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Notes: Summary The influence of the oxygen partial pressure on the morphogenesis of thylakoids, the biosynthesis of bacteriochlorophyll, and the activity of photophosphorylation is investigated. 1. The growth rate (doubling time of protein) in aerobically grown dark cultures of Rhodopseudomonas capsulata is in a relative wide range independent of the oxygen partial pressure. The protein doubling time in a complex medium at 30° C amounts to 2.5–2.75 hrs in the range of 4–100 mm Hg (pO2). On conditions that the pO2 is saturated (150–160 mm Hg) the growth will be reduced about 10%. Underneath a critical oxygen partial pressure (4 mm Hg) the oxydative metabolism and the growth rate are strongly limited. 2. The rate of bacteriochlorophyll synthesis in the log phase of aerobic dark cultures is strongly related to the oxygen partial pressure. The production of bacteriochlorophyll comes to a maximum in the range of 3 to 5 mm Hg (pO2). But bacteriochlorophyll is produced up to 60–70 mm Hg (pO2). The rate of bacteriochlorophyll synthesis is relatively high below 20 mm Hg. 3. The production rate of bacteriochlorophyll is greatly enhanced when the oxygen partial pressure is suddenly lowered. Therefore the specific bacteriochlorophyll content of the cells (μg BChl./mg Protein) is strongly increased until a characteristic new level has been attained after 8 hrs. 4. The regulation of the bacteriochlorophyll synthesis in relation to the oxygen partial pressure in the medium is between 4 to 100 mm Hg (pO2) during log phase growth independent of the growth rate. 5. The biosynthesis of the photochemical apparatus (measured as photophosphorylating activity) of Rhodopseudomonas capsulata is increased likewise by lowering of the oxygen partial pressure. But the kinetic is different from that of bacteriochlorophyll synthesis. The increase in activity of light induced phosphorylation (PP) is enhanced lineary to the protein content of the membrane fraction. But the PP/bacteriochlorophyll ratio is decreased after a short increase. The bacteriochlorophyll content of the intracellular membranes is increased from 3.3 to 33.9 μg/mg protein two hours after induction (pO2: 160 mm Hg→5 mm Hg). 6. Micrographs of thin sections show intracellular membrane structures in high aerated (pO2 150–160 mm Hg) as well as in low aerated cultures (pO2 5 mm Hg). In aerobic cultures tubular membrane structures and in semiarobic cultures vesicular thylakoids (chromatophores) are to be seen. 7. The results demonstrate clearly that the production rate of the bulk of bacteriochlorophyll is not strictly paralled with the biosynthesis of the photochemical apparatus and the membrane morphogenesis.

Notes: Summary Cultures of Rps. capsulata were grown anaerobically in the light and at different oxygen partial pressures (5, 150, and 400 Torr) in the dark. The respiratory and photosynthetic activities as well as the protein patterns of the membranes are influenced significantly by the oxygen partial pressure. These variations give an idea of the process of membrane differentiation in the membrane system of Rps. capsulata in vivo. The bacteriochlorophyll content of intracytoplasmic membranes is found to vary between 5 μg bacteriochlorophyll per mg membrane protein (tubules from cells grown aerobically in the dark) to 195 μg bacteriochlorophyll per mg membrane protein (vesicles from cells grown anaerobically in the light). Vesicles from cells grown semiaerobically in the dark exhibit a bacteriochlorophyll content of about 110 μg per mg membrane protein. The bacteriochlorophyll values for light grown cells decrease to values below 1% (1.5 μg per mg membrane protein) by cultivation at 400 Torr oxygen partial pressure in the dark. In the cytoplasmic membrane of semiaerobically grown cells the bacteriochlorophyll content increases up to 10 μg per mg membrane protein. The bacteriochlorophyll content of the intracytoplasmic membranes varies in proportion to the bacteriochlorophyll content of the whole cells. Photophosphorylation of intracytoplasmic membranes is found to be highest in vesicles from light grown cells (19.5 μmoles PO4 3- per mg membrane protein per 30 min). According to the bacteriochlorophyll content the rates decrease to 10% in tubular membrane fractions from aerobically grown cells. The activity of the NADH oxidase were determined as (μmoles NADH per mg protein per min): 0.83 in tubules from dark grown cells (150 mm pO2), 0.17 in vesicles from dark grown cells (5 mm pO2), and 0.011 in vesicles from cells grown anaerobically in the light. The activities of oxidative phosphorylation do not exactly run parallel to the respiratory values. They were determined as (μmoles PO4 3- per 30 min per mg protein): 0.74 in vesicles from anaerobically light grown cells, 1.2 in vesicles from dark grown cells (5 mm pO2) and 2.01 in tubules (150 mm pO2). Variations of the photosynthetic and respiratory activities are also found in the cytoplasmic membrane, inferring specific processes of membrane differentiation in these structures, other than those in intracytoplasmic membranes. The state of membrane differentiation of intracytoplasmic membranes can be described by the ratio of photophosphorylation to oxidative phosphorylation. In vesicles isolated from light grown cells, the ratio is 25, from dark grown cells it is 5, and in tubules from aerobically grown cells it is 1. When treated with phenol, urea and acetic acid, membranes are split into 10 typical protein bands which can be obtained by polyacrylamide gel electrophoresis. According to the culture conditions, the pattern of specific protein bands is changed in relation to the variation of enzymatic activities and bacteriochlorophyll content of the membranes. During transient experiments, membrane proteins can be labelled specifically if cells are incubated with 14C (U) protein hydrolysate. In this way, membrane differentiation may be demonstrated by incorporation of specific newly synthesized proteins.

Notes: Summary The membrane system of Rhodopseudomonas capsulata, strain 37b4 is investigated in cells cultivated anaerobically in the light and in the dark, respectively, at different oxygen partial pressures (pO2). The intracytoplasmic membrane vesicles of anaerobically light grown and semiaerobically [5 mm (Hg) pO2] dark grown cells show similar diameters (30–50 nm). Growing aerobically in darkness the cells contain tubular intracytoplasmic membranes with comparable diameters. An increase of the pO2 up to 400 mm (Hg) results in a slightly decreased growth rate and in a complete inhibition of bacteriochlorophyll synthesis and intracytoplasmic membrane formation. After 8 h of cultivation under these conditions tubular membranes are still found. However, they are restricted to one cell pole only. In order to isolate the membranes, cells were broken by means of a French pressure cell. The crude, membrane fractions (sedimented at 104000-314000xg, 60 min) are purified by centrifugation on a Ficoll density gradient. This results in the formation of three membrane fractions. The light fraction consists of small vesicular particles derived from the cytoplasmic membrane. Crude membrane fractions of all cells sedimented at 314 000xg contain a relative high percentage of these particles. Intracytoplasmic membranes of aerobically grown cells are found in a middle band. Their tubular structure remains unaffected if the cells are treated by lower pressures during homogenization. The heavy band contains the intracytoplasmic membrane vesicles from light grown and from semiaerobically dark grown cells in a highly purified form. After treatment with butanol or NaCl and subsequently with lysozyme plus EDTA cells release flat membrane fragments which still exhibit invaginations. This shows once more that the membranes are connected to each other. Pulse chase experiments with (2-14C)-acetate support the hypothesis that the cytoplasmic membrane and intracytoplasmic membranes are transformable into each other. So they should be looked at as a morphogenetical unit.