Abstract
Syntrophic butyrate metabolism involves the thermodynamically unfavorable production of hydrogen and/or formate from the high potential electron donor, butyryl-CoA. Such redox reactions can occur only with energy input by a process called reverse electron transfer. Previous studies have demonstrated that hydrogen production from butyrate requires the presence of a proton gradient, but the biochemical machinery involved has not been clearly elucidated. In this study, the gene and enzyme systems involved in reverse electron transfer by Syntrophomonas wolfei were investigated using proteomic and gene expression approaches. S. wolfei was grown in co-culture with Methanospirillum hungatei or Dehalococcoides mccartyi under conditions requiring reverse electron transfer and compared to both axenic S. wolfei cultures and co-cultures grown in conditions that do not require reverse electron transfer. Blue native gel analysis of membranes solubilized from syntrophically grown cells revealed the presence of a membrane-bound hydrogenase, Hyd2, which exhibited hydrogenase activity during in gel assays. Bands containing a putative iron-sulfur (FeS) oxidoreductase were detected in membranes of crotonate-grown and butyrate grown S. wolfei cells. The genes for the corresponding hydrogenase subunits, hyd2ABC, were differentially expressed at higher levels during syntrophic butyrate growth when compared to growth on crotonate. The expression of the FeS oxidoreductase gene increased when S. wolfei was grown with M. hungatei. Additional membrane-associated proteins detected included FoF1 ATP synthase subunits and several membrane transporters that may aid syntrophic growth. Furthermore, syntrophic butyrate metabolism can proceed exclusively by interspecies hydrogen transfer, as demonstrated by growth with D. mccartyi, which is unable to use formate. These results argue for the importance of Hyd2 and FeS oxidoreductase in reverse electron transfer during syntrophic butyrate degradation.
Highlights
Syntrophy is a thermodynamically based metabolic coupling between two or more microorganisms
To identify membrane proteins potentially involved in reverse electron transfer in S. wolfei, we performed blue native polyacrylamide gel electrophoresis (BN-PAGE) of solubilized membrane proteins from cells grown in axenic culture and co-culture with M. hungatei on crotonate, and in co-culture with M. hungatei on butyrate
Several Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE) protein bands were more pronounced in membranes prepared from butyrate-grown S. wolfei cells relative to membranes of S. wolfei cells grown on crotonate (Supplementary Figures S1A,B)
Summary
Syntrophy is a thermodynamically based metabolic coupling between two or more microorganisms. In co-culture with M. hungatei, S. wolfei syntrophically metabolizes short chain fatty acids of four to eight carbon atoms to acetate, using the beta-oxidation pathway (McInerney et al, 1979, 1981; Müller et al, 2009; Sieber et al, 2010, 2015; Schmidt et al, 2013). Beta-oxidation of fatty acids generates NADH and reduced electron transfer flavoprotein (Etf), which must be reoxidized by hydrogen or formate production (Müller et al, 2009; Sieber et al, 2012, 2015; Schmidt et al, 2013). Hydrogen and formate production from electrons derived from the oxidation of acyl-CoA intermediates requires energy input by a process called reverse electron transfer even at low hydrogen or formate concentrations (Schink, 1997; Sieber et al, 2012). Wallrabenstein and Schink (1994) showed that hydrogen production from butyrate by cell suspensions of S. wolfei required chemiosmotic energy consistent with the involvement of reverse electron transfer
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