Viability and Endogenous Substrates Used During Starvation Survival of Rhodospirillum rubrum

Cells of Rhodospirillum rubrum were grown photoorganotrophically and chemoorganotrophically and then starved for organic carbon and combined nitrogen under four conditions: anaerobically in the light and dark and aerobically in the light and dark. Illumination prolonged viability and suppressed the net degradation of cell material of phototrophically grown cells, but had no effect on chemotrophically grown cells that did not contain bacteriochlorophyll. The half-life survival times of carbohydrate-rich phototrophically grown cells during starvation anaerobically or aerobically in the light were 17 and 14.5 days, respectively. The values for starvation aerobically and anaerobically in the dark were 3 and 0.5 days, respectively. Chemotrophically grown cells had half-life survival times of 3 and 4 days during starvation aerobically in the light and dark, respectively, and 0.8 day during starvation anaerobically in the light or dark. Of all cell constituents examined, carbohydrate was most extensively degraded during starvation, although the rate of degradation was slowest for phototrophically grown cells starved anaerobically in the light. Phototrophically grown cells containing poly-beta-hydroxybutyrate as carbon reserve were less able to survive starvation anaerobically in the light than were carbohydrate-rich cells starved under comparable conditions. Light intensity had a significant effect on viability of phototrophically grown cells starving anaerobically. At light intensities of 320 to 650 lx, the half-life survival times were 17 to 24 days. At 2,950 to 10,500 lx, the survival times decreased to 1.5 to 5.5 days. The kinetics of cell death correlated well with the rate of loss of cell mass of starving cells. However, the cause of death could not be attributed to degradation of any specific cell component.     

Activity and characterization of mixed organic compounds extracted from Rhodobacter sphaeroides as alternative materials to serum for mammalian cell growth

Photosynthetic microorganisms produce relatively large amounts of physiologically active materials which stimulate the physiological activity of other organisms. In this study, mammalian HeLa cells were cultured in different culture media which were Dulbecco's modified Eagle medium (DMEM) with newborn calf serum (NCS), and DMEM including different types of physiologically activating compounds (PACs) extracted from Rhodobacter sphaeroides grown under various culture conditions. R. sphaeroides was grown under the following five different culture conditions: anaerobically in the light, anaerobically in the dark and treated with dimethyl sulfoxide, aerobically in the dark for 48 h, in the light for 48 h, and in the light for 24 h and changed after previous culturing in the dark for 24 h. The growth of HeLa cell was measured by cell counting using a hemocytometer, and the fluorescent intensities of cellular lysosomes were measured to check the level of cellular stress caused by adding PACs. The growth of HeLa cells cultured in DMEM with PACs extracted from R. sphaeroides aerobically grown under dark conditions was enhanced compared to that of cells grown with NCS. We also found that a high concentration of pigments such as bacteriochlorophylls and carotenoids and a high concentration of arginine produced by R. sphaeroides aerobically grown in the dark were implicated in increased growth of the HeLa cells. Therefore, our results suggest that PACs extracted from R. sphaeroides aerobically cultured in dark conditions can enhance the physiological activity of mammalian cells and serve as nontoxic and bioavailable materials.     

Porphyrin biosynthesis in Rhodopseudomonas palustris--XII. delta-Aminolevulinate synthetase switch-off/on regulation

The high levels of delta-aminolevulinate synthetase (ALA-S) in Rhodopseudomonas palustris cells grown anaerobically in the light (Ph) decrease to those found in cells grown aerobically in the dark (A), when the former cultures were vigorously oxygenated; simultaneously bacteriochlorophyll (Bchl) synthesis abruptly halted leading to diminished steady-state specific Bchl content. When flushing oxygen was interrupted, enzymic activity increased, whether chloramphenicol was present or not in the medium; if the protein synthesis inhibitor was added when oxygenation started, ALA-S declined in the same fashion as in its absence, but thereafter reactivation of the enzyme was lower than before. Succinyl-CoA-synthetase and ALA-dehydratase activities were also measured under the conditions described, and no changes at all have been observed. The existence of different forms of ALA-S in R. palustris depending on growth conditions is postulated along with the formation of low molecular weight factors which can modulate ALA-S activity by binding to the enzyme; a widespread mechanism in the adaptation of micro-organisms to changes in environment. It is also proposed that this particular regulatory phenomenon, could be referred to as a switch off/on mechanism controlling ALA-S activity in R. palustris.     

Semiaerobic induction of bacteriochlorophyll synthesis in the green bacterium Chloroflexus aurantiacus.

Comparison of Chloroflexus aurantiacus J-10-fl cells by freeze-fracture electron microscopy showed that cell shape and dimensions did not depend on oxygen tension or light intensity during growth. The major morphological difference between cells cultured anaerobically in the light and aerobically in the dark was the absence of chlorosomes in aerobically grown cells. C. aurantiacus cells cultured aerobically in the dark began bacteriochlorophyll synthesis immediately when shifted to either phototrophic or semiaerobic conditions. Cells adapting to phototrophic conditions grew to the same density and synthesized as much bacteriochlorophyll as nonadapting phototrophic cultures grown at the same light intensity. Cells adapting to reduced oxygen tension (semiaerobic conditions) in the dark entered an 8- to 12-h growth lag during which the bacteriochlorophyll content increased significantly. Despite variations in the initial bacteriochlorophyll content and in the length of the growth lag, the amounts of bacteriochlorophyll a and c were constant at the end of the semiaerobic growth lag. At later times during adaptation to semiaerobic conditions, after growth resumed, variations in the ratio of bacteriochlorophyll c/bacteriochlorophyll a were observed and suggested independent regulation of the two bacteriochlorophylls.     

Metabolic differences betweenEscherichia coli cultures growing aerobically and anaerobically in the presence of fluoroacetate

The amount of citrate and pyruvate increased during the stationary phase of anEscherichia coli B culture growing in a synthetic medium, aerobically in the presence of glucose. In an anaerobic culture the amount of citrate was minute and did not rise during the stationary phase; the level of pyruvate in the stationary phase rose only slightly. Fluoroacetate blocked the growth of cells both aerobically and anaerobically. Cultures growing aerobically in the presence of fluoroaeetate displayed an increased accumulation of citrate as compared with the control. In anaerobic cultures citrate did not accumulate in the presence of inhibitory concentrations of fluoroacetate. In the presence of 2mm fluoroacetate cells grew aerobically somewhat more slowly at first, then inhibition ceased and finally the growth yield was greater than in the control. The obtained data indicate that citrate accumulated at first was partly utilized for growth under these conditions. The results are discussed from the point of view of differences in the metabolism of aerobically and anaerobically grown cells.     

Changes in the cytochrome composition of Rhodopseudomonas sphaeroides grown aerobically, photosynthetically and on dimethyl sulphoxide.

Several strains and mutants of Rhodopseudomonas sphaeroides can be grown anaerobically in the dark in the presence of dimethyl sulphoxide as an electron acceptor. During adaptation to this fermentative mode of growth, two major c-type cytochromes are synthesized, one with Mr 45 000 and the second with Mr 20 000 and a midpoint potential of +120 mV. These cytochromes are barely detectable in membranes prepared from cells grown in aerobic or photosynthetic conditions. An electrophoretic method is presented for the detection of the b-type and c-type cytochromes of pigmented or unpigmented membranes. The method resolves three b-type cytochromes and four c-type cytochromes in membranes from aerobically and photosynthetically grown cells.     

Adenylate energy charge in Saccharomyces cerevisiae during starvation.

Bakers' yeast cells, Saccharomyces cerevisiae, if grown aerobically on ethanol or if grown aerobically on glucose and allowed to pass into stationary phase, with utilization of accumulated ethanol, maintain a normal value (0.8 to 0.9) of the adenylate energy charge during prolonged starvation. In contrast, cells grown anaerobically on glucose and cells in the early stages of aerobic growth on glucose exhibit a rapid decrease of energy charge if transferred to medium lacking on energy source. These results suggest that functional mitochondria or enzymes of balance of adenine nucleotides during starvation. Yeast cells remain viable at energy charge values below 0.1, in marked contrast to results previously obtained with Escherichia coli. In other respects, the engery charge responses of yeast to starvation and refeeding are generally similar to those previously reported for E. coli.     

Ribonucleic Acid from Aerobically and Anaerobically Grown Rhodopseudomonas spheroides: Comparison by Hybridization to Chromosomal and Satellite Deoxyribonucleic Acid

Ribonucleic acid (RNA) species from aerobically and anaerobically grown Rhodopseudomonas spheroides were compared via hybridization to deoxyribonucleic acid (DNA). Both long-labeled and stable RNA bound to chromosomal DNA to the same extent, regardless of derivation. About 4% of the chromosomal DNA hybridized with total cell RNA and about 0.08% with stable RNA. About 4% of the mixed satellite DNA could be hybridized to total cell RNA from aerobic or anaerobic cultures, whereas essentially no stable RNA formed a hybrid with this DNA. Hybridization competition experiments with aerobic and anaerobic pulse-labeled RNA and chromosomal or satellite DNA demonstrated that no qualitative differences existed between the RNA species. It is concluded that identical species of RNA in the same relative amounts are synthesized by R. spheroides during aerobic or anaerobic growth on the same medium.     

Long-term oxidant deficiency in Pseudomonas aeruginosa PAO303 results in cells which are non-culturable under aerobic conditions

Abstract The effect of long-term energy starvation (lack of electron acceptor in respiration) on the culturability of Pseudomonas aeruginosa PAO303 was studied by subsequent incubations for growth on aerobic medanaerobic media. A batch culture was grown on O₂-free citrate minimal medium containing NO₃⁻ as oxidant. Stationary phase was reached when NO₃⁻ was exhausted. This was followed by a rapid loss of cell culturability as tested by aerobic growth on agar plates (colony forming units, cfu) or on 0.2 μm membrane filters (epifluorescence technique) using the citrate minimal medium. However, energy-starved cells could form ten times more colonies when incubated anaerobically with NO₃⁻ (denitrifying conditions) than when incubated aerobically. Hence the energy starvation resulted in a subpopulation of cells, which were detectable under denitrifying, but not under aerobic growth conditions.     

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.     

Comparative studies of two membrane fractions isolated from chemotrophically and phototrophically grown cells of Rhodopseudomonas capsulata.

Light and heavy membrane fractions have been isolated by equilibrium sucrose density centrifugation from Rhodopseudomonas capsulata 938 GCM grown aerobically in the dark (chemotrophically) and anaerobically in the light (phototrophically). The densities of the light and heavy fractions from phototrophic cells were 1.1004 to 1.1006 and 1.1478, respectively, and the densities of the light and heavy fractions from chemotrophic cells were 1.0957 to 1.0958 and 1.1315, respectively. Both fractions were active in photochemical and respiratory functions and in electron transport-coupled phosphorylation. The light membrane fraction isolated from chemotrophic cells contained the reaction center and the light-harvesting pigment-protein complex B 870, but not the variable light-harvesting complex B 800-850. A small amount of the complex B 800-850 was present in the light fraction isolated from phototrophically grown cells, but it was not energetically coupled to the photosynthetic apparatus. From inhibitor studies, difference spectroscopy, and measurement of enzyme activities it was tentatively concluded that the light membrane fraction contains only the reduced nicotinamide adenine dinucleotide-oxidizing electron transport chain having a KCN-insensitive, low-potential cytochrome c oxidase, whereas the heavy fraction contains additionally the succinate dehydrogenase and a high-potential cytochrome b terminal oxidase sensitive to KCN. The light membrane fraction was more labile than the heavy fraction in terms of phosphorylating activity.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Effect of aerobically generated inoculum on xylose fermentation by an ethanologenic recombinantE. coli

Summary The anaerobic conversion of xylose to ethanol by a genetically engineredE. coli B (pLOI297) was investigated using anaerobically and aerobically grown cultures as inocula. Using anaerobically grown cells, an increase in the inoculation density from 50 to 340 mg dry wt. cells/L resulted in an increase in the overall volumetric productivity from 0.57 to 0.71 g/L/h. At the higher inoculation density, substitution of the anaerobic inoculum by aerobically grown cells resulted in a 15% reduction in volumetric productivity (0.61 g/L/h) that was caused by the introduction of a lag period during which the aerobic inoculum adapted to the anaerobic environment. In all cases, the ethanol yield from xylose approached the theoretical maximum and seemed unaffected by the physiological history of the inoculum with respect to aeration. It is concluded that aeration should be avoided in the production of high performance starter cultures.     

Early Stages of Conjugation in Escherichia coli

We initiated these studies to learn more about the initial events during bacterial conjugation and to optimize conditions for their occurrence. We found that cells in donor cultures grown anaerobically prior to mating have (i) a higher mean number of F pili per cell, (ii) longer F pili, (iii) a higher probability of forming specific pairs with F(-) cells, and (iv) a faster rate of initiation of chromosome transfer than cells grown aerobically. The growth medium for the donor culture also influences these same parameters: a rich medium is superior to a completely synthetic medium. Starvation of donor cells in buffered saline or for a required amino acid results in (i) a loss of F pili, (ii) a loss in the ability of donor-specific phages to adsorb, (iii) a loss of ability to form specific pairs with F(-) cells and to yield recombinants, and (iv) an increase in recipient ability. These changes occur as a function of starvation time, and at rates which are dependent on the conditions of prior growth and starvation of the donor culture. Either treatment provides a rapid method for the production of F(-) phenocopies from donor cultures. Resynthesis of F pili by cells within a starved donor culture commences very soon after restoration of normal growth conditions, but full restoration of donor ability, as measured by recombinant yield, occurs at a slower rate. We found, along with other investigators, that F pili are essential for specific pair formation. We also found, however, that the presence of F pili is not sufficient for display of donor ability, nor is the absence of F pili enough for cells to exhibit recipient ability. This suggests, therefore, that one or more components, in addition to F pili, are necessary for the conversion of specific pairs to effective pairs (or for chromosome mobilization, or both) and for preventing donor cells from acting as recipients. On the basis of our results, we suggest optimal conditions for achieving high mating efficiencies.     

Metabolism of the phase variants of the phototrophic bacterium Rhodobacter sphaeroides

Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobacter sphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2–3 times lower in the cells of the M variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa ₃, acting as cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient at the cytochrome aa ₃ level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.     

Hydrogenase activity in Rhodopseudomonas capsulata: relationship with nitrogenase activity.

Hydrogenase activity was found in cells of Rhodopseudomonas capsulata strain B10 cultured under a variety of growth conditions either anaerobically in the light or aerobically in the dark. The highest activities were found routinely in cells grown in the presence of H2. The hydrogenase of R. capsulata was localized in the particulate fraction of the cells. High hydrogenase activities were usually observed in cells possessing an active nitrogenase. The hydrogen produced by the nitrogenase stimulated the activity of hydrogenase in growing cells. However, the synthesis of hydrogenase was not closely linked to the synthesis of nitrogenase. Hydrogenase was present in dark-grown cultures, whereas nitrogenase synthesis was not significant in the absence of light. Unlike nitrogenase, hydrogenase was present in cultures grown on NH4+. Conditions were established which allowed the synthesis of either nitrogenase or hydrogenase by resting cells. We concluded that hydrogenase can be synthesized independently of nitrogenase.     

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria

Abstract From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.     

Anaerobic iron uptake by Escherichia coli.

Assimilation and uptake of iron in anaerobic cultures of Escherichia coli were supported by iron supplied as ferrienterobactin, ferrichrome, and ferrous ascorbate; however, as in the aerobic cultures, ferrichrome A was a poor iron source. Albomycin inhibited both aerobically and anaerobically grown cells. The siderophore outer membrane receptor proteins FepA and FhuA were produced under anaerobic iron-deficient conditions. Anaerobic transport of ferrienterobactin and ferrichrome was inhibited by KCN and dinitrophenol. The Km for ferrienterobactin uptake in anaerobically grown cells was 0.8 microM, and the Vmax was 38 pmol/min per mg, compared with 0.1 microM and 80 pmol/min per mg, respectively, in aerobically grown cells.     

Photooxidative production of carbon monoxide by phototrophic microorganisms

Net photosynthesis and CO production were measured in cell suspensions of Chlorella fusca. The rate of net photosynthesis showed saturation curves with increasing radiation intensities and CO₂-mixing ratios. Maximum rates were found at 35° C with a sharp decrease at higher temperatures. By contrast, the rate of CO production was proportional to the radiation intensity and did not show any saturation up to 1.5 kW · m^?2 white light. The CO-production rate was higher in blue than in red light and was independent of the CO₂-mixing ratio of the carrier gas within a range of 0–1000 ppmv. We found that the CO-production rate was constant within the physiological temperature range of 10–35° C, but increased considerably at higher temperatures and that CO production by the chlorophyll-deficient mutant of C. fusca was 5 times that of the wild type. In addition, we measured CO production in cell suspensions of Chromatium vinosum, Rhodopseudomonas sphaeroides and Rhodopseudomonas acidophila, which were grown either anaerobically in the light or aerobically in the dark. CO production could only be observed when the cells were incubated in the presence of oxygen and light. Under these conditions more CO was produced by aerobically grown cells than by phototrophically grown cells of R. sphaeroides and R. acidophila. The results obtained indicate that CO was produced by photosensitized oxidations and not by metabolic processes.