Differential carotenoid composition of the B875 and B800-850 photosynthetic antenna complexes in Rhodobacter sphaeroides 2.4.1: involvement of spheroidene and spheroidenone in adaptation to changes in light intensity and oxygen availability.

Rhodobacter sphaeroides 2.4.1 is a member of the nonsulfur purple facultative photosynthetic proteobacteria, capable of growth under a variety of cultivation conditions. In addition to the structural polypeptides and bacteriochlorophyll, the two major antenna complexes, B875 and B800-850, contain a variety of carotenoids which are an important structural and functional component of the membrane-bound photosynthetic complexes of this bacterium. Two major carotenoids, spheroidene and its keto derivative, spheroidenone, are differentially synthesized by R. sphaeroides, depending on the growth conditions. Spheroidene prevails during growth under anaerobic conditions and low light intensities, whereas spheroidenone is predominant in semiaerobically grown cells or during anaerobic growth at high light intensities. In this study, we demonstrate that in wild-type cells, spheroidene is predominantly associated with the B800-850 photosynthetic antenna complex and spheroidenone is more abundant in the B875 complex. Exploiting mutants defective in the biosynthesis of either the B875 or B800-850 light-harvesting complex, we demonstrate an association between the formation of either the B875 or B800-850 complex, on the one hand, and the accumulation of spheroidenone or spheroidene, on the other. The possible involvement of the conversion of spheroidene to spheroidenone as a significant control mechanism involved in the adaptation of R. sphaeroides to changes in light intensity and oxygen tension is discussed.     

Differential protein insertion into developing photosynthetic membrane Regions of Rhodopseudomonas sphaeroides

Previous studies have suggested that much of the B800-850 light-harvesting bacteriochlorophyll a-protein complex is inserted directly into the intracytoplasmic photosynthetic membrane of Rhodopseudomonas sphaeroides. In contrast, the B875 light-harvesting and reaction center complexes are assembled preferentially at peripheral sites of photosynthetic membrane growth initiation. The basis for this apparent site-specific polypeptide insertion was examined during the inhibition of RNA and protein syntheses. The pulse labeling of polypeptides at the membrane growth initiation sites was significantly less sensitive to inhibition by rifampicin, chloramphenicol, or kasugamycin than in the intfacytoplasmic or outer membranes. This suggests increased stability for the translation machinery at these membrane invagination sites. Similar differential effects in polypeptide insertion were observed during inhibition of bacteriochlorophyll synthesis through deprival of δ-aminolevulinate to R sphaeroides mutant H-5, which requires this porphyrin precursor. The pulse-labeling patterns observed during the inhibition of both RNA and pigment syntheses were consistent with the uncoupling of polypeptide insertion into the membrane invagination sites from their growth and maturation into intracytoplasmic membranes.     

Role of apparent membrane growth initiation sites during photosynthetic membrane development in synchronously dividing Rhodopseudomonas sphaeroides.

Sites of intracytoplasmic membrane growth and temporal relations in the assembly of photosynthetic units were examined in synchronously dividing Rhodopseudomonas sphaeroides cells. After rate-zone sedimentation of cell-free extracts, apparent sites of initiation of intracytoplasmic membrane growth formed an upper pigmented band that sedimented more slowly than the intracytoplasmic membrane-derived chromatophore fraction. Throughout the cell cycle, the levels of the peripheral B800-850 light-harvesting pigment-protein complex relative to those of the core B875 complex in the upper pigmented fraction were only about half those of chromatophores. Pulse-labeling studies with L-[35S]methionine indicated that the rates of assembly of proteins in the upper pigmented fraction were much higher than those of chromatophores throughout the cell cycle; rates for the reaction center polypeptides were estimated to be approximately 3.5-fold higher than in chromatophores when the two membrane fractions were equalized on a protein basis. In pulse-chase studies, radioactivity of the reaction center and B875 polypeptides increased significantly in chromatophores and decreased in the upper pigmented band during cell division. These data suggest that the B875 reaction center cores of the photosynthetic units are inserted preferentially into sites of membrane growth initiation isolated in the upper pigmented band and that the incomplete photosynthetic units are transferred from their sites of assembly into the intracytoplasmic membrane during cell division. These results suggested further that B800-850 is added directly to the intracytoplasmic membrane throughout the cell cycle.     

Characterization of light-harvesting mutants of Rhodopseudomonas sphaeroides. I. Measurement of the efficiency of energy transfer from light-harvesting complexes to the reaction center

Light-harvesting mutants of Rhodopseudomonas sphaeroides lacking either the B800-850 complex or the B875 complex have been characterized by their absorption spectra in the visible and near-infrared region, and by their ability to transfer energy from the light-harvesting complexes to the reaction center. A new method of measuring the relative efficiency of energy transfer from the light-harvesting complexes to the reaction center is described. The B875- mutant had absorption maxima in the near-infrared at 800 and 849 nm with no evidence of an 875-nm shoulder. The efficiency of energy transfer from the light-harvesting complexes to the reaction center in the B875- mutant was 24% of the value measured for the wild-type strain and the B800-850- mutant. Yet, despite the fact that the efficiency of energy transfer for the B800-850- mutant and the wild-type strain were the same, there was a large difference in their photosynthetic unit size. These results are discussed in the context of a model in which light energy captured by the B800-850 complexes is transferred through the B875 complexes to the reaction center.     

Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting B800-850 complex and its dependence on the spectral composition of the light is discussed.     

Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting the B800-850 complex and its dependence on the spectral composition of the light is discussed.     

Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides.

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.9 nmol/mg of protein. This was strictly proportional to a decrease in the ratio of B800-850 to B875 light-harvesting complexes. MgPME levels increased from 1.5 to 5.3 nmol/mg of protein, while cytochrome as well as coproporphyrin levels stayed constant at 0.46 and 1.95 nmol/mg of protein, respectively. Since in chemostat cultures steady-state levels of a product represent the rate of synthesis, these results infer only slight control of the rate-limiting step of total tetrapyrrol formation by light. In substrate-limited cultures MgPME was accumulated when growth and Bchl formation approached substrate saturation. This suggests that light controls a second step, i.e., MgPME conversion, whenever too much precursor is available, owing to the low sensitivity of the initial step of control. MgPME was preferentially localized in a subcellular fraction with high contents of B875 complexes. A second fraction exhibiting increased contents of B800-850 complexes lacked significant levels of MgPME. These results are discussed in terms of localization of Bchl synthesis in the membrane system of R. sphaeroides.     

Induction of the photosynthetic membranes of Rhodopseudomonas sphaeroides: biochemical and morphological studies

Cells of Rhodopseudomonas sphaeroides grown in a 25% O2 atmosphere were rapidly subjected to total anaerobiosis in the presence of light to study the progression of events associated with the de novo synthesis of the inducible intracytoplasmic membrane (ICM). This abrupt change in physiological conditions resulted in the immediate cessation of cell growth and whole cell protein, DNA, and phospholipid accumulation. Detectable cell growth and whole cell protein accumulation resumed ca. 12 h later. Bulk phospholipid accumulation paralleled cell growth, but the synthesis of individual phospholipid species during the adaptation period suggested the existence of a specific regulatory site in phospholipid synthesis at the level of the phosphatidylethanolamine methyltransferase system. Freeze-fracture electron microscopy showed that aerobic cells contain small indentations within the cell membrane that appear to be converted into discrete ICM invaginations within 1 h after the imposition of anaerobiosis. Microscopic examination also revealed a series of morphological changes in ICM structure and organization during the lag period before the initiation of photosynthetic growth. Bacteriochlorophyll synthesis and the formation of the two light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides (B800-850 and B875) occurred coordinately within 2 h after the shift to anaerobic conditions. Using antibodies prepared against various ICM-specific polypeptides, the synthesis of reaction center proteins and the polypeptides associated with the B800-850 complex was monitored. The reaction center H polypeptide was immunochemically detected at low levels in the cell membrane of aerobic cells, which contained no detectable ICM or bacteriochlorophyll. The results are discussed in terms of the oxygen-dependent regulation of gene expression in R. sphaeroides and the possible role of the reaction center H polypeptide and the cell membrane indentations in the site-specific assembly of ICM pigment-protein complexes during the de novo synthesis of the ICM.     

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata.

Cells of Rhodopseudomonas capsulata, strain 37b4, leu-, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W.m-2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W.m-2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased. The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.     

Fusion of liposomes and chromatophores of Rhodopseudomonas capsulata: effect on photosynthetic energy transfer between B875 and reaction center complexes.

The photosynthetic chromatophore membranes of Rhodopseudomonas capsulata were fused with liposomes to investigate the effects of lipid dilution on energy transfer between the bacteriochlorophyll-protein complexes of this membrane. Phosphatidylcholine-containing liposomes were mixed with chromatophores at pH 6.0 to 6.2, and the mixture was fractionated on discontinuous sucrose gradients into four membrane fractions with lipid-to-protein ratios that varied 11-fold. Freeze-fracture electron microscopy revealed that the fractions contained closed vesicles formed by the fusion of liposomes to chromatophores. Particles with 9-nm diameters on the P fracture faces did not appear to change in size with increasing lipid content, but the number of particles per membrane area decreased proportionally with increases in the lipid-to-protein ratio. The bacteriochlorophyll-to-protein ratios, electrophoretic polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gels, and light-induced absorbance changes at 595 nm caused by photosynthetic reaction centers were not altered by fusion. The relative fluorescence emission intensities due to the B875 light-harvesting complex increased significantly with increasing lipid content, but no increases in fluorescence due to the B800-B850 light-harvesting complex were observed. Electron transport rates, measured as succinate-cytochrome c reductase activities, decreased with increased lipid content. The results indicate an uncoupling of energy transfer between the B875 light-harvesting and reaction center complexes with lipid dilution of the chromatophore membrane.     

Turnover of the B870-α pigment-binding protein in a mutant of Rhodopseudomonas capsulata which is defective in assembling reaction center and B870 into membranes

Abstract The photosynthetically negative mutant strain Y142 of Rhodopseudomonas capsulata , which synthesizes bacteriochlorophyll (Bchl), carotenoids and the light-harvesting (LH) complex B800–850, but no reaction center and LH complex B870, is capable of synthesizing the Bchl-binding polypeptide (α, 12 kDa) of B870. In contrast to the high stability of the polypeptides of the B800–850 complex, the 12 kDa polypeptide was rapidly degraded after synthesis and insertion into the membrane.     

Spectroscopic studies of two spectral variants of light-harvesting complex 2 (LH2) from the photosynthetic purple sulfur bacterium Allochromatium vinosum

Two spectral forms of the peripheral light-harvesting complex (LH2) from the purple sulfur photosynthetic bacterium Allochromatium vinosum were purified and their photophysical properties characterized. The complexes contain bacteriochlorophyll a (BChl a) and multiple species of carotenoids. The composition of carotenoids depends on the light conditions applied during growth of the cultures. In addition, LH2 grown under high light has a noticeable split of the B800 absorption band. The influence of the change of carotenoid distribution as well as the spectral change of the excitonic absorption of the bacteriochlorophylls on the light-harvesting ability was studied using steady-state absorption, fluorescence and femtosecond time-resolved absorption at 77K. The results demonstrate that the change of the distribution of the carotenoids when cells were grown at low light adapts the absorptive properties of the complex to the light conditions and maintains maximum photon-capture performance. In addition, an explanation for the origin of the enigmatic split of the B800 absorption band is provided. This spectral splitting is also observed in LH2 complexes from other photosynthetic sulfur purple bacterial species. According to results obtained from transient absorption spectroscopy, the B800 band split originates from two spectral forms of the associated BChl a monomeric molecules bound within the same complex.     

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata

Cells of Rhodopseudomonas capsulata, strain 37b4, leu^?, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W · m^?2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W · m^?2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased.The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.     

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.     

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala⁺) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala⁺ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.     

Quantum efficiency of the minor channel for excitation migration from B800 to B875 bacteriochlorophyll fractions in purple bacteria

Excitation migration in the light-harvesting bacteriochlorophyll complexes LH1 and LH2 of purple bacteria has been studied in many experimental and theoretical works. According to the widely accepted notions, it proceeds along the descending energy stairway, B800* → 850* → 875* → P870*, where symbol * stands for excitations in the corresponding BChl fraction. In this paper we demonstrate the existence of one more way of direct excitation delivery from B800 to B875, bypassing the main route via B850. The comparative modeling enables the estimation of the mean portion of excitation that uses this minor migration way. In some real cases it may reach 9–9.5%. The values of the critical distances for excitation migration from B800 to B850 and from B800 to B875, as well as their values for arbitrary spectral shifts in BChl molecules, are determined.     

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.     

Reconstitution of carotenoids into the light-harvesting complex B800-850 of Chromatium minutissimum

Chromatophores and peripheral light-harvesting complexes B800–850 with a trace of carotenoids were isolated from Chromatium minutissimum cells in which carotenoid biosynthesis was inhibited by diphenylamine. Three methods previously used for the reconstitution of carotenoids into either the light-harvesting (LH1) type complexes or reaction centers (RC) of carotenoidless mutants were examined for the possibility of carotenoid reconstitution into the carotenoid depleted chromatophores. All these methods were found to be unsuitable because carotenoid depleted complex B800–850 from Chr. minutissimum is characterized by high lability. We have developed a novel method maintaining the native structure of the complexes and allowing reconstitution of up to 80% of the carotenoids as compared to the control. The reconstituted complex has a similar CD spectrum in the carotenoid region as the control, and its structure restores its stability. These data give direct proof for the structural role of carotenoids in bacterial photosynthesis.     

The effect of different levels of the B800-850 light-harvesting complex on intracytoplasmic membrane development in Rhodobacter sphaeroides

A role for the peripheral (B800-850) light-harvesting complex in vesicularization of the Rhodobacter sphaeroides intracytoplasmic membrane (ICM), suggested from studies in mutant strains lacking one or more of the pigment-protein complexes, was examined further in the wild-type strain NCIB 8253 grown at high (∼1000 W m^–2), moderate (∼300 W m^–2), and low (∼100 W m^–2) light intensities. The resulting ICM vesicles (chromatophores) had B800-850 levels related inversely to irradiance and banded in rate-zone sedimentation at ∼1.10, 1.09, and 1.07 g ml^–1, respectively. Equilibrium centrifugation on iso-osmotic gradients indicated that this distinct sedimentation behavior resulted solely from differences in hydrodynamic radii. These size differences were confirmed by gel-exclusion chromatography and in electron micrographs of thin-sectioned cells. A pulse-chase study of ICM growth following a tenfold reduction in light intensity showed a relatively slow equilibration of membrane proteins during adaptation, and that new protein was incorporated largely into additional ICM formed at the lowered illumination level, giving rise to chromatophores of reduced size and elevated B800-850 content. These results provide further evidence for a model in which the B800-850 complex both drives development of vesicular ICM in Rba. sphaeroides and determines the size of resulting vesicles. Received: 12 October 1995 / Accepted: 21 December 1995