Genes downstream from pucB and pucA are essential for formation of the B800-850 complex of Rhodobacter capsulatus.

The formation of the light-harvesting complex B800-850 (LH-II) of Rhodobacter capsulatus requires, in addition to the synthesis of the polypeptides alpha and beta (the gene products of pucA and pucB), the synthesis of bacteriochlorophyll and carotenoids and the expression of at least one gene localized downstream from the pucBA operon. This was concluded from the observation that a Tn5 insertion downstream from pucBA inhibited the formation of the LH-II complex and the formation of the pucBA mRNA. The Tn5 insertion point was mapped and found to be over 500 base pairs (bp) downstream from the end of the pucA gene, suggesting the presence of additional puc genes. A region of about 3,000 bp including the pucB and pucA genes and DNA downstream from pucA was sequenced and found to contain three open reading frames (ORFs C, D, and E). The polypeptide deduced from the first ORF (C) contains 403 amino acids with strongly hydrophobic stretches and one large and three small hydrophilic domains carrying many charged residues. The other two ORFs contain 113 (D) and 118 (E) codons. The amino acid sequences of the N terminus and two tryptic peptides of an alkaline-soluble Mr-14,000 subunit of the isolated LH-II complex were identical with the deduced amino acid sequence of ORF E.     

Phosphatidylcholine is required for the efficient formation of photosynthetic membrane and B800-850 light-harvesting complex in Rhodobacter sphaeroides

No phosphatidylcholine (PC) was detected in the membrane of Rhodobacter sphaeroides pmtA mutant (PmtA1) lacking phosphatidylethanolamine (PE) N-methyltransferase, whereas PE in the mutant was increased up to the mole % comparable to the combined level of PE and PC of wild type. Neither the fatty acid composition nor the fluidity of membrane was altered by pmtA mutation. Consistently, aerobic and photoheterotrophic growth of PmtA1 were not different from wild type. However, PmtA1 showed an extended lag phase (15 h) after the growth transition from aerobic to photoheterotrophic conditions, indicating the PC requirement for the efficient formation of intracytoplasmic membrane (ICM). Interestingly, the B800-850 complex of PmtA1 was decreased more than twofold in comparison with wild type, whereas the level of the B875 complex comprising the fixed photosynthetic unit was not changed. Since puc expression is not affected by pmtA mutation, PC appears to be required for the proper formation of the B800-850 complex in the ICM of R. sphaeroides.     

Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 s for the B800-850 complex, 14±2 s for the B850 complex incorporated with spheroidene, and 19±2 s for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 s. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49–55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.     

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.     

Fusion of proteoliposomes containing the B800-850 light-harvesting complex with membranes of the mutant strain NK3 of Rhodopseudomonas capsulata lacking B800-850

Intracytoplasmic membranes of the mutant strain NK3 of Rhodopseudomonas capsulata lacking the lightharvesting complex B800-850 were fused with proteoliposomes containing the B800-850 complex. Fluorescence emission spectroscopy at 77K showed that after fusion the fluorescence of the B850 bacteriochlorophyll disappeared nearly completely and the B870 fluorescence became prominent. This result and control experiments with proteoliposome-chromatophore mixture and with chromatophore and solubilized B800-850 complexes, respectively, indicate that in fused membranes a reorientation of membrane particles took place and excitons migrated from B850 to B870 bacteriochlorophyll.In fused proteoliposome-chromatophore vesicles a light-induced carotenoid band shift was observed, reflecting the building of an electrical membrane potential due to chargeseparation. Carotenoid band shift was not observed in separated proteoliposomes and NK3 chromatophores.It is concluded that by membrane fusion and lateral diffusion of membrane particles reaction center-light-harvesting B870 complexes came in functional contact with B800-850 antenna complexes.Abbreviations Bchl bacteriochlorophyll - LDAO lauryl dimethylamine oxide - RC reaction center Dedicated to Professor R. Clinton Fuller, Amherst, MA, USA, on the occasion of his 60th birthday in recognition of his work on photosynthetic bacteria and the cooperation between our laboratories     

Chemical cross-linking studies of the light-harvesting pigment-protein complex B800-850 of Rhodopseudomonas capsulata

The spatial relationship of the three polypeptides contained in the B800-850 light-harvesting complex of Rhodopseudomonas capsulata has been studied with chemical cross-linking of crude membrane preparations of the phototrophic negative mutant strain Y5. Samples were cross-linked with the cleavable reagent dithiobis (succinimidyl propionate) (1.1 nm chain length) and analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membranes labelled with 14C-amino acids were used to determine the compositional stoichiometry of cross-linked products. It was found that the two polypeptides with an apparent Mr of 8000 and 10 000, respectively, that are associated with the pigments bacteriochlorophyll a and carotenoid form homooligomers as well as heterooligomers. The data support the idea that these polypeptides are closely arranged in clusters probably containing at least four of each species. The third subunit with an Mr of 14 000, which is not associated with pigments, was found to be most susceptible to cross-linking and formed homooligomers but no heterooligomers with the other two subunits, and is thus likely to be loosely attached to these clusters. Comparative studies with the phototrophic positive wild type strain indicated that the results found with the phototrophic negative mutant strain Y5 reflect the organization of the B800-850 complex in the membrane of Rhodopseudomonas capsulata. Studies with the isolated B800-850 complex revealed that the sterical arrangement of the three constituent polypeptides in dodecyl dimethylamine-N-oxide containing solutions must be very similar to that in the membrane.     

Femtosecond energy-transfer processes in the B800-850 light-harvesting complex of Rhodobacter sphaeroides 2.4.1

The B800-to-B850 energy transfer time in the purified B800-850 light-harvesting complex of Rhodobacter sphaeroides 2.4.1 is determined to be 0.7 ps at room temperature. The electronic state dynamics of the principal carotenoid of this species, spheroidene, are examined, both in vivo and in vitro, by direct femtosecond time-resolved experiments and by fluorescence emission yield studies. Evidence is presented which suggests that carotenoid-to-bacteriochlorophyll energy transfer may occur directly from the initially excited carotenoid S2 state, as well as from the carotenoid S1 state. Further support for this conjecture is obtained from calculations of energy transfer rates from the carotenoid S2 state. Previous measurements of in vivo carotenoid and B800 dynamics are discussed in light of the new results, and currently unresolved issues are described.     

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

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

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     

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.     

B800-->B850 energy transfer mechanism in bacterial LH2 complexes investigated by B800 pigment exchange

Femtosecond transient absorption measurements were performed on native and a series of reconstituted LH2 complexes from Rhodopseudomonas acidophila 10050 at room temperature. The reconstituted complexes contain chemically modified tetrapyrrole pigments in place of the native bacteriochlorophyll a-B800 molecules. The spectral characteristics of the modified pigments vary significantly, such that within the B800 binding sites the B800 Q(y) absorption maximum can be shifted incrementally from 800 to 670 nm. As the spectral overlap between the B800 and B850 Q(y) bands decreases, the rate of energy transfer (as determined by the time-dependent bleaching of the B850 absorption band) also decreases; the measured time constants range from 0.9 ps (bacteriochlorophyll a in the B800 sites, Q(y) absorption maximum at 800 nm) to 8.3 ps (chlorophyll a in the B800 sites, Q(y) absorption maximum at 670 nm). This correlation between energy transfer rate and spectral blue-shift of the B800 absorption band is in qualitative agreement with the trend predicted from F?rster spectral overlap calculations, although the experimentally determined rates are approximately 5 times faster than those predicted by simulations. This discrepancy is attributed to an underestimation of the electronic coupling between the B800 and B850 molecules.     

The complete amino-acid sequence of the large bacteriochlorophyll-binding polypeptide from light-harvesting complex II (B800-850) of Rhodopseudomonas capsulata

The large bacteriochlorophyll-a-binding polypeptide of the light-harvesting complex II (B800-850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16-35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C-terminal part is also hydrophobic while the N-terminal part consists of hydrophilic amino acids. The polarity of the total amino acids was determined to be 28.3%.     

Multiple copies of the coding regions for the light-harvesting B800-850 alpha- and beta-polypeptides are present in the Rhodopseudomonas palustris genome.

A reverse-phase HPLC System for isolation of the water insoluble alpha- and beta-polypeptides of the light-harvesting complex II (LH II) of Rhodopseudomonas (Rps.) palustris without employment of any detergent was developed. The material obtained was of high purity and suitable for direct microsequence analysis. Chromatographic analysis could resolve at least two major beta-polypeptides, beta a and beta b, two major alpha-polypeptides, alpha a and alpha b, and two additional minor polypeptides. N-terminal amino acid sequencing shows that the resolved peaks correspond to different polypeptide species and that the minor species have an N-terminal sequence identical to that of the alpha b polypeptide. An oligonucleotide derived from the amino terminal sequence of the alpha a polypeptide was utilized to screen a genomic library from Rps.palustris. Several independent clones have been characterized by Southern blot and nucleotide sequence analysis. We show that Rps.palustris contains at least four different clusters of beta and alpha genes. Two clones contain sequences potentially coding for beta a-alpha a and beta b-alpha b polypeptides; and two additional clones potentially coding for beta and alpha peptides which we named beta c-alpha c and beta d-alpha d, which did not correspond to the major purified polypeptides. In addition to the protein chemistry data, the conservation at the amino acid level and the presence of canonical ribosomal binding sites upstream of each of the identified genes strongly suggest that all four coding regions are expressed.