Assimilation of exogenous fructose, aspartate and some organic acids during the growth of methylotrophs.

The percentage of bacterial carbon that was derived from exogenous labelled compounds present in the medium during the growth of some methylotrophs on trimethylamine or on non-C1 compounds was determined. Less than 10% of bacterial carbon was derived from acetate during the growth of the obligate methylotrophs 4B6 and C2A1, and of the restricted facultative methylotroph PM6; the other restricted facultative methylotroph W3A1 gave a value of 18%. Corresponding values for three typical facultative methylotrophs growing on trimethylamine were 41, 42 and 52%. Aspartate, fructose, pyruvate and succinate made only a small percentage contribution (0-4 to 12%) to bacterial carbon in 4B6, C2A1, W3A1 and PM6. Washed suspensions of 4B6, C2A1, W3A1 and PM6, all grown on trimethylamine, assimilated labelled acetate only in the presence of trimethylamine and there was a linear relationship between the amount of acetate assimilated and the amount of trimethylamine oxidized. Organisms 4B6, C2A1, W3A1 and PM6 assimilated 14C from labelled acetate predominantly into lipid (except PM6), glutamate, arginine, proline and leucine, whereas the typical facultative methylotrophs assimilated 14C from acetate into lipid, nucleic acid and all the protein amino acids. These results are consistent with the presence of a deficient tricarboxylic acid cycle in the obligate methylotrophs and in the restricted facultative methylotrophs.

Regulation of citrate synthase activity in methylotrophs by reduced nicotinamide-adenine dinucleotide, adenine nucleotides and 2-oxoglutarate.

Citrate synthase from two typical facultative methylotrophs, but not from four obligate methylotrophs or from two restricted facultative methylotrophs, is inhibited by 0.1 mM-NADH. ATP or ADP (both at 10mM) inhibits all the citrate synthases, whereas 2-oxoglutarate (10mM) inhibits those from only three of the eight methylotrophs.

Enzymological aspects of the pathways for trimethylamine oxidation and C1 assimilation of obligate methylotrophs and restricted facultative methylotrophs.

Extracts of trimethylamine-grown W6A and W3A1 (type M restricted facultative methylotrophs) contain trimethylamine dehydrogenase whereas similar extracts of Bacillus PM6 and Bacillus S2A1 (type L restricted facultative methylotrophs) contain trimethylamine mono-oxygenase and trimethylamine N-oxide demethylase but no trimethylamine dehydrogenase. Extracts of the restricted facultatives and of the obligate methylotroph C2A1 contain hexulose phosphate synthase-hexulose phosphate isomerase activity; hydroxypyruvate reductase was not detected. Neither the restricted facultatives nor the obligates 4B6 and C2A1 contain all the enzymes of the hexulose phosphate cycle of formaldehyde assimilation as originally proposed by Kemp & Quayle (1967). Organisms PM6 and S2A1 lack transaldolase and use a modified cycle involving sedoheptulose 1,7-diphosphate and sedoheptulose diphosphatase. The obligates 4B6 and C2A1, and the type M organisms W6A and W3A1, use a different modification of the assimilatory hexulose phosphate cycle involving the Entner-Doudoroff-pathway enzymes phosphogluconate dehydratase and phospho-2-keto-3-deoxygluconate aldolase. The lack of fructose diphosphate aldolase and hexose diphosphatase in these organisms may be a partial explanation of their restricted growth-substrate range. Enzymological evidence suggests that all the obligates and the restricted facultatives use a dissimilatory hexulose phosphate cycle to accomplish the complete oxidation of formaldehyde to CO2 and water.

Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs.

The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.

Purification and properties of the trimethylamine dehydrogenase of bacterium 4B6.

1. The trimethylamine dehydrogenase of bacterium 4B6 was purified to homogeneity as judged by analytical polyacrylamide-gel electrophoresis. The specific activity of the purified enzyme is 30-fold higher than that of crude sonic extracts. 2. The molecular weight of the enzyme is 161000. 3. The kinetic properties of the purified enzyme were studied by using an anaerobic spectrophotometric assay method allowing the determination of trimethylamine dehydrogenase activity at pH8.5, the optimum pH. The apparent K(m) for trimethylamine is 2.0+/-0.3mum and the apparent K(m) for the primary hydrogen acceptor, phenazine methosulphate, is 1.25mm. 4. Of 13 hydrogen acceptors tested, only Brilliant Cresyl Blue and Methylene Blue replace phenazine methosulphate. 5. A number of secondary and tertiary amines with N-methyl and/or N-ethyl groups are oxidized by the purified enzyme; primary amines and quaternary ammonium salts are not oxidized. Of the compounds that are oxidized by the purified enzyme, only trimethylamine and ethyldimethylamine support the growth of bacterium 4B6. 6. Trimethylamine dehydrogenase catalyses the anaerobic oxidative N-demethylation of trimethylamine with the formation of stoicheiometric amounts of dimethylamine and formaldehyde. Ethyldimethylamine is also oxidatively N-demethylated yielding ethylmethylamine and formaldehyde; diethylamine is oxidatively N-de-ethylated. 7. The activity of the purified enzyme is unaffected by chelating agents and carbonyl reagents, but is inhibited by some thiol-binding reagents and by Cu(2+), Co(2+), Ni(2+), Ag(+) and Hg(2+). Trimethylamine dehydrogenase activity is potently inhibited by trimethylsulphonium chloride, by tetramethylammonium chloride and other quaternary ammonium salts, and by monoamine oxidase inhibitors of the substituted hydrazine and the non-hydrazine types. 8. Inhibition by the substituted hydrazines is time-dependent, is prevented by the presence of trimethylamine or trimethylamine analogues and in some cases requires the presence of the hydrogen acceptor phenazine methosulphate. The inhibition was irreversible with the four substituted hydrazines that were tested.

Hexose phosphate synthase in trimethylamine-grown bacterium 2B2, a facultative methylotroph.

Hexose phosphate synthase and hexulose phosphate isomerase activities were found in trimethylamine-grown bacterium 2B2, a facultative methylotroph. The activities were separated by column chromatography of cell extracts on DEAE-cellulose. Hexulose phosphate isomerase activity was measured spectrophotometrically by using the product of the hexose phosphate synthase reaction as substrate.

Trimethylamine metabolism in obligate and facultative methylotrophs.

1. Twelve bacterial isolates that grow with trimethylamine as sole source of carbon and energy were obtained in pure culture. All the isolates grow on methylamine, dimethylamine and trimethylamine. One isolate, bacterium 4B6, grows only on these methylamines whereas another isolate, bacterium C2A1, also grows on methanol but neither grows on methane; these two organisms are obligate methylotrophs. The other ten isolates grow on a variety of C(i) and other organic compounds and are therefore facultative methylotrophs. 2. Washed suspensions of the obligate methylotrophs bacteria 4B6 and C2A1, and of the facultative methylotrophs bacterium 5B1 and Pseudomonas 3A2, all grown on trimethylamine, oxidize trimethylamine, dimethylamine, formaldehyde and formate; only bacterium 5B1 and Ps. 3A2 oxidize trimethylamine N-oxide; only bacterium 4B6 does not oxidize methylamine. 3. Cell-free extracts of trimethylamine-grown bacteria 4B6 and C2A1 contain a trimethylamine dehydrogenase that requires phenazine methosulphate as primary hydrogen acceptor, and evidence is presented that this enzyme is important for the growth of bacterium 4B6 on trimethylamine. 4. Cell-free extracts of eight facultative methylotrophs, including bacterium 5B1 and Ps. 3A2, do not contain trimethylamine dehydrogenase but contain instead a trimethylamine monooxygenase and trimethylamine N-oxide demethylase. It is concluded that two different pathways for the oxidation of trimethylamine occur amongst the isolates.

Two benzaldehyde dehydrogenases in bacterium N.C.I.B. 8250. Distinguishing properties and regulation.

Evidence is presented for the existence in bacterium N.C.I.B. 8250 of two inducible NAD(+)-linked benzaldehyde dehydrogenases. They may be distinguished in crude extracts by their different thermal stabilities at high pH values, benzaldehyde dehydrogenase I being much more heat-stable than benzaldehyde dehydrogenase II. Only benzaldehyde dehydrogenase I is activated by K(+) and certain other univalent cations. Gel-filtration experiments indicate that both enzymes have molecular weights of about 180000. Both enzymes are induced by growth on l-mandelate or phenylglyoxylate; only benzaldehyde dehydrogenase I is gratuitously induced by thiophenoxyacetate and only benzaldehyde dehydrogenase II is induced by benzyl alcohol, by benzaldehyde, and by a number of heterocyclic compounds which do not support growth. Mutants have been isolated that lack either benzaldehyde dehydrogenase II or benzyl alcohol dehydrogenase, or both of the enzymes. Results obtained in induction experiments with the wild-type bacterium N.C.I.B. 8250 and with the mutants show that benzaldehyde dehydrogenase II and benzyl alcohol dehydrogenase are co-ordinately regulated. Overall, the results suggest that benzaldehyde dehydrogenase I is associated with the metabolism of l-mandelate whereas benzaldehyde dehydrogenase II is associated with the metabolism of benzyl alcohol.

The microbial oxidation of methanol. Purification and properties of the alcohol dehydrogenase of Pseudomonas sp. M27.

1. A method for the purification of the nicotinamide nucleotide-independent alcohol dehydrogenase of Pseudomonas sp. M27 is described. 2. In the analytical ultracentrifuge, the purified enzyme shows a single major component of molecular weight 146000. 3. On electrophoresis in polyacrylamide gels between pH5.0 and 9.55, it shows only one protein band and the isoelectric point appears to be between pH7.0 and 8.0. 4. Spectrographic analysis indicates no significant metal content. 5. Amino acid analysis shows an unusually small number of cysteine/cystine residues per molecule as well as about 4.1% of glucosamine. 6. The role of ammonia as enzyme activator has been investigated.

The microbial oxidation of methanol. The prosthetic group of the alcohol dehydrogenase of Pseudomonas sp. M27: a new oxidoreductase prosthetic group.

1. The purified alcohol dehydrogenase of Pseudomonas sp. M27, whose action is independent of nicotinamide nucleotides, has absorption peaks at 280mmu and at 350mmu with little or no absorption at or above 450mmu. 2. It does not fluoresce, but green-fluorescent material, diffusible on dialysis, is produced when the enzyme is treated with acid or alkali or when it is boiled. 3. Evidence is presented that the enzyme is not a flavoprotein. 4. Kinetic studies show a correlation between enzyme inactivation by acid, alkali or heat and liberation of the fluorescent material. 5. Some purification of the fluorescent material was achieved, but definite identification was not possible; the major component has a fluorescence maximum at about 460mmu with excitation maxima at about 260mmu and 365mmu. 6. Data are given (including absorption and fluorescence spectra) that support the suggestion that the prosthetic group of the enzyme is a pteridine derivative. 7. Possible mechanisms of action of the enzyme are discussed.