New dimensions in microbiology: an introduction.

Microbiology--a century on from Koch's pioneering development of monoculture techniques--is steadily changing from traditional studies of pure cultures and their growth on single substrates to those in which new dimensions are being added. These new dimensions include the analysis of mixed substrates, mixed cultures and multiphase systems in which microorganisms grow on or within solid substrates. While it has long been recognized that metabolism of complex substrate mixtures, such as the contents of the rumen, may require a complex community of microorganisms, a new concept is the metabolism of single substrates, whether complex or simple, by stable communities of different organisms, so stable that some have been handled for years as monocultures and named accordingly. Finally, the widespread occurrence of genetic exchange between microorganisms has introduced an entirely new dimension to the older ideas of stable organisms only altering their metabolic potentialities by rare mutational events.

Simultaneous assay of dihydroxyacetone synthase and transketolase in a methylotrophic yeast grown in continuous culture. A cautionary note.

The methylotrophic yeast Candida boidinii CBS 5777 was grown in continuous culture under carbon limitation on glucose, glucose plus methanol, and methanol as carbon and energy sources. During adaptation from glucose to methanol there was a rapid rise in the specific activities of triokinase, fructose-1,6-bisphosphatase and dihydroxyacetone synthase, which are key enzymes of the xylulose phosphate cycle of formaldehyde fixation. The specific activity of classical transketolase fell during this adaptation. Extracts from carbon-limited C. boidinii contained an enzyme which catalysed oxidation of NADH when some preparations or ribose 5-phosphate were added, which was not a transketolase. This enzyme activity was dependent on an impurity in such ribose 5-phosphate preparations and can be confused with transketolase activity.

Purification and properties of the methanol dehydrogenase from Methylophilus methylotrophus.

1. A dye-linked methanol dehydrogenase, resembling many others from a variety of methylotrophic bacteria, was purified to homogeneity from extracts of methanol-grown Methylophilus methylotrophus. 2. The enzyme was very stable in the presence of methanol; in the absence of methanol it had a half-life of 1-2 days at 4 degrees C. 3. The value of A1% 1cm,280 was 17.5. 4. The enzyme retained bound methanol after passage through Sephadex G-25. This tightly-bound methanol slowly exchanged with free [14C]-methanol from a value of 0.27 mol of [14C]methanol/mol of enzyme after 48 h incubation at 4 degrees C to a limiting value of approx. 2.5 mol of [14C]methanol/mol of enzyme after 3 weeks incubation at 4 degrees C. 5. One mol of this enzyme reduced 89.4 mol of 2,6-dichlorophenol-indophenol (via phenazine methosulphate) in the absence of any additional methanol in the assay mixture. The source of the electrons involved in this reduction is not known.

The interrelation transketolase and dihydroxyacetone synthase activities in the methylotrophic yeast Candida boidinii.

Crude extracts of Candida boidinii grown on glucose, xylose or ethanol gave single peaks of classical transketolase activity following chromatography, on columns of hydroxylapatite; the enzyme was heat-stable and showed no appreciable activity with formaldehyde as acceptor in place of ribose 5-phosphate. Extracts of methanol-grown cells showed two peaks of transketolase activity following chromatography on both hydroxylapatite and DEAE-cellulose. One peak was identified with that found for the cells grown on substrates other than methanol; the other peak showed dihydroxyacetone synthase activity in addition to transketolase activity. Both activities in the latter peak were very unstable and have been ascribed to one enzyme on the basis of identical rates of denaturation at all temperatures tested between 0 and 40 degrees C. It is suggested that this enzyme is a special transketolase synthesized only during methylotrophic growth of the yeast and in contrast to classical transketolase, is capable of using equally well either formaldehyde or ribose 5-phosphate as glycolaldehyde acceptor. A method based on heat treatment has been suggested for the simultaneous assay of both transketolases present in crude extracts of a methylotrophically grown yeast.

Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila.

1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties.

Aerobic and anaerobic growth of Paracoccus denitrificans on methanol.

1. The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate. 2. Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme. 3. Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions. 4. Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured. 5. From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.

The dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila. Comparison with dye-linked methanol dehydrogenases.

1. A dye-linked alcohol dehydrogenase was purified 20-fold from extracts of Rhodopseudomonas acidophila 10050 grown anaerobically in the light on methanol/HCO3-. 2. The enzyme resembled many previously reported methanol dehydrogenases from other methylotrophic organisms in coupling to phenazine methosulphate, requiring ammonia as an activator, possessing a pH optimum of 9 and a mol.wt. of approx. 116000. In many other respects the enzyme showed singular properties. 3. The stability of the enzyme under various conditions of temperature and pH was studied. 4. Primary aliphatic amines containing up to nine carbon atoms (the longest tested) were better activators than ammonia. 5. A wide range of primary alcohols and aldehydes served as substrates, with apparent Km values ranging from 57 mM for methanol to 6 micron for ethanol. 6. O2 was an inhibitor competitive with respect to the alcohol substrate. In the presence of O2, apparent Km values of 145 mM were recorded for methanol. 6. Cyanide and alphaalpha'-bipyridine were inhibitors competitive with respect to the amine activator. 7. The properties of the enzyme from Rhodopseudomonas acidophila are compared with those of similar enzymes from other organisms, and implications of the salient differences are discussed.

Fructose metabolism in four Pseudomonas species.

1. ATP-Dependent phosphorylation of fructose could not be detected in extracts of fructose-grown cells of Pseudomonas extorquens strain 16, Pseudomonas 3A2, Pseudomonas acidovorans and Pseudomonas fluorescens. Instead, phosphorylation of fructose to fructose-1-phosphate was found to occur when cell-free extracts were incubated with fructose and phosphoenolpyruvate. Such an activity could not be detected in cell-free extracts of succinate-grown cells. 2. High levels of 1-phosphofructokinase were found in extracts of the above organisms when growth on fructose. 3. Mutants of Pseudomonas extorquens strain 16 lacking 1-phosphofructokinase were unable to grow on fructose. Revertants to growth on fructose had regained the capacity to synthesize this enzyme, indicating its necessary involvement in fructose metabolism. 4. A survey has been carried out of enzymes involved in carbohydrate metabolism in the species listed above.

Synthesis and hydrolysis of malyl-coenzyme A by Pseudomonas AM1: an apparent malate synthase activity.

The malate synthase activity detectable in crude extracts of Pseudomonas AM1 has been shown to be due to a coupling of a malyl-CoA hydrolase with malyl-CoA lyase and not due to a discrete malate synthase enzyme. The partial purification of this malyl-CoA hydrolase from Pseudomonas AM1 has shown that it is distinct from citrate synthase which also hydrolyses malyl-CoA. The malyl-CoA hydrolase has a low Km for malyl-CoA (7-0 muM). A mutant of Pseudomonas AM1, ICT51 (Taylor & Anthony, 1975), which is unable to grow on ethanol, malonate or 3-hydroxybutyrate, has been shown to have an altered malyl-CoA hydrolase with a Km for malyl-CoA 30 times higher than that of the enzyme present in the wild-type organism. Two classes of revertants to growth on these substrates have been isolated: (i) those with a malyl-CoA hydrolase of similar Km to the wild-type and (ii) those in which the malyl-CoA hydrolase activity remains the same as in the mutant ICT51. The nature of the mutation leading to the latter class of revertants is unknown.

Metabolism of methanol by Rhodopseudomonas acidophila.

Rhodopseudomonas acidophila strain 10050, grown anaerobically in the light on methanol, contained a methanol and formaldehyde dehydrogenase which could be coupled to phenazine methosulphate; an NAD-linked formaldehyde dehydrogenase which required GSH for activity; and an NAD-linked formate dehydrogenase. The specific activities of these enzymes varied in a non-coordinate manner when the organism was grown on different alcohols, formate or succinate. The affinity of the phenazine methosulphate linked methanol dehydrogenase for methanol was increased 10-fold if the cell-free extract was prepared and assayed in the absence of oxygen. Pulse-labelling experiments with [14C5methanol and [14C]bicarbonate indicated that fixation of carbon dioxide occurred via the ribulose diphosphate cycle and C3 + CO 2 fixation reaction(s). No evidence was obtained for operation of a reduced C1 fixation sequence. This conclusion was borne out by the enzyme content of cell-free extracts of the organism.

Oxidation of carbon monoxide and methane by Pseudomonas methanica.

The oxidation of carbon monoxide and methane by suspensions and ultrasonic extracts of Pseudomonas methanica was studied. A continuous assay for the oxidation of CO to CO2 was devised, using O2 and CO2 electrodes in combination. Stoicheiometries of CO-dependent CO2 formation, O2 consumption and NADH oxidation, and the partial stoicheiometries of methane-dependent NADH oxidation, suggest the involvement of a mono-oxygenase in these oxidations. Evidence is presented suggesting methane and CO oxidation are catalysed by a single enzyme system, distinct, at least in part, from the NADH oxidase present in extracts. Ethanol was able to provide the reductant necessary for CO oxidation by cell suspensions, though the metabolism of ethanol by P. methanica was found unlikely to result in substrate-level formation of NADH; the means whereby alcohol oxidation could supply reductant for the mono-oxygenase are discussed.

The autotrophic growth of Micrococcus denitrificans on Methanol.

Ribulose bisphosphate carboxylase is present at a high specific activity in extracts of methanol-grown Microccus denitrificans. Enzymic and physiological evidence indicates that, during growth on methanol, the ribulose bisphosphate cycle is the route of carbon assimilation.

Utilization of methanol by rhodospirillaceae.

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophia strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or fomaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.