Mixotrophic and autotrophic growth of Thiobacillus acidophilus on tetrathionate

Thiobacillus acidophilus can grow in batch and chemostat culture as a heterotroph on glucose, a chemolithoautotroph on tetrathionate and CO₂, or as a mixotroph. Mixotrophically it obtains energy from the simultaneous oxidation of tetrathionate and glucose, and carbon from both glucose and CO₂. Mixotrophic cultures contain lower activities of ribulose 1,5-bisphosphate carboxylase and exhibit lower specific rates of tetrathionate oxidation than do autotrophic cultures. Mixotrophic cultures with low concentrations of glucose have growth rates that are intermediate between slow autotrophic growth and fast heterotrophic growth. Slightly more glucose-carbon is assimilated by mixotrophic cultures than by heterotrophic ones provided with the same concentrations of glucose. Mixotrophic yield in the chemostat is also slightly greater than predicted from autotrophic and heterotrophic yields. These observations indicate that there is preferential assimilation of glucose, at the expense of energy from tetrathionate oxidation, during mixotrophy, resulting in an overall energy saving that produces enhanced growth yield. These observations are relevant to understanding the regulatory behaviour of T. acidophilus in its acidic, mineral-leaching habitats.     

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.     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells

Chlorobium limicola has been proposed to assimilate CO₂ autotrophically via a reductive tricarboxylic acid cycle rather than via the Calvin cycle. This proposal has been a matter of considerable controversy. In order to determine which pathway is operative, the bacterium was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C-pyruvate, and the incorporation of ¹⁴C into alanine (intracellular pyruvate), aspartate (oxaloacetate), glutamate (-ketoglutarate), and glucose (hexosephosphate) was measured in exponentially growing cells in long term labeling experiments. During growth in presence of pyruvate, 20% of the cell carbon were derived from pyruvate in the medium, 80% from CO₂. Since pyruvate was not oxidized to CO₂, only those compounds should become labeled which were synthesized from CO₂ via pyruvate.The three amino acids and glucose were found to be labeled. Alanine had one fifth the specific radioactivity of the extracellular pyruvate, indicating that 20% of the intracellular pyruvate pool were derived from pyruvate in the medium, 80% were synthesized from CO₂. Glucose had twice the specific radioactivity of alanine, showing that hexosephosphate synthesis from CO₂ proceeded via the pyruvate pool. The latter finding is not consistent with the operation of the Calvin cycle, in which pyruvate is not an intermediate. The specific radioactivities of aspartate (oxaloacetate) and of glutamate (-ketoglutarate) were practically identical but considerably lower than that of alanine ( intracellular pyruvate). These findings are compatible with the operation of a reductive tricarboxylic acid cycle as mechanism of autotrophic CO₂ fixation. Degradation studies of the cell components support this interpretation. Offprint requests to: G. Fuchs     

CO2 fixation in halobacteria

Seven strains of extremely halophilic bacteria (Halobacterium spp., Halococcus spp., and Haloarcula sp.) fixed CO₂ under light and dark conditions. Light enhanced CO₂ fixation in Halobacterium halobium but inhibited it in Halobacterium volcanii and Haloarcula strain GN-1. Propionate stimulated ¹⁴CO₂ incorporation in some strains, but inhibited it in others. Semi-starvation in basal salts plus glycerol induced enhanced CO₂ fixation rates. ¹⁴CO₂ fixation in semi-starved cells was stimulated by NH ₄ ⁺ or pyruvate and inhibited by succinate and acetate in most strains. No possible reductant was found. In cell-free extracts of H. halobium, NH ₄ ⁺ but not propionate stimulated ¹⁴CO₂ fixation. No RuBP carboxylase activity was detected. The main ¹⁴C-labeled -keto acid detected after a 2-min incubation with ¹⁴CO₂ and pyruvate was pyruvate. Little or no -ketobutyrate was detected among the early products of propionate-stimulated CO₂ fixation. Glycine was the major amino acid synthesized during a 2-min incubation with NH ₄ ⁺ , propionate, and ¹⁴CO₂. Propionate-stimulated CO₂ fixation was sensitive to trimethoprim and insensitive to avidin. A novel pathway for non-reductive CO₂ fixation involving a glycine synthase reaction with CO₂, NH ₄ ⁺ , and a methyl carbon derived from the -carbon cleavage of propionate is tentatively proposed.Abbreviations used BBS buffered basal salts - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - DNPH 2,4-dinitrophenylhydrazine - DNP dinitrophenyl - TLC thin-layer chromatography - FH₄ tetrahydrofolate This work was supported by National Science Foundation grant PCM-8116330 and Petroleum Research Fund grant PRF 13704-AC2     

Heterotrophic growth of Thiobacillus A2 on sugars and organic acids

Thiobacillus A2 grew on a number of organic acids, pentoses, hexoses and -linked disaccharides, but not on -linked disaccharides or galactosides. Growth was slow on glucose, although fast-growing strains were selectively isolated. Additive growth rates occurred on glucose and galactose; growth on glucose with fructose, pyruvate or gluconate was biphasic rather than diauxic; fructose was used preferentially over glucose; slow growth on glucose was accelerated by some disaccharides; growth on acetate, fumarate or succinate with glucose gave diauxic growth with preferential use of the acid and repression of glucose incorporation. Acetate and succinate tended to be used preferentially even with cultures grown on them in mixture with fructose or sucrose.     

The influence of acetate on the oxidation of sulfide by Rhodopseudomonas capsulata

The capacity to oxidize sulfide and the influence of the simultaneous presence of acetate in heterotrophically (acetate) and autotrophically (sulfide/CO₂) grown Rhodopseudomonas capsulata was investigated.Sulfide oxidation of acetate-limited cultures was found inversely related to the specific growth rate. Upon acetate deprevation (metering pump stopped) increased rates of sulfide oxidation were observed. This points to the existence of a constitutive acceptor for the electrons from sulfide. It is suggested that a carrier functional in the light-induced cyclic electron flow operates as such. The rate of sulfide oxidation, however, is low when compared to autotrophically-grown cells. This is probably due to the low levels of Calvin cycle enzymes present in the acetate-grown cells.In cells growing on sulfide/CO₂, the addition of acetate resulted in less sulfide being oxidized. Upon depletion of the acetate, the rate of sulfide oxidation again increased, however, insufficiently to maintain the accelerated growth rate. This indicates that under mixotrophic conditions the enzymes of the Calvin cycle are being synthesized to a far lesser extent.Non-Standurd Abbreviations PHB poly--hydroxybutyric acid - D dilution rate - TCA Tri carboxylic acid cycle - RubPcase ribulose 1,5-bisphosphate carboxylase - RP reducing power     

Evidence for an incomplete reductive carboxylic acid cycle in Methanobacterium thermoautotrophicum

The involvement of reactions of the tricarboxylic acid cycle in autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum was investigated. The incorporation of succinate into glutamate (=-ketoglutarate), aspartate (=oxaloacetate) and alanine (=pyruvate) was studied. The organism was grown on H₂ plus CO₂ at pH 6.5 in the presence of 1 mM [U-¹⁴C-]succinate. Significant amounts of the dicarboxylic acid were incorporated into cellular material under these conditions. Alanine, aspartate, and glutamate were isolated and their specific radioactivities were determined. Only glutamate was found to be labelled. Degradation of glutamate revealed that C-1 of glutamate was derived from CO₂ and C-2-C-5 from succinate indicating that in M. thermoautotrophicum -ketoglutarate is synthesized via reductive carboxylation of succinyl CoA. The finding that succinate was not incorporated into alanine and aspartate excludes that oxaloacetate and pyruvate are synthesized from -ketoglutarate via isocitrate or citrate. This is taken as evidence that a complete reductive carboxylic acid cycle is not involved here in autotrophic CO₂ fixation.     

Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h.The mechanism of CO₂ fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (¹³C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term ¹⁴CO₂ labelling. Thus CO₂ is not fixed by the Calvin cycle.Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO₂ fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised.Glyoxylate inhibited CO₂ fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO₂ by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and -ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO₂ fixation process, but the mechanism of its synthesis is not clear.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase - TCA cycle tricarboxylic acid cycle - FAc monofluoroacetate - PEP phosphoenolpyruvate - MV methyl viologen - TTC triphenyltetrazolium chloride - PMS phenazine methosulfate     

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.     

Fructose utilization by the cyanobacterium Anabaena variabilis studied using whole filaments and isolated heterocysts

We have investigated the utilization of [¹⁴C]-fructose by whole filaments and isolated heterocysts of Anabaena variabilis ATCC 29413, a strain which is capable of fructose-dependent heterotrophic growth. The experimental conditions were chosen such that both transport and subsequent metabolism were studied. The apparent K_m for fructose was 60 mM, close to the results of previous studies. Rates of fructose utilization were the same in light and darkness. When photosynthetic CO₂ fixation was possible, almost all the label appeared as cell-carbon. In darkness or in the presence of DCMU appreciable amounts of label were released as CO₂. Isolated heterocysts with high rates of endogenous metabolism were not capable of utilizing added fructose at significant rates. The effects of oxygen concentration on the metabolism of added fructose in darkness showed that uptake was saturated at low pO₂ values. Increasing the pO₂ values lead to an increase in the ratio between the lable released as CO₂ and that recovred as cell-carbon. These results suggest that fructose is taken up only by the vegetative cells but carbon derived from added fructose can be released as CO₂ as a result of respiration in the heterocysts. Fructose utilization was inhibited by uncouplers. The greatest inhibition was found when both (delta) (psi) and (delta) pH were abolished. High concentrations of erythrose inhibited fructose utilization. None of the other potential analogs tested had any effect.     

Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle

The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO₂ and H₂S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO₂/H₂S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP⁺-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD⁺-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO₂ with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO₂ proceeds via the citric acid cycle.     

Optimization of growth and fatty acid composition of a unicellular marine picoplankton,Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO₂-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO₂ of 2800 l CO₂ l^–1 and aeration gave the highest biomass yield (634 mg dry wt l^–1), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g^–1 dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:53) (16 mg g^–1 dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO₂ in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.     

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)     

Growth effects and assimilation of organic acids in chemostat and batch cultures of <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis>

The ability of Acidithiobacillus caldus to grow aerobically using pyruvate, acetate, citrate, 2-ketoglutarate, succinate, and malate as either an electron donor and carbon source (heterotrophic growth), or as a carbon source when potassium tetrathionate was added as an electron donor (mixotrophic growth), was tested in chemostat cultures. Under both heterotrophic and mixotrophic conditions, organic acids were added to a sub-lethal concentration (50 μM). Under mixotrophic conditions, potassium tetrathionate was added to an excess concentration (10 mM). No cell growth was observed under heterotrophic conditions; however, effluent cell concentrations increased over threefold when pyruvate was coupled with potassium tetrathionate. Under these conditions, the effluent pyruvate concentration was reduced to below the detection limit (2 μM), and oxygen consumption increased by approximately 100%. Although pyruvate provided a carbon source in these experiments, ambient carbon dioxide was also available to the cells. To test whether At. caldus could grow mixotrophically using pyruvate as a sole carbon source and potassium tetrathionate as an electron donor, cells were batch cultured in a medium free of dissolved inorganic carbon, and with no carbon dioxide in the headspace. These experiments showed that At. caldus was able to convert between 65 ± 8 and 82 ± 15% of the pyruvate carbon to cellular biomass, depending on the initial pyruvate concentrations. This work is the first to identify a defined organic-carbon source, other than glucose, that At. caldus can assimilate. This has important implications, as mixotrophic and heterotrophic activity has been shown to increase mineral leaching in acidic systems.     

Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes malic enzyme, NAD(P)H: ferredoxin oxidoreductase, pyruvate: ferredoxin oxidoreductase, hydrogenase, acetate: succinate CoA transferase and succinate thiokinase leading to the formation of H₂, CO₂, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O₂-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role-especially in zoospores — as H₂-evolving, ATP-generating and O₂-scavenging organelles.Abbrevations DTT Dithiotreitol - PEP Phosphoenol pyruvate     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

Treponema succinifaciens sp. nov., an anaerobic spirochete from the swine intestine

The morphology, the general physiological characteristics, and the energy-yielding metabolism of an obligately anaerobic spirochete isolated from the colon of a swine were studied. Electron microscopy showed that the helical spirochetal cells possessed an outer sheath, a protoplasmic cylinder, and 4 periplasmic fibrils in a 2-4-2 arrangement. The spirochete grew in an atmosphere of N₂ in prereduced media containing a carbohydrate, NaHCO₃, rumen fluid, yeast extract, peptone, l-cysteine, and inorganic salts. The spirochete fermented carbohydrates and required substrate amounts of CO₂ (HCO ₃ ^- ) for growth. Amino acids were not fermented. Major fermentation products of cells growing with glucose as the substrate and in the presence of CO₂ were acetate, formate, succinate, and lactate. Small amounts of 2,3-butanediol, pyruvate, and acetoin were also formed. Determinations of enzymatic activities in cell extracts, and of radioactivity in products formed by growing cells from [1-¹⁴C]glucose, indicated that this sugar was dissimilated to pyruvate via the Embden-Meyerhof pathway. The spirochetes used a coliform-type clastic reaction to metabolize pyruvate. Determinations of radioactivity in products formed from [¹⁴C]NaHCO₃ indicated that CO₂ was assimilated and used in succinate production. The guanine+cytosine content of the DNA was 36 mol%. This study indicates that this intestinal spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema succinifaciens.Abbreviations cpm counts per minute - DTT dithiothreitol - EM Embden-Meyerhof - GC guanine plus cytosine - IgG immunoglobulin G - PC protoplasmic cylinder - PF periplasmic fibrils (axial fibrils) - OS outer sheath     

Acetyl CoA,a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

The pathway of autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H₂ plus CO₂. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO₂. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO₂. M. thermoautotrophicum growing in presence of U-¹⁴C acetate incorporated ¹⁴C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine. M. thermoautotrophicum growing in presence of 3-¹⁴C pyruvate, however, did not incorporate ¹⁴C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO₂ fixation pathway hexosephosphates and pentosephosphates are synthesized from CO₂ via acetyl CoA and pyruvate.