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Microbial growth on C1 compounds. Incorporation of C1 units into allulose phosphate by extracts of Pseudomonas methanica.

ABSTRACT: 1. Incubation of cell-free extracts of methane- or methanol-grown Pseudomonas methanica with [(14)C]formaldehyde and d-ribose 5-phosphate leads to incorporation of radioactivity into a non-volatile product, which has the chromatographic properties of a phosphorylated compound. 2. Treatment of this reaction product with a phosphatase, followed by chromatography, shows the presence of two compounds whose chromatographic properties are consistent with their being free sugars. 3. The minor component of the dephosphorylated products has been identified as fructose. The major component has been identified as allulose (psicose) on the basis of co-chromatography, co-crystallization of the derived phenylosazone and dinitrophenylosazone with authentic derivatives of allulose and behaviour towards oxidation with bromine water. 4. It is suggested that the bacterial extracts catalyse the condensation of a C(1) unit identical with, or derived from, formaldehyde with ribose 5-phosphate to give allulose 6-phosphate. 5. Testing of hexose phosphates and pentose phosphates as substrates has so far shown the reaction to be specific for ribose 5-phosphate. 6. The condensation reaction is not catalysed by extracts of methanol-grown Pseudomonas AM1. 7. A variant of the pentose phosphate cycle, involving this condensation reaction, is suggested as an explanation for the net synthesis of C(3) compounds from C(1) units by P. methanica.

SUBMITTER: Kemp MB

PROVIDER: S-EPMC1264954 | biostudies-other | 1966 Apr

REPOSITORIES: biostudies-other

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MICROBIAL GROWTH ON C1 COMPOUNDS. SYNTHESIS OF CELL CONSTITUENTS BY METHANE- AND METHANOL-GROWN PSEUDOMONAS METHANICA.

Project description:1. A study has been made of the incorporation of carbon from [(14)C]methane, [(14)C]methanol and [(14)C]bicarbonate by cultures of Pseudomonas methanica growing on methane, and [(14)C]methanol by cultures of the same organism growing on methanol. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compound for periods up to 3min., has been analysed by chromatography and radioautography. 3. Over 90% of the radioactivity fixed from [(14)C]methane or [(14)C]methanol at the earliest times of sampling appeared in phosphorylated compounds. Glucose phosphate and fructose phosphate together constituted the largest part of the radioactive phosphates (70-90%); phosphoglycerate was a relatively minor component (2-17%). Other compounds becoming labelled during the incubation included glycine, serine, glutamate, aspartate, malate, citrate and alanine. 4. The first stable products of [(14)C]bicarbonate fixation were malate and aspartate (containing between them over 90% of the total radioactivity fixed at the earliest times of sampling). 5. The percentage of the total radioactivity fixed that was contained in each of the radioactive compounds has been plotted against time. The slopes of the curves obtained show that hexose phosphates are primary stable products of [(14)C]methane and [(14)C]methanol incorporation and that aspartate and malate are primary stable products of [(14)C]bicarbonate incorporation. 6. No carboxydismutase activity has been found in cell-free extracts of the organism. This fact, together with the other findings, shows that an autotrophic metabolism involving the ribulose diphosphate cycle of carbon dioxide fixation cannot be operating.

| S-EPMC1206817 | biostudies-other

Microbial growth on C1 compounds. The role of folat in the metabolism of Pseudomonas AM1.

Project description:1. The growth of Pseudomonas AM1 is much more sensitive to inhibition by sulphanilamide when methanol, rather than succinate, acts as the sole carbon and energy source; a sulphanilamide concentration of 1mm, which causes almost complete inhibition of growth on methanol, has little effect in a succinate medium. 2. Similar results have been obtained with sulphadiazine and sulphathiazole. Sulphanilic acid has little effect. 3. A similar differential sensitivity to sulphanilamide is shown by Protaminobacter ruber and Pseudomonas extorquens when grown on methanol media as compared with succinate. 4. Sulphanilamide inhibited the growth of Pseudomonas oxalaticus on formate, oxalate and succinate media to about the same extent. 5. Inhibition of growth of Pseudomonas AM1 by sulphanilamide is accompanied by an accumulation of glycine in the cells. 6. Inhibition of growth by sulphanilamide can be reversed by p-aminobenzoic acid. 7. Microbiological assays of the folate content of Pseudomonas AM1 have been performed after growth on both methanol and succinate, and the results are discussed in terms of differences in metabolism.

| S-EPMC1265007 | biostudies-other

Microbial metabolism of C1 and C2 compounds. The role of acetate during growth of Pseudomonas AM1 on C1 compounds, ethanol and beta-hydroxybutyrate.

Project description:Pseudomonas AM1 grows on beta-hydroxybutyrate and methanol at similar rates. beta-Hydroxybutyrate is not metabolized by way of the glyoxylate bypass, but is assimilated by the novel route (with acetate as an intermediate) that operates during growth of this organism on ethanol. Evidence from short-term labelling experiments indicates that acetate, which is a possible intermediate in the assimilation of C(1) compounds, is rapidly metabolized to glycine during growth of Pseudomonas AM1 on methanol.

| S-EPMC1177654 | biostudies-other

Microbial growth on C1 compounds. Uptake of [14C]formaldehyde and [14C]formate by methane-grown Pseudomonas methanica and determination of the hexose labelling pattern after brief incubation with [14C]methanol.

Project description:1. A study has been made of the incorporation of carbon from [(14)C]formaldehyde and [(14)C]formate by cultures of Pseudomonas methanica growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compounds for periods of up to 1min., has been analysed by chromatography and radioautography. 3. Radioactivity was fixed from [(14)C]formaldehyde mainly into the phosphates of the sugars, glucose, fructose, sedoheptulose and allulose. 4. Very little radioactivity was fixed from [(14)C]formate; after 1min. the only products identified were serine and malate. 5. The distribution of radioactivity within the carbon skeleton of glucose, obtained from short-term incubations with [(14)C]methanol of Pseudomonas methanica growing on methane, has been investigated. At the earliest time of sampling over 70% of the radioactivity was located in C-1; as the time increased the radioactivity spread throughout the molecule. 6. The results have been interpreted in terms of a variant of the pentose phosphate cycle, involving the condensation of formaldehyde with C-1 of ribose 5-phosphate to give allulose phosphate.

| S-EPMC1270214 | biostudies-other

The microbial metabolism of C1 compounds. The electron-transport chain of Pseudomonas am1.

Project description:Pseudomonas AM1, Hyphomicrobium X and Pseudomonas MS all contain cytochrome a/a(3) and a b-type cytochrome able to react with CO. Pseudomonas AM1 and Hyphomicrobium X also have a CO-binding cytochrome c. The purified cytochrome c (redox potential 0.26V) of Pseudomonas AM1 was not susceptible to oxidation by molecular oxygen. CO reacted slowly with the reduced form giving a CO difference spectrum with a peak at 412nm and troughs at 420nm and 550nm. Similar results were obtained with the cytochrome c of Hyphomicrobium (aerobically grown or anaerobically grown with nitrate) and with that of Pseudomonas extorquens. The results given in the present paper are incompatible with an oxygenase or oxidase function for the soluble cytochrome c of methylotrophs. Studies with whole cells of Pseudomonas AM1 and a cytochrome c-deficient mutant have demonstrated that cytochrome b (redox potential 0.009V) is the first cytochrome in the electron-transport chain for oxidation of all substrates except methanol (and ethanol) whose oxidation does not involve this cytochrome. All substrates are usually oxidized by way of cytochrome c and cytochrome oxidase (cytochrome a/a(3)), but there is an alternative route for the reduction of cytochrome a/a(3) in the mutant lacking cytochrome c. Results of experiments on cyanide inhibition of respiration and cytochrome oxidation support the suggestion that the susceptibility of cytochrome b to oxidation by molecular oxygen (reflected in its ability to react with CO) is probably irrelevant to the normal physiology of Pseudomonas AM1.

| S-EPMC1172477 | biostudies-other

The microbial metabolism of C1 compounds. Oxidative phosphorylation in membrane preparations of Pseudomonas AM1.

Project description:A method is described for preparation of membrane vesicles (diameter 80nm) capable of respiration-linked ATP synthesis. Vesicles prepared from succinate-grown bacteria oxidized NADH, succinate and ascorbate plus NNN'N'-tetramethylphenylenediamine; vesicles prepared from methanol-grown bacteria also oxidized methanol and formaldehyde, but they were otherwise identical. The uncoupling agent carbonyl cyanide chlorophenylhydrazone and the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide both inhibited ATP synthesis, whereas they had no effect on the rate of respiration. Rotenone inhibited ATP synthesis and respiration with NADH as substrate; antimycin A inhibited with succinate as substrate, and cyanide inhibited with all substrates. P/O ratios were usually 0.7-1.3 with NADH, 0.6-1.0 with succinate and 0.2-0.6 with reduced NNN'N'-tetramethylphenylenediamine or methanol as respiratory substrate. When 2,6-dichlorophenol-indophenol was used as an alternative electron acceptor to O(2) (NADH as donor) the P/2e ratio was 1.65. Although these P/O ratios are minimum values, because they do not take into account unknown amounts of uncoupled O(2) consumption, they are consistent with previous proposals [O'Keeffe & Anthony (1978) Biochem, J.170, 561-567] based on measurements of proton translocation in whole cells. The results also confirm that methanol dehydrogenase and cytochromes c and a/a(3) are arranged so that the first step in methanol oxidation is coupled to synthesis of ATP.

| S-EPMC1186522 | biostudies-other

Pathways leading to and from serine during growth of Pseudomonas AM1 on C1 compounds or succinate.

Project description:1. The following enzymes of the phosphorylated pathway of serine biosynthesis have been found in methanol- and succinate-grown Pseudomonas AM1: phosphoglycerate dehydrogenase, phosphoserine-alpha-oxoglutarate aminotransferase and phosphoserine phosphohydrolase. Their specific activities were similar in the organism grown on either substrate. 2. A procedure for preparation of auxotrophic mutants of Pseudomonas AM1 is described involving N-methyl-N'-nitro-N-nitrosoguanidine as mutagen and a penicillin enrichment step. 3. A mutant, M-15A, has been isolated that is unable to grow on methanol and that lacks phenazine methosulphate-linked methanol dehydrogenase. The mutant is able to grow on methylamine, showing that the amine is not oxidized by way of methanol. 4. Loss of methanol dehydrogenase activity in mutant M-15A led to loss of phenazine methosulphate-linked formaldehyde dehydrogenase activity showing that the same enzyme is probably responsible for both activities. 5. A mutant, 20B-L, has been isolated that cannot grow on any C(1) compound tested but can grow on succinate. 6. Mutant 20B-L lacks hydroxypyruvate reductase, and revertants that regained the ability to grow on methanol, methylamine and formate contained hydroxypyruvate reductase activity at specific activities similar to that of the wild-type organism. This shows that hydroxypyruvate reductase is necessary for growth on methanol, methylamine and formate but not for growth on succinate. 7. The results suggest that during growth of Pseudomonas AM1 on C(1) compounds, serine is converted into 3-phosphoglycerate by a non-phosphorylated pathway, whereas during growth on succinate, phosphoglycerate is converted into serine by a phosphorylated pathway.

| S-EPMC1178961 | biostudies-other

The microbial metabolism of C1 compounds. The cytochromes of Pseudomaonas AM1.

Project description:Pseudomonas AM1 contains cytochromes a, b and c and more than one CO-binding pigment (cytochrome a3, cytochrome c and possibly a cytochrome o). The soluble cytochrome c has been purified; its isoelectric point is low and its molecular weight is 20000. This cytochrome is reduced in whole bacteria by all oxidizable substrates at rates determined by the primary dehydrogenases. A mutant lacking cytochrome c oxidizes all substrates except methanol, ethanol and methylamine; these no longer support growth. The role of cytochrome c in electron transport in Pseudomonas AM1 is discussed.

| S-EPMC1165305 | biostudies-other

The biosynthesis of serine and glycine in Pseudomonas AM1 with special reference to growth on carbon sources other than C1 compounds.

Project description:1. A mutant, 20S, of Pseudomonas AM1 was obtained that requires a supplement of serine to grow on succinate, lactate or ethanol. This mutant lacks phosphoserine phosphatase and revertants to wild-type phenotype regained this enzymic activity showing that the phosphorylated pathway of serine biosynthesis is necessary for growth on these three substrates. 2. The requirement for supplemental serine by mutant 20S could be met by glycine, suggesting that Pseudomonas AM1 can obtain C(1) units from glycine. 3. Mutant 20S grows on C(1) compounds at a lower rate compared with the wild type. Supplementation with serine stimulated the growth rate of the mutant suggesting that the phosphorylated pathway of serine biosynthesis plays some role, but not an essential role, during growth on C(1) compounds. 4. A mutant, 82G, was obtained that requires a supplement of glycine to grow on succinate, lactate or ethanol. When grown in such supplemented media, the mutant lacks serine hydroxymethyltransferase and revertants to wild-type phenotype regained enzymic activity showing that during growth on succinate, lactate or ethanol, glycine is made from serine via serine hydroxymethyltransferase, and that the organism can obtain C(1) units from glycine. 5. Mutant 82G grew on methanol and then contained serine hydroxymethyltransferase suggesting that this enzyme is necessary for growth on C(1) compounds and that Pseudomonas AM1 may synthesize two such enzymes, one used in growth on C(1) compounds, the other used in growth on other substrates. Mutant 82G might lack the latter enzyme. 6. Phosphoglycerate dehydrogenase is specifically inhibited by l-serine and the regulatory implications of this are discussed.

| S-EPMC1176663 | biostudies-other

Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates.

Project description:The use of plant growth promoting bacterial inoculants as live microbial biofertilizers provides a promising alternative to chemical fertilizers and pesticides. Inorganic phosphate solubilization is one of the major mechanisms of plant growth promotion by plant associated bacteria. This involves bacteria releasing organic acids into the soil which solubilize the phosphate complexes converting them into ortho-phosphate which is available for plant up-take and utilization. The study presented here describes the ability of endophytic bacteria to produce gluconic acid (GA), solubilize insoluble phosphate, and stimulate the growth of Pisum sativum L. plants. This study also describes the genetic systems within three of these endophyte strains thought to be responsible for their effective phosphate solubilizing abilities. The results showed that many of the endophytic strains produced GA (14-169 mM) and have moderate to high phosphate solubilization capacities (~400-1300 mg L(-1)). When inoculated into P. sativum L. plants grown in soil under soluble phosphate limiting conditions, the endophytes that produced medium-high levels of GA displayed beneficial plant growth promotion effects.

| S-EPMC4510416 | biostudies-literature

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