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.     

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.     

Characterization and complementation of a mutant of Rhodobacter sphaeroides with a chromosomal deletion in the light-harvesting (LH2) genes

An LH2- strain of Rhodobacter sphaeroides, DBC1, has been constructed by deleting the puc operon, which encodes the LH2 alpha and beta polypeptides, from the chromosome and replacing it with a kanamycin resistance gene. Southern blot analysis indicates that the 950 bp BamHI restriction fragment which contains the puc operon has been lost and has been replaced by the 1.25 kb Km(R) cassette derived from Tn903. Strain DBC1 lacked the LH2 complex, as shown by loss of the characteristic absorbance bands at 800 and 850 nm. The LH2 polypeptides were also found to be absent after SDS-PAGE. The wild-type phenotype was restored to DBC1 by the transfer of a 3.8 kb BscI fragment containing the puc operon in plasmid pMA81. Transconjugants possessed a wild-type absorbance spectrum and LH2 polypeptides.     

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.     

Oxido-reduction of B800-850 and B880 holochromes isolated from three species of photosynthetic bacteria as studied by electron-paramagnetic resonance and optical spectroscopy

Certain redox properties of bacteriochlorophyll alpha were used to probe the structure of several light-harvesting pigment-protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800-850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll alpha could be oxidized by ferricyanide as witnessed by the bleaching of their near-infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800-850 and the B880 holochromes is that oxidation of the latter gives rise to a g = 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37 mT to 0.48 mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800-850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800-nm and 850-nm bands. This indicates that, contrary to the B880 holochromes, the B800-850 holochromes may not form a homogeneous class.     

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     

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.     

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.     

Polypeptides and bacteriochlorophyll organization in the light-harvesting complex B850 of Rhodobacter sphaeroides R-26.1

The light-harvesting complex (LHC) B850 from Rhodobacter sphaeroides was dissociated into several fragments by treatment with sodium dodecyl sulfate. The molecular weight of each fragment was determined by using transverse polyacrylamide gel electrophoresis under nondenaturing conditions and gel filtration techniques. Four B850 LHCs were observed, having molecular weights of 60,000, 72,000-75,000, 105,000, and 125,000-145,000, and two small bacteriochlorophyll (Bchl)-polypeptide complexes having molecular weights of 6000-8000 and 12,000-14,000. Each of the B850 complexes contains ca. one Bchl a for each 6.5-kDa protein. The optical absorption and circular dichroism of the B850 LHCs recorded directly from the gels are similar to those measured previously for a 22-24-kDa B850 LHCs by Sauer and Austin [(1978) Biochemistry 17, 2011-2019]. These data, combined with studies of other groups, indicate that the smallest LHC in LH1 and LH2 is a Bchl-polypeptide tetramer. Each tetramer contains two Bchl dimers that probably have the structure of P-860, the primary electron donor in Rhodobacter sphaeroides, and two alpha-beta-polypeptide pairs. Interactions among the paired Bchls shift their individual Qy transitions from 780-800 to 850-860 nm, and interactions among two such pairs induce the circular dichroism signal of the LHCs. Three Bchl-polypeptide tetramers probably form a dodecamer having C3 symmetry, and six such dodecamers organize into a large hexagon that can accommodate one or two reaction center complexes.     

Organization of the genes coding for the reaction-centre L and M subunits and B870 antenna polypeptides α and β from the aerobic photosynthetic bacterium Erythrobacter species OCH114

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light-harvesting (LH) complex B870 and the reaction-centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728 kbp EcoRI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 beta), 52 (B850 alpha), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a 'hairpin' structure (delta G degrees = -26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728 kbp EcoRI fragment reconstituted a photoinactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.