Novel aspects of benzoate and crotonate metabolism by the strictly anaerobic bacterium Syntrophus aciditrophicus strain SB.

Although enzyme activities and metabolites detected in S. aciditrophicus indicated that benzoate was degraded by the pathway found in Rhodopseudomonas palustris, proteomic data detected the gene products homologous to that found in Thauera aromatica. Such observation supports the hypothesis of two routes for benzoate degradation exist in S. aciditrophicus, one involved in benzoate degradation to acetate and CO2 and the other involved in cyclohexane carboxylate formation from benzoate.S. aciditrophicus was shown to ferment benzoate to acetate and cyclohexane carboxylate via a dismutation process where reducing equivalents produced during benzoate oxidation to acetate and CO2 are used to reduce benzoate to cyclohexane carboxylate. The ability to ferment benzoate suggests that benzoate could serve as an electron acceptor if a suitable electron donor was present. To test whether benzoate can be respired, S. aciditrophicus was grown with crotonate and benzoate. Benzoate was stoichiometrically reduced to cyclohexane carboxylate while crotonate was oxidized to acetate. Cultures grown with [ring-13C]-benzoate and unlabeled crotonate formed ring-labeled 13C-cyclohexane carboxylate. No [13C]-labeled acetate was detected. The molar growth yield was 22.7 +/- 2.1 g (dry wt) cell per mol of crotonate compared to 14.0 +/- 0.1g per mol of crotonate when crotonate was used as a sole substrate. Furthermore, benzoate fermentation occurred only if traces amounts of crotonate were present.The metabolism of crotonate in the strictly anaerobic bacteria Syntrophus aciditrophicus was studied. S. aciditrophicus produced 1.4 +/- 0.24 moles of acetate and 0.16 +/- 0.02 moles of cyclohexane carboxylate per mole of crotonate degraded. [U- 13C] Crotonate was metabolized to [1, 2-13C] acetate and [1, 2, 3, 4, 5, 7-13C] cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [13C] sodium bicarbonate formed [6-13C] cyclohexane carboxylate. Cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates. These are the same intermediates as that detected during syntrophic or fermentative benzoate metabolism by S. aciditrophicus . When S. aciditrophicus was grown with [1, 2- 13C] acetate and unlabeled crotonate, the m/z-15 of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, and the m/z-15 of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4 and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO2 addition, rather than the use of the intact four-carbon skeleton of crotonate.Fluorobenzoates and hydroxybenzoates were tested as substrates for S. aciditrophicus in order to detect potential intermediates of interest. The utilization of 3-fluorobenzoate allowed the detection of a metabolite, which had a mass ion increase of 2 units greater than the parent compound, or 3 or 4 units greater than the parent compound when deuterated water was used. These results were consistent with the formation of a fluorinated diene intermediate. The transient accumulation of benzoate when 2-hydroxybenzoate was the substrate showed that hydroxylation of the ring was not required for ring reduction. The metabolites detected with fluoro- and hydroxy-benzoates are consistent with the hypothesis that benzoyl-CoA reduction involves a two-electron reduction forming a diene intermediate, rather than a four- or six-electron reduction.Phototrophic and denitrifying bacteria couple the hydrolysis of two ATP molecules to reduce benzoyl-CoA to cyclohex-1,5-diene carboxyl-CoA. The use of such an energy intensive reaction by fermentative bacteria such as S. aciditrophicus has been questioned since it is not clear how net ATP synthesis would occur. Rather, a four- or six-electron reduction which thermodynamic calculations indicate is exergonic under standard conditions, or hydroxylation of the ring prior to its reduction have been proposed.Proteomic analysis of S. aciditrophicus grown with crotonate or crotonate and benzoate allowed the identification of gene products involved in benzoate metabolism. Two benzoyl-CoA ligases and a possible novel benzoyl-CoA reductase, a tungsten/molybdenum-containing aldehyde ferredoxin oxidoreductase associated with heterodisulfide reductase components similar to the benzoate-induced proteins found in G. metallireducens were identified. Cyclohex-1,5-diene carboxyl-CoA hydratase and the enzymes needed to form 3-hydroxypimelyl-CoA from the diene were also detected. The detection of subunits of ATP synthase, cytoplasmic and periplasmic formate dehydrogenases, a sodium-translocating glutaconyl-CoA, and sodium-driven membrane-bound NADH:ferredoxin oxidoreductase indicates that S. aciditrophicus has the potential to create and use both sodium and proton gradients. ATP synthesis from acetyl-CoA appears to occur by an archaeal-like acetyl-CoA synthetase (ADP-forming) rather than the typical bacterial phosphotransacetylaseacetate kinase system

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UnlabelledSyntrophus aciditrophicus is 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 by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves 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 that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and 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) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has 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 aciditrophicus apparently 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 that S. aciditrophicus uses 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

Cyclohexane Carboxylate and Benzoate Formation from Crotonate in Syntrophus aciditrophicus

The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 ± 0.24 mol of acetate and 0.16 ± 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U-(13)C]crotonate was metabolized to [1,2-(13)C]acetate and [1,2,3,4,5,7-(13)C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [(13)C]sodium bicarbonate formed [6-(13)C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U-(13)C]crotonate, the m/z-15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [(13)C]sodium bicarbonate and unlabeled crotonate, the m/z-15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2-(13)C]acetate and unlabeled crotonate, the m/z-15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO(2) addition, rather than the use of the intact four-carbon skeleton of crotonate

Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp. Strain X514 via Isotopomer-Assisted Metabolite Analysis▿ †

Thermoanaerobacter sp. strain X514 has great potential in biotechnology due to its capacity to ferment a range of C5 and C6 sugars to ethanol and other metabolites under thermophilic conditions. This study investigated the central metabolism of strain X514 via 13C-labeled tracer experiments using either glucose or pyruvate as both carbon and energy sources. X514 grew on minimal medium and thus contains complete biosynthesis pathways for all macromolecule building blocks. Based on genome annotation and isotopic analysis of amino acids, three observations can be obtained about the central metabolic pathways in X514. First, the oxidative pentose phosphate pathway in X514 is not functional, and the tricarboxylic acid cycle is incomplete under fermentative growth conditions. Second, X514 contains (Re)-type citrate synthase activity, although no gene homologous to the recently characterized (Re)-type citrate synthase of Clostridium kluyveri was found. Third, the isoleucine in X514 is derived from acetyl coenzyme A and pyruvate via the citramalate pathway rather than being synthesized from threonine via threonine ammonia-lyase. The functionality of the citramalate synthase gene (cimA [Teth514_1204]) has been confirmed by enzymatic activity assays, while the presence of intracellular citramalate has been detected by mass spectrometry. This study demonstrates the merits of combining 13C-assisted metabolite analysis, enzyme assays, and metabolite detection not only to examine genome sequence annotations but also to discover novel enzyme activities

Complete genome sequence of Methanospirillum hungatei type strain JF1.

Methanospirillum hungatei strain JF1 (DSM 864) is a methane-producing archaeon and is the type species of the genus Methanospirillum, which belongs to the family Methanospirillaceae within the order Methanomicrobiales. Its genome was selected for sequencing due to its ability to utilize hydrogen and carbon dioxide and/or formate as a sole source of energy. Ecologically, M. hungatei functions as the hydrogen- and/or formate-using partner with many species of syntrophic bacteria. Its morphology is distinct from other methanogens with the ability to form long chains of cells (up to 100 μm in length), which are enclosed within a sheath-like structure, and terminal cells with polar flagella. The genome of M. hungatei strain JF1 is the first completely sequenced genome of the family Methanospirillaceae, and it has a circular genome of 3,544,738 bp containing 3,239 protein coding and 68 RNA genes. The large genome of M. hungatei JF1 suggests the presence of unrecognized biochemical/physiological properties that likely extend to the other Methanospirillaceae and include the ability to form the unusual sheath-like structure and to successfully interact with syntrophic bacteria

Complete genome sequence of Methanospirillum hungatei type strain JF1.

Methanospirillum hungatei strain JF1 (DSM 864) is a methane-producing archaeon and is the type species of the genus Methanospirillum, which belongs to the family Methanospirillaceae within the order Methanomicrobiales. Its genome was selected for sequencing due to its ability to utilize hydrogen and carbon dioxide and/or formate as a sole source of energy. Ecologically, M. hungatei functions as the hydrogen- and/or formate-using partner with many species of syntrophic bacteria. Its morphology is distinct from other methanogens with the ability to form long chains of cells (up to 100 μm in length), which are enclosed within a sheath-like structure, and terminal cells with polar flagella. The genome of M. hungatei strain JF1 is the first completely sequenced genome of the family Methanospirillaceae, and it has a circular genome of 3,544,738 bp containing 3,239 protein coding and 68 RNA genes. The large genome of M. hungatei JF1 suggests the presence of unrecognized biochemical/physiological properties that likely extend to the other Methanospirillaceae and include the ability to form the unusual sheath-like structure and to successfully interact with syntrophic bacteria

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

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 13 C-atoms from 1-[13 C]-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

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

Syntrophus aciditrophicus is 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 by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves 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 that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and 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) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. Bacteria 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 aciditrophicus apparently 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 that S. aciditrophicus uses 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