Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids

Abstract

Summary Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane‐1‐carboxylate and catalyses the novel synthesis of benzoate and cyclohexane‐1‐carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane‐1‐carboxylate synthesis. Enzymes involved in the metabolism of cyclohex‐1,5‐diene carboxyl‐CoA to acetyl‐CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane‐1‐carboxylate. Incorporation of 13C‐atoms from 1‐[13C]‐acetate into crotonate, benzoate and cyclohexane‐1‐carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl‐CoA intermediates to formate was detected. Ligases and membrane‐bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl‐CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases

“…The use of a non-BF [FeFe]-hydrogenase instead of a BF [FeFe]-hydrogenase is advantageous to syntrophic organisms as the syntroph does not have to use energy established in the form of an electrochemical gradient across the membrane to generate reduced ferredoxin for every NADH that is oxidized. Non-BF NADH-dependent hydrogen production is consistent with the known physiological properties of the syntrophic fatty, aromatic and alicyclic acid oxidizers, which use degradative pathways that do not generate reduced ferredoxin (McInerney et al, 2007;Sieber et al, 2010Sieber et al, , 2015James et al, 2019). In support of this concept, peptides matching ferredoxins were not detected in the proteome of S. wolfei (Sieber et al, 2015;Losey et al, 2017).…”

“…In support of this concept, peptides matching ferredoxins were not detected in the proteome of S. wolfei (Sieber et al, 2015;Losey et al, 2017). While S. aciditrophicus encodes both ferredoxin and the ferredoxin-generating Rnf complex (McInerney et al, 2007), it is likely that Rnf in S. aciditrophicus functions to make reduced ferredoxin for benzoyl-CoA reduction and biosynthetic reactions (McInerney et al, 2007;Fuchs et al, 2011;Kung et al, 2013;James et al, 2019).…”

“…S. aciditrophicus syntrophically metabolizes benzoate and cyclohexane-1-carboxylate to acetate, CO 2 , H 2 and formate via a 6-substituted cyclohex-1-ene-1-carboxyl-CoA intermediate using a common core set of enzymes for all its substrates (James et al, 2019). S. aciditrophicus uses an unique mechanism for energy generation from acetyl-CoA where an AMP-forming, acetyl-CoA synthetase is used to make acetate, CoA and ATP from acetyl-CoA, AMP and pyrophosphate (James et al, 2016).…”

“…The latter is predicted to use NADH as the electron donor. The genes for both hydrogenases were expressed when S. aciditrophicus was grown in pure culture on crotonate and in coculture with methanogen on crotonate, benzoate, and cyclohexane-1-carboxylate (Sieber et al, 2014) but only HydAB peptides were detected in the proteome under these growth conditions (James et al, 2019).…”

Enoyl-Coenzyme A Respiration via Formate Cycling in Syntrophic Bacteria

“…The presence of two soluble NADH-dependent FDHs ([sFDHs], FDH-2 and FDH-4) and a NADH-dependent [Fe-Fe]-hydrogenase (HydAB) has been reported in genomic and proteomic studies ( 15 , 31 ). The latter has been characterized after heterologous expression of the encoding genes as a non-electron-bifurcating, NADH-dependent enzyme ( 12 ).…”