Tungstate does not support synthesis of active formylmethanofuran dehydrogenase in Methanosarcina barkeri

Cell extracts of Methanosarcina barkeri grown on methanol in media supplemented with molybdate exhibited a specific activity of formylmethanofuran dehydrogenase of approximately 1 U (1 mol/min)/mg protein. When the growth medium was supplemented with tungstate rather than with molybdate, the specific activity was only 0.04 U/mg. Despite this reduction in specific activity growth on methanol was not inhibited. An inhibition of both growth and synthesis of active formylmethanofuran dehydrogenase was observed, however, when H₂ and CO₂ were the energy substrates. The results indicate that, in contrast to Methanobacterium wolfei and Methanobacterium thermoautotrophicum, M. barkeri possesses only a molybdenum containing formylmethanofuran dehydrogenase and not in addition a tungsten isoenzyme.     

Diffusion of the Interspecies Electron Carriers H2 and Formate in Methanogenic Ecosystems and Its Implications in the Measurement of Km for H2 or Formate Uptake

We calculated the potential H₂ and formate diffusion between microbes and found that at H₂ concentrations commonly found in nature, H₂ could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H₂ concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H₂ concentration at the cell surface of H₂-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 μm from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the K_m for H₂ or formate.     

Cytolytic T lymphocytes: an overview of their characteristics

Cloned T cells have been useful for assessing the lytic potential of distinct T cell subsets and for determining the relative contribution of different effector mechanism involved in the lytic process. Alloreactive CD8⁺ murine T cell clones and cloned murine CD4⁺ T_H1 and T_H2 T cells reactive with nominal antigen (ovalbumin) lysed nucleated target cells bearing antigen or coated with anti-CD3 monoclonal antibody in a short term⁵¹Cr-release assay. These clones were also evaluated for their ability to lyse efficiently sheep erythrocyte (SRBC) target cells coated with anti-CD3 mAb by a mechanism (presumably involving membrane damage) that does not involve nuclear degradation. Three patterns of lysis were observed: CD8⁺ and some CD4⁺ T_H2 effector cells lysed efficiently nucleated target cells and anucleated SRBC coated with anti-CD3 mAb. However, CD4⁺ T_H1 (and a few T_H2) T cells which lysed nucleated target cells bearing antigen or coated with anti-CD3 mAb didnotlyse efficiently the SRBC coated with anti-CD3 mAb. One CD4 bearing T_H2 cell failed to lyse efficiently either nucleated target cells or anucleated SRBC coated with anti-CD3 mAb. These results indicate that both T_H1 and T_H2 clones have lytic capabilities. Furthermore, they suggest that some but not all T_H2 murine T cell clones have lytic characteristics similar to those of conventional CD8⁺ CTL. However, it is not certain how these patterns of lysis of target cellsin vitro relates to the capacity of CTL to lyse such target cellsin vivo.     

Syntrophomonas wolfei gen. nov. sp. nov., an Anaerobic, Syntrophic, Fatty Acid-Oxidizing Bacterium

An anaerobic, nonphototrophic bacterium that β-oxidizes saturated fatty acids (butyrate through octanoate) to acetate or acetate and propionate using protons as the electron acceptor (H₂ as electron sink product) was isolated in coculture with either a non-fatty acid-degrading, H₂-utilizing Desulfovibrio sp. or methanogens. Three strains of the bacterium were characterized and are described as a new genus and species, Syntrophomonas wolfei. S. wolfei is a gram-negative, slightly helical rod with round ends that possesses between two to eight flagella laterally inserted along the concave side of the cell. It has a multilayered cell wall of the gram-negative type. The presence of muramic acid, inhibition of growth by penicillin, and increased sensitivity of the cells to lysis after treatment with lysozyme indicate that peptidoglycan is present in the cell wall. Cells of S. wolfei contain poly-β-hydroxybutyrate. Isoheptanoate was degraded to acetate, isovalerate, and H₂. Carbohydrates, proteinaceous materials, alcohols, or other tested organic compounds do not support growth. Common electron acceptors are not utilized with butyrate as the electron donor. Growth and degradation of fatty acids occur only in syntrophic association with H₂-using bacteria. The most rapid generation time obtained by cocultures of S. wolfei with Desulfovibrio and Methanospirillum hungatei is 54 and 84 h, respectively. The addition of Casamino Acids but neither Trypticase nor yeast extract stimulated growth and resulted in a slight decrease in the generation time of S. wolfei cocultured with M. hungatei. The addition of H₂ to the medium stopped growth and butyrate degradation by S. wolfei.     

Simultaneous butyrate oxidation by Syntrophomonas wolfei and catalytic olefin reduction in absence of interspecies hydrogen transfer

Methanogenesis by a Syntrophomonas wolfei/ Methanospirillum hungatei coculture was inhibited in presence of ethylene and the hydrogenation catalyst Pd-BaSO₄. However, butyrate oxidation by S. wolfei continued and ethylene was reduced to ethane. Per mol of butyrate oxidized, 2.4 mol acetate was produced and 0.8 mol ethylene was reduced. Acetylene, propylene and butene were less effective as H₂ acceptors than ethylene, and addition of bromoethanesulfonic acid was necessary to inhibit methanogenesis in the presence of the two longer-chain olefins. Other hydrogenation catalysts were less effective in the order Pd-charcoal < PE-asbestos < Pd-PEI beads < Pt-Al₂O₃, Pd-CaCO₃. Optimal ethylene hydrogenation was achieved with still incubation in presence of 7.2 mg Pd-BaSO₄ and 0.7 g sand per ml medium. The higher catabolic rate of S. wolfei in presence of the methanogen indicated that the biological H₂ removal mechanism was more efficient than the catalytic olefin reduction.Abbreviations BES bromoethane sulfonic acid - VFA volatile fatty acid     

Short communication: Butyrate-degrading syntrophic culture from an anaerobic digester treating cattle waste

A co-culture of bacteria responsible for the conversion of butyrate to methane and CO₂ was isolated from a cattle-waste treatment plant. The non-methanogenic partner of the co-culture was Syntrophomonas wolfei and the methanogenic partner was Methanobacterium formicicum. Although butyrate degradation occurred at pH<6.0 and below 45°C, methanogenesis was observed at pH>6.5 and above 40°C.     

Formate utilization by members of the genus Methanobacterium

Washed cell suspensions of Methanobacterium formicicum MF, Methanobacterium bryantii M.o.H.G. and Methanobacterium strain FR-2 but not Methanobacterium bryantii M.o.H., were shown to produce hydrogen and methane from formate. Levels of dissolved gases (H₂ and CH₄) were continuously and simultaneously monitored within a closed reaction vessel using membrane inlet mass spectrometry. Growth on formate (0–50mM), measured by methane production and increase in absorbance, was observed for both M. formicicum MF and M. bryantii M.o.H.G. but not with Methanobacterium strain FR-2 or M. bryantii M.o.H.     

Methanobacterium thermoalcaliphilum spec. nov., a new moderately alkaliphilic and thermophilic autotrophic methanogen

An autotrophic moderately alkaliphilic and thermophilic nonmotile rod-shaped methanogen was isolated from a biogas plant. The isolate grows only on H₂+CO₂ and requires yeast extract. Growth optimum is at 60°C with a generation time of 4 h. In the absence of substrates complete lysis occurs. The pH range for growth is 7.5–8.5. Growth was also observed at pH values above 9.0. The DNA base composition is 38.8 mol% G+C. According to its physiological properties the nameMethanobacterium thermoalcaliphilum is proposed.Abbreviations G+C Guanine+cytosine     

Physical and genetic map of the Methanobacterium wolfei genome and its comparison with the updated genomic map of Methanobacterium thermoautotrophicum Marburg

A physical and genetic map of the chromosome of Methanobacterium wolfei was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by digestion with NotI and NheI. The chromosome was found to be circular and 1,729 kb in size. Twenty-eight genes were mapped to specific restriction enzyme fragments by performing hybridization experiments with gene probes from various Methanobacterium strains. The genomic map obtained was compared with the updated genomic map of Methanobacterium thermoautotrophicum Marburg. In spite of major restriction pattern dissimilarities, the overall genetic organization seemed to be conserved between the genomes of the two strains. In addition, the two rRNA operons of strain Marburg were precisely mapped on the chromosome, and it was shown that they are transcribed in the same direction.     

Nickel,cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum on H₂ and CO₂ as sole energy and carbon sources was found to be dependent on Ni, Co, and Mo. At low concentrations of Ni (<100 nM), Co (<10 nM) and Mo (<10 nM) the amount of cells formed was roughly proportional to the amount of transition metal added to the medium; for the formation of 1 g cells (dry weight) approximately 150 nmol NiCl₂, 20 nmol CoCl₂ and 20 nmol Na₂MoO₄ were required. A dependence of growth on Cu, Mn, Zn, Ca, Al, and B could not be demonstrated. Conditions are described under which the bacterium grew exponentially with a doubling time of 1.8 h up to a cell density of 2 g cells (dry weight)/1.     

An improved assay method for a pseudomurein-degrading enzyme of Methanobacterium wolfei and the Protoplast formation of Methanobacterium thermoautotrophicum by the enzyme

An improved assay method of a pseudomurein-degrading enzyme and its properties are described. The pseudomurein-degrading enzyme purified from Methanobacterium wolfei autolysate under an anoxic condition was assayed with the cell wall of Methanobacterium thermoautotrophicum as a substrate. By this improved method the enzyme activity was measured quantitatively and reproducibly. Moreover, the cell wall substrate can be stored in a freezer and used as needed, and the time required for an assay was as short as 1 h. The optimum pH and temperature of the enzyme was pH 6.8-7.4 and 75°C, respectively. Although the enzyme lost 50% of the activity upon heating at 75°C for 10 min in the absence of the cell wall substrate, it was more stable against heat inactivation in the presence of the substrate. Furthermore the inactivated enzyme recovered some of the activity by incubating with the substrate. Although the enzyme lost most of the activity under aerobic conditions, the activity was recovered under reducing conditions with Na₂S·9H₂O or DTT (dithiothreitol). The enzyme was also purified under aerobic conditions retaining the same specific activity as the anoxically purified enzyme. Using the partially purified enzyme the conditions preparing protoplasts of M. thermoautotrophicum was established.     

Isolation and characterization of a new thermophilic and autotrophic methane producing bacterium:Methanobacterium thermoaggregans spec. nov.

Methanobacterium thermoaggregans is a new thermophilic autotrophic rod-shaped methane producing bacterium. The organism likes to form aggregates during growth and utilizes only H₂ and CO₂ as substrates. Growth optimum is at 65°C with a doubling time of 3.5 h. Optimal growth occurs at pH-values between 7 and 7.5. The addition of yeast extract to the mineral salt medium stimulates growth. The DNA base composition is 42 mol% G+C. The organism was isolated from mud taken from a cattle pasture. Because of its optimal growth temperature and its tendency to form aggregates the nameMethanobacterium thermoaggregans is suggested.Abbreviations G+C Guanine+cytosine     

Oxidative Stress and Antioxidant Cell Protection Systems in the Microaerophilic Bacterium Spirillum winogradskii

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O₂) aerobic conditions (21% O₂ of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes that defend against toxic forms of O₂—peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)—massive lysis of cells starts in the midexponential phase and leads to culture death in the stationary phase because of H₂O₂ accumulation in the periplasm (up to 10 g/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H₂O₂, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H₂O₂ accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H₂O₂, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.     

Acetate thiokinase and the assimilation of acetate in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum growing on H₂ plus CO₂ as sole carbon and energy source was found to contain acetate thiokinase (Acetyl CoA synthetase; EC 6.2.1.1): Acetate+ATP+CoA Acetyl CoA+AMP+PPi. The apparent K _m value for acetate was 40 M. Acetate kinase (EC 2.7.2.1) and phosphotransacetylase (EC 2.3.1.8) could not be detected. The specific activity of acetate thiokinase was high in cells grown with limited H₂ and CO₂ supply (approximately 100nmol/min · mg protein), it was low in exponentially grown cells (2 nmol/min·mg protein). This corresponded with the finding that cells growing linearly in the presence of acetate assimilated the monocarboxylic acid in high amounts (>10% of the cell carbon was derived from acetate), whereas exponentially growing cells did not (<1% of cell carbon was derived from acetate). These latter observations indicated that acetate thiokinase and free acetate are not involved in autotrophic CO₂ fixation in M. thermoautotrophicum. The presence and some kinetic properties of succinate thiokinase (EC 6.2.1.5), adenylate kinase (EC 2.7.4.3), and inorganic pyrophosphatase (EC 3.6.1.1.) are also described.     

Growth of Syntrophomonas wolfei in pure culture on crotonate

A Synthrophomonas wolfei-Methanospirillum hungatei coculture was adapted to catabolize crotonate. S. wolfei was then isolated in axenic culture using agar spread plates and roll tubes with crotonate as the sole energy source. S. wolfei catabolized crotonate via a disproportionation mechanism similar to that of some Clostridium species. Growth on crotonate was very slow (specific growth rate of 0.029 h^–1) but the conversion of energy into cell material was very efficient with cell yields of 14.6 g (dry wt.) per mol of crotonate. S. wolfei alone did not catabolize butyrate, but butyrate was stoichiometrically degraded to acetate and presumably methane when S. wolfei was reassociated with M. hungatei. S. wolfei-M. hungatei cocultures accumulated some butyrate during growth on crotonate indicating that protons were not the sole electron acceptors used for crotonate oxidation by the coculture.     

Structural characterization and physiological function of component B from Methanosarcina thermophila

The electron donor (component B) to the methyl coenzyme M methylreductase system from Methanosarcina thermophila was isolated as the 7-methyl derivative and characterized. Gas chromatography-mass spectrometry and ¹H NMR analyses identified this derivative as 7-methylthioheptanoylthreonine phosphate (CH₃-S-HTP), indicating that the original component B had the same structure (HS-HTP) as previously determined for component B from Methanobacterium thermoautotrophicum. The heterodisulfide of HS-HTP and coenzyme M (HS-CoM, 2-mercaptoethanesulfonate) was enzymatically reduced in cell extracts using electrons supplied by either H₂ or CO, confirming that HS-HTP was a functional molecule in M. thermophila.     

Function of fumarate reductase in methanogenic bacteria (Methanobacterium)

Methanogenic bacteria contain high activities of fumarate reductase. An interesting hypothesis has recently been advanced that this enzyme, in cooperation with a succinate dehydrogenase, functions in a fumarate-succinate cycle for ATP synthesis. This hypothesis was tested by determining whether [2, 3-³H] succinate loses³H when taken up by growing cells.Methanobacterium thermoautotrophicum was grown on H₂ plus CO₂ in the presence of [U-¹⁴C, 2,3-³H] succinate. The double labelled dicarboxylic acid was found to be incorporated into cell material with the loss of only 30% of tritium. Neither was³H released into H₂O in significant amounts. This finding excludes a catabolic oxidation of succinate to fumarate in the growing cells and thus the operation of a fumaratesuccinate cycle. It is shown that the function of fumarate reductase inM. thermoautotrophicum is to provide the cells with succinate for the synthesis of -ketoglutarate, an intermediate in glutamate, arginine and proline synthesis.     

Extinction of cells of cyanobacterium Anabaena circinalis in the presence of humic acid under illumination

Laboratory experiments targeting the effect of humic acid (HA) on the cell lysis of cyanobacterium Anabaena circinalis have been performed. Light irradiation was found to be an important factor for the cell lysis phenomenon, whereas intracellular hydrogen peroxide (H₂O₂) might be a chemical factor for the process. An exogenous H₂O₂ concentration of 1.0 mg l⁻¹ was determined as the threshold for cell survival. Our results indicated that HA or its possible product(s) of photochemical reaction can induce damage to intracellular catalase under artificial illumination, which leads intracellular H₂O₂ to be accumulated to an abnormally high concentration, eventually resulting in cell death. Moreover, H₂O₂ released into the culture from dead cells can damage other cells, which in turn brings about the population extinction.     

Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain ΔH) in a fed-batch fermentor

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 K _m ^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.