The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards.13 C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-13 C 6 ]benzoate. None of the metabolites mentioned above was detected in non-substrateamended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 M, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of "S. aciditrophicus" and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of "S. aciditrophicus" grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATPdependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxylCoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of "S. aciditrophicus"-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of "S. aciditrophicus". These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four-or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.

Three mutants deficient in hydrogen/formate uptake were obtained through screening of a transposon mutant library containing 5,760 mutants of Desulfovibrio desulfuricans G20. Mutations were in the genes encoding the type I tetraheme cytochrome c 3 (cycA), Fe hydrogenase (hydB), and molybdopterin oxidoreductase (mopB). Mutations did not decrease the ability of cells to produce H 2 or formate during growth. Complementation of the cycA and mopB mutants with a plasmid carrying the intact cycA and/or mopB gene and the putative promoter from the parental strain allowed the recovery of H 2 uptake ability, showing that these specific genes are involved in H 2 oxidation. The mop operon encodes a periplasm-facing transmembrane protein complex which may shuttle electrons from periplasmic cytochrome c 3 to the menaquinone pool. Electrons can then be used for sulfate reduction in the cytoplasm.

Syntrophomonas wolfei is an anaerobic fatty acid degrader that can only be grown in coculture with H2-using bacteria such as Methanospirillum hungatei. Cells of S. wolfei were selectively lysed by lysozyme treatment, and unlysed cells of M. hungatei were removed by centrifugation. The cell extract of S. wolfei obtained with this method had low levels of contamination by methanogenic cofactors. However, lysozyme treatment was not efficient in releasing S. wolfei protein; only about 15% of the L-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase activity was found in the lysozyme supernatant. Cell extracts of S. wolfei obtained with this method had high specific activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. These activities were not detected in cell extracts of M. hungatei grown alone, confirming that these activities were present in S. wolfei. The acyl-CoA dehydrogenase activity was high when a C4 but not a C8 or C16 acyl-CoA derivative served as the substrate. S. wolfei cell extracts had high CoA transferase specific activities and no detectable acyl-CoA synthetase activity, indicating that fatty acid activation occurred by transfer of CoA from acetyl-CoA. Phosphotransacetylase and acetate kinase activities were detected in cell extracts of S. wolfei, indicating that S. wolfei is able to perform substrate-level phosphorylation.The complete anaerobic degradation of organic matter to CH4 and CO2 involves the concerted action of four major metabolic groups of bacteria (9,27,45). First, fermentative bacteria hydrolyze the primary substrate polymers such as polysaccharides and proteins and ferment the products mainly to volatile fatty acids, C02, and CH4. The H2-producing acetogenic bacteria degrade propionate and longer-chain fatty acids and some aromatic acids to acetate, H2, and sometimes CO2. The methanogenic bacteria use H2 to reduce CO2 to CH4, and some species cleave acetate to CO2 and CH4. A fourth group of bacteria, H2-using acetogens, produces acetate and some butyrate from H2/CO2, methanol, CO, and methoxy moieties of some aromatic compounds (1, 2, 38).Propionate and longer-chain acids are important intermediates in the complete degradation of organic matter to CO2 and CH4 (21,23,25), and the degradation of these compounds is often the rate-limiting step in methane fermentation (26). Only recently have the bacteria responsible for the degradation of these compounds been isolated in coculture with H2-using bacteria (7,29,32 of fatty acids with H2 production is energetically unfavorable unless the H2 concentration is maintained at a very low level by the H2-using bacterium (9, 27). Because of this, the anaerobic fatty acid-degrading syntrophic bacteria can only be grown in coculture with H2-using bacteria.S. wolfei does not use any other common bacterial energy source or combination of electron donor and acceptor that would enable it to grow in pure culture (28,29). Growth of S. wolfei in coculture with an H2-using sulfate reducer or met...

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.

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