Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii.

Azotobacter vinelandii was grown diazotrophically in chemostat cultures limited by sucrose, citrate, or acetate. Specific activities of cellular oxygen consumption (qO2) and nitrogenase (acetylene reduction) were measured in situ at different dilution rates (D, representing the specific growth rate mu at steady state). Sucrose-limited cultures exhibited linear relationships between qO2 and D, each of which, however, depended on the dissolved oxygen concentration in the range of 12 to 192 microM O2. From these plots, qO2 required for maintenance processes (mO2) were extrapolated. mO2 values did not increase linearly with increasing dissolved oxygen concentrations. With citrate- or acetate-limited cultures qO2 also depended on D. At 108 microM O2, however, qO2 and mO2 of the latter cultures were significantly lower than those of sucrose-limited cultures. Specific rates of acetylene reduction increased linearly with D, irrespective of the type of limitation and of the dissolved oxygen concentration (J. Kuhla and J. Oelze, Arch. Microbiol. 149:509-514, 1988). The reversible switch-off of nitrogenase activity under oxygen stress also depended on D and was independent of qO2, mO2, or the limiting substrate. Increased switch-off effects resulting from increased stress heights could be compensated for by increasing D. Since D represents not only the supply of the carbon source but also the supply of electrons and energy, the results suggest that the flux of electrons to the nitrogenase complex, rather than qO2, stabilizes nitrogenase activity against oxygen inactivation in aerobically growing A. vinelandii.     

Temperature dependence of growth and membrane-bound activities of Chloroflexus aurantiacus energy metabolism

The temperature dependence of various activities related to the energy metabolism of isolated membranes and whole cells of the thermophilic bacterium Chloroflexus aurantiacus was determined after phototrophic growth at either 40, 50, or 60 degrees C. The data obtained were expressed by use of Arrhenius plots. Maximum activities were determined at about 65 degrees C for succinate 2,4-dichlorophenol-indophenol reductase as well as NADH oxidase and at about 70 degrees C for Mg-ATPase and for light-induced proton extrusion by cells. Activation energies for Mg-ATPase and light-induced proton extrusion were about 40 kJ mol-1 from 30 degrees C to about 50 degrees C and they increased significantly at higher temperatures. Essentially the same dependency was detectable with NADH oxidase, except for an increase in activation energy below 41 degrees C. All of these responses were independent of growth temperature. Succinate-2,4-dichlorophenol-indophenol reductase showed a change in activation energy around 41 degrees C only with cells grown at 60 degrees C. Differences in the responses of cells grown at different temperatures were identified on the basis of changes from sigmoidal to hyperbolic kinetics for light saturation of proton extrusion. Moreover, the thermostability of proton extrusion was maximal when assayed at the corresponding growth temperatures. In any case, thermostability was lowest at the 65 and 68 degrees C assay temperatures. Differential scanning calorimetry with membranes revealed irreversible heat uptake from about 60 to 72 degrees C. The results are discussed in light of the activation energy for the specific growth rate, which is lowest at temperatures from 40 degrees C to the optimum at 60 degrees C.     

Bacteriochlorophyllgehalt und Proteinmuster der Thylakoide von Rhodospirillum rubrum während der Morphogenese des Photosynthese-Apparates

Zusammenfassung Der Zusammenhang zwischen dem spezifischen Bacteriochlorophyllgehalt der Zellen und der Thylakoidmorphogenese wird an Rhodospirillum rubrum untersucht. Bei der Induktion des Photosynthese-Apparates wird zunächst Bacteriochlorophyll synthetisiert, obgleich noch keine Thylakoide gebildet werden (1. Phase). Werden Thylakoide ausgebildet, so steigt der spezifische Bacteriochlorophyllgehalt der Thylakoide in Abhängigkeit vom spezifischen Bacteriochlorophyllgehalt der Zellen an, bis ein Wert von 12–13 g Bacteriochlorophyll je mg Zellprotein erreicht ist (2. Phase). Während der Wert für die Zellen darüber hinaus weiter erhöht werden kann, bleibt der Wert der Thylakoide konstant bei 100 g Bacteriochlorophyll je mg Thylakoid-Protein (3. Phase). Isolierte Thylakoide aus Zellen mit niedrigem Bacteriochlorophyllgehalt besitzen geringere Durchmesser als Thylakoide aus Zellen mit höheren Werten. Auch hinsichtlich der Zusammensetzung der Thylakoide in Abhängigkeit von den steigenden Bacteriochlorophyllgehalten bei induzierten Zellen konnten Unterschiede festgestellt werden. Ähnlich wie die spezifischen Bacteriochlorophyllgehalte der Thylakoide, nähern sich die verschiedenen Proteinbausteine der Thylakoide einem festen Verhältnis zueinander, das sich oberhalb von 10–14 g Bacteriochlorophyll je mg Zellprotein nicht mehr ändert. Mit Zunahme des Bacteriochlorophyllgehaltes der Zellen steigt der Gehalt an thylakoidspezifischen Proteinen in den Membranen an und der Anteil der für die Cytoplasmamembran spezifischen Komponenten nimmt ab.

---

The bacteriochlorophyll content and protein composition of chromatophores (=thylakoids) of Rhodospirillum rubrum during morphogenesis of the photosynthetic apparatus

Summary The correlation between the specific bacteriochlorophyll content of whole cells and the morphogenesis of chromatophores was investigated in Rhodospirillum rubrum. During the first phase after induction of the photosynthetic apparatus bacteriochlorophyll is synthesized without formation of chromatophores. In a second phase chromatophores increases in a linear correlation to the specific bacterichlorophyll content of the cells. In a third phase, when the specific bacteriochlorophyll content of the cells has reached 12–13 g/mg protein, the specific bacteriochlorophyll content of the chromatophores remains constant (100 g/mg protein). Isolated chromatophores from the second phase have smaller diameters, than chromatophores from the third phase. The composition of the protein compounds of isolated chromatophores changes during the development of the chromatophores in a similar fashion as the specific bacteriochlorophyll content of chromatophores does change. With increasing bacteriochlorophyll content of the cells the chromatophore specific proteins in the membranes increase whereas proteins specific for the cytoplasmic membrane decrease until both reach a constant level.

---

Verwendete Abkürzungen BChl. Bacteriochlorophyll     

Morphological and ultrastructural variations inAzotobacter vinelandii growing in oxygen-controlled continous culture

Membrane development as a response to growth at different oxygen tensions (from about 1% to 100% saturation of the medium with air) was determined inAzotobacter vinelandii strain OP. The organisms were grown in a carbonlimited chemostat either on atmospheric nitrogen or on ammonium as nitrogen sources. Both types of cultures increased not only the number of intracytoplasmic membrane vesicles per cell but also the cell volume with aeration. As the ratio of length per width stayed largely constant increases of volume resulted in decreases of the cell surface area, representing the surface area of the peripheral cytoplasmic membrane, per cell volume. While in nitrogen-fixing cells the proportion of intracytoplasmic membrane surface area per cytoplasmic membrane surface area increased from 1:2 to 3:1 the ratio stayed almost constant in ammonium-assimilating cells. The data suggest that oxygen controls changes in the ratio of intracytoplasmic to cytoplasmic membrane surface areas only under conditions of nitrogen fixation.Abbreviations CM Cytoplasmic membrane - ICM intracytoplasmic membrane     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

The effect of transfer from low to high light intensity on electron transport in Rhodospirillum rubrum membranes

The effects of transfer from low to high ligh intensity on membrane bound electrontransport reactions of Rhodospirillum rubrum were investigated. The experiments were performed with cultures which did not form bacteriochlorophyll (Bchl) for about two cell mass doublings during the initial phase of adaptation to high light intensity. Lack of Bchl synthesis causes a decrease of Bchl contents of cells and membranes. Also, the cellular amounts of photosynthetically active intracytoplasmic membranes decrease.In crude membrane fractions containing both cytoplasmic and intracytoplasmic membranes the initial activities of NADH oxidizing reactions increase only slightly (about 1.2 times) per protein, but the initial activities of succinate oxidizing reactions decrease (multiplied by a factor of 0.7). On a Bchl basis activities of NADH oxidizing reactions increase 3.4 times while activities of succinate dependent reactions increase 1.9 times. With isolated intracytoplasmic membranes activities of NADH as well as succinate dependent reactions increase to a comparable extent on a Bchl basis (about 1.8 times) and stay nearly constant on a protein basis. Cytochrome c oxidase responds like succinate dependent reactions. The data indicate that in cells growing under the conditions applied NADH oxidizing electron transport systems are incorporated into both, cytoplasmic and intracytoplasmic membranes, while incorporation of succinate oxidizing systems is confined to intracytoplasmic membranes only.Activities of photophosphorylation and succinate dependent NAD⁺ reduction in the light increase per Bchl about 1.8 times. On a Bchl basis increases of the fast light induced on reactions at 422 nm and increases of soluble cytochrome c ₂ levels are comparable to increases of photophosphorylations and succinate dependent activities. But increases of slow light off reactions at 428 nm and of b-type cytochrome levels become three times greater then increases of cytochrome c ₂ reactions and levels. These results infer that although electrontransport reactions of intracytoplasmic membranes change correlated to each other, Bchl, cytochrome c ₂ and b-type cytochromes cellular levels are independent of each other. Furthermore, the data indicate that cytochrome c ₂ rather than b-type cytochrome is involved with steps rate limiting for photophosphorylation.Abbreviations Bchl bacteriochlorophyll - DCIP 2,6-dichlorophenolindophenol     

Multi-isotopic analysis reveals individual mobility and diet at the Early Iron Age monumental tumulus of Magdalenenberg, Germany

For the Early Iron Age western Hallstatt culture, which includes the site of Magdalenenberg in southwest Germany, it has been proposed that people were mobile and maintained far reaching social and trading networks throughout Europe. We tested this hypothesis by analyzing multiple isotopes (strontium, oxygen, sulfur, carbon, and nitrogen) of the preserved skeletons from the Magdalenenberg elite cemetery to determine diets and to look for evidence of mobility. The analysis of carbon, nitrogen, and sulfur isotope ratios in collagen of humans (n = 50) and associated domestic fauna (n = 10) indicates a terrestrial-based diet. There was a heterogeneous range of isotope values in both strontium (0.70725 to 0.71923, n = 76) and oxygen (13.4‰ to 18.5‰, n = 78) measured in tooth enamel. Although many of the individuals had values consistent with being from Hallstatt culture sites within southwest Germany, some individuals likely originated from further afield. Possible areas include the Alps of Switzerland and Austria or even locations in Italy. Our study strongly supports the assumption of far reaching social and economic networks in the western Hallstatt culture.