nearly 10% formed propionate, succinate, and acetate. Over 40% of species carrying out this fermentation also had genes for Rnf. This work shows Rnf is important to propionate formation in many bacteria from the environment, and it provides fundamental knowledge for manipulating fermentative propionate production.

The phenotype and genotype of fermentative microbes

“…We collected information from Bergey’s Manual of Systematic of Archaea and Bacteria using an approach similar to ref. 19,20,58 . We downloaded all n = 1,751 articles for genera.…”

“…Following ref. 19,20,58 , we searched GOLD database for a GOLD organism ID, GOLD project ID, and IMG genome ID for each organism. Organisms were matched to GOLD organism ID by their strain ID plus genus or species name.…”

The phenotype and genotype of fermentative prokaryotes

“…We collected information from Bergey’s Manual of Systematic of Archaea and Bacteria using an approach similar to ( 18 , 19 , 56 ). We downloaded all n = 1751 articles for genera.…”

“…We obtained information on genomes and genes from GOLD ( 23 ) and IMG/M ( 24 ) databases. Following ( 18 , 19 , 56 ) , we searched GOLD database for a GOLD organism ID, GOLD project ID, and IMG genome ID for each organism. Organisms were matched to GOLD organism ID by their strain ID plus genus or species name.…”

“…Total gas production (milliliter per culture) was measured with a syringe. Aliquots of gas (1 ml) were then collected and analyzed for H 2 by gas chromatography ( 56 ). Production of H 2 (millimole per liter of culture) was then calculated.…”