Membranes of Rhodopseudomonas spheroides: Interactions of Chromatophores with the Cell Envelope

Under carefully controlled ionic conditions, large-scale preparations of highly purified chromatophores and cell envelopes were obtained from phototrophically grown Rhodopseudomonas spheroides by zonal ultracentrifugation. The majority of the bacteriochlorophyll a was located in a single, discrete chromatophore band, whereas the envelopes were nearly devoid of photopigment. The envelope fraction contained substantial quantities of succinic dehydrogenase and cytochromes, confirming that phototrophically grown cells contain a photopigment-deficient cytoplasmic membrane. Magnesium at concentrations of 1.0 mM or higher caused chromatophores to reversibly aggregate with the cell envelope. Significant aggregation was also promoted by other divalent metals (Co(2+) > Mn(2+) > Ca(2+) > Mg(2+)), but aggregation was less extensive with monovalent cations. These results account for the distribution of photopigments in two bands reported by others and further suggest that the photosynthetic apparatus of R. spheroides is located on membranes largely distinct from the cell wall-cytoplasmic membrane complex.     

Localization of ferrochelatase and of newly synthesized haem in membrane fractions from Rhodopseudomonas spheroides.

Cells of Rhodopseudomonas spheroides, strains R-26 or GVP, were grown photosynthetically, disrupted and two particulate fractions separated by sucrose-density-gradient centrifugation. The upper particulate fraction, enriched in bacteriochlorophyll, was identified as containing the chromatophores; the lower particulate fraction had the characteristics of the cell envelope. The two fractions differed in cytochrome content and cytochrome spectra. Ferrochelatase was found almost exclusively in the chromatophore fraction and was located on the outer face of the chromatophores, i.e. in contact with the cytosol in intact cells. The addition of 59FeCl3 to cells growing in low-iron media resulted in labelling of the protohaem fraction (probably arising from cytochrome b) of the membranes. The specific radioactivity of the haem of the chromatophores rose more rapidly than that of the envelope fraction and then after 2 h declined to approximately the same value, suggesting that haems of the chromatophore may act as precursors of haem of the envelope.     

Asymmetry of an energy transducing membrane the location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata.

Monospecific antibodies have been prepared against cytochrome c2 from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c' from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth. Cytochrome c2 is located in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.     

Isolation and fractionation of the photosynthetic membranous organelles from Rhodopseudomonas spheroides

Molecular sieve chromatography and sucrose gradient centrifugation were used to prepare large quantities of purified chromatophores from Rhodopseudomonas spheroides. Electron micrographs of these chromatophores revealed that the final preparations were very homogeneous and free of non-chromatophore particulate material. As an additional check on purity, (14)C-l-phenylalanine-labeled aerobic cells, devoid of chromatophores, were mixed with unlabeled photosynthetic cells. The resulting preparation contained less than 1% of the radioactivity, originally located in non-chromatophore protein. The purified chromatophores were solubilized in 2-chloroethanol and separated into two fractions. Fraction P(1) contained 3 to 5% of the total chromatophore protein and could be resolved into 10 electrophoretic components. The second fraction, P(II), contained five electrophoretic components. One of these components had associated with it all of the pigment and phospholipid present in P(II). Preliminary immunochemical studies on these fractions are also reported.     

Isolation and Characterization of a Temperate Bacteriophage Specific for Rhodopseudomonas spheroides

The isolation of a temperature phage specific for the photosynthetic bacterium Rhodopseudomonas spheroides is reported. This phage, Rphi-1, establishes a state of lysogeny and can be induced from the prophage state by exposure to mitomycin C or UV irradiation. Mutants of Rphi-1 which grow on a standard laboratory strain (2.4.1) of Rhodopseudomonas spheroides were isolated. Although the original Rphi-1 isolated was chloroform sensitive, the mutant which plates on strain 2.4.1 is chloroform resistant. Rphi-1 does not grow on closely related bacteria, such as Rhodopseudomonas palustris or Rhodopseudomonas capsulata. Rphi-1 mutants forms plaques with the same efficiency whether the plates are incubated under aerobic conditions in the dark or under anaerobic conditions in the light (phototropic conditions).     

Deficiencies of Chromatophore Proteins in Some Mutants of Rhodopseudomonas spheroides with Altered Carotenoids

Chromatophore proteins of a wild type and three mutant strains of Rhodopseudomonas spheroides were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mutants consisted of a green and a blue-green one, whose phenotypes were essentially the same as those of known mutants, and a brown one, which may be a double mutant and represents a new phenotype. Wild-type chromatophores contained at least six major and seven minor protein bands, with molecular weights ranging from 10,000 to 65,000. The green mutant contained the same protein bands in the same relative quantities. The brown mutant had one protein completely missing and no other alterations. The blue-green mutant was deficient in a different protein, and had reduced quantities of all proteins with molecular weights less than 25,000. Chromatophores were separated into a fraction containing the reaction centers and a fraction containing the light-harvesting bacteriochlorophyll by treatment with sodium dodecyl sulfate. Eight of the proteins were found only in the reaction center fraction, one was only in the light-harvesting fraction, and the remainder were present in both fractions. The protein missing from the brown mutant was found to be a component of the reaction center fraction, whereas the proteins which were missing from the blue-green mutant were all components of the light-harvesting fraction. Some implications for the structure and biogenesis of chromatophores are discussed.     

Localisation of the subunits of the photosynthetic reaction centers in the chromatophore membrane of Rhodospirillum rubrum.

Reaction centers were isolated with the detergent lauryl dimethyl amine oxide from chromatophore membranes of Rhodospirillum rubrum. The subunit composition of these reaction centers is similar to the one obtained from Rhodopseudomonas spheroides: three subunits with the molecular weights of 21 000, 24 000 and 29 000. Reaction centers prepared from chromatophores labeled with 131I were heavely labeled in their large subunit (H). The smaller subunits (L and M) contained only little label. Sonication during labeling yielded a slightly higher incorporation of 131I in subunit H compared to the smaller ones. It is concluded that the H protein is largely exposed at the cytoplasmic side of the membrane but might also be accessible for iodination on the inside of the membrane while the L and M proteins are almost completely embedded in the membrane. Iodination of spheroplasts results in only a slight binding of 131I to chromatophores and reaction centers.     

On the state of carotenoids bound to reaction centers of photosynthetic bacteria: a resonance Raman study.

The carotenoids bound to reaction centers of wild, Ga and GIC strains of Rhodopseudomonas spheroides, of Rhodospirrillum rubrum, strain S1 and of Rhodopseudomonas viridis, yield very similar, but unusual resonance Raman spectra. Through a comparison with resonance Raman spectra of 15,15'-cis-beta-carotene, these carotenoids are shown to assume cis conformations, while the corresponding chromatophores contain all-trans forms only. These cis conformations likely are identical for all the carotenoids studied. They remain unaffected by variations of temperature from 20 to 300 K as well as by the redox state of P-870. They are unstable, being rapidly isomerised towards the all-trans forms when extracted from the reaction centers. The possible nature of these conformers is discussed on the basis of their electronic and vibrational spectra.     

Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b(560) was reduced; the dark oxidation of cytochrome b(560) was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b(560), another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b(560). This pigment, tentatively identified as cytochrome b(565), was also detected in spectra at 77 degrees k, after brief illumination at room temperature; the maxima at 77 degrees k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b(565) and an oxidation of cytochrome b(560). Dark oxidation of b(565) was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77 degrees k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77 degrees k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.     

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.     

Photoreduction of Pyridine Nucleotide by Subcellular Preparations from Rhodopseudomonas spheroides

We have investigated the photoreduction of pyridine nucleotides by crude extracts and chromatophores of Rhodopseudomonas spheroides.

Our findings are as follows:

NADP is preferentially photoreduced by crude extracts (37,000 × g supernatant fraction) and there is no requirement for the addition of exogenous substrates. Crude extracts also catalyze a nonphotosensitive reduction of NAD.

NADP photoreduction is completely inhibited if an NADH trapping system is present and indicates that NADH is required for NADP photoreduction.

Washed chromatophores (150,000 × g pellet) do not catalyze NADP photoreduction unless the supernatant fraction is added. The restoring effect of supernatant fraction is lost upon boiling and dialysis. However, supernatant materials can be replaced by an NADH generating system. There is no requirement for anaerobic conditions.

Evidence has been presented which suggests that Rhodopseudomonas spheroides contains an energy-linked transhydrogenase that can be driven by a high energy intermediate generated by light or ATP. This intermediate may also be functional in ATP synthesis. The synthesis of ATP and the ATP-supported transhydrogenase is inhibited by oligomycin. This inhibitor does not affect the light-mediated reaction.

     

Light- and diffusion-potential-induced shift of carotenoid spectrum in reconstituted vesicles of Rhodopseudomonas sphaeroides.

Proteoliposomes were reconstituted from detergent-solubilized pigment.protein complexes of chromatophores of Rhodopseudomonas sphaeroides and soybean phospholipids. The reconstituted vesicles showed a photooxidation of reaction center bacteriochlorophyll and a light-induced spectral shift of carotenoid to longer wave-lengths. The red shift similar to that in intact cells or chromatophores, indicates the generation of local fields in the membrane of proteoliposomes. When inside-positive membrane potential was induced by adding valinomycin and potassium salt, a shift of carotenoid spectrum to shorter wavelengths was observed. Therefore, the reconstituted vesicles, at least in the major part of population, produced the light-induced local field in the same direction as in intact cells, which is inside negative. Sidedness of the membrane structure and the direction of electric field formation in reconstituted vesicles were opposite to those in chromatophores (inside-out vesicles.     

16s and 23s components in the ribosomal ribonucleic acid of Rhodopseudomonas spheroides

1. Ribosomal RNA was extracted from lysates of Rhodopseudomonas spheroides without prior isolation of ribosomes. 2. The composition of this RNA was investigated by using gradient centrifugation, showing that the proportion present as 23s component depended on the method of extraction. 3. The highest proportion of 23s component was found when cells were disrupted by ultrasonic treatment in the presence of ribonuclease inhibitors. 4. The results indicated that a ribonuclease is active in the cell lysate; this could account for the previous report (Lessie, 1965) that ribosomes of Rhodopseudomonas spheroides do not contain a 23s component.     

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.     

Control of porphyrin biosynthesis in Rhodopseudomonas spheroides and Propionibacterium shermanii. A direct 13C nuclear-magnetic-resonance spectroscopy study.

The facultative anaerobes Rhodopseudomonas spheroides and Propionibacterium shermanii were grown under anaerobic and aerobic conditions. The effect of light was studied with the photosynthetic R. spheroides, and the adaptation of both species to dark anaerobic life was monitored by direct observation of 5-amino[5-13C]laevulinic acid metabolism by using 13C nuclear-magnetic-resonance spectroscopy.     

Topology and Growth of the Intracytoplasmic Membrane System of Rhodopseudomonas spheroides: Protein, Chlorophyll, and Phospholipid Insertion into Steady-State Anaerobic Cells

An equilibrium density gradient centrifugation study involving the separation of "old" and "new" membranes has been developed to determine the manner in which protein, lipid, and chlorophyll are incorporated into growing intracytoplasmic membranes (chromatophores) of Rhodopseudomonas spheroides. Chromatophores derived from cells grown in an H(2)O-medium had a density of 1.175 to 1.180 g/cm(3) and were readily separable from chromatophores having a density of 1.220 to 1.230 isolated from cells grown in a 70% D(2)O-medium. After a shift from "D(2)O-" to "H(2)O"-based media, only hybrid chromatophores derived from a combination of "heavy" (old) and "light" (new) chromatophore material could be detected. The experimentally determined, median density values for the growing intracytoplasmic membrane system followed a theoretically determined profile which was calculated from the density of full "heavy" and full "light" material assuming random, homogeneous incorporation of new material into old membrane. The distribution of the radioactive labels for protein (leucine) and chlorophyll (delta-aminolevulinic acid) were identical and showed a reproducible displacement of the "old" material to the heavy side of the optical density at 365 nm (OD(365)) absorbance and a displacement of the "new" material to the light side of the OD(365) absorbance profile. Specific phospholipid growth showed no displacement for either the "old" or "new" material from the median absorbance profile.     

Biosynthesis of Isoleucine and Valine in Rhodopseudomonas spheroides: Regulation of Threonine Deaminase Activity

The activities of threonine deaminase, acetohydroxy acid synthetase, acetohydroxy acid reductoisomerase, dihydroxy acid dehydrase, and transaminase B were detected in cell-free extracts of Rhodopseudomonas spheroides. No significant repression or derepression of threonine deaminase activity was observed.     

Preliminary characterization by X-ray diffraction of crystals of photochemical reaction centres from wild-type Rhodopseudomonas spheroides

Reaction centres from wild-type Rhodopseudomonas spheroides (strain Y) in a solution of octylglucoside have been crystallized with polyethylene glycol as precipitant, either by vapour diffusion or dialysis. Orthorhombic crystals (space group P2(1)2(1)2(1)) diffract to 3.5 A resolution. The unit cell parameters are a = 142.5 A, b = 141.5 A, c = 80 A; they are compatible with the presence of one reaction centre per asymmetric unit.     

The Separation of Chromatophores from the Cell Envelope in Rhodopseudomonas Spheroides

When subcellular particles from. Rhodopseudophas spheroices wore laced on sucrose density gradients, the separation of chromatophores from the cell envelope was markedly affected by the presence of ionic species. In gradients that contained Tris buffer plus 0.01’ magnesium, chromatophores were distributed nearly equally between an upper and a lower pigmented band. About half of the chronatopnores were release from the lower band when magnesium was excluded from the gradients. exclusion of both Tris and magnesium resulted in a quantitative separation of chromatophores (upper band) from the cell envelope (lower ‘and). Thus, the photosynthotic apparatus in Rps. spheroides resides on a membrane system separable from the cell wall-cell membrane complex.     

Bacteriochlorophyll fluorescence changes related to the bacteriopheophytin photoreduction in the chromatophores of purple sulfur bacteria]

It is shown that illumination of chromatophores of sulfur bacterium Chromatium minutissimum at Eh of the medium --200 mV divided by --620 mV (when the photooxidation of pigment P890 is completely inhibited) induces a decrease in bacteriochlorophyll fluorescence yield, reversible in the dark. Under these conditions a reversible photoreduction of bacteriopheophytin is detected (bleaching of absorption bands at 543 and 760 nm and development of a band at 650 nm), which is accompanied by a blue shift of the absorption band at 8 nm. As a possible interpretation of these effects the suggestion is made on the function of bacteriopheophytin as a primary electron acceptor in reaction centers of bacteria. The bacteriopheophytin photoreduction, followed by a decrease in fluorescence yield, is also observed in other sulfur bacteria, Thiocapsa roseopersicina and Ectothiorodospira shaposhnikovii, but it is not detected in nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides. This is considered as an evidence for the difference in the functional organization of the reaction centers of these two groups of bacteria,