Heterologous expression of genes encoding bacterial light-harvesting complexes in Rhodobacter sphaeroides.

One of the major problems in structural work on membrane-spanning proteins is the identification of an expression system which will allow the production of enough pure protein for structural studies; an inadequate expression system can lead, for example, to the formation of unwanted protein inclusion bodies. In the present work we report the expression of genes encoding the light-harvesting 2 (LH2) membrane-spanning proteins from a number of species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna. The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas acidophila and Rubrivivax gelatinosus were amplified and tailed by polymerase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb.

Studies on the light-harvesting complexes from the thermotolerant purple bacterium Rhodopseudomonas cryptolactis.

The antenna complexes from Rps. cryptolactis have been isolated and purified. Rps.

Complete DNA sequence, specific Tn5 insertion map, and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides.

The carotenoid biosynthesis genes form a cluster within the genome of Rhodobacter sphaeroides, lying in the middle of a larger cluster and 45 kb in length, which contains genes for bacteriochlorophyll biosynthesis and for the reaction center and light-harvesting apoproteins. The positions and approximate limits of the carotenoid genes were determined previously by localized transposon Tn5 mutagenesis and by comparison with the closely related Rhodobacter capsulatus carotenoid gene cluster. In this report, analysis of the DNA and deduced amino acid sequences of the carotenoid genes in R.

Structure and properties of the bacteriochlorophyll binding site in peripheral light-harvesting complexes of purple bacteria.

In this paper, we have examined, using FT resonance Raman spectroscopy, the bacteriochlorophyll (BChl) binding sites in the peripheral light-harvesting complexes extracted from a number of purple bacterial strains. A comparison of interactions of the BChl molecules with their binding sites in these LH2 complexes, together with the primary sequences of the alpha and beta polypeptides, allows three amino acids to be proposed to be involved in the hydrogen bonding of the 9-keto carbonyl of one of the 850-nm-absorbing pair of BChl molecules. Specifically, we show that one keto carbonyl group, which is strongly hydrogen bonded in Rhodobacter sphaeroides LH2, is involved in much weaker interactions in the LH2 complexes from all the other species studied (i.

Early steps in carotenoid biosynthesis: sequences and transcriptional analysis of the crtI and crtB genes of Rhodobacter sphaeroides and overexpression and reactivation of crtI in Escherichia coli and R. sphaeroides.

In the purple photosynthetic bacterium Rhodobacter sphaeroides, the desaturation of phytoene has already been implicated in the assembly of the light-harvesting 2 complex (H.P. Lang and C.

Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides.

Carotenoids have two major functions in bacterial photosynthesis, photoprotection and accessory light harvesting. The genes encoding many carotenoid biosynthetic pathways have now been mapped and cloned in several different species, and the availability of cloned genes which encode the biosynthesis of carotenoids not found in the photosynthetic genus Rhodobacter opens up the possibility of introducing a wider range of foreign carotenoids into the bacterial photosynthetic apparatus than would normally be available by producing mutants of the native biosynthetic pathway. For example, the crt genes from Erwinia herbicola, a gram-negative nonphotosynthetic bacterium which produces carotenoids in the sequence of phytoene, lycopene, beta-carotene, beta-cryptoxanthin, zeaxanthin, and zeaxanthin glucosides, are clustered within a 12.

The effect of growth conditions on the light-harvesting apparatus in Rhodopseudomonas acidophila.

The detailed effect on the light-harvesting apparatus of three different wild-type strains of Rhodopseudomonas acidophila in response to changes in both light-intensity and temperature have been investigated. In all three strains at high light-intensities (160 μmol s m(2) and above) the only LH2 antenna complex synthesised is the B800-850 complex. In strains 7050 and 7750 as the light-intensity is lowered the B800-850 complex is gradually replaced by another type of LH2 the B800-820 complex.

Predicting the point at which transmembrane helices protrude from the bilayer: a model of the antenna complexes from photosynthetic bacteria.

We describe a method for predicting the point at which a transmembrane helix leaves the bilayer and enters the more polar region of the aqueous exterior. This is achieved by comparing the relative directions of the hydrophobic and internal faces of the transmembrane helices which should be opposite for the regions within the bilayer but equivalent for the regions on the outside. This information provides a strong constraint in the process of modelling membrane proteins.

A progress report on the crystallographic studies on the B800-850 antenna complex from Rhodopseudomonas acidophila strain 10050.

Large single crystals (up to 1 mm in each dimension) of the B800-850 antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown in the presence of beta-octyl-glucoside. These crystals have the space group R32 and unit cell dimensions of a = b = 119.9 A and c = 297.

Picosecond Raman spectroscopy of the B830 LH2 complex ofChromatium purpuratum BN 5500.

A setup for generating the Stokes Raman lines of benzene (556, 588 and 624 nm, ∼50 ps) by the use of the second harmonic of a Nd: YLF regenerative amplifier system (527 nm, 70 ps, 1 kHz) has been built. This was then used to detect, for the first time, the picosecond Raman spectrum of a carotenoid bound to an isolated light-harvesting complex of a photosynthetic bacterium. The 527 and 588 nm pulses have been used, respectively, for pumping and probing (delay ∼0 ps) the S1 and T1 states of okenone which is bound to both the isolated B830 LH2 complex and the chromatophores fromChromatium purpuratum BN 5500.

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts.

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC.

Crystallization of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7750.

The B800-820 light-harvesting complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 121.8 A and c = 283.

Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050.

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Qx (550-650 nm) and carotenoid (450-550 nm) regions similar to that observed from the random phase solution.

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 complete amino acid sequences of the B800-850 antenna polypeptides from Rhodopseudomonas acidophila strain 7750.

Spectrally pure B800-850 light harvesting complexes of Rhodopseudomonas acidophila 7750 were prepared by chromatography of LDAO-solubilised photosynthetic membranes on Whatmann DE-52 ion exchange resin. Two low molecular mass polypeptides (alpha, beta) have been isolated by organic solvent extraction of the lyophilised B800-850 light harvesting complexes. Their primary structures were determined by liquid phase sequencer runs, by the sequence analyses of C-terminal o-iodosobenzoic acid fragments, by hydrazinolysis and by carboxypeptidase degradation.

How carotenoids function in photosynthetic bacteria.

Carotenoids are essential for the survival of photosynthetic organisms. They function as light-harvesting molecules and provide photoprotection. In this review, the molecular features which determine the efficiencies of the various photophysical and photochemical processes of carotenoids are discussed.

Pigment-protein complexes of purple photosynthetic bacteria: an overview.

A minireview of antenna and reaction center pigment-protein complexes of purple bacteria is presented. Advances in our knowledge of their structure and composition during the past 3 yr are emphasized and some new thoughts are introduced.

Reconstitution of carotenoids into the light-harvesting pigment-protein complex from the carotenoidless mutant of Rhodopseudomonas as sphaeroides R26.

Two carotenoids, neurosporene and spheroidene, have been successfully added to chromatophores from the carotenoidless mutant of Rhodopseudomonas sphaeroides R26. Carotenoids reconstituted in this way into the B-850 light-harvesting pigment-protein complex both sensitive bacteriochlorophyll fluorescence and protect the complex from the photodynamic reaction.

Energy transfer between the carotenoid and the bacteriochlorophyll within the B-800-850 light-harvesting pigment-protein complex of Rhodopseudomonas sphaeroides.

Energy transfer between carotenoid and bacteriochlorophyll has been studied in isolated B-800-850 antenna pigment-protein complexes from different strains of Rhodopseudomonas sphaeroides which contain different types of carotenoid. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll is efficient (75-100%) and is rather insensitive to carotenoid type, over the range of carotenoids tested. The yield of carotenoid triplets is low (2-15%) but this arises from a low yield of bacteriochlorophyll triplet formation rather than from an inefficient triplet-triplet exchange reaction.