G. H. Tait

Metabolic turnover of the lipids of Rhodopseudomonas spheroides

Experiments with L-[Me-14C]methionine and inorganic 32P indicate that the lipids of this micro-organism are metabolically stable. Lipids of unpigmented cells were incorporated into chromatophores when these cells were allowed to adapt to semi-anaerobic conditions in the light. The specific radioactivity of the phospholipid of the chromatophores was the same as that of the adapted cells; thus it appears that no distinction can be made between different parts of the cytoplasmic membrane system with respect to the labelling of the lipids. It is concluded that chromatophores originate from the cytoplasmic membrane and remain structurally a part of it.

The Isolation and Characterization of Subcellular Fractions from Pigmented and Unpigmented Cells of Rhodopseudomonas spheroides

Current ideas on the nature and development of the chromatophores of photosynthetic bacteria are reviewed. A simple method of obtaining purified chromatophores by sucrose density gradient centrifuging of cell-free extracts of Rhodopseudomonas spheroides is described. Such preparations consist of about 60% protein, 20% phospholipid and 10% pigment, most of which is bacteriochlorophyll. Small quantities of carbohydrate, but only traces of nucleic acid, are found. The material was fairly homogeneous on electron microscopy. Rps. spheroides was also grown under pure oxygen in the dark. A particulate preparation from cells cultured under these conditions was similar to the chromatophores with respect to its high content of protein and of phospholipid but had a much greater content of nucleic acid and no bacteriochlorophyll. In addition, it contained amino sugars and diaminopimelic acid which are not found in chromatophores. These differences in chemical composition were correlated with the electron microscope appearances of the different subcellular fractions. It is concluded that the particulate preparation from cells grown under oxygen represents the cytoplasmic membrane of the micro-organism, but as isolated it is heavily contaminated with fragments of the cell wall from which it cannot be readily separated.

Adaptation of Rhodopseudomonas spheroides

The composition of Rhodopseudomonas spheroides grown under oxygen or under air in the dark, or semi-anaerobically in the light was studied. The amounts of various constituents were expressed per cell. Anaerobically grown cells were 20 % lighter than cells grown under oxygen but contained approximately 50 % more protein and 85 % more phospholipid. The differences in weight were largely due to greater amounts of reserve material (poly-B-hydroxybutyrate and carbohydrate) in oxygen-grown cells. The major increase in the protein was due to a doubling of the 4particulate5 protein, the ‘soluble’ protein increasing by only 20% . The values of certain constituents in cells grown in air were intermediate between those of oxygen-grown cells and semi-anaerobically grown cells. The changes in composition were followed during adaptation from growth under oxygen to semi-anaerobic conditions in the light. Particulate protein, phospholipid, and enzymes concerned in bacteriochlorophyll synthesis increased markedly before photopigments or chromatophores were formed. These results indicate the sequence of some of the steps concerned in the differentiation of the cytoplasmic membrane of pigment-free microorganisms into the fully formed photosynthetic apparatus.

Incorporation of radioactivity from [me-14C]methionine and [2-14C]glycine into the lipids of Rhodopseudomonas spheroides.

Radioactivity from L-[Me-14C]methionine, in addition to being incorporated into pigments, as found by other workers, was incorporated into phosphatidylcholine, into phosphatidyl N-methylethanolamine and into phosphatidyl N, N-dimethylethanolamine. The latter two compounds were present in trace amounts only. Of the radioactivity in the phospholipid fraction, 90% was in the bases with only minor amounts in the fatty acids. Incorporation was also found into ornithine lipid. The presence of the pathway of phosphatidylcholine biosynthesis which proceeds by the successive methylation of phosphatidylethanolamine was shown by demonstrating a precursor-product relationship between the N-methylated phospholipids and phosphatidylcholine. Evidence for the presence of the enzymes concerned in the biosynthesis of phosphatidylcholine by the pathway mentioned and also for the incorporation of the methyl group into ornithine lipid has been obtained in cell-free systems from Rhodopseudomonas spheroides. The methyl donor was S-adenosylmethionine. Radioactivity from [2-14C]glycine was incorporated into ethanolamine, into choline and into fatty acids. Ethionine markedly reduced the incorporation into choline without affecting the labelling of ethanolamine. Thus it seems likely that the methyl carbons of choline were radioactive and that ethionine acted by inhibiting transmethylation from S-adenosylmethionine. It is concluded that the α-carbon of glycine gives rise to ‘one carbon’ fragments and also to acetate and the pathways by which these reactions occur are discussed.

Intracellular location of enzymes in rhodopseudomonas spheroides.

By differential centrifugation of extracts of pigmented Rhodopseudomonas spheroides a number of constituents, phospholipid and lipid ornithine, and enzymes, zinc protoporphyrin chelatase, succinic dehydrogenase and S-adenosylmethionine-magnesium protoporphyrin methyltransferase, have been found to be associated both with chromatophores and with non-pigmented particulate material. These components are present in both types of material at about the same level. In extracts of non-pigmented organisms the particulate material contains some of the above components, but others are only present in low amounts. The subcellular structures present in the particulate material—ribosomes, cell wall and cytoplasmic membrane—have only been partially separated but, by comparing the distribution of the components listed above with those of known components of ribosomes and cell wall, it is probable that they are associated with cytoplasmic membrane. These studies suggest that the cytoplasmic membrane, apart from lacking the photosynthetic pigments, has a composition similar to that of chromatophores. The data are consistent with the conclusion drawn from electron microscopic studies that chromatophores are derived by invagination of the cytoplasmic membrane.