Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides.

Triton extracts of intracytoplasmic photosynthetic membranes (chromatophores) purified from Rhodopseudomonas sphaeroides were subjected to crossed immunoelectrophoresis with antiserum raised in rabbits to purified chromatophores. A total of 31 immunoprecipitates was visualized; 2 of the immunoprecipitates were identified as reduced nicotinamide adenine dinucleotide (EC 1.6.99.3) and L-lactate dehydrogenases by enzyme staining techniques. Reaction with a monospecific antiserum identified the photochemical reaction center. Photopigments were associated with a major precipitate in the pattern which was identified on the basis of immunological identity as light-harvesting bacteriochlorophyll a . protein complex. These results provide the basis for a detailed structural and functional analysis of the chromatophore membrane by crossed immunoelectrophoresis.     

Assessment of Rhodopseudomonas sphaeroides chromatophore membrane asymmetry through bilateral antiserum adsorption studies.

The asymmetric structure of the Rhodopseudomonas sphaeroides chromatophore membrane was examined in detail by crossed immunoelectrophoresis techniques. Because these methods are quantitative and allow increased resolution and sensitivity, it was possible to analyze simultaneously the relative transmembrane distribution of a number of previously identified antigenic components. This was demonstrated by analysis of immunoglobulin samples that were adsorbed by preincubation with either isolated chromatophores or osmotically protected spheroplasts. The photochemical reaction center, the light-harvesting bacteriochlorophyll a-protein complex, the L-lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3) were found to be exposed on the chromatophore surface (cytoplasmic aspect of the membrane within the cell). Other antigenic components were found to be exposed on the surface of spheroplasts (periplasmic aspect of the in vivo chromatophore membrane). Antigens with determinants expressed on both sides of the chromatophore membrane were also identified. Charge shift crossed immunoelectrophoresis confirmed the suggested amphiphilic character of the pigment-protein complexes and identified several additional amphiphilic membrane components.     

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.     

Average distance between bacteriochlorophyll molecules on chromatophore membranes of Rhodopseudomonas sphaeroides

The average distance betweeen bacteriochlorophyll moleculeson the chromatophore membrane of Rhodopseudomonas sphaeroideswas estimated by two methods: measurement of the ratio of thenumber of chromatophores to the number of polystyrene-latexparticles mixed in a chromatophore suspension and measurementof the packed volume of chromatophores in the suspension. Theaverage distance was obtained under the assumption that bacteriochlorophyllmolecules are positioned at hexagonal lattice points. The valuewas 34 A{ring} when the bacteriochlorophyll molecules were assumedto be on one side of the membranes and 48 A{ring} when theywere assumed to be on both sides. (Received February 20, 1978; )     

Bacteriochlorophyll a cation radical in solution and in reaction centers of Rhodopseudomonas sphaeroides. Resonance Raman scattering

Resonance Raman spectra of the π-cation of bacterio-chlorophyll a in solution at 30 K are reported and discussed. Outer C

C bonds of the pyrroles and the methine bridges are weakened by the ionization, while C

N and Mg-N bonds remain essentially unaffected. Resonance Raman spectra of reaction centers suggest that the positive charge on P-870⁺ should be localized on a single bacteriochlorophyll molecule by the lifetime of the scattering process (≈ 10^?13 s).     

Nitrate reductase from Rhodopseudomonas sphaeroides.

The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed.     

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.     

Phytol and Bacteriochlorophyll Synthesis in Rhodopseudomonas spheroides

Phytol has been separated and identified in the unsaponifiable lipid fraction from wild type Rhodopseudomonas spheroides, but it was not detected in mutant strains blocked at various stages of bacteriochlorophyll synthesis. Incorporation of ¹⁴C-acetate into phytol paralleled bacteriochlorophyll synthesis in suspensions of the wild type incubated anaerobically in the light. The addition of chloramphenicol inhibited both processes. It is concluded that phytol formation is tightly coupled to the synthesis of the pyrrole component of bacteriochlorophyll.     

Nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023.

Chemical analysis of the lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023, isolated by the phenol-chloroform-petroleum ether method, revealed the presence of glucuronic acid, 2-keto-3-deoxyoctonate, threonine, and phosphorus in the polysaccharide moiety. The lipid A component contained glucosamine, glucosamine phosphate, amide-bound 3-oxotetradecanoic acid and 3-hydroxytetradecanoic acid, and ester-bound 3-hydroxydecanoic acid and 7-tetradecenoic acid. Structural similarity of the lipid A from R. sphaeroides ATCC 17023 to enterobacterial lipid A is indicated by the existence of a serological cross-reaction occurring between the lipid A from R. sphaeroides ATCC 17023 and that from Salmonella minnesota R595. The lipopolysaccharide and lipid A of R. sphaeroides, however, were found to be neither toxic in mice nor pyrogenic in rabbits.     

Glycerol dissimilation in Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides followed a diauxic growth curve when grown on a malate-glycerol medium, the first phase of growth being supported by malate and the second by glycerol. A soluble glycerokinase and a particulate, pyridine nucleotide-independent glycerophosphate dehydrogenase, were induced by the presence of glycerol in the medium, but neither was fully expressed nor functional until all malate had been consumed.     

Delayed fluorescence from Rhodopseudomonas sphaeroides following single flashes.

Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.     

Plasmid transfer and expression in Rhodopseudomonas sphaeroides.

Plasmid RP4 (among others) has been transferred from Escherichia coli to Rhodopseudomonas sphaeroides. Data bearing on the physical presence of the plasmid and its expression of drug resistance determinants in R. sphaeroides are presented. Conditions of transfer between R. sphaeroides strains, between R. sphaeroides and R. capsulata, and between R. sphaeroides and E. coli have been carefully defined.     

Denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO ₃ ^- under anaerobic conditions accumulated NO ₂ ^- in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO ₃ ^- , NO ₂ ^- and N₂O to N₂ gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N₂O reductase was inhibited by NaN₃, C₂H₂ and KCNS. Cells incubated with either K¹⁵NO₃ or K¹⁵NO₂ produced ¹⁵N₂O and ¹⁵N₂. A stoichiometry of 2:1 was recorded for the reduction of either NO ₃ ^- or NO ₂ ^- to N₂O and N₂ and for N₂O to N₂ it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen     

Diffusion-potential-induced oxidation and reduction of cytochromes in chromatophores from Rhodopseudomonas sphaeroides.

A membrane potential jump was induced by the addition of valinomycin in the presence of a KCl concentration gradient across the membrane of Rhodopseudomonas sphaeroides chromatophores. As well as a carotenoid band shift, which is known to be an indicator of membrane potential, absorbance changes due to the oxidation-reduction reactions of cytochromes accompanied the jump. Under aerobic conditions with no reductant added, a part of cytochrome c2 was reduced by an inside-positive potential jump of about 100 mV in the time range of tens of seconds. This can be explained by the location of the cytochrome on the inner side of the chromatophore membrane and electrophoretic flow of electrons across the membrane. On the other hand, in the presence of 1 mM ascorbate, a similar jump of membrane potential induced a rapid oxidation of cytochrome c2 and a subsequent reduction. A rapid reduction of b-type cytochrome was also observed. Antimycin A inhibited the c2 oxidation, but did not inhibit the b reduction. The oxidation of cytochrome c2 may be explained by a diffusion-potential-induced electron flow to cytochrome b and a simultaneous electron donation by cytochrome b and cytochrome c2 to a common electron acceptor, possibly a quinone.     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Fusion of chromatophores derived from Rhodopseudomonas sphaeroides

Fusion of chromatophores, the photosynthetic membrane vesicles isolated from the intracytoplasmic membranes of Rhodopseudomonas sphaeroides, was achieved by the use of poly(ethylene glycol) 6000 as fusogen. Ultracentrifugation, electron microscopy, intrinsic density and isotope labeling were used to demonstrate chromatophore fusion. Although studies of the flash-induced shift in the carotenoid absorbance spectrum indicated that the membrane was rendered leaky to ions by either the fusion procedure or the increased size of the fused products, the orientation and integrity of fused chromatophores were otherwise demonstrated to be identical to control chromatophores by freeze-fracture electron microscopy, proteolytic enzyme digestion, enzymatic radioiodination, and transfer of chromatophore phospholipids mediated by phospholipid exchange protein extracted from Rps. sphaeroides.     

Purification and properties of the adenylate kinases from Rhodopseudomonas palustris,Rhodopseudomonas sphaeroides and Rhodopseudomonas rubrum

The adenylate kinases (EC 2.7.4.3) from photosynthetically grown Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum were purified to homogeneity by the same procedure. The purified enzymes showed optimal rates of activity with MgCl₂ at 25° C and pH 8.0. They were found to be heat labile and were characterized by pI-values of 4.5. Apparent molecular weights of 33 500 for R. palustris, 34 400 for R. sphaeroides and 32 100 for R. rubrum were determined by high performance liquid chromatography. No separation into subunits was observed by use of sodium dodecylsulfate polyacrylamide gel electrophoresis. The apparent K _m -values for ADP corresponded to 0.26 mM for R. palustris, 0.27 mM for R. sphaeroides and 0.24 mM for R. rubrum. ADP in excess had a strong inhibitory effect. Competitive product inhibition was found for AMP, with K _i-values of 0.017 mM for R. palustris, 0.018 mM for R. sphaeroides and 0.014 mM for R. rubrum. A competitive inhibitor likewise was P¹,P⁵-di(adenosine-5)pentaphosphate with K _i-values of 0.020 M for R. palustris and R. sphaeroides, and 0.017 M for R. rubrum. Sulfhydryl-reacting reagents like p-chloromercuribenzoate and iodoacetic acid were found to be non-inhibitory. All measurements of adenylate kinase activity were carried out with the stabilized and most sensitive luciferin-luciferase system.