Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol

Studies were performed to determine if the growth of Methylomicrobium album BG8 on methanol could be enhanced through the provision of chloromethane. M. album BG8 was found to be able to oxidize chloromethane via the particulate methane monooxygenase with an apparent K(s) of 11+/-3 microM and V(max) of 15+/-0.6 nmol (min mg protein)(-1). When up to 2.6 mM chloromethane was provided with 5 mM methanol, methanotrophic growth was significantly enhanced in comparison to the absence of chloromethane, indicating that methanotrophs can utilize chloromethane to support growth, although it could not serve as a sole growth substrate. [(14)C]chloromethane was found to be oxidized to [(14)C]CO(2) as well as incorporated into biomass. These results indicate that reactions previously thought to be cometabolic may actually provide some benefit to methanotrophs and that these cells can use multiple compounds to enhance growth.     

Oxidative decarboxylation of glycollate and glyoxylate by leaf peroxisomes

1. The conditions under which peroxisomal preparations from leaves of spinach beet and spinach catalyse the release of (14)CO(2) from [1-(14)C]glycollate and [1-(14)C]glyoxylate were investigated. 2. At pH8, (14)CO(2) production from [1-(14)C]glyoxylate was accompanied by equivalent quantities of formate. The accumulation of oxalate and the effects of various reagents, especially catalase inhibitors, show that glyoxylate is non-enzymically oxidized by H(2)O(2), which is generated by the oxidation of glyoxylate to oxalate by the action of glycollate oxidase. 3. (14)CO(2) is shown to be generated from [1-(14)C]glycollate at pH8 by a similar reaction, but the H(2)O(2) is generated mainly by the oxidation of glycollate to glyoxylate. 4. The physiological significance of these reactions is discussed, with special reference to their role in photorespiration.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide.

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Comparison of EPR-visible Cu(2+) sites in pMMO from Methylococcus capsulatus (Bath) and Methylomicrobium album BG8

X-band (9.1 GHz) and S-band (3.4 GHz) electron paramagnetic resonance (EPR) spectra for particulate methane monooxygenase (pMMO) in whole cells from Methylococcus capsulatus (Bath) grown on (63)Cu and (15)N were obtained and compared with previously reported spectra for pMMO from Methylomicrobium album BG8. For both M. capsulatus (Bath) and M. album BG8, two nearly identical Cu(2+) EPR signals with resolved hyperfine coupling to four nitrogens are observed. The EPR parameters for pMMO from M. capsulatus (Bath) (g( parallel) = 2.244, A( parallel) = 185 G, and A(N) = 19 G for signal one; g( parallel) = 2.246, A( parallel) = 180 G, and A(N) = 19 G for signal two) and for pMMO from M. album BG8 (g( parallel) = 2.243, A( parallel) = 180 G, and A(N) = 18 G for signal one; g( parallel) = 2. 251, A( parallel) = 180 G, and A(N) = 18 G for signal two) are very similar and are characteristic of type 2 Cu(2+) in a square planar or square pyramidal geometry. In three-pulse electron spin echo envelope modulation (ESEEM) data for natural-abundance samples, nitrogen quadrupolar frequencies due to the distant nitrogens of coordinated histidine imidazoles were observed. The intensities of the quadrupolar combination bands indicate that there are three or four coordinated imidazoles, which implies that most, if not all, of the coordinated nitrogens detected in the continuous wave spectra are from histidine imidazoles.     

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis-dichloroethylene (cis-DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans-dichloroethylene (trans-DCE), and 1,1,1-trichloroethane (1,1,1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans-DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis-DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans-DCE, cis-DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans-DCE oxidation products were also toxic but to a lesser extent. cis-DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity. Received: 17 June 1999 / Accepted: 3 September 1999     

Methanol toxicity and formate oxidation in NEUT2 mice

NEUT2 mice are deficient in cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH; EC 1.5.1.6) which catalyzes the oxidation of excess folate-linked one-carbon units in the form of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (Champion et al., Proc. Natl. Acad. Sci. USA 91, 11338-11342, 1994). The absence of FDH should impair the oxidation of formate via the folate-dependent pathway and as a consequence render homozygous NEUT2 mice more susceptible to methanol toxicity. Normal (CB6-F1) and NEUT2 heterozygous and homozygous mice had essentially identical LD(50) values for methanol, 6.08, 6.00, and 6.03 g/kg, respectively. Normal mice oxidized low doses of [(14)C]sodium formate (ip 5 mg/kg) to (14)CO(2) at approximately twice the rate of homozygous NEUT2 mice, indicating the presence of another formate-oxidizing system in addition to FDH. Treatment of mice with the catalase inhibitor, 3-aminotriazole (1 g/kg ip) had no effect on the rate of formate oxidation, indicating that at low concentrations formate was not oxidized peroxidatively by catalase. High doses of [(14)C]sodium formate (ip 100 mg/kg) were oxidized to (14)CO(2) at identical rates in normal and NEUT2 homozygous mice. Pretreatment with 3-aminotriazole (1 g/kg ip) in this instance resulted in a 40 and 50% decrease in formate oxidation to CO(2) in both normal and homozygous NEUT2 mice, respectively. These results indicate that mice are able to oxidize formate to CO(2) by at least three different routes: (1) folate-dependent via FDH at low levels of formate; (2) peroxidation by catalase at high levels of formate; and (3) by an unknown route(s) which appears to function at both low and high levels of formate. The implications of these observations are discussed in terms of the current hypotheses concerning methanol and formate toxicity in rodents and primates.     

Reassessment of 14CO2 compartmentation and of [14C]formate oxidation in rat liver

Our previous report (Marsolais, C., Huot, S., David, F., Garneau, M., and Brunengraber, H. (1987) J. Biol. Chem. 262, 2604-2607) had concluded that a fraction of [14C]formate oxidation in liver occurs in the mitochondrion. This conclusion was based on the labeling patterns of urea and acetoacetate labeled via 14CO2 generated from [14C]formate and other [14C]substrates. We reassessed our interpretation in experiments conducted in (i) perifused mitochondria and (ii) isolated livers perfused with buffer containing [14C]formate, [14C]gluconolactone, 14CO2, or NaH13CO3, in the absence and presence of acetazolamide, an inhibitor of carbonic anhydrase. Our data show that the cytosolic pools of bicarbonate and CO2 are not in isotopic equilibrium when 14CO2 is generated in the cytosol or is supplied as NaH14CO3. We retract our earlier suggestion of a mitochondrial site of [14C]formate oxidation.     

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V_max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K_s) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, V_max/K_s, doubled from 4.9 × 10⁻³ to 9.9 × 10⁻³ liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V_max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V_max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K_s from 36 to 7.9 μM. V_max/K_s for TCE degradation by pMMO also increased from 6.9 × 10⁻⁵ to 5.2 × 10⁻⁴ liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.     

The bacterial oxidation of N-methylisonicotinate, a photolytic product of Paraquat

Two bacteria have been isolated that are capable of oxidizing N-methylisonicotinate, a photodegradation product of Paraquat (1.1'-dimethyl-4,4'-bipyridylium ion). N-Methylisonicotinate-grown cells of strain 4C1, a Gram-positive rod, oxidized 2-hydroxy-N-methylisonicotinate without lag. Cell-free extracts of these cells converted 2-hydroxyisonicotinate into 2,6-dihydroxyisonicotinate; the reaction did not require molecular oxygen. Maleamate was deamidated and maleate isomerized to fumarate by soluble enzyme systems. [(14)C]Formaldehyde was isolated as the dimedone derivative from the supernatant of a cell suspension oxidizing N-[(14)C]methylisonicotinate, and no [(14)C]-methylamine was detected. Whole cells incubated with N-methyl[carboxy-(14)C]isonicotinate released 95% of the radioactivity as (14)CO(2). The second bacterium, strain 4C2, a Gram-negative rod, did not oxidize any of the mono- or di-hydroxypyridines or their N-methyl derivatives that were available or could be synthesized; nor did cell-free extracts oxidize any of these compounds. Methylamine was oxidized by whole cells without lag; cell-free extracts converted methylamine into formaldehyde when a soluble enzyme system requiring an electron acceptor was used; formaldehyde was oxidized to formate and formate to CO(2) by enzyme systems requiring NAD(+).     

Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions.

A biological process for remediation of groundwater contaminated with tetrachloroethylene (PCE) and trichloroethylene (TCE) can only be applied if the transformation products are environmentally acceptable. Studies with enrichment cultures of PCE- and TCE-degrading microorganisms provide evidence that, under methanogenic conditions, mixed cultures are able to completely dechlorinate PCE and TCE to ethylene, a product which is environmentally acceptable. Radiotracer studies with [14C]PCE indicated that [14C]ethylene was the terminal product; significant conversion to 14CO2 or 14CH4 was not observed. The rate-limiting step in the pathway appeared to be conversion of vinyl chloride to ethylene. To sustain reductive dechlorination of PCE and TCE, it was necessary to supply an electron donor; methanol was the most effective, although hydrogen, formate, acetate, and glucose also served. Studies with the inhibitor 2-bromoethanesulfonate suggested that methanogens played a key role in the observed biotransformations of PCE and TCE.     

Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions

A biological process for remediation of groundwater contaminated with tetrachloroethylene (PCE) and trichloroethylene (TCE) can only be applied if the transformation products are environmentally acceptable. Studies with enrichment cultures of PCE- and TCE-degrading microorganisms provide evidence that, under methanogenic conditions, mixed cultures are able to completely dechlorinate PCE and TCE to ethylene, a product which is environmentally acceptable. Radiotracer studies with [14C]PCE indicated that [14C]ethylene was the terminal product; significant conversion to 14CO2 or 14CH4 was not observed. The rate-limiting step in the pathway appeared to be conversion of vinyl chloride to ethylene. To sustain reductive dechlorination of PCE and TCE, it was necessary to supply an electron donor; methanol was the most effective, although hydrogen, formate, acetate, and glucose also served. Studies with the inhibitor 2-bromoethanesulfonate suggested that methanogens played a key role in the observed biotransformations of PCE and TCE.     

Concentration of Cu, EPR-detectable Cu, and formation of cupric-ferrocyanide in membranes with pMMO

EPR spectra were obtained for the type 2 Cu(2+) site in particulate methane monooxygenase, pMMO, from membrane fractions of Methylomicrobium album BG8. In addition to the EPR signal with g parallel = 2.24 and A parallel = 185 G found in both cells and membrane fractions, a second EPR signal with g parallel = 2.29 and A parallel = 146 G was found in membrane fractions and attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by atomic absorption suggests that there are two or three EPR-silent coppers for every EPR-detectable copper and that there are approximately four coppers per enzyme composed of the 47, 27, and 25 kDa subunits. Treatment of membrane fractions loaded with pMMO with Fe(CN)6(3-) results in a new EPR signal that is attributed to CuFe(CN)6(2-), not to an intrinsic trimeric copper cluster as previously reported in studies with a related bacterium.     

Metabolism of methylamine in the tea plant (Thea sinensis L.)

1. The metabolism of methylamine in excised shoot tips of tea was studied with micromolar amounts of [(14)C]methylamine. Of the [(14)C]methylamine supplied 57% was utilized by tea shoots during the 10h experimental period. 2. The main products of [(14)C]methylamine metabolism in tea shoots were serine, gamma-glutamylmethylamide, theobromine, caffeine and CO(2). There was also incorporation of the label into glutamate, aspartate, RNA purine nucleotides and S-adenosylmethionine. 3. The formation of methylamine from gamma-glutamylmethylamide was confirmed by feeding tea shoots with gamma-glutamyl[(14)C]methylamide. The products of gamma-glutamyl[(14)C]methylamide metabolism in tea plants were serine, theobromine, caffeine, glutamate and aspartate. 4. The results indicate that the oxidation of methylamine to formaldehyde is the first step of methylamine utilization. Labelled formaldehyde released by the metabolism of methylamine leads to the incorporation of the label into metabolites on the C(1) pathways of this compound. It is also suggested that formaldehyde is further oxidized via formate to CO(2). 5. The role of gamma-glutamylmethylamide in methylamine metabolism in tea plants is discussed. 6. Results support the view that theobromine is the immediate precursor of caffeine.     

Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

Influence of nitrate on oxalate- and glyoxylate-dependent growth and acetogenesis by Moorella thermoacetica

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [(14)C]bicarbonate or [(14)C]oxalate showed that CO(2) reduction to acetate and biomass or oxalate oxidation to CO(2) was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO(2) into biomass than oxalate-grown cells. CO(2) conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO(2) as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.     

Metabolism of [1-14C]glyoxylate, [1-14C]-glycollate, [1-14C]glycine and [2-14C]-glycine by homogenates of kidney and liver tissue from hyperoxaluric and control subjects

1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.     

Relationships between glycollate and formate metabolism in greening barley leaves

The activities of enzymes catalysing glycollate oxidation, formate production and folate-dependent formate utilization were examined in the primary leaves of Hordeum vulgare cv Galt. Seedlings were grown for 6 days in darkness and then transferred to continuous light (500 μinsteins/m² per sec) for up to 5 days. Cell-free extracts of the primary leaves contained glycollate oxidase (EC 1.1.3.1), 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5, 10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and ability to enzymically decarboxylate glyoxylate. These activities increased during greening and at the end of the light treatment were 70–450% higher than etiolated controls. Greened primary leaves also incorporated [¹⁴C]formate at rates that were three- to four-fold higher than shown by etiolated leaves. The specific activity of 10-formyltetrahydrofolate synthetase was decreased by 20–35% when the leaves were greened in the presence of 10 mM hydroxysulphonate. This inhibitor also reduced the incorporation of [¹⁴C]formate by up to 45%. A potential flow of carbon from glycollate to 10-formyltetrahydrofolate via glyoxylate and formate was suggested by the data.     

Oxidation of C1-compounds in Pseudomonas C.

Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.