Transcriptional responses to four weak organic acids (benzoate, sorbate, acetate and propionate) were investigated in anaerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. To enable quantitative comparison of the responses to the acids, their concentrations were chosen such that they caused a 50% decrease of the biomass yield on glucose. The concentration of each acid required to achieve this yield was negatively correlated with membrane affinity. Microarray analysis revealed that each acid caused hundreds of transcripts to change by over twofold relative to reference cultures without added organic acids. However, only 14 genes were consistently upregulated in response to all acids. The moderately lipophilic compounds benzoate and sorbate and, to a lesser extent, the less lipophilic acids acetate and propionate showed overlapping transcriptional responses. Statistical analysis for overrepresented functional categories and upstream regulatory elements indicated that responses to the strongly lipophilic acids were focused on genes related to the cell wall, while acetate and propionate had a stronger impact on membrane-associated transport processes. The fact that S. cerevisiae exhibits a minimal generic transcriptional response to weak organic acids along with extensive specific responses is relevant for interpreting and controlling weak acid toxicity in food products and in industrial fermentation processes.

Coenzyme F 420 is the central low-redox-potential electron carrier in methanogenic metabolism. The coenzyme is reduced under hydrogen by the action of F 420 -dependent hydrogenase. The standard free-energy change at pH 7 of F 420 reduction was determined to be "15 kJ mol "1 , irrespective of the temperature (25-65 6C). Experiments performed with methane-forming cell suspensions of Methanothermobacter thermautotrophicus incubated under various conditions demonstrated that the ratios of reduced and oxidized F 420 were in thermodynamic equilibrium with the gas-phase hydrogen partial pressures. During growth in a fed-batch fermenter, ratios changed in connection with the decrease in dissolved hydrogen. For most of the time, the changes were as expected for thermodynamic equilibrium between the oxidation state of F 420 inside the cells and extracellular hydrogen. Also, methanol-metabolizing, but not acetate-converting, cells of Methanosarcina barkeri maintained the ratios of reduced and oxidized coenzyme F 420 in thermodynamic equilibrium with external hydrogen. The results of the study demonstrate that F 420 is a useful probe to assess in situ hydrogen concentrations in H 2 -metabolizing methanogens.

The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H 2 -dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( p H 2 ). Formylmethanofuran synthesis is an endergonic reaction (DG°¢ ¼ +16 kJAEmol )1 ). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (DG¢) were found to range between +10 and +35 kJAEmol )1 depending on the p H 2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions.Heterodisulfide reduction (DG°¢ ¼ )40 kJAEmol )1 ) was associated with free energy changes as high as )55 to )80 kJAEmol )1 . The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at p H 2 <0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow p H 2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations.Keywords: energy conservation; methanogenesis; proton motive force; sodium motive force; Methanothermobacter thermautotrophicus.Methanothermobacter thermautotrophicus is a methanogenic Archaeon that derives the energy for autrophic growth from the reduction of CO 2 with molecular hydrogen as the electron donor. The process of methanogenesis consists of a series of reduction reactions at which the one-carbon unit derived from CO 2 is bound to C 1 carriers of unique nature (for recent reviews see [1,2]). From a bioenergetic point of view, three reactions are of importance, notably the formation of formyl-methanofuran, the N 5 -methyl-tetrahydromethanopterin:coenzyme M methyl transfer step and the H 2 -dependent reduction of CoM-S-S-CoB [1,[3][4][5].Formyl-methanofuran (MFR-NH-CHO; f-MFR) synthesis represents the first step in methanogenesis. In this step, CO 2 is bound to methanofuran (MFR-NH 3 + ; MFR) and subsequently reduced to the formyl state with electrons derived from hydrogen (reaction 1). MFR-NHThe reaction is endergonic under thermodynamic standard conditions [1,6]. Studies with cell suspensions of Methanosarcina barkeri and Methanothermobacter marburgensis indicated that reaction (1) is driven by a sodium motive

In nature, H 2 -and CO 2 -utilizing methanogenic archaea have to couple the processes of methanogenesis and autotrophic growth under highly variable conditions with respect to the supply and concentration of their energy source, hydrogen. To study the hydrogen-dependent coupling between methanogenesis and growth, Methanothermobacter thermautotrophicus was cultured in a fed-batch fermentor and in a chemostat under different 80% H 2 -20% CO 2 gassing regimens while we continuously monitored the dissolved hydrogen partial pressures (p H2 ). In the fed-batch system, in which the conditions continuously changed the uptake rates by the growing biomass, the organism displayed a complex and yet defined growth behavior, comprising the consecutive lag, exponential, and linear growth phases. It was found that the in situ hydrogen concentration affected the coupling between methanogenesis and growth in at least two respects. (i) The microorganism could adopt two distinct theoretical maximal growth yields (Y CH4 max ), notably approximately 3 and 7 g (dry weight) of methane formed mol ؊1 , for growth under low (p H2 < 12 kPa)-and high-hydrogen conditions, respectively. The distinct values can be understood from a theoretical analysis of the process of methanogenesis presented in the supplemental material associated with this study. (ii) The in situ hydrogen concentration affected the "specific maintenance" requirements or, more likely, the degree of proton leakage and proton slippage processes. At low p H2 values, the "specific maintenance" diminished and the specific growth yields approached Y CH4 max , indicating that growth and methanogenesis became fully coupled.Most methanogenic archaea, including the Methanothermobacter thermautrophicus used in the present study, derive their energy for autotrophic growth from the H 2 -dependent reduction of CO 2 into methane. The pathways of methane formation, CO 2 fixation, and ATP synthesis are highly conserved among the different H 2 -utilizing (hydrogenotrophic) methanogens (for reviews, see references 5, 6, 9, and 32 and additional information in the supplemental material). Nevertheless, different species display remarkable differences in specific growth yields (Y CH4 ), i.e., the amount of biomass formed per mole of methane produced at a given growth condition (Table 1). Y CH4 values can be variable for a given species. Even maximal growth yields (Y CH4 max ) seem to differ. Y CH4 max represents the theoretical maximal growth yield that would be obtained if methanogenesis and growth are fully coupled.Methanogens have to couple the processes of energy generation (methanogenesis) and biomass formation under highly diverse concentrations of their energy source, hydrogen. In environments such as anaerobic sediments and sewage digestors, hydrogen formed by obligate proton reducers is available at only very low levels (11, 37). In contrast, hydrogen concentrations can be high at sites where methanogens obtain the gas from H 2 -producing fermentative microorganisms (29, 37). Under labo...

During growth of Methanobacterium thermoautotrophicum in a fed-batch fermentor, the cells are confronted with a steady decrease in the concentration of the hydrogen energy supply. In order to investigate how the organism responds to these changes, cells collected during different growth phases were examined for their methanogenic properties. Cellular levels of the various methanogenic isoenzymes and functionally equivalent enzymes were also determined. Cells were found to maintain the rates of methanogenesis by lowering their affinity for hydrogen: the apparent Km(H2) decreased in going from the exponential to the stationary phase. Simultaneously, the maximal specific methane production rate changed. Levels of H2-dependent methenyl-tetrahydromethanopterin dehydrogenase (H2-MDH) and methyl coenzyme M reductase isoenzyme II (MCR II) decreased upon entry of the stationary phase. Cells grown under conditions that favored MCR II expression had higher levels of MCR II and H2-MDH, whereas in cells grown under conditions favoring MCR I, levels of MCR II were much lower and the cells had an increased affinity for hydrogen throughout the growth cycle. The use of thiosulfate as a medium reductant was found to have a negative effect on levels of MCR II and H2-MDH. From these results it was concluded that M. thermoautotrophicum responds to variations in hydrogen availability and other environmental conditions (pH, growth temperature, medium reductant) by altering its physiology. The adaptation includes, among others, the differential expression of the MDH and MCR isoenzymes.