Mixotrophic and Autotrophic Growth of Thiobacillus acidophilus on Glucose and Thiosulfate

Mixotrophic growth of the facultatively autotrophic acidophile Thiobacillus acidophilus on mixtures of glucose and thiosulfate or tetrathionate was studied in substrate-limited chemostat cultures. Growth yields in mixotrophic cultures were higher than the sum of the heterotrophic and autotrophic growth yields. Pulse experiments with thiosulfate indicated that tetrathionate is an intermediate during thiosulfate oxidation by cell suspensions of T. acidophilus. From mixotrophic growth studies, the energetic value of thiosulfate and tetrathionate redox equivalents was estimated to be 50% of that of redox equivalents derived from glucose oxidation. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) activities in cell extracts and rates of sulfur compound oxidation by cell suspensions increased with increasing thiosulfate/glucose ratios in the influent medium of the mixotrophic cultures. Significant RuBPCase and sulfur compound-oxidizing activities were detected in heterotrophically grown T. acidophilus. Polyhedral inclusion bodies (carboxysomes) could be observed at low frequencies in thin sections of cells grown in heterotrophic, glucose-limited chemostat cultures. Highest RuBPCase activities and carboxysome abundancy were observed in cells from autotrophic, CO₂-limited chemostat cultures. The maximum growth rate at which thiosulfate was still completely oxidized was increased when glucose was utilized simultaneously. This, together with the fact that even during heterotrophic growth the organism exhibited significant activities of enzymes involved in autotrophic metabolism, indicates that T. acidophilus is well adapted to a mixotrophic lifestyle. In this respect, T. acidophilus may have a competitive advantage over autotrophic acidophiles with respect to the sulfur compound oxidation in environments in which organic compounds are present.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Autotrophic growth of four Sulfolobus strains on tetrathionate and the effect of organic nutrients

Autotrophic growth yields of four strains of Sulfolobus using tetrathionate as sole energy substrate fell in the range 6.2–7.8 g dry weight (mol tetrathionate oxidized)^-1. Autotrophic organisms lacked ribulose 1,5-bis-phosphate carboxylase, but contained pyruvate and phosphoenolpyruvate carboxylases. S. brierleyi and strains B6-2 and LM exhibited mixotrophic growth, with tetrathionate oxidation, CO₂-fixation and organic substrate assimilation occurring concurrently, using media containing glucose or acetate. Yeast extract or succinate supported heterotrophic growth and showed strain-dependent repression of one or both of tetrathionate oxidation and CO₂-fixation resulting in biphasic growth. All four carbon atoms of succinate were assimilated to cell-carbon during growth. Acetate was the major source of cell-carbon during mixotrophic growth. These observations are not inconsistent with the possibility of a reductive carboxylic acid cycle in these organisms. Radiorespirometric analysis of glucose oxidation indicated CO₂ release to occur by means of an Entner-Doudoroff pathway (followed by pyruvate decarboxylation) and oxidative pentose phosphate pathway reactions. There was little evidence from the glucose radiorespirometry of the large-scale use of an oxidative tricarboxylic acid cycle for terminal oxidation of acetate derived from pyruvate. These results demonstrate the considerable metabolic versatility of Sulfolobus strains and show that there is significant variation among them.Abbreviations PIPES Piperazine-N,N-bis (2-ethane sulphonic acid)     

Autotrophic growth and inorganic sulphur compound oxidation by Sulfolobus sp. in chemostat culture

Sulfolobus strain LM was grown in tetrathionate and thiosulphate-limited continuous culture. CO₂ limitation resulted in a decrease of the steady-state biomass and an increase in the specific rate of thiosulphate oxidation so that substrate did not accumulate in the medium. The initial step in thiosulphate utilization appeared to be its conversion to tetrathionate. The affinity for tetrathionate oxidation appeared to increase with prolonged continuous culture giving an apparent K _m of about 6 M tetrathionate, a higher affinity than for thiosulphate oxidation and in the same range as values observed with acidophilic, sulphur-oxidizing eubacteria.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Oxidation of reduced sulphur compounds by intact cells of Thiobacillus acidophilus

Oxidation of reduced sulphur compounds by Thiobacillus acidophilus was studied with cell suspensions from heterotrophic and mixotrophic chemostat cultures. Maximum substrate-dependent oxygen uptake rates and affinities observed with cell suspensions from mixotrophic cultures were higher than with heterotrophically grown cells. ph Optima for oxidation of sulphur compounds fell within the pH range for growth (pH 2–5), except for sulphite oxidation (optimum at pH 5.5). During oxidation of sulphide by cell suspensions, intermediary sulphur was formed. Tetrathionate was formed as an intermediate during aerobic incubation with thiosulphate and trithionate. Whether or not sulphite is an inter-mediate during sulphur compound oxidation by T. acidophilus remains unclear. Experiments with anaerobic cell suspensions of T. acidophilus revealed that trithionate metabolism was initiated by a hydrolytic cleavage yielding thiosulphate and sulphate. A hydrolytic cleavage was also implicated in the metabolism of tetrathionate. After anaerobic incubation of T. acidophilus with tetrathionate, the substrate was completely converted to equimolar amounts of thiosulphate, sulphur and sulphate. Sulphide- and sulphite oxidation were partly inhibited by the protonophore uncouplers 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) and by the sulfhydryl-binding agent N-ethylmaleimide (NEM). Oxidation of elemental sulphur was completely inhibited by these compounds. Oxidation of thiosulphate, tetrathionate and trithionate was only slightly affected. The possible localization of the different enzyme systems involved in sulphur compound oxidation by T. acidophilus is discussed.     

Chemolithotrophic metabolism of the newly-isolated moderately thermophilic,obligately autotrophic Thiobacillus tepidarius

Thiobacillus tepidarius, isolated from the hot springs at Bath, Avon, UK, grew optimally at 43–45°C and pH 6.0–7.5 on thiosulphate or tetrathionate. In batch culture, thiosulphate was oxidized stoichiometrically to tetrathionate, with a rise in pH. The tetrathionate was then oxidized to sulphate, supporting growth and producing a fall in pH to a minimum of ph 4.8. The organism contained high levels of thiosulphate-oxidizing enzyme, rhodanese and ribulose bisphosphate carboxylase. It was obligately chemolithotrophic and autotrophic. In chemostat culture, T. tepidarius grew autotrophically with the following sole energy-substrates: sulphide, thiosulphate, trithionate, tetrathionate, hexathionate or heptathionate. Thiocyanate, dithionate and sulphite were not used as sole substrates, although sulphite enhanced growth yields in the presence of thiosulphate. Maximum specific growth rate on tetrathionate was 0.44 h^-1. True growth yields (Y _max) and maintenance coefficients (m) were calculated for sulphide, thiosulphate, trithionate and tetrathionate and observed yields at a single fixed dilution rate compared with those on hexathionate and heptathionate. Mean values for Y _max, determined from measurements of absorbance, dry wt, total organic carbon and cell protein, were similar for sulphide, thiosulphate and trithionate (10.9 g dry wt/mol substrate) as expected from their equivalent oxygen consumption for oxidation. Y _max for tetrathionate (20.5) and the relative Y _o values (as g dry wt/g atom oxygen consumed) for thiosulphate and all four polythionates indicated that substrate level phosphorylation did not contribute significantly to energy conservation. These Y _max values were 40–70% higher than any of those previously reported for obligately aerobic thiobacilli. Mean values for m were 6.7 mmol substrate oxidized/g dry wt·h for sulphide, thiosulphate and trithionate, and 2.6 for tetrathionate.Abbreviation PIPES Piperazine-N,N-bis(ethane sulphonic acid)     

Glucose metabolism in Acetobacter aceti

Acetobacter aceti NCIB 8554 grows on a minimal medium with ethanol but not with glucose as carbon and energy source. Addition of glucose to a wild type culture on ethanol has no influence on growth of the organism. Growth of a glucose sensitive mutant A5 is inhibited by the addition of glucose until all glucose has disappeared from the medium. In order to determine the routes by which glucose is metabolised in wild type and mutant, radiorespirometric, enzymatic, and uptake experiments have been performed. For the radiorespirometric experiments of the continuous substrate feeding type an apparatus has been constructed.Of the glucose entering the cells about 30% is excreted as gluconate and 6% metabolised with liberation of C-1 as CO₂. The rest is accumulated intracellularly. No differences were found between wild type and mutant.Under different growth conditions and with different enzymatic assay methods no pyruvate kinase activity (EC 2.7.1.40) could be detected. This might explain the inability of A. aceti to grow on glucose.Abbreviations PPO 2,5-diphenyloxazole - DM-POPOP 2,2-p-phenylene bis(4-methyl-5-phenyloxazole) - TCA trichloroacetic acid     

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 mol quanta·m^-2·s^-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO₂. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO₂), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed ¹⁴CO₂ at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of ¹⁴CO₂ or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.Abbreviations Chl chlorophyll - DW dry weight - FW fresh weight - SGAT Serine:glyoxylate aminotransferase - WT wild-type     

Isolation and physiological characterisation of Thiobacillus thyasiris sp. nov., a novel marine facultative autotroph and the putative symbiont of Thyasira flexuosa

A novel facultatively chemolithoautotropic Thiobacillus, isolated from the gill tissue of the marine bivalve Thyasira flexuosa, is described. It is believed to be the symbiont from this animal, providing the animal with carbon fixed by the Calvin cycle. The organism grows lithoautotrophically on thiosulphate, tetrathionate and elemental sulphur, which are oxidised to sulphate. It oxidizes sulphide, thiosulphate, trithionate, tetrathionate and hexathionate, but not thiocyanate. Kinetic constants for these substrates are presented. In autotrophic batch culture it produces yields that are among the lowest reported for thiosulphate or tetrathionate as energy substrates (1.25 and 2.5 g cell-carbon per mol substrate, respectively). Autotrophic cultures contain ribulose bisphosphate carboxylase and excreted 20% of their fixed carbon into the medium during growth. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of the carboxylase. The organism is slightly halophilic and markedly halotolerant, showing optimum growth at about pH 7.5 and maximum growth rate at 37° C. It contains ubiquinone Q-10 and its DNA contains 52 mol % G+C. These characteristics distinguish it from any other Thiobacillus or Thiomicrospira species previously described. The organism is formally described and named as Thiobacillus thyasiris.     

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose,cellobiose and cellulose

Summary The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N₂/CO₂ or N₂ gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h^–1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and ¹⁴C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (=0.03h^–1) and molar growth yield (Y_glucose=21.5) were considerably lower than those obtained (=0.17 h^–1, Y_cellobiose=68.9) in cellobiose media. A Y_ATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H₂, 2.64 CO₂, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.National Research council of Canada No. 20826     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

Physiological factors determining formation of plastocyanin and plastidic cytochrome c-553 in Scenedesmus

Physiological conditions necessary for the formation of plastocyanin and the concurrent cessation of cytochrome c-553 formation were studied in cells of copper-deficient Scenedesmus acutus after the addition of copper. Plastocyanin is formed after a lag-phase, leaving constant the content of plastidic cytochrome c-553. Therefore, the concentration of plastocyanin per cell increases and the concentration of cytochrome c-553 decreases during growth. Formation of plastocyanin during the induction period studied is dependent on light intensity. In the dark, there is a 90% inhibition, whereas under light intensities above 50 Wm^-2, a ratio of 1.3 molecules plastocyanin per 1,000 molecules chlorophyll is attained.Plastocyanin formations is inhibited by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), but not by moderate concentrations of 3-[3,4-dichlorophenyl]-1,1-dimethylurea (DCMU), and by keeping the algae under a nitrogen atmosphere without CO₂. Concurrently, the cultures treated with FCCP show a decreased endogenous ATP level. The ATP is necessary for plastocyanin formation.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-[3,4-dichlorophenyl]-1,1-dimethylurea - pcv packed cell volume     

Efficiency of CO2 fixation in a glycollate oxidoreductase mutant of Alcaligenes eutrophus which exports fixed carbon as glycollate

Mutant strains of the facultative autotrophic bacterium Alcaligenes eutrophus blocked in glycollate utilization were isolated and characterized. One of the strains, AE161, which lacked glycollate oxidoreductase activity, excreted up to 1.2mol glycollate/mg cell protein per hour during autotrophic growth. This mutant strain was used to study the efficiency of CO₂ fixation in terms of how much of the fixed carbon was excreted as glycollate under different conditions. Glycollate excretion was not detected during heterotrophic growth. Only 1% of the total CO₂ fixed was excreted as glycollate in an atmosphere of 4% CO₂ plus 20% O₂. The rate of glycollate excretion showed a large increase and CO₂ fixation decreased as the CO₂ concentration was lowered. Almost half (40–50%) of the total CO₂ fixed was excreted as glycollate in an atmosphere of 0.07% CO₂ plus 20% O₂.Abbreviations HPMS 2-pyridyl-hydroxymethane sulphonic acid - RuBP ribulose 1,5-bisphosphate To whom offprint requests are to be sent     

Glycollate production and excretion byAlcaligenes eutrophus

Autotrophic cultures of the facultative chemolithotrophAlcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO₂ to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic conditions.Extracts from autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction ofd0ribulose 1,5-diphosphate carboxylase under the same conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - HPMS 2-pyridyl-hydroxymethane sulphonie acid - RuDP d-ribulose 1,5-diphosphate     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of oxalate and formate in continuous culture

Pseudomonas oxalaticus was grown in carbon- and energy-limited continuous cultures either with oxalte or formate or with mixtures of these substrates. During growth on the mixtures, simultaneous utilization of the two substrates occurred at all dilution rates tested. Under these conditions oxalate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and the ratio of oxalate and formate in the medium reservoir. At a fixed oxalate/formate ratio repression was greatest at intermediate dilution rates, whereas derepression occurred at both low and high dilution rates. Progressive depression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation at low dilution rates was attributed to the decreasing concentration of intracellular repressor molecule(s), parallel to the decreasing concentration of the growth-limiting substrates in the culture. To account for the derepression at higher dilution rates, it is proposed that the rate of oxalyl-CoA production from oxalate limits the supply of metabolic intermediates and that additional energy and reducing power generated from formate drains the pools of metabolic intermediates sufficiently to lower the intracellular concentration of the repressor(s). During growth of Pseudomonas oxalaticus on the heterotrophic substrate oxalate alone, at dilution rates below 10% of the maximum specific growth rate, derepression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation was observed to a level which was 50% of that observed during growth on formate alone at the same dilution rate. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO₂ fixation via the Calvin cycle is regulated by a repression/derepression mechanism and that the contribution of autotrophic CO₂ fixation to the biosynthesis of cell material in this organism is mainly controlled via the synthesis of these enzymes.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenolindophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

Lactate conversion to acetate,CO2 and H2 in cell suspensions of Desulfovibrio vulgaris (Marburg): indications for the involvement of an energy driven reaction

Cell suspensions of Desulfovibrio vulgaris were found to catalyze, in the absence of sulfate, the complete conversion of 1 lactate to 1 acetate, 1 CO₂, and 2 H₂ (G₀=-8.8 kJ/mol) and of 1 pyruvate to 1 acetate, 1 CO₂, and 1 H₂ (G₀=-52 kJ/mol). Protonophores, the proton translocating ATPase inhibitor N,N-dicyclohexylcarbodiimide, and arsenate specifically inhibited H₂ formation from lactate but not from pyruvate. The results suggest that lactate oxidation to pyruvate and H₂ (G ₀=+43.2 kJ/mol) is energy driven.     

Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO₂, H₂, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H₂S. Enzymes of the autotrophic CO₂ assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 mol · min^-1 · mg cell protein^-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO₂ fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO₂ fixation, two different autotrophic pathways occur in the same physiological group.Dedicated to Prof. H. G. Wood on the occasion of his 80th birthday     

Plasmids required for utilization of molecular hydrogen by Alcaligenes eutrophus

Alcaligenes eutrophus and three other hydrogen bacteria exposed to plasmid-curing agents generated autotrophic-minus mutants at high frequency. These mutants were blocked in the metabolism of H₂ as an energy source and had normal levels of enzymes involved in CO₂ fixation. The loss of hydrogenase activity in A. eutrophus was accompanied by the loss or alteration of a plasmid that had molecular weight of approximately 200×10⁶. Mobilization of this plasmid from wild-type A. eutrophus strains into cured hydrogenase-minus derivatives restored hydrogenase function. It is concluded that A. eutrophus contains a large plasmid required for hydrogen metabolism and thereby autotrophic growth.Abbreviations Aut autotrophic - Hup hydrogen uptake - NTG N-methyl-N-nitro-N-nitrosoguanidine - RuBP ribulose bisphosphate - RuMP ribulose monophosphate - Kan kanamycin - Nal nalidixic acid - Rif rifampicin - Tet tetracycline     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine