CH, formation from C 0 2 and H2 rather than from formaldehyde and H2 in methanogenic bacteria is inhibited by uncouplers, indicating that C 0 2 reduction to the formaldehyde level is energy-driven. We report here that in Methanosarcina hurkeri the driving force is a primary electrochemical sodium potential (AjiNa') generated by formaldehyde reduction to CH,. This is concluded from the following findings.1. C 0 2 reduction to CH, was insensitive towards protonophores, when the N a + / H t antiporter was inhibited; under these conditions A,iINa.+ was 120 mV (inside negative), whereas both dBH+ and the cellular ATP content were low.2. C 0 2 reduction to CH,, rather than foiinaldehyde reduction, was sensitive towards Na' ionophorcs. which dissipated AjiNa'. 3. C 0 2 reduction to CH,, in the presence of protonophores and Na+/H+ antjport inhibitors, was coupled with the extrusion of 1 -2 mol Na+/mol CH,, and formaldehyde reduction to CH4 was coupled with the extrusion of 3 -4 mol Na+/mol CH,. Thus during C 0 2 reduction to the formaldehyde level 2 -3 mol Na ' were consumed.Most methanogenic bacteria are able to reduce C 0 2 to CH, via the redox level of formate, formaldehyde and methanol. The one-carbon intermediates are not free but all coenzyme-bound [I, 21. CH, formation from H2 and CO, is an exergonic process (reaction 1) that is coupled with the synthesis of ATP as evidenced by growth of the bacteria on these substrates. Thermodynamic calculations of thc partial reactions indicate that only the reduction of formaldehyde to the methanol level (reaction 2) and the reduction of the methanol level to CH, (reaction 3) are exergonic reactions, whereas the reduction of C 0 2 to the formaldchyde level (reaction 4) is an endergonic process. The free-energy changes of the reactions 2-4 are given for free formaldehyde and free methanol [3] and are probably not significantly different from those of coenzyme-bound C1 intermediates [2, 41. It has been possible to study the partial reactions experimentally since methanogenic bacteria have been shown to generate CH, from H2 and formaldehyde; in addition, a few species including Methaiio.surcina are also able to form CH, by the reduction of methanol with H2. Formaldehyde reacts non-enzymatically with tetrahydromethanopterin (H4MPT) to form methylene-H,MPT (CH2 = H,MPT) and methanol is transferred to coenzyme M (CoM) via specific transferases [l, 21. Thus formaldehyde and methanol can be used as substitutes for the corrcsponding coenzyme-bound intcrmediates. In accordance with the thermodynamics it was found that CH, formation from C 0 2 and H2, rather than from formaldehyde and H2 or from methanol and H2, is sensitive towards uncouplers [5 -71. From this result it was concluded that C 0 2 reduction to the formaldehyde level is energy-driven and can proceed only when coupled Lo formaldehyde reduction to CH4 [8]. The mcchanism of coupling is not known. In principle coupling might proceed via either ATP. the electrochemical H + potential or the electrochemical Nat potential. We have...

lsocitrate lyase (ICL) was assayed during batch cultivations of Ashbya gossypii on soybean oil or glucose as carbon source. On soybean oil, a correlation between enzyme activity and riboflavin synthesis was observed. On glucose as carbon source, riboflavin overproduction started in the late growth phase when glucose was exhausted. ICL activity appeared in parallel and reached a maximum of 041 U (mg protein)-'. This suggested synthesis of vitamin B, from the intracellular reserve fat. ICL specific activity correlated with the enzyme concentration detected by specific antibodies. Itaconate, an efficient inhibitor of ICL, was used as an antimetabolite to screen mutants with enhanced ICL activity. Cultivations of an itaconate-resistant mutant on soybean oil revealed a 15 O/ o increase in enzyme specific activity and a 25-fold increase in riboflavin yield compared to the wild-type. On the other hand, growth experiments on glucose resulted in an eightfold increase in riboflavin yield but showed a 33% reduction in ICL specific activity compared to the wild-type grown on the same medium. These results support the idea of an ICL bottleneck in the riboflavin overproducer A. gossypii when plant oil is used as the substrate.

Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to C 0 2 and 2H2 (AGO' = -27 kJ/mol C02), when methanogenesis was inhibited by 2-bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na' translocation at a stoichiometry of 2-3 Na'/C02, (b) with secondary H + translocation via a Na'/H' antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings.Formaldehyde oxidation to C 0 2 and 2H2 was dependent on Na' ions, 2-3 mol Na'/mol formaldehyde oxidized were extruded. Na' translocation was inhibited by Na' ionophores, but not affected by protonophores or Na'/H' antiport inhibitors.Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores.Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na' ionophores, N a + / H + antiport inhibitors, by protonophores and by the H'-translocating-ATP-synthase inhibitor, dicyclohexylcarbodiimide.With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.Most methanogenic bacteria form methane from H2 and C 0 2 and couple this exergonic reaction with the phosphorylation of ADP (4H2 + C 0 2 --f CH4 + 2H20, AGO' = -131 kJ/mol CH4). The pathway of C 0 2 reduction to CH4 involves coenzyme-bound single-carbon intermediates at the redox level of formate, formaldehyde and methanol. Formylmethanofuran, formyltetrahydromethanopterin (formyl-H4MPT), methenyl-H4MPT, methylene-H,MPT (CH2 = H4MPT), methyl-H4MPT and methyl-coenzyme M have been identified as intermediates [I -31. Methanogenesis from H2 and C 0 2 is a Na+-dependent process [4]. For the study of the role of sodium ions the finding has been exploited that cell suspensions of methanogens catalyze the reduction of HCHO via CHI = H4MPT to CH4 (2H2 + HCHO + CH4 + H20, AGO' = -158 kJ/mol CH4).

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.

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