The missing enzymatic link in syntrophic methane formation from fatty acids

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from β-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop–driven reduction of CO2 by acyl–coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of β-oxidation in (methyl)menaquinone-containing organisms.

Microbial analysis and enrichment of anaerobic phenol and p-cresol degrading consortia with addition of AQDS

Quinones and humus are ubiquitous in the biosphere and play an important role in the anaerobic biodegradation and biotransformation of organic acids, poisonous compounds as well as inorganic compounds. The impact of humic model compound, anthraquinone-2, 6-disulfonate (AQDS) on anaerobic phenol and p-cresol degradation were studied. Four methanogenic AQDS-free phenol and p-cresol enrichments and two phenol-AQDS enrichments were obtained using two sludges with potential biodegradability of phenol and cresol isomers as inoculum. 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that syntrophic aromatic compound degrading bacterium Syntrophorhabdus aromaticivorans was dominant in four AQDS-free enrichments, whereas phenol degrading Cryptanaerobacter phenolicus was dominant in two phenol-AQDS enrichments. Neither co-culture of Syntrophorhabdus aromaticivorans with Methanospirillum hungatei nor two phenol-AQDS enrichments could metabolize phenol using AQDS as the terminal electron acceptor. Further degradation experiments suggested that C. phenolicus related microbes in two phenol-AQDS enrichments were responsible for the conversion of phenol to benzoate, and benzoate was further degraded by benzoate degraders of Syntrophus aciditrophicus or Sporotomaculum syntrophicum to acetate.

SyntrophusConductive Pili Demonstrate that Common Hydrogen-Donating Syntrophs can have a Direct Electron Transfer Option

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H2and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron sharing because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report thatSyntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A inSyntrophus aciditrophicus, a New Twist on ATP Formation

ABSTRACTSyntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.IMPORTANCEBacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show thatS. aciditrophicususes an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Fermentative Cyclohexane Carboxylate Formation in Syntrophus aciditrophicus

Short-chain fatty acids such as acetic, propionic, butyric or lactic acids are typical primary fermentation products in the anaerobic feeding chain. Fifteen years ago, a novel fermentation type was discovered in the obligately anaerobic Deltaproteobacterium <i>Syntrophus aciditrophicus</i>. During fermentative growth with crotonate and/or benzoate, acetate is formed in the oxidative branch and cyclohexane carboxylate in the reductive branch. In both cases cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) is a central intermediate that is either formed by a class II benzoyl-CoA reductase (fermentation of benzoate) or by reverse reactions of the benzoyl-CoA degradation pathway (fermentation of crotonate). Here, we summarize the current knowledge of the enzymology involved in fermentations yielding cyclohexane carboxylate as an excreted product. The characteristic enzymes involved are two acyl-CoA dehydrogenases specifically acting on Ch1,5CoA and cyclohex-1-ene-1-carboxyl-CoA. Both enzymes are also employed during the syntrophic growth of <i>S. aciditrophicus</i> with cyclohexane carboxylate as the carbon source in coculture with a methanogen. An investigation of anabolic pathways in <i>S. aciditrophicus</i> revealed a rather unusual pathway for glutamate synthesis involving a <i>Re</i>-citrate synthase. Future work has to address the unresolved question concerning which components are involved in reoxidation of the NADH formed in the oxidative branch of the unique cyclohexane carboxylate fermentation pathway in <i>S. aciditrophicus</i>.

Two Pathways for Glutamate Biosynthesis in the Syntrophic Bacterium Syntrophus aciditrophicus

ABSTRACTThe anaerobic metabolism of crotonate, benzoate, and cyclohexane carboxylate bySyntrophus aciditrophicusgrown syntrophically withMethanospirillum hungateiprovides a model to study syntrophic cooperation. Recent studies revealed thatS. aciditrophicuscontainsRe-citrate synthase but lacks the commonSi-citrate synthase. To establish whether theRe-citrate synthase is involved in glutamate synthesis via the oxidative branch of the Krebs cycle, we have used [1-13C]acetate and [1-14C]acetate as well as [13C]bicarbonate as additional carbon sources during axenic growth ofS. aciditrophicuson crotonate. Our analyses showed that labeled carbons were detected in at least 14 amino acids, indicating the global utilization of acetate and bicarbonate. The labeling patterns of alanine and aspartate verified that pyruvate and oxaloacetate were synthesized by consecutive carboxylations of acetyl coenzyme A (acetyl-CoA). The isotopomer profile and13C nuclear magnetic resonance (NMR) spectroscopy of the obtained [13C]glutamate, as well as decarboxylation of [14C]glutamate, revealed that this amino acid was synthesized by two pathways. Unexpectedly, only the minor route usedRe-citrate synthase (30 to 40%), whereas the majority of glutamate was synthesized via the reductive carboxylation of succinate. This symmetrical intermediate could have been formed from two acetates via hydration of crotonyl-CoA to 4-hydroxybutyryl-CoA. 4-Hydroxybutyrate was detected in the medium ofS. aciditrophicuswhen grown on crotonate, but an active hydratase could not be measured in cell extracts, and the annotated 4-hydroxybutyryl-CoA dehydratase (SYN_02445) lacks key amino acids needed to catalyze the hydration of crotonyl-CoA. BesidesClostridium kluyveri, this study reveals the second example of a microbial species to employ two pathways for glutamate synthesis.

Metabolism of Hydroxylated and Fluorinated Benzoates by Syntrophus aciditrophicus and Detection of a Fluorodiene Metabolite

ABSTRACT Transformations of 2-hydroxybenzoate and fluorobenzoate isomers were investigated in the strictly anaerobic Syntrophus aciditrophicus to gain insight into the initial steps of the metabolism of aromatic acids. 2-Hydroxybenzoate was metabolized to methane and acetate by S. aciditrophicus and Methanospirillum hungatei cocultures and reduced to cyclohexane carboxylate by pure cultures of S. aciditrophicus when grown in the presence of crotonate. Under both conditions, transient accumulation of benzoate but not phenol was observed, indicating that dehydroxylation occurred prior to ring reduction. Pure cultures of S. aciditrophicus reductively dehalogenated 3-fluorobenzoate with the stoichiometric accumulation of benzoate and fluorine. 3-Fluorobenzoate-degrading cultures produced a metabolite that had a fragmentation pattern almost identical to that of the trimethylsilyl (TMS) derivative of 3-fluorobenzoate but with a mass increase of 2 units. When cells were incubated with deuterated water, this metabolite had a mass increase of 3 or 4 units relative to the TMS derivative of 3-fluorobenzoate. 19F nuclear magnetic resonance spectroscopy (19F NMR) detected a metabolite in fluorobenzoate-degrading cultures with two double bonds, either 1-carboxyl-3-fluoro-2,6-cyclohexadiene or 1-carboxyl-3-fluoro-3,6-cyclohexadiene. The mass spectral and NMR data are consistent with the addition of two hydrogen or deuterium atoms to 3-fluorobenzoate, forming a 3-fluorocyclohexadiene metabolite. The production of a diene metabolite provides evidence that S. aciditrophicus contains dearomatizing reductase that uses two electrons to dearomatize the aromatic ring.