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

ABSTRACT: 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.

SUBMITTER: JOHNSON PA

PROVIDER: S-EPMC1206817 | biostudies-other | 1965 Jun

REPOSITORIES: biostudies-other

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Project description:1. A study was made of the incorporation of carbon from [(14)C]methanol by cultures of Methylococcus capsulatus and Methanomonas methanooxidans 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 substrate for periods of up to 3min, was analysed by chromatography and radioautography. 3. Over 80% of the radioactivity fixed by Methylococcus capsulatus at 30 degrees C at the earliest times of sampling appeared in phosphorylated compounds, of which glucose phosphate constituted 60%. 4. Most of the radioactivity fixed by Methanomonas methanooxidans at 30 degrees C at the earliest times of sampling appeared in serine, malate, aspartate and an unknown compound(s) tentatively suggested to be folate derivative(s). At 16 degrees C, [(14)C]methanol was fixed predominantly into serine and the unknown compound(s). 5. Extracts of Methylococcus capsulatus contain an enzyme system that catalyses the condensation of formaldehyde and ribose 5-phosphate to give a mixture consisting mainly of fructose phosphate and allulose phosphate. No similar activity was detected in extracts of Methanomonas methanooxidans. A convenient method was developed for assay of this enzyme system. 6. The enzyme system catalysing the condensation of formaldehyde with ribose 5-phosphate is particle-bound in both Methylococcus capsulatus and Pseudomonas methanica and is unstable in the absence of Mg(2+). 7. Extracts of Methanomonas methanooxidans contain high activities of d-glycerate-NAD oxidoreductase, whereas extracts of Methylococcus capsulatus and Pseudomonas methanica contain negligible activities of this enzyme. 8. These results indicate that during growth of Methylococcus capsulatus on methane, as with Pseudomonas methanica, cell constituents are made by the ribose phosphate cycle of formaldehyde fixation. This contrasts with Methanomonas methanooxidans, whose assimilation pathway resembles in some features that of Pseudomonas AM1 growing on methanol.

Project description:Microbial communities in deep subsurface environments comprise a large portion of Earth's biomass, but the microbial activity in these habitats is largely unknown. Here, we studied how microorganisms from two isolated groundwater fractures at 180 and 500 m depths of the Outokumpu Deep Drillhole (Finland) responded to methane or methanol amendment, in the presence or absence of sulfate as an additional electron acceptor. Methane is a plausible intermediate in the deep subsurface carbon cycle, and electron acceptors such as sulfate are critical components for oxidation processes. In fact, the majority of the available carbon in the Outokumpu deep biosphere is present as methane. Methanol is an intermediate of methane oxidation, but may also be produced through degradation of organic matter. The fracture fluid samples were incubated in vitro with methane or methanol in the presence or absence of sulfate as electron acceptor. The metabolic response of microbial communities was measured by staining the microbial cells with fluorescent redox sensitive dye combined with flow cytometry, and DNA or cDNA-derived amplicon sequencing. The microbial community of the fracture zone at the 180 m depth was originally considerably more respiratory active and 10-fold more numerous (105 cells ml-1 at 180 m depth and 104 cells ml-1 at 500 m depth) than the community of the fracture zone at the 500 m. However, the dormant microbial community at the 500 m depth rapidly reactivated their transcription and respiration systems in the presence of methane or methanol, whereas in the shallower fracture zone only a small sub-population was able to utilize the newly available carbon source. In addition, the composition of substrate activated microbial communities differed at both depths from original microbial communities. The results demonstrate that OTUs representing minor groups of the total microbial communities play an important role when microbial communities face changes in environmental conditions.

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

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