Microbial metabolism of C1 and C2 compounds. The role of acetate during growth of Pseudomonas AM1 on C1 compounds, ethanol and beta-hydroxybutyrate.
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ABSTRACT:
SUBMITTER: Dunstan PM
PROVIDER: S-EPMC1177654 | biostudies-other | 1973 Apr
REPOSITORIES: biostudies-other
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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
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
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
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
Project description:Pseudomonas AM1 grows on ethanol with a mean generation time of about 10h. A single alcohol dehydrogenase is responsible for oxidation of both methanol and ethanol. It is proposed that the glyoxylate bypass does not operate in Pseudomonas AM1 during growth on ethanol. Although malate synthase is present in extracts of ethanol-grown Pseudomonas AM1, the activity of isocitrate lyase is negligible. Short-term incubation experiments with [(14)C]ethanol and [(14)C]acetate indicate that a novel pathway operates during growth of Pseudomonas AM1 on ethanol. Glycollate, glyoxylate and malate are probably intermediates in this pathway.
| S-EPMC1173574 | biostudies-other
Project description:Succinate (or a product of succinate metabolism) is a catabolite repressor of some enzymes of the serine pathway (hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glycerate kinase) but not of methanol dehydrogenase nor methylamine dehydrogenase. A mutant (PCT64) of Pseudomonas AM1, which is unable to grow on C(1) compounds, lacks glycerate kinase, showing that this enzyme is essential for the operation of the serine pathway. Mutant PCT48, unable to convert acetate into glycollate, has lost the ability to grow both on C(1) compounds and on ethanol. The properties of a third mutant (PCT57) show that Pseudomonas AM1 contains enzymes catalysing the conversion of acetate into glyoxylate. Evidence is presented that hydroxypyruvate reductase is involved in the oxidation of glycollate to glyoxylate during growth on ethanol. A scheme is proposed for the conversion of ethanol and of C(1) compounds into glyoxylate in which acetate (or a derivative) and glycollate are intermediates.
| S-EPMC1173575 | biostudies-other
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
Project description: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.
| S-EPMC1264954 | biostudies-other
Project description:This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.
| S-EPMC1183932 | biostudies-other
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