Lipid biosynthesis in synchronized cultures of the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid biosynthesis has been studied in photosynthetic cultures of Rhodopseudomonas sphaeroides that had been synchronized by stationary-phase cycling or by a centrifugation selection procedure. Synchrony index values in the range 0.70-0.80 were obtained for the first cell cycle with both synchronization methods. The major membrane lipids phosphatidylethanolamine and phosphatidylglycerol were accumulated discontinuously during the cell cycle, their mass doubling immediately before cell division. This accumulation of lipid corresponded to peaks in incorporation of radioactivity from either [1-14C]acetate or [2-3H]glycerol into individual acyl lipids as measured in individual portions of bacteria. For phosphatidylglycerol an additional peak of incorporation of radioactivity from [2-3H]glycerol was found midway through the cell cycle. In spite of their rather similar endogenous fatty acid compositions, the individual phosphoacylglycerols showed distinctive patterns of incorporation of radioactivity from [1-14C]acetate into their acyl moieties. The discontinuous synthesis of acyl lipids observed in cultures of Rhodopseudomonas sphaeroides synchronized by either stationary-phase cycling or centrifugation selection procedures contrasted with the accumulation of chlorophyll-protein complexes whose amounts were found to increase throughout the cell cycle. The implications of these findings for the control of lipid synthesis in bacterial photosynthetic membranes are discussed.     

Phospholipid composition of photosynthetic membranes of Rhodopseudomonas capsulata

The phospholipids and the fatty acids present in membranes of cells of Rhodopseudomonas capsulata, grown photosynthetically in anaerobiosis, were analyzed by thin layer chromatography and gas chromatography-mass spectrometry. The three phospholipids detected, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol, contained about 80% of a single monounsaturated C18 fatty acid, cis-vaccenic acid. These membranes offer therefore a naturally occurring model system endowed with an extremely simplified phospholipid complement. The data indicate moreover that the biosynthetic pathway of unsaturated fatty acids present in these facultative aerobic bacteria proceeds only via the 3-hydroxydecanoyl acyl carrier protein dehydratase (E.C. 4.2.1.60).     

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.

Strains of all 18 species of the family Rhodospirillaceae (nonsulfur photosynthetic bacteria) were studied for their comparative nitrogen-fixing abilities. All species, with the exception of Rhodocyclus purpureus, were capable of growth with N2 as the sole nitrogen source under photosynthetic (anaerobic) conditions. Most rapid growth on N2 was observed in strains of Rhodopseudomonas capsulata. Within the genus Rhodopseudomonas, the species R. capsulata, R. sphaeroides, R. viridis, R. gelatinosa, and R. blastica consistently showed the highest in vivo nitrogenase rates (with the acetylene reduction technique); nitrogenase rates in other species of Rhodopseudomonas and in most species of Rhodospirillum were notably lower. Chemotrophic (dark microaerobic) nitrogen fixation occurred in all species with the exception of one strain of Rhodospirillum fulvum; oxygen requirements for dark N2 fixation varied considerably among species and even within strains of the same species. We conclude that the capacity to fix molecular nitrogen is virtually universal among members of the Rhodospirillaceae but that the efficacy of the process varies considerably among species.     

Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.     

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.     

Acyl lipid changes in photosynthetic mutants of Rhodopseudomonas sphaeroides correlate with blocks in bacteriochlorophyll synthesis

Abstract In comparison with the wild-type, mutants of Rhodopseudomonas sphaeroides defective in bacteriochlorophyll synthesis fail to alter their lipid composition on shifting from non-photosynthetic to photosynthetic growth conditions. The earlier the lesion in the bacteriochlorophyll synthetic pathway, the more severe the effect on membrane lipid composition, indicating that acyl lipid and pigment syntheses are co-ordinated and linked to pigment-protein complex assembly.     

Identification of nitrogenase and carboxylase genes in the photosynthetic bacteria and cloning of a carboxylase gene from Rhodopseudomonas sphaeroides

The presumptive genes for the ribulose 1,5-bisphosphate carboxylase large subunit and for nitrogenase-specific components from Rhodopseudomonas sphaeroides and several other photosynthetic bacteria were identified and located by interspecific probing. Restriction digests of R. sphaeroides genomic DNA were hybridized under stringent conditions to cloned DNA from Rhodospirillum rubrum (plasmid pRR2119 carrying the carboxylase gene) and Klebsiella pneumoniae (pSA30 carrying the nitrogenase genes). The nitrogenase probe hybridized with different signal intensities to several distinct HindIII, BglII, EcoRI, BamHI and PvuII fragments of R. sphaeroides 2.4.1.DNA. The carboxylase probe hybridized to only single R. sphaeroides 2.4.1.DNA fragments produced with all five restriction enzymes. A 3000-bp EcoRI-BamHI R. sphaeroides 2.4.1.DNA fragment carrying the presumptive gene for the large subunit of ribulose 1,5-bisphosphate carboxylase was cloned into pBR322 and positively identified by probing with a 32P-labeled internal PstI fragment of the Rhodospirillum carboxylase gene.     

Fermentation of pyruvate by 7 species of phototrophic purple bacteria]

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.     

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.     

Effect of oxygen on acetylene reduction by photosynthetic bacteria

The effect of dissolved oxygen concentration on nitrogenase activity was studied in three species of photosynthetic bacteria. The O2 concentration in the cell suspension was measured with an O2 electrode inserted into the reaction vessel. Acetylene reduction by whole cells of Rhodopseudomonas capsulata, Rhodospirillum rubrum, and Chromatium vinosum strain D was inhibited 50% by 0.73, 0.32, and 0.26 microM O2, respectively. The inhibition of the activity by O2 in R. capsulata usually was reversed completely by reestablishing anaerobic conditions. In R. rubrum and C. vinosum the inhibition was only partially reversible. The respiration rate of R. capsulata was the highest of the three, that of R. rubrum was intermediate, and that of C. vinosum was lowest. R. capsulata and R. rubrum cells were broken after their acetylene reduction activity in vivo had been completely inhibited by O2, and nitrogenase was found to be active in vitro. A concentration of cyanide that did not affect acetylene reduction activity, but which inhibited 75 to 90% of the O2 uptake by whole cells of R. capsulata, shifted the O2 concentration causing 50% inhibition of nitrogenase activity from 0.73 microM to 2.03 microM. These results are in accordance with the assumption that within a limited range of O2 concentrations, the respiratory activity of the cells is enough to scavenge the O2 and to keep the interior of the cells essentially anaerobic. It is suggested that O2 inhibits nitrogenase activity by competing for a limited supply of electrons. When cyanide is present, respiration is slower but is adequate to keep the nitrogenase environment in the cell anaerobic. The lower respiration rate may allow a greater proportion of the electrons to be used for acetylene reduction.     

Hybridization of cloned Rhodopseudomonas capsulata photosynthesis genes with DNA from other photosynthetic bacteria

The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or bacteriochlorophyll biosynthesis genes showed either weak or undetectable heterospecific hybridization under the conditions employed.     

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. II. Comparison of diffraction patterns of photosynthetic units from various purple bacteria

Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dichroism of bacteriochlorophyll in chromatophores of photosynthetic bacteria.

The dichroism was measured in films of air-dried and, consequently, flattened chromatophores of Chromatium vinosum, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum. The values (deltaA/A) of dichroism in C. vinosum were found to be -1.05 at 590 nm and 0.75 in the near infrared region. The values of dichroism in R. sphaeroides were -0.70 at 590 nm and 0.80 at 870 nm. The values of dichroism in R. rubrum were -1.45 at 590 nm and 0.97 at 870 nm.     

Thiosulfate oxidation by nonsulfur purple bacteria

The capability to oxidize thiosulfate was studied in 11 cultures of purple bacteria belonging to Rhodomicrobium vannielii, Rhodopseudmonas viridis, Rh. sphaeroides, Rh. capsulata, and Rhodospirillum rubrum. All the bacteria oxidized thiosulfate under aerobic conditions in the dark. The strains 2R, 8259, A1, A2 and D1 of Rh. sphaeroides oxidized thiosulfate under anaerobic conditions in the light, and the process was coupled with carbon dioxide fixation. All the strains contained thiosulfate reductase, and the majority of them possessed also the activity of thiosulfate oxidase and sulfite oxidase.     

Generic interrelations of purple bacteria in the genus Rhodopseudomonas

The technique of DNA--DNA hybridization was used to study relations offween purple nonsulfur bacteria (the family Rhodospirillaceae). The level of homologies with Rhodopseudomonas sphaeroides 8259 was nearly the same for different species (8-17%) in the genus Rhodopseudomonas under the conditions optimal for hybridization. The same level of homologies was found for the DNA of Rhodospirillum rubrum, a species belonging to another genus of purple nonsulfur bacteria (13%). Rhodomicrobium vannielli was most remote from R. sphaeroides 8259 (3%). Similar results were obtained under other conditions of hybridization. The intraspecial heterogeneity of R. sphaeroides was studied in this work. The thermal stability of hybrid duplexes was analysed. The results are indicative of a considerable divergence of different R. sphaeroides strains (delta T50 = 2.1-11.6).     

<Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> has a family II pyrophosphatase: comparison with other species of photosynthetic bacteria

The cytoplasmic pyrophosphatase from Rhodobacter sphaeroides was purified and characterized. The enzyme is a homodimer of 64 kDa. The N-terminus was sequenced and used to obtain the complete pyrophosphatase sequence from the preliminary genome sequence of Rba. sphaeroides, showing extensive sequence similarity to family II or class C pyrophosphatases. The enzyme hydrolyzes only Mg-PP(i) and Mn-PP(i) with a K(m) of 0.35 mM for both substrates. It is not activated by free Mg (2+), in contrast to the cytoplasmic pyrophosphatase from Rhodospirillum rubrum, and it is not inhibited by NaF, methylendiphosphate, or imidodiphosphate. This work shows that Rba. sphaeroides and Rhodobacter capsulatus cytoplasmic pyrophosphatases belong to family II, in contrast to Rsp. rubrum, Rhodopseudomonas palustris, Rhodopseudomonas gelatinosa, and Rhodomicrobium vannielii cytoplasmic pyrophosphatases which should be classified as members of family I. This is the first report of family II cytoplasmic pyrophosphatases in photosynthetic bacteria and in a gram-negative organism.     

Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)

Abstract The evolutionary relationship of the RuBisCO large subunit gene(s) ( rbcL ) of several prokaryotes was examined using the technique of heterologous DNA hybridization. Restriction fragments of cloned rbcL from Anacystis nidulans 6301, Chlamydomonas reinhardtii, Rhodospirillum rubrum , and maize were nick-translated and used as probes. The C. reinhardtii and maize probes hybridized with restriction fragment(s) only from cyanobacteria: Agmenellum quadruplicatum, Fremyella diplosiphon , and Mastigocladus laminosus . In addition, the A. nidulans probe hybridized with restriction fragment(s) from Alcaligenes eutrophus, Chromatium vinosum, Nitrobacter hamburgensis, Paracoccus denitrificans, Pseudomonas oxalaticus, Rhodomicrobium vannielii, Rhodopseudomonas capsulata, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides, Thiobacillus intermedius, Thiobacillus neapolitanus , and Thiothrix nivea . The elucidated fragment of Rhodopseudomonas species is presumably for the Form I RuBisCO LSU of these organisms. The R. rubrum probe hybridized only to a restriction fragment(s) from R. capsulata, R. palustris, R. sphaeroides, T. neapolitanus , and T. nivea . The fragment(s) of Rhodopseudomonas species is the Form II rbcL of these organisms. The restriction fragments of T. neapolitanus and T. nivea were also different from those elucidated by the A. nidulans probe, suggesting the presence of a second (different) rbcL in these organisms. Positive hybridization was not obtained using any of the probes with DNA from Beggiatoa alba, Chlorobium vibrioforme or Chloroflexus aurantiacus . It appears that all rbcL have evolved from a common ancestor. Our data are consistent with and supportive of the evolutionary scheme for RuBisCO proposed by Akazawa, Takabe, and Kobayashi [1].     

Phospholipid Composition of Gluconobacter cerinus

Phospholipids of Gluconobacter cerinus were investigated. The cells of this bacterium were found to have cardiolipin, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine together with three ornithine-containing lipids. Phosphatidylcholine was the most abundant of these phospholipids, and calculated at 31.0% of total phospholipids in the cells at the late logarithmic phase. The fatty acid of this lipid was composed of palmitate, stearate and vaccenate, and there were no remarkable differences in quality among fatty acids of these four phospholipids. The phosphatidylcholine was also found in other acetic acid bacteria.     

Uptake of methionine sulfoximine by some N2 fixing bacteria, and its effect on ammonium transport

The N2 fixing bacteria Klebsiella pneumoniae, Azospirillum brasilense, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum, but not Azotobacter vinelandii accumulate the glutamine analogue methionine sulfoximine in the cell. In the accumulating cells methionine sulfoximine inhibits ammonium transport. Accumulation and inhibition are prevented by glutamine.     

Influence of pH, O2, and temperature on the absorption properties of the secondary light-harvesting antenna in members of the family Rhodospirillaceae

In some Rhodospirillaceae, the primary light-harvesting (LH I) antenna absorbs near-infrared light around 870 nm, whereas LH II (holochrome B800-860) has a major absorption band between 850 and 860 nm (B860) and a minor absorbancy around 800 nm (B800). Results show that, unlike LH I, holochrome B800-860 (LH II) exhibits unstable light absorption properties in whole cells. This was observed in Rhodopseudomonas capsulata grown anaerobically in light in weakly buffered carbohydrate medium; cultures lost both carotenoid-dependent brown-yellow pigmentation and LH II absorbancy. The whole cell spectrophotometric changes were attributed to mild acid conditions generated during sugar metabolism. LH II absorbancy was also destroyed in both R. capsulata and Rhodopseudomonas gelatinosa when cultures growing at neutral pH were acidified to a pH value around 5.0 with HCl. In contrast, during the same time period of exposure to pH 5.0, only a 50% decrease in Rhodopseudomonas sphaeroides LH II B800 absorbancy was measured. At neutral pH, LH II absorbancy in suspensions of nongrowing Rhodopseudomonas spp. was also sensitive to O2 exposure and to incubation at 30 to 40 degrees C. During treatment with O2, the rate of LH II B800 absorption decrease in R. gelatinosa and R. sphaeroides was 60 and 40% per h, respectively, compared with their absorbancy maximum around 860 nm. Both 860-nm absorbancy and the total bacteriochlorophyll content of the cells remained unchanged. On the other hand, no significant decrease in B800 if LH II in R. capsulata occurred during O2 exposure, but a 20% absorption decay rate per h of B800 was observed in cells incubated anaerobically at 40 degrees C. These B800 LH II spectral changes Rhodopseudomonas spp. were prevented by maintaining cells at neutral pH and at 10 degrees C. The near-infrared absorption spectrum of Rhodospirillum rubrum, which does not form LH II, was not significantly influenced by these different pH, aerobic, or temperature conditions.