Purification and properties of F420- and NADP(+)-dependent alcohol dehydrogenases of Methanogenium liminatans and Methanobacterium palustre, specific for secondary alcohols

The F420-dependent alcohol dehydrogenase (ADH) of Methanogenium liminatans and the NADP(+)-dependent ADH of Methanobacterium palustre were purified to homogeneity. The native F420-dependent ADH of Mg. liminatans had a molecular mass of 150 kDa and consisted of four (presumably identical) subunits with a mass of 39 kDa. The temperature optimum was 42 degrees C, the optimum pH 6.0 and NaCl or KCl were inhibitory. The NADP(+)-dependent ADH of Mb. palustre had a molecular mass of 175 kDa and consisted also of four (presumably identical) subunits with a mass of 44 kDa. The temperature optimum was 60 degrees C, the optimum pH 8.0 and optimal activity was observed in the presence of 500 mM NaCl or KCl. The ADHs of both organisms catalysed the oxidation of various secondary and cyclic alcohols to the corresponding ketones and the reverse reaction. No primary alcohols were apparently oxidized. The NADP(+)-dependent ADH of Mb. palustre contained 4-8 mol atoms zinc/mol enzyme and was inhibited by low concentrations of iodoacetate and 4-hydroxymercuribenzoate, whereas the F420-dependent ADH of Mg. liminatans presumably contained no zinc ions and was inhibited by 1,10-phenanthroline or high concentrations (e.g. 100 microM) of 4-hydroxymercuribenzoate. Polyclonal antibodies against the NADP(+)-dependent ADH of Mb. palustre precipitated only the homologous ADH. A precipitation of the NADP(+)-dependent ADH of Methanocorpusculum parvum required a 10-fold higher antibody concentration, showing at least a distant relationship of both ADHs. Antibodies against the NADP(+)-dependent ADH of Mcp. parvum, however, formed precipitates with the homologous ADH of Mcp. parvum and with the NADP(+)-dependent ADH of Mb. palustre. They also formed precipitates with the ADH of Thermoanaerobium brockii, which is not related to methane bacteria. Antibodies against the F420-dependent ADH of Mg. liminatans reacted only with the homologous enzyme and did not form precipitates with NADP(+)-dependent ADHs. No immunological relation of the NADP(+)- or F420-dependent ADHs of methanogens with ADH of yeast or horse liver was found. In accordance with the immunological data, the N-terminal amino acid sequences of the NADP(+)-dependent ADHs of Mb. palustre and Mcp. parvum had a high degree of similarity, whereas the N-terminal amino acid sequence of the ADH of Mg. liminatans revealed no similarity with the two NADP(+)-dependent enzymes.     

Electron transfer reactions for the reduction of NADP+ in Methanosphaera stadtmanae

Abstract Methanosphaera stadtmanae , a member of the Methanobacteriales reduces methanol, but not CO₂ with H₂ or 2-propanol to produce methane. In cell-free extracts of M. stadtmanae the activities of several enzymes involved in electron transfer were measured. The activities of an F₄₂₀-nonreactive hydrogenase, NADP⁺: F₄₂₀ oxidoreductase, NADP⁺-dependent 2-propanol dehydrogenase, and a methyl viologen dependent F₄₂₀ dehydrogenase were observed. Based on the presence of these particular enzyme activities, their cofactor requirements and the absence of F₄₂₀-dependent hydrogenase activity, a model of the electron transport pathway through the coenzyme F₄₂₀ to provide electrons for biosynthesis, was formulated.     

Secondary alcohols as hydrogen donors for CO2-reduction by methanogens

Out of 22 methanogens Methanobacterium formicicum, Methanobacterium bryantii M.o.H., Methanogenium marisnigri, Methanomicrobium paynteri, Methanocorpusculum parvum and the new coccoid methanogenic isolates GKZPZ and SZSXXZ were found to grow at the expense of 2-propanol and 2-butanol + CO₂. 2-Propanol was oxidized to acetone and 2-butanol was converted to 2-butanone during CO₂-reduction to methane. Growth was poor compared to that on H₂/CO₂, and in the presence of both, 2-propanol and H₂, molecular hydrogen was the preferred reductant. Acetone, formed during oxidation of 2-propanol in the absence of hydrogen, was reduced again to 2-propanol, when the culture was supplied with H₂/CO₂. Ethanol, 1-propanol, 1-butanol, 2-pentanol and cyclohexanol could not serve as hydrogen donors for methanogenesis.     

Accumulation and utilization of triacylglycerol and polysaccharides in Liposcelis bostrychophila (Psocoptera, Liposcelididae) selected for resistance to carbon dioxide

Abstract: Two populations of the psocid, Liposcelis bostrychophila Badonnel, were exposed to two CO₂-enriched atmospheres (35% CO₂ + 21% O₂, and 55% CO₂ + 21% O₂, balance N₂) for 30 generations. Controls were reared in normal atmospheres. The reserves of triacylglycerol and polysaccharides were evaluated in adults of the two experimental and the control populations in generations F₁₅ and F₃₀. The utilization rate of triacylglycerol and polysaccharides in the CO₂-enriched atmospheres were also determined in generation F₃₀. The results indicated that the reserves of triacylglycerol and polysaccharides increased significantly during selection for CO₂ resistance; the higher the resistance level, the greater the reserves. Exposure of these populations to controlled atmosphere was associated with a steady utilization of the reserves. By contrast, the unselected population responded to controlled atmospheres by accelerated utilization of triacylglycerol and polysaccharides. Comparison of the utilization rates during CO₂ exposure showed that triacylglycerol is the main energy source, and polysaccharides contribute to metabolic energy supply only to a small extent.     

The ATP-dependent synthesis of factor 390 by cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH)

Abstract Cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH) converted the 8-OH-5-deazaflavin coenzyme F₄₂₀ to factor 390, a 8-adenylyl derivative (F₄₂₀-AMP). Activity was only observed upon exposure of the crude cell-free extract to oxygen. The ability to synthesize F₃₉₀ was lost when crude cell-free extract was subsequently brought to an anaerobic reducing environment. The enzymatic reaction used ATP and oxidized coenzyme F₄₂₀ as substrates and inorganic pyrophosphate was formed next to F₃₉₀. GTP could be used instead of ATP resulting in a guanylylated derivative. The crude cell-free extract showed K _m values of 154 μM for coenzyme F₄₂₀ and 2.4 mM for ATP. A partially purified enzyme preparation exhibited a K _eq of 0.32. In accordance, coenzyme F₄₂₀ and ATP could be synthesized from F₃₉₀ and PPi by the reverse reaction.     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Biomass Production and Carbohydrate Content of Arabidopsis thaliana at Atmospheric CO2 Concentrations from 390 to 1680 μl l-1

Abstract: The concentration dependency of the impact of elevated atmospheric CO₂ concentrations on Arabidopsis thaliana L. was studied. Plants were exposed to nearly ambient (390), 560, 810, 1240 and 1680 μl I^-1 CO₂ during the vegetative growth phase for 8 days. Shoot biomass production and dry matter content were increased upon exposure to elevated CO₂. Maximal increase in shoot fresh and dry weight was obtained at 560 μl I^-1 CU₂, which was due to a transient stimulation of the relative growth rate for up to 3 days. The shoot starch content increased with increasing CO₂ concentrations up to two-fold at 1680 μl I^-1 CO₂, whereas the contents of soluble sugars and phenolic compounds were hardly affected by elevated CO₂. The chlorophyll and carotenoid contents were not substantially affected at elevated CO₂ and the chlorophyll a/b ratio remained unaltered. There was no acclimation of photosynthesis at elevated CO₂; the photosynthetic capacity of leaves, which had completely developed at elevated CO₂ was similar to that of leaves developed in ambient air. The possible consequences of an elevated atmospheric CO₂ concentration to Arabidopsis thaliana in its natural habitat is discussed.     

Time-resolved microfluorescence for measuring coenzyme F420 in Methanobacterium thermoautotrophicum

Abstract The time course of photobleaching and the nanosecond fluorescence decay have been measured from microscopic samples of methanogenic bacteria, to our knowledge the first application of these methods in this field. Decay times of about 1 ns and 3 ns were obtained for the specific coenzymes F₄₂₀ and 7-methylpterin, respectively. In contrast to methylpterin and other fluorescent compounds the intensity of F₄₂₀ fluorescence was reduced selectively due to photobleaching. This effect, as well as the different decay time constants could be used to discriminate F₄₂₀ from other fluorescent components. In addition, active and inactive bacterial cells could be differentiated following the course of photobleaching.     

Distribution of transition metal ions in multiple forms of Methanosarcina hydrogenase

There were significant levels of in vitro hydrogenase activity in Methanosarcina strains. The multiple forms of hydrogenase were observed in cell free extracts of cells grown on methanol. Strains having poor growth on H₂ : CO₂ had four forms while strains having normal growth on all substrates contained two forms of hydrogenase. These multiple forms differ in their charges as well as in their composition of transition metal ions. The strain having normal growth showed higher incorporation of ⁶³Ni²⁺ and ⁶⁵Zn²⁺. Both hydrogenases, A and D, of strain P₃ had methylviologen and F₄₂₀-reducing activity and contained Zn²⁺ and Co²⁺ respectively. Hydrogenases A and D of strains P₁ and P₄ also had similar characteristics whereas hydrogenases B and C had only methylviologen reducing activity.     

Some rumen ciliates have endosymbiotic methanogens

Abstract Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F₄₂₀ autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H₂ evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.     

Spectrophotometric identification of 8-hydroxy-5-deazaflavin: NADPH oxidoreductase activity in streptomycetes producing tetracyclines

Abstract Cell-free extracts of vegetative mycelia of Streptomyces aureofaciens and Streptomyces rimosus were found to reduce streptomycete-origin 8-hydroxy-5-deazaisoalloxazine derivatives (SF₄₂₀) using NADPH as a dnor of hydrogen and electrons. 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F₀) also was a substrate, although with a lower reaction rate than that for SF₄₂₀. NADH could not substitute for NADPH. The F₄₂₀-reductase activity was also observed in homogenates of S. aureofaciens spores.     

Nitrogen nutrition of C3 plants at elevated atmospheric CO2 concentrations

The atmospheric CO₂ concentration has risen from the preindustrial level of approximately 290 μl l⁻¹ to more than 350 μl l⁻¹ in 1993. The current rate of rise is such that concentrations of 420 μl l⁻¹ are expected in the next 20 years. For C₃ plants, higher CO₂ levels favour the photosynthetic carbon reduction cycle over the photorespiratory cycle, resulting in higher rates of carbohydrate production and plant productivity. The change in balance between the two photosynthetic cycles appears to alter nitrogen and carbon metabolism in the leaf, possibly causing decreases in nitrogen concentrations in the leaf. This may result from increases in the concentration of storage carbohydrates of high molecular weight (soluble or insoluble) and/or changes in distribution of protein or other nitrogen containing compounds. Uptake of nitrogen may also be reduced at high CO₂ due to lower transpiration rates. Decreases in foliar nitrogen levels have important implications for production of crops such as wheat, because fertilizer management is often based on leaf chemical analysis, using standards estimated when the CO₂ levels were considerably lower. These standards will need to be re-evaluated as the CO₂ concentration continues to rise. Lower levels of leaf nitrogen will also have implications for the quality of wheat grain produced, because it is likely that less nitrogen would be retranslocated during grain filling.     

Development of Vacuoles and Vacuolar H+-ATPase Activity under Extremely High CO2 Conditions in Chiorococcum littorale Cells

Abstract: The number and cross-sectional area of vacuoles in Chlorococcum littorale cells visualized with a differential interfer ence fluorescence microscope increased after their transfer from air to 40% CO₂. An immunological observation indicated that the level of subunit B of vacuolar H*ATPase also increased under 40% CO₂ conditions. The activity of nitrate-sensitive ATP-ase associated with the vacuolar membrane was 2–fold higher in 40% CO₂-_-grown cells than in air-grown cells. The effects of inhi bitors on the ATPase activity confirmed that these activities were derived from vacuolar-type H-ATPase. These results sug gest that vacuole development associated with that of vacuolar H⁺-ATPase occurred during the acclimatization of C. littorale cells to extremely high CO₂ conditions.     

Sensing of atmospheric CO2 by plants

Abstract. Despite recent interest in the effects of high CO₂ on plant growth and physiology, very little is known about the mechanisms by which plants sense changes in the concentration of this gas. Because atmospheric CO₂ concentration is relatively constant and because the conductance of the cuticle to CO₂ is low, sensory mechanisms are likely to exist only for intercellular CO₂ concentration. Therefore, responses of plants to changes in atmospheric CO₂ will depend on the effect of these changes on intercellular CO₂ concentration. Although a variety of plant responses to atmospheric CO₂ concentration have been reported, most of these can be attributed to the effects of intercellular CO₂ on photosynthesis or stomatal conductance. Short-term and long-term effects of CO₂ on photosynthesis and stomatal conductance are discussed as sensory mechanisms for responses of plants to atmospheric CO₂. Available data suggest that plants do not fully realize the potential increases in productivity associated with increased atmospheric CO₂. This may be because of genetic and environmental limitations to productivity or because plant responses to CO₂ have evolved to cope with variations in intercellular CO₂ caused by factors other than changes in atmospheric CO₂.     

Acetogenesis from H2 and CO2 by methane- and non-methane-producing human colonic bacterial communities

Abstract: The purpose of the study was to define the potential for reductive acetogenesis of colonic microflora from six non-methane- and four methane-excreting human subjects in relation to numbers of the different H₂-utilizing microorganisms. Faecal bacterial suspensions were incubated in the presence of NaH¹³CO₃ and under a gas phase composed of either 100% N₂ (control) or 80% H₂–20% N₂. The effects of a specific methanogenesis inhibitor or of sulfate supplementation were also determined. Quantitative nuclear magnetic resonance showed the presence of both single- and double-labelled acetate in all incubations under hydrogen. H₂/CO₂-acetogenesis appears to be a quantitatively important activity only in the presence of very low numbers of methanogens. Inhibition of methanogenesis induced a large increase in ¹³CO₂ incorporation into acetate in CH₄-producing samples. These results showed that methanogens can efficiently outcompete acetogens in human colonic contents. In contrast, no clear-cut competition for H₂ between acetogenesis and dissimilatory sulfate-reduction could be demonstrated. A slight reduction of the acetogenic activity was only observed at the highest sulfate addition (100 mM).     

Affinity for CO2 in relation to the ability of freshwater macrophytes to use HCO–3

1. The affinity of photosynthesis for CO₂ is calculated here as the initial slope of net-photosynthetic rate against concentration of CO₂. The affinity for CO₂ for pairs of freshwater macrophytes with similar leaf morphology but able or unable to use HCO^–₃ as a carbon source was compared.
2. Species restricted to CO₂ had a higher affinity for CO₂ than species that were also able to use HCO^–₃ when rates were expressed on the basis of area, dry mass and content of chlorophyll a .
3. Published values for the affinity for CO₂ and the concentration of CO₂ which half-saturated rate of photosynthesis were compiled and compared. Despite a large range of values, affinity for CO₂ was greater for species restricted to CO₂ than for those also able to use HCO^–₃ and statistically different when the slope was expressed on the basis of dry mass and chlorophyll a content.
4. The difference in affinity is consistent with predicted benefits of a high permeability to CO₂ for species relying on passive diffusion of CO₂ and a lower permeability for species able to use HCO^–₃ in order to reduce efflux of CO₂ from a high internal concentration generated by active transport.
5. The implications of the different affinities are discussed in terms of species distribution.     

Comparison of gas use efficiency and treatment uniformity in a forest ecosystem exposed to elevated [CO2] using pure and prediluted free-air CO2 enrichment technology

A direct comparison of treatment uniformity and CO₂ use of pure and prediluted free-air CO₂ enrichment (FACE) systems was conducted in a forest ecosystem. A vertical release pure CO₂ fumigation system was superimposed on an existing prediluted CO₂ fumigation system and operated on alternate days. The FACE system using prediluted CO₂ fumigation technology exhibited less temporal and spatial variability than the pure CO₂ fumigation system. The pure CO₂ fumigation system tended to over-fumigate the upwind portions of the plot and used 25% more CO₂ than the prediluted CO₂ fumigation system. The increased CO₂ use by the pure CO₂ system was exacerbated at low wind speeds. It is not clear if this phenomenon will also be observed in plots with smaller diameters and low-stature vegetation.     

What physiological acclimation supports increased growth at high CO2 conditions?

Chlamydomonas acidophila Negoro is a green algal species abundant in acidic waters (pH 2–3.5), in which inorganic carbon is present only as CO₂. Previous studies have shown that aeration with CO₂ increased its maximum growth rate, suggesting CO₂ limitation under natural conditions. To unravel the underlying physiological mechanisms at high CO₂ conditions that enables increased growth, several physiological characteristics from high- and low-CO₂-acclimated cells were studied: maximum quantum yield, photosynthetic O₂ evolution (P_max), affinity constant for CO₂ by photosynthesis (K_0.5,p), a CO₂-concentrating mechanism (CCM), cellular Rubisco content and the affinity constant of Rubisco for CO₂ (K_0.5,r). The results show that at high CO₂ concentrations, C. acidophila had a higher K_0.5,p, P_max, maximum quantum yield, switched off its CCM and had a lower Rubisco content than at low CO₂ conditions. In contrast, the K_0.5,r was comparable under high and low CO₂ conditions. It is calculated that the higher P_max can already explain the increased growth rate in a high CO₂ environment. From an ecophysiological point of view, the increased maximum growth rate at high CO₂ will likely not be realised in the field because of other population regulating factors and should be seen as an acclimation to CO₂ and not as proof for a CO₂ limitation.     

Growth and physiological responses of canola (Brassica napus) to UV-B and CO2 under controlled environment conditions

Few studies have investigated the interaction of ultraviolet (UV)-B radiation and CO₂ concentration on plants. We studied the combined effects of UV-B radiation and CO₂ concentration on canola ( Brassica napus cv. 46A65) under four growth conditions – ambient CO₂ with UV-B (control), elevated CO₂ with UV-B, ambient CO₂ without UV-B, and elevated CO₂ without UV-B – to determine whether the adverse effects of UV-B are mitigated by elevated CO₂. Elevated CO₂ significantly increased plant height and seed yield, whereas UV-B decreased them. Elevated CO₂ ameliorated the adverse effects of UV-B in plant height. UV-B did not affect the physical characteristics of leaf but CO₂ did. Certain flower and fruit characteristics were affected negatively by UV-B and positively by CO₂. UV-B did not affect net photosynthesis, transpiration and stomatal conductance but decreased water use efficiency (WUE). Elevated CO₂ significantly increased net photosynthesis and WUE. Neither UV-B nor CO₂ affected chlorophyll (Chl) fluorescence. UV-B significantly decreased Chl b and increased the ratio of Chl a / b . Elevated CO₂ decreased only the ratio of Chl a / b . UV-B significantly increased UV-absorbing compounds while CO₂ had no effect on them. Both UV-B and CO₂ significantly increased epicuticular wax content. Many significant relationships were found between morphological, physiological, and chemical parameters. This study showed that elevated CO₂ can partially ameliorate some of the adverse effects of UV-B radiation in B . napus .     

Influence of soil O2 and CO2 on root respiration for Agave deserti

Respiration measured as CO₂ efflux was determined at various soil O₂ and CO₂ concentrations for individual, attached roots of a succulent perennial from the Sonoran Desert, Agave deserti Engelm. The respiration rate increased with increasing O₂ concentration up to about 16% O₂ for established roots and 5% O₂ for rain roots (fine branch roots on established roots induced by wetting of the soil) and then remained fairly constant up to 21% O₂. When O₂ was decreased from 21 to 0%, the respiration rates were similar to those obtained with increasing O₂ concentration. The CO₂ concentration in the root zone, which for the shallow-rooted A. deserti in the field was about 1 000 μl l^-1, did not affect root respiration at concentrations up to 2 000 μl l^-1, but higher concentrations reduced it, respiration being abolished at 20 000 μl l^-1 (2%) CO₂ for both established and rain roots. Upon lowering CO₂ to 1 000 μl l^-1 after exposure to concentrations up to 10000 μl l^-1 CO₂, inhibition of respiration was reversible. Uptake of the vital stain neutral red by root cortical cells was reduced to zero, indicating cell death, in about 4 h at 2% CO₂, substantiating the detrimental effects of high soil CO₂ concentrations on roots of A. deserti . This CO₂ response may explain why roots of desert succulents tend to occur in porous, well-aerated soils.