Localization and characterization of the sn-glycerol-3-phosphate acyltransferase in Rhodopseudomonas sphaeroides

The membrane localization and properties of the Rhodopseudomonas sphaeroides sn-glycerol-3-phosphate acyltransferase have been examined utilizing enzymatically prepared acyl-acyl carrier protein (acyl-ACP) substrates as acyl donors for sn-glycerol-3-phosphate acylation. Studies conducted with membranes prepared from chemotrophically and phototrophically grown cells show that sn-glycerol-3-phosphate acyltransferase activity is predominantly (greater than 80%) associated with the cell's cytoplasmic membrane. Enzyme activity associated with the intracytoplasmic membranes present in phototrophically grown R. sphaeroides was within the range attributable to cytoplasmic membrane contamination of this membrane fraction. Enzyme activity was optimal at 40 degrees C and pH 7.0 to 7.5, and required the presence of magnesium. No enzyme activity was observed with any of the long-chain acyl-CoA substrates examined. Vaccenoyl-ACP was the preferred acyl-ACP substrate and vaccenoyl-ACP and palmitoyl-ACP were independently utilized to produce lysophosphatidic and phosphatidic acids. With either vaccenoyl-ACP or palmitoyl-ACP as sole acyl donor substrate, the lysophosphatidic acid formed was primarily 1-acylglycerol-3-phosphate and the Km(app) for sn-glycerol-3-phosphate utilization was 96 microM. The implications of these results to the mode and regulation of phospholipid synthesis in R. sphaeroides are discussed.     

Stringency in the absence of ppGpp accumulation in Rhodobacter sphaeroides.

Leucine deprivation of either phototrophically or chemotrophically growing cells of Rhodobacter sphaeroides resulted in a restriction in the continued accumulations of cellular RNA, phospholipids, and protein. Phototrophically growing cells also displayed restrictions in the accumulations of cellular carotenoids and bacteriochlorophyll. Leucine deprivation, however, did not provoke the accumulation of cellular ppGpp or alter the steady-state levels of ppGpp, ATP, or GTP in cells of R. sphaeroides.     

Electron-transport chains of phototrophically and chemotrophically grown Chloroflexus aurantiacus

The membrane-bound electron-transfer chain components of both phototrophically and chemotrophically grown Chloroflexus aurantiacus have been characterized. Membranes isolated from chemotrophically grown Chloroflexus have been shown to contain at least three c-type cytochromes and at least three b-type cytochromes. In addition, these cells appear to lack a photochemical reaction center and the high potential (E_m = +260 mV) cytochrome c-554 that serves as the immediate donor to the reaction center in phototrophically grown Chloroflexus. Phototrophically grown cells contain a CO-binding c-type cytochrome, apparently absent in the chemotrophically grown cells. However, a different CO-binding component, which may function as the terminal oxidase, is present in chemotrophically grown cells.     

The light-harvesting complex II (B800-850) of Rhodobacter sulfidophilus: Characterization and formation under different growth conditions

Abstract The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800–850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 μg Bchl per mg cell protein when grown at high light intensity (20 000 lux) to 60 μg Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographie analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of a and one type of β polypeptide in the purified complex, of which the N-terminal sequences have been determined.     

Porin from Rhodopseudomonas sphaeroides

A protein homooligomer was purified from both the cell envelope fractions and the saline extracts of Rhodopseudomonas sphaeroides cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with egg phosphatidylcholine. In the saline extracts of both chemotrophically and phototrophically grown cells, the porin oligomer was the most predominant polypeptide, which produced pores whose behavior toward various sugars could be approximated by hollow cylinders of 0.62 nm in radius. The oligomer was dissociated, in the presence of EDTA, into monomers that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as though their molecular weight was about 47,000. The monomer was active in the reconstitution assay and produced pores with sizes comparable to those produced by the oligomer. Circular dichroism spectra indicated the predominance of beta-sheet structure in both the oligomeric and EDTA-dissociated monomeric forms. Drastic conditions, for example, precipitation with 10% trichloroacetic acid or heating for a few hours at 100 degrees C in sodium dodecyl sulfate, were necessary to denature the protein into a form with a reduced content of beta-sheet structure.     

Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria.

Antibodies were raised against the succinate dehydrogenase (SDH) present in the chromatophores of phototrophically grown Rhodopseudomonas sphaeroides. Crossed immunoelectrophoresis experiments indicated that the SDH present in the cytoplasmic membranes of heterotrophically grown R. sphaeroides is probably the same enzyme observed in the chromatophores. The enzyme was extracted by Triton X-100 in a form which consisted of only two subunits (molecular weight, 68,000 and 30,000) and was not associated with a cytochrome b. The antibodies directed against SDH from R. sphaeroides showed no immunocross-reactivity with SDH from phylogenetically related bacterial species, including Rhodopseudomonas capsulata, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodospirillum rubrum, and Rhodospirillum fulvum.     

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.     

Disappearence of the intracytoplasmic membranes inRhodopseudomonas sphaeroides

The photosynthetic bacterium,Rhodopseudomonas sphaeroides, can be grown phototrophically (light, anaerobiosis), of chemotrophically (dark, aerobiosis). In the first case, it contains intracytoplasmic membranes with photosynthetic pigments. When shifted from phototrophy to chemotrophy these membranes disappear in an unknown fashion. In the present experiment, samples were taken for electron microscopy, cell density and bacteriochlorophyll determinations after shift from phototrophy to chemotrophy. The density of intracytoplasmic vesicles was measured on micrographs. During the first 2h growth is very slow and the ultrastructure remains unaltered. As growth resumes, the vesicles disappear at a rate which implies that they are not incorportated into the cytoplasmic membrane, nor actively digested, but remain intact and become increasingly diluted in the cytoplasm as the culture grows. The size of the vesicles was estimated to about 500 Å. The number of vesicles in phototrophically grown cells was calculated to about 575 per cell, and after 6h chemotrophic growth to about 100. The areas of the cytoplasmic and intracytoplasmic membranes are roughly calculated.Abbreviations Bchl bacteriochlorophyll - CM cytoplasmic membranes - ICM intracytoplasmic membranes     

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.     

Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides has a pyridine nucleotide-independent L-lactate dehydrogenase associated with the membrane fraction of cells grown either aerobically or phototrophically. The dehydrogenase is present in cells grown on a variety of carbon sources, but at levels less than 20% of that found in cells grown with DL-lactate. The dehydrogenase has been purified 45-fold from membranes of strain L-57, a non-photosynthetic mutant, by steps involving solubilization with lauryl dimethylamine oxide and three anion-exchange chromatography steps. The purified enzyme was specific for the L-isomer of lactate. The Km of the purified enzyme for L-lactate is 1.4 mM, whereas that of the membrane-associated enzyme is 0.5 mM. The enzyme activity was inhibited competitively by D-lactate and non-competitively by oxalate and oxamate. Quinacrine, a flavin analog, also inhibited the activity. The inducible enzyme may serve as a marker of membrane protein in studies of membrane development.     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll alpha-depleted cytoplasmic membrane in phototrophically grown cells.

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll alpha-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by less than 9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is condluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

Rhodopseudomonas sphaeroides membranes: alterations in phospholipid composition in aerobically and phototrophically grown cells.

The effects of growth conditions on phospholipid composition in Rhodopseudomonas sphaeroides have been reexamined. The levels of phosphatidylethanolamine (27 to 28%), phosphatidylglycerol (23 to 24%), and phosphatidylcholine (11 to 18%) were very similar in cells grown aerobically or phototrophically at a high light intensity, consistent with findings for another member of Rhodospirillaceae. In addition, an unknown phospholipid species was detected which comprised 20 to 30% of the total phospholipid in these cells. In cells growing phototrophically at low-intensity illumination, the level of phosphatidylethanolamine increased by about 1.6-fold and that of the unknown phospholipid markedly decreased. Although the synthesis of photosynthetic pigments, light-harvesting protein, and intracytoplasmic photosynthetic membranes also increased markedly, the ratios of individual phospholipid species were essentially identical in photosynthetic membrane and cell wall fractions purified from these cells. Since a significant exchange of lipids apparently did not occur during the isolation of these fractions, it was suggested that the changes in cellular phospholipid accumulation were not due to a unique composition within the photosynthetic membrane. Instead, these phosphoglyceride changes were found to be related to overall phospholipid metabolism and could be accounted for principally by differences in biosynthetic rates. These results, together with studies in nutrient-restricted aerobic cells, suggested that the mechanism by which phospholipid levels are regulated may be related to radiant energy flux rather than cellular energy limitation.     

Broad-host-range plasmid vector for the in vitro construction of transcriptional/translational lac fusions

A broad-host-range plasmid was constructed that allows the in vitro formation of beta-galactosidase fusions. DNA from the photosynthetic bacterium Rhodopseudomonas sphaeroides was cloned into this plasmid and a number of R. sphaeroides isolates were recovered that had varying levels of beta-galactosidase activity. beta-galactosidase antigenic activity from the fusion strains could be localized immunologically in polypeptides with an Mr of 120 000 or greater. Expression of beta-galactosidase activity under control of fusion derivatives was either very low or nonexistent in Escherichia coli relative to R. sphaeroides, indicating that R. sphaeroides promoters or translational start signals function poorly in E. coli.     

Membranes of Rhodopseudomonas sphaeroides: effect of cerulenin on assembly of chromatophore membrane.

The effects of cerulenin were investigated in Rhodopseudomonas sphaeroides to elucidate further the mechanisms controlling the assembly of the chromatophore membrane. When this potent inhibitor of fatty acid biosynthesis was added to photosynthetically grown cultures, there was an immediate cessation of phospholipid, bacteriochlorophyll a, carotenoid, and ubiquinone formation. Concurrently, there was also a marked decrease in the rate of incorporation of protein into the chromatophore membrane. In contrast, only a small decrease in the rate of soluble and cell envelope protein synthesis was observed and, in chemotrophically grown cells, protein continued to be incorporated into both the cytoplasmic and outer membranes. The removal of delta-aminolaevulinate from mutant H-5 of R. sphaeroides, which requires this porphyrin precursor, was reexamined to determine whether cerulenin-induced cessation of chromatophore protein incorporation was due solely to blocked bacteriochlorophyll a synthesis. In the deprived H-5 cells, inhibition of [35S]methionine incorporation into chromatophores was confined mainly to apoproteins of bacteriochlorophyll a complexes. Other minor chromatophore proteins continued to be inserted to a greater extent than in cerulenin-treated wild type where phospholipid synthesis has also ceased. These results indicated that the assembly of the chromatophore membrane is under strict regulatory control involving concomitant phospholipid, pigment, and protein syntheses.     

Characterization of cell-envelope fractions of chemotrophically and phototrophically grown Rhodospirillum tenue

Envelope preparations of chemotrophically and phototrophically grown Rhodospirillum tenue were isolated and characterized on the basis of their contents and composition in phospholipids and fatty acids, as well as on the basis of their enzyme activities, absorption spectra and polypeptide patterns. Both preparations were similar in their contents in phospholipids and fatty acids. Their total fatty acids were characterized by a rather high percentage of saturated fatty acids. The activities of the respiratory reactions were considerably higher in chemotrophic than in phototrophic membranes. This is true especially for activities of the terminal oxidase which were over 20 times greater. The affinities of the corresponding enzymes to their respective substrates (K _m ) differed with differences in the culture conditions. Under chemotrophic conditions the K _m values for the NADH-dependent reactions were lower than those values under phototrophic conditions, whereas the K _m values for the succinate dependent reactions were higher. The low temperature (77°K) near infrared spectrum of the phototrophic membrane showed a peak at 875 nm which was not detectable in the chemotrophic membrane. The polypeptide patterns, in the presence of sodium dodecyl sulfate, of both preparations were quite similar except for the presence of two low molecular weight proteins (M. Wt. 12,000 and 10,000) in the phototrophic membrane which were absent in the chemotrophic membrane. Both envelope preparations were further fractionated into enriched cytoplasmic membrane and outer membrane fractions which were identified on the basis of their contents in 2-keto-3-deoxyoctonate, ubiquinone 8 and bacteriochlorophyll (in the case of the phototrophic membrane) and their enzyme activities. The buoyant densities of the corresponding fractions from both envelope preparations were found to be equal. The data presented in this paper demonstrate that envelope preparations of chemotrophically and phototrophically grown R. tenue are similar in their contents in 2-keto-3-deoxyoctonate, ubiquinone 8, phospholipids and fatty acids, yet differ significantly in their spectra, protein patterns and enzyme activities.Abbreviations BChl bacteriochlorophyll - UQ_s ubiquinone 8 - KDO 2-keto-3-deoxyoctonate - PG phosphatidyl glycerol - PE phosphatidyl ethanolamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Effects of ionophores and TPMP+ on light-induced responses in Dictyostelium discoideum

In spite of previous reports, the activities of respiratory oxygen uptake by whole cells are higher with chemotrophically than with phototrophically grown cells of Rhodospirillum rubrum and Rhodospirillum tenue. The same applies to NADH dependent respiratory reactions as determined with isolated crede membrane preparations. This is largely, but not only, due to an outstandingly high increase in activity of cytochrome c-oxidase measurable upon adaptation of phototrophically grown cells to chemotrophic conditions. In R. rubrum the dependency of the total respiratory chain on the activities of different sections of this chain becomes confused by the presence of differently composed membranes (i.e. cytoplasmic and intracytoplasmic membranes) which under the experimental conditions become functionally differentiated to different extents. But in R. tenue, which does not produce intracytoplasmic membranes, respiration at low activities parallels clearly cytochrome c oxidase activities while high respiratory activities parallel the activities of NADH dehydrogenase. The data are interpreted to indicate that, in cells of facultative phototrophic bacteria, the formation of the respiratory chain, up to certain stages, depends on the formation of the terminal oxidase. At least in R. tenue this is comparable to the role of bacteriochlorophyll in the formation of the photosynthetic apparatus.Abbreviation Bchl bacteriochlorophyll     

Plasmid rearrangements in the photosynthetic bacterium Rhodopseudomonas sphaeroides

Mu d1(Ap lac) was introduced into the photosynthetic bacterium Rhodopseudomonas sphaeroides 2.4.1. via the R-plasmid R751 in an attempt to isolate fusion derivatives involving photosynthetic operons. The selection system is potentially very powerful since R. sphaeroides is normally Lac negative. Among the exconjugants, photosynthesis-deficient mutants were recovered, some of which had elevated beta-galactosidase levels. Among the mutants examined, beta-galactosidase expression was linked exclusively to R751 . Many of the photosynthesis-deficient mutants were found to have alterations in their indigenous plasmids which apparently involved the exchange of DNA from one plasmid to another. Southern blot analysis revealed that there are extensive DNA sequences which are shared by the two plasmids that are involved in the rearrangements and that no exogenous DNA sequences appear to be involved. It was further discovered that plasmid rearrangement is a general phenomenon which can occur spontaneously in R. sphaeroides 2.4.1 and shows a high correlation with a photosynthesis minus phenotype.     

Localization and environmental regulation of MCP-like proteins in Rhodobacter sphaeroides

Chemotaxis to many compounds by Rhodobacter sphaeroides requires transport and at least partial metabolism of the chemoeffector. Previous investigations using phototrophically grown cells have failed to find any homologues of the MCP chemoreceptors identified in Escherichia coli. However, using an antibody raised against the highly conserved domain of E. coli Tsr, MCP-like proteins were identified in R. sphaeroides WS8N. Analysis using Western blotting and immunogold electron microscopy showed that expression of these MCP-like proteins is environmentally regulated and that receptors are targeted to two different cellular locations: the poles of the cells and the cytoplasm. In aerobically grown cells, these proteins were shown by immunoelectron microscopy to localize predominantly to the cell poles and to an electron-dense body in the cytoplasm. Western blot analysis indicated a 17-fold reduction in protein concentration when cells were grown in the light. The number of immunogold particles was also dramatically reduced in anaerobically light-grown cells and their cellular distribution was altered. Fewer receptors localized to the cell poles and more particles randomly distributed within the cell, but the cytoplasmic cluster remained. These trends were more pronounced in cells grown anaerobically under dim light than in those grown anaerobically under bright light, suggesting that expression is controlled by redox state and either light intensity or the extent of photosynthetic membrane synthesis. Recent work on E. coli chemosensing suggests that oligomerization of receptors and chemosensory proteins is important for sensory signalling. The data presented here suggest that this oligomerization can occur with cytoplasmic receptors and also provides an explanation for the multiple copies of chemosensory proteins in R. sphaeroides.     

Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide.

Rhodobacter sphaeroides 2.4.1, which is incapable of denitrification, has been found to carry nnrR, the nor operon, and nnrS, which are utilized for denitrification in R. sphaeroides 2.4.3. The gene encoding nitrite reductase was not found in 2.4.1. Expression of beta-galactosidase activity from a norB-lacZ fusion was activated when cells of 2.4.1 were incubated with NO-producing bacteria. This result indicates that the products of nnrR and the genes flanking it are utilized when 2.4.1 is growing in an environment where denitrification occurs.