The pathway of fermentative benzoate degradation by the syntrophically fermenting bacterium Syntrophus gentianae was studied by measurement of enzyme activities in cell-free extracts. Benzoate was activated by a benzoate-CoA ligase reaction, forming AMP and pyrophosphate, which was subsequently cleaved by a membrane-bound proton-translocating pyrophosphatase. Glutaconyl-CoA (formed from hypothetical pimelyl-CoA and glutaryl-CoA intermediates) was decarboxylated to crotonyl-CoA by a sodium-ion-dependent membrane-bound glutaconyl-CoA decarboxylase, a biotin enzyme that could be inhibited by avidin. The overall energy budget of this fermentation could be balanced only if the dearomatizing reduction of benzoyl-CoA is assumed to produce cyclohexene carboxyl-CoA rather than cyclohexadiene carboxyl-CoA, although experimental evidence of this reaction is still insufficient. With this assumption, benzoate degradation by S. gentianae can be balanced to yield onethird to two-thirds of an ATP unit per benzoate degraded, in accordance with earlier measurements of whole-cell energetics.

Syntrophus gentianae is a strictly anaerobic bacterium which ferments benzoate to acetate, CO 2 and H 2 in the presence of hydrogen-utilizing partner bacteria. Benzoate is activated by a benzoyl CoA ligase enzyme which forms AMP and pyrophosphate as coproducts. Pyrophosphatase activity was found to be largely membrane bound. Pyrophosphate hydrolysis was associated with proton translocation across the cytoplasmic membrane. Proton translocation could be abolished by the protonophor carbonylcyanide pchlorophenylhydrazone, and could also be coupled to ATP formation in membrane vesicle preparations. The ratio of ATP formation/pyrophosphate hydrolysis was 1:3. The reverse reaction, ATP-dependent pyrophosphate synthesis, was possible with the same coupling stoichiometry. Pyrophosphatase was 90% saturated at 1 mM pyrophosphate ; pyrophosphate concentrations higher than 5 mM inhibited enzyme activity. Inhibition studies with ATP and EDTA indicated that MgPP Ϫ i was probably the physiological substrate. The optimum temperature was 35°C. In the presence of Mg 2ϩ , the enzyme was remarkably heat stable, with 50% of its maximum activity after 10 min at 60°C. Exogenously added pyrophosphate could not be used for energy conservation.Keywords : Syntrophus gentianae; pyrophosphatase ; proton translocation ; ATP synthase ; energy conservation.Pyrophosphate is formed in several reactions for substrate activation by anaerobic bacteria, e.g. in the activation of sulfate by sulfate-reducing bacteria [1] or in the activation of benzoate derivatives during anaerobic degradation of such compounds by nitrate-reducing [2] phototrophic [3] or sulfate-reducing bacteria [4]. In these reactions, pyrophosphate is formed by group transfer from ATP as a side product, and is supposed to be hydrolyzed subsequently by a pyrophosphatase enzyme to shift the overall reaction equilibrium towards product formation [1]. This strategy is efficient but implies a considerable loss of metabolic energy that is released as heat [5].Alcohols and fatty acids longer than two carbon atoms, benzoate and certain other aromatic compounds are degraded to methane and CO 2 in so-called syntrophic associations of fermenting bacteria with methanogenic partners. The energetical situation of the syntrophic fermenting organisms is comparably difficult; they obtain energy amounts in the range of only fractions of 1 ATP/reaction run in such cooperations [6Ϫ8]. These organisms have to save every fraction of an ATP unit, therefore. Assessment of the energetical situation of syntrophically benzoate-degrading bacteria on the basis of substrate, product and hydrogen concentrations in syntrophic cocultures revealed that these organisms have only about 40Ϫ45 kJ available/reaction Correspondence to B. Schink,

Summary: Growing cocultures of Syntrophus gentianae with Methanospirillum hungatei degraded benzoate to CH4 and acetate. During growth, the change of free energy available for Syntrophus gentianae ranged between -50 and -55 kJ mol−1. At the end-point of benzoate degradation, a residual concentration of benzoate of 0.2 mM was found, correlating with a free energy change of -45 kJ mol−1 available to the fermenting bacterium. Benzoate thresholds were also observed in dense cell suspensions. They corresponded 1 a final energy situation in the range -31.8 to -45.8 kJ mol−1 for the fermentin bacterium. Addition of a H2-oxidizing sulfate reducer to the methanogenic coculture inhibited by bromoethanesulfonate (BES) resulted in benzoate degradation to below the limit of benzoate detection (10 μM). Accumulated acetate proved to be thermodynamically inhibitory; removal of acetate by Methanosaeta concilii in methanogenic or molybdate-inhibited sulfate-reducing cocultures led to degradation of residual benzoate with a final δG’ -45.8 kJ mol−1. In methanogenic cocultures, the residual Gibbs free energy (δG’) available for the fermenting bacterium at the end of benzoate degradation correlated with the concentration of acetate built up during the course of benzoate degradation; higher concentrations led to more positive values for δG’. Addition of different concentrations of propionate resulted in different values for δG when benzoate degradation had ceased; higher concentrations led to more positive values for δG’. Addition of acetate or propionate to benzoate-degrading cocultures also lowered the rate of benzoate degradation. The protonophore carbonylcyanide chlorophenylhydrazone (CCCP) facilitated further benzoate degradation in methanogenic BES-inhibited cocultures until a δG’ of -31 kJ mol−1 was reache We conclude that the minimum energy required for growth and energy conservation of the benzoate-fermenting bacterium S. gentianae is approximately -45 kJ (mol benzoate)−1, equivalent to two-thirds of an ATP unit. Both hydrogen and acetate inhibit benzoate degradation thermodynamically, and acetate also partly uncouples substrate degradation from energy conservation.

Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of approximately 66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn2+ and stabilization of the nucleotide substrate by Mg2+. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 micromol min-1 (mg enzyme)-1. The apparent Km values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent Km values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium.