Interspecies Hydrogen Transfer and Its Effects on Global Transcript Abundance in Ruminococcus albus, a Predominant Fiber‐Degrading Species in the Rumen

Abstract

The mutually‐beneficial interdependence of hydrogen‐producing and hydrogen‐utilizing bacteria was discovered by M. P. Bryant, M. J. Wolin and R. S. Wolfe at the University of Illinois in 1967. Based on thermodynamic principles, interspecies hydrogen transfer is a central process in anaerobic environments linking transfer of reducing power from fermentation of organic molecules to inorganic electron acceptors via hydrogen. Interspecies hydrogen transfer is the most significant example of unidirectional substrate supply enabling the syntrophic metabolic association between interacting microbial species and plays a significant role in the global methane cycle. R. albus 7 is a hydrogen‐producing, fermentative bacterium with two known hydrogenase complexes (HydABC and HydA2) as well as a putative hydrogen‐sensing protein, HydS. HydABC is the only chromosomal hydrogenase, while HydA2 and HydS form a transcriptional unit in R. albus 7 on its plasmid pRumal01. The electron‐bifurcating ferredoxin‐ and NAD‐dependent [FeFe]‐hydrogenase, HydABC, couples proton reduction using nicotinamide adenine dinucleotide (NADH) to proton reduction using reduced ferredoxin, producing molecular hydrogen: 3H+ + NADH + Fdred ‐> 2H2 + NAD+ + Fdox. HydA2, a ferredoxin‐dependent [FeFe]‐hydrogenase, reduces protons to molecular hydrogen using only reduced ferredoxin: 2H+ + Fdred ‐> H2 + Fdox. HydS contains a C‐terminal PAS domain, which often are present on sensory proteins. In addition, HydS contains a putative redox‐sensing [4Fe:4S] cluster. We hypothesized that HydS transcriptionally regulates HydA2 in a manner dependent on the presence of a hydrogen‐utilizing skyntroph. To test this hypothesis, we grew R. albus 7 and a hydrogen‐utilizing bacterium, W. succinogenes DSM‐1740, in mono‐ and bi‐culture. We monitored cell growth by optical density (OD600) and quantitative polymerase chain reaction (qPCR), as well as gas and fermentation product production. Lastly, based on qPCR growth data, we determined mid‐log phase (ΔOD~0.20 for R. albus, 0.14 for W. succinogenes, and 0.35 for the bi‐culture) and extracted RNA for sequencing to compare whole genome transcriptomic profiles. In bi‐culture with the hydrogen‐utilizing bacterium, R. albus produced 1.11 moles acetate and 0.03 moles ethanol per mole available glucose. In monoculture, R. albus produced 0.75 moles acetate and 0.30 moles ethanol per mole available glucose. We confirmed that hydrogen accumulated in the R. albus monoculture, but not in the bi‐culture. From RNA‐Seq analysis, we identified that R. albus, in bi‐culture, had a lowered transcript abundance of HydA2 (90‐fold), relative to monoculture. Interestingly, the electron‐bifurcating hydrogenase, HydABC, had a similar transcript abundance in bi‐culture to monoculture (1.2–1.3‐fold change). This suggests that HydS might be sensing hydrogen levels and regulating the transcription of HydA2. These results also suggest the electron‐bifurcating hydrogenase (HydABC) functions in central metabolism regardless of external hydrogen concentration.Support or Funding InformationAgriculture and Food Research Initiative Competitive Grant 2012‐67015‐19451 from the US Department of Agriculture National Institute of Food and Agriculture