Regulation of formate dehydrogenase activity in Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus can use either H2 or formate as the electron donor for methanogenesis from CO2. Resuspended-cell experiments revealed that the ability to use H2 as the source of electrons for methanogenesis was constitutive; cells grown on formate or H2-CO2 were equally capable of H2-CO2 methanogenesis. The ability to metabolize formate at high rates was observed only in cells previously grown on formate. Two such strains were distinguished: strain F and strain HF. Strain F was repeatedly grown exclusively on formate for over 3 years; this strain showed a constitutive capacity to metabolize formate to methane, even after subsequent repeated transfers to medium containing only H2-CO2. Strain HF could only metabolize formate to methane when grown in the presence of formate with no H2 present; this strain was recently derived from another strain (H) that had been exclusively grown on H2-CO2 and which upon initial transfer to formate medium could only metabolize formate to methane at a very slow rate. Initial adaptation of strain H to growth on formate was preceded by a long lag. The specific activities of hydrogenase and formate dehydrogenase in cell extracts derived from these different strains confirmed these findings. Similar levels of hydrogenase were observed in all strains, independent of the presence of H2 in the growth medium medium. High levels of formate dehydrogenase were also constitutive in strain F. Only low formate dehydrogenase activities were observed in strain H. High levels of formate dehydrogenase were observed in strain HF only when these cells were grown with formate in the absence of H2. In all strains the two- to threefold fluctuations of both hydrogenase and formate dehydrogenase cell-free activities were observed during growth, with peak activities reached in the middle of the exponential phase.     

Effects of Formate on Fermentative Hydrogen Production by Enterobacter aerogenes

This paper describes the effects of formate on fermentative hydrogen production by Enterobacter aerogenes by way of batch culture. When 20 mM formate was added to pH 6.3 and pH 5.8 E. aerogenes glucose cultures (formate culture) at the beginning of cultivation, hydrogen evolution through both glucose consumption and decomposition of the extrinsic formate occurred together, while hydrogen evolution occurred only through glucose consumption in the control cultures. The hydrogen evolution rates in the formate cultures were faster than in the control cultures, although cell growth and glucose consumption rates in the formate cultures were slower than the control cultures’. The decomposition rate of the extrinsic formate in the pH 5.8 formate culture was faster than in the pH 6.3 fomiate culture. The hydrogen yield from glucose in the pH 6.3 formate culture increased due to the increasing amount of the nicotinamide adenine dinucleotide for hydrogen production.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes

Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (Mr 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (Em = -224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is able to produce methane either from H2-CO2 or from formate. The route of formate entry into the methanogenic pathway was investigated by using 2H2O or [13C]formate and analysis by mass spectrometry. When cells (H2-CO2 or formate grown) were transferred to formate medium in 95% 2H water, the proportion of 2H in methane was 95%. When cells (H2-CO2 or formate grown) were transferred to media containing [13C]formate in the presence of H2-CO2 or He-CO2, the ratio of 13CH4 to 12CH4 increased over time parallel to the ratio of 13CO2 to 12CO2. The cells catalyzed a significant exchange of label between [13C]formate and 13CO2.     

Evidence for Isotopic Exchange during Metabolism of Stable-Isotope-Labeled Formate in a Methanogenic Sediment

The disappearance of stable-isotope-labeled formate in freshwater sediment from Lake Vechten was studied by gas chromatography-mass spectrometry. Both deuterium- and (sup13)C-labeled formate were removed from pore water at a high rate (400 (plusmn) 35 [standard deviation] and 1,700 (plusmn) 600 (mu)M h(sup-1), respectively), with concomitant formation of nonlabeled formate. The removal rate of labeled and nonlabeled formate amounted to 240 (plusmn) 15 and 400 (plusmn) 40 (mu)M h(sup-1), respectively. The formation of nonlabeled formate is explained by isotopic exchange due to the activity of formate dehydrogenase rather than by the turnover of formate.     

Electrochemical study of proton translocation function of hydrogenase 3

The oxidation of formate associated with fast acidification of medium by whole Escherichia coli cells lacking both hya and hyb hydrogenases was studied. The extent of acidification was dependent on the amount of formate added. An average H+/formate ratio of 1.3 was obtained. The proton release was inhibited by carbonyl cyanide m-chlorophenylhydrazone. Inverted vesicles of E. coli were found to translocate protons upon oxidation of formate at pH 6.5. The extent of alkalization was also dependent on the amount of formate added. The maximum H+/formate ratio for this reaction was close to 0.6. Formate oxidation by inverted vesicles from E. coli (delta hya delta hyb) was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. It was supposed that the hydrogenase 3 (hyc) component of E. coli formate hydrogen lyase is responsible for the translocation of protons at low pH.     

In Vivo Kinetics of Formate Metabolism in Folate-deficient and Folate-replete Rats

It is now established that the mitochondrial production of formate is a major process in the endogenous generation of folate-linked one-carbon groups. We have developed an in vivo approach involving the constant infusion of [¹³C]formate until isotopic steady state is attained to measure the rate of endogenous formate production in rats fed on either a folate-replete or folate-deficient diet. Formate was produced at a rate of 76 μmol·h⁻¹·100 g of body weight⁻¹ in the folate-replete rats, and this was decreased by 44% in folate-deficient rats. This decreased formate production was confirmed in isolated rat liver mitochondria where formate production from serine, the principal precursor of one-carbon groups, was decreased by 85%, although formate production from sarcosine and dimethylglycine (choline metabolites) was significantly increased. We attribute this unexpected result to the demonstrated production of formaldehyde by sarcosine dehydrogenase and dimethylglycine dehydrogenase from their respective substrates in the absence of tetrahydrofolate and subsequent formation of formate by formaldehyde dehydrogenase. Comparison of formate production with the ingestion of dietary formate precursors (serine, glycine, tryptophan, histidine, methionine, and choline) showed that ∼75% of these precursors were converted to formate, indicating that formate is a significant, although underappreciated end product of choline and amino acid oxidation. Ingestion of a high protein diet did not result in increased production of formate, suggesting a regulation of the conversion of these precursors at the mitochondrial level to formate.     

The effect of mixtures of glucose and formate on the yield and respiration of a chemostat culture of Beneckea natriegens

Beneckea natriegens oxidizes sodium formate constitutively when grown on glucose or glycerol in chemostat culture, but cannot utilize formate as the sole source of carbon and energy for growth. However, when grown on a mixture of glucose and formate (D=0.37 h^-1, pH 7.6) the yield is higher than on glucose alone.The yield, expressed in terms of g bacterial dry weight g^-1 glucose plus formate carbon utilized, gave a linear relationship when plotted against the total heat of combustion of glucose plus formate utilized. Extrapolation of the plot cut the abscissa at a value equivalent to the heat of combustion of formate, which suggests that formate is not utilised as a source of carbon but only energy.In cultures with nitrate as the sole source of nitrogen the yield from glucose was lower than that observed with ammonia but the addition of formate to the culture utilizing nitrate resulted in an increase in the yield from glucose to a value similar to that observed with ammonia.At a culture pH value of 7.65 unused formate (<0.15–227 mM) in the culture supernatant had no effect on respiration spiration or yield, but at a culture pH of 6.7 excess formate caused a marked increase in respiration rate and a large decrease in the yield from glucose; further decrease in the pH value caused washout of the culture. This may be explained by undissociated formic acid causing uncoupling of oxidative phosphorylation.     

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.     

Formate auxotroph of Methanobacterium thermoautotrophicum Marburg.

A formate-requiring auxotroph of Methanobacterium thermoautotrophicum Marburg was isolated after hydroxylamine mutagenesis and bacitracin selection. The requirement for formate is unique and specific; combined pools of other volatile fatty acids, amino acids, vitamins, and nitrogen bases did not substitute for formate. Compared with those of the wild type, cell extracts of the formate auxotroph were deficient in formate dehydrogenase activity, but cells of all of the strains examined catalyzed a formate-carbon dioxide exchange activity. All of the strains examined took up a small amount (200 to 260 mumol/liter) of formate (3 mM) added to medium. The results of the study of this novel auxotroph indicate a role for formate in biosynthetic reactions in this methanogen. Moreover, because methanogenesis from H2-CO2 is not impaired in the mutant, free formate is not an intermediate in the reduction of CO2 to CH4.     

Formate dehydrogenase activity in cells and outer membrane blebs of Desulfovibrio gigas

Formate dehydrogenase in Desulfovibrio gigas was measured by following the release of 14CO2 from radiolabeled formate. Experiments with whole cells using sulfate as the electron acceptor revealed optimal formate dehydrogenase activity at pH 7.0 and formate utilization followed saturation kinetics. While formate dehydrogenase was constitutively produced in pyruvate or lactate media, the formate dehydrogenase activity was markedly increased in cells grown with formate as the electron donor. In cell-free experiments with methyl viologen or 2,6 dichlorophenolindophenol, about 1% of the cellular formate dehydrogenase activity was present in blebs from the outer membrane. Electron microscopy revealed that these blebs were closed structures with diameters ranging from 80-800A and were not induced by changes in osmotic pressure or cellular autolysis. Analysis of blebs revealed the presence of lipopolysaccharides and two proteins with molecular masses of 70 and 53 kDa.     

A physiological study of formate dehydrogenase,formate oxidase and hydrogenlyase fromEscherichia coli K-12

Escherichia coli was grown under various culture conditions. Variations in the levels of formate dehydrogenase which reacts with methylene blue (MB) or phenazine methosulfate (PMS) (N enzyme), formate dehydrogenase which reacts with benzyl viologen (BV) (H enzyme), formate oxidase and hydrogenlyase were analyzed. It was observed that formate dehydrogenase N and formate oxidase were induced by nitrate and repressed by oxygen. Synthesis of formate dehydrogenase H and hydrogenlyase was induced by formate and repressed by nitrate and oxygen. Selenite was required for the biosynthesis of formate dehydrogenase H and hydrogenlyase. Activity of both formate oxidase and hydrogenlyase was inhibited by azide and KCN but not by N-heptyl hydroxyquinoline-N-oxide (HOQNO); on the other hand, formate oxidase was extremely sensitive to HOQNO. Data were obtained which suggest that cytochromes are not involved in hydrogen formation from formate. Part of this work was carried out when the senior author was visiting Research Biologist in the Laboratory of Dr. J. A. de Mosss at the University of California, San Diego. Thanks are given to Dr. De Moss for his hospitality and advise and to Dr. Warren Butler of the University of California, San Diego for making available his spectrophotometer to carry out cytochrome analyses. Most of this work was sustained by a grant from the Research Corporation, Brown Hazen Fund and the financial help of the C.O.F.A.A. from the Instituto Politécnico Nacional.     

Effects of Molybdate and Selenite on Formate and Nitrate Metabolism in Escherichia coli

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.     

Acetate and formate uptake into vesicles isolated from the basolateral region of the plasma membrane of ovine parotid acinar cells.

The transport of acetate and formate into plasma membrane vesicles derived from the basolateral face of the ovine parotid acinar cell has an absolute requirement for an anion to be present within the intravesicular space: bicarbonate, formate, acetate, propionate, and butyrate support the uptake of either acetate or formate. A pH gradient across the vesicle membrane, pHi 7.4, pH0 5.5, enhances the uptake of formate, but not acetate. There is no direct relationship between the rate of exchange and the degree of protonation of formate or acetate in the extravesicular medium. The process is saturable and can be inhibited by a range of functional group reagents. When mannitol is the main external osmoticum, the uptake of acetate and formate is still rapid; thus, no other ions are involved in the process apart from the external formate or acetate and the intravesicular anion. This activity could play a major role in the provision of energy in ruminant tissues.     

Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum.

The catabolism of sodium formate to acetate and carbon dioxide by the anaerobic acetogen Butyribacterium methylotrophicum was analyzed by fermentation time course and 13C nuclear magnetic resonance studies. Significant hydrogen production and consumption fluxes were observed during formate catabolism but not during the catabolism of formate plus CO. In the latter case, formate and CO were simultaneously consumed and label distribution studies with mixtures of 13C-labeled CO and formate demonstrated their preferential incorporation into the acetate carboxyl and methyl groups, respectively. Hydrogen consumption was inhibited by CO when both were present, whereas hydrogen and formate were simultaneously consumed when CO2 was supplied. Carbon dioxide was required for the conversion of CO to acetate, but a similar need was not observed when methanol plus CO or formate plus CO was present. These analyses indicate a bifurcated single-carbon catabolic pathway in which CO2 is the sole single-carbon compound that directly supplies the carbonyl and methyl group synthesis pathways leading to the formation of acetyl coenzyme A, the primary reduced product. We discuss causes for the reported inability of B. methylotrophicum to use formate as a sole substrate.     

Growth of Thiobacillus ferrooxidans on Formic Acid

A variety of acidophilic microorganisms were shown to be capable of oxidizing formate. These included Thiobacillus ferrooxidans ATCC 21834, which, however, could not grow on formate in normal batch cultures. However, the organism could be grown on formate when the substrate supply was growth limiting, e.g., in formate-limited chemostat cultures. The cell densities achieved by the use of the latter cultivation method were higher than cell densities reported for growth of T. ferrooxidans on ferrous iron or reduced sulfur compounds. Inhibition of formate oxidation by cell suspensions, but not cell extracts, of formate-grown T. ferrooxidans occurred at formate concentrations above 100 μM. This observation explains the inability of the organism to grow on formate in batch cultures. Cells grown in formate-limited chemostat cultures retained the ability to oxidize ferrous iron at high rates. Ribulose 1,5-bisphosphate carboxylase activities in cell extracts indicated that T. ferrooxidans employs the Calvin cycle for carbon assimilation during growth on formate. Oxidation of formate by cell extracts was NAD(P) independent.     

甲酸乙酯对松材线虫的熏蒸效果

【背景】在溴甲烷面临禁用的情形下,探讨新熏蒸剂甲酸乙酯对松材线虫的处理效果,可以科学地评估甲酸乙酯的使用前景。【方法】采用松材线虫分离液和带疫松木段,设置甲酸乙酯5个剂量梯度、5个处理温度和5个处理时间,测定其对松材线虫的毒力及CT值。【结果】在25℃下处理3、6、12、24、48h,甲酸乙酯对松材线虫的Lc50分别为2．63、1．60、0．99、0．41、0．20mg·L-1。温度对毒力有显著影响,在10～29℃,随温度升高,甲酸乙酯对松材线虫的毒力降低,19和29℃下,1．85mg·L-1甲酸乙酯处理松材线虫的死亡率分别为63％和100％。甲酸乙酯熏蒸12h内,能完全杀灭木段中的松材线虫。在23℃下处理6和12h,松材线虫死亡率达到99％时,甲酸乙酯的cr值分别为453．94和424．14mg·h-1·L-1。【结论与意义】甲酸乙酯可用于松材线虫的检疫处理。