Adaptation of Rhodopseudomonas acidophila strain 7050 to growth at different light intensities: what are the benefits to changing the type of LH2?

Typical purple bacterial photosynthetic units consist of light harvesting one/reaction centre 'core' complexes surrounded by light harvesting two complexes. Factors such as the number and size of photosynthetic units per cell, as well as the type of light harvesting two complex that is produced, are controlled by environmental factors. In this paper, the change in the type of LH2 present in the Rhodopsuedomonas acidophila strain 7050 is described when cells are grown at a range of different light intensities. This species contains multiple pucBA genes that encode the apoproteins that form light-harvesting complex two, and a more complex mixture of spectroscopic forms of this complex has been found than was previously thought to be the case. Femto-second time resolved absorption has been used to investigate how the energy transfer properties in the membranes of high-light and low-light adapted cells change as the composition of the LH2 complexes varies.

Towards quantification of vibronic coupling in photosynthetic antenna complexes.

Photosynthetic antenna complexes harvest sunlight and efficiently transport energy to the reaction center where charge separation powers biochemical energy storage. The discovery of existence of long lived quantum coherence during energy transfer has sparked the discussion on the role of quantum coherence on the energy transfer efficiency. Early works assigned observed coherences to electronic states, and theoretical studies showed that electronic coherences could affect energy transfer efficiency--by either enhancing or suppressing transfer. However, the nature of coherences has been fiercely debated as coherences only report the energy gap between the states that generate coherence signals. Recent works have suggested that either the coherences observed in photosynthetic antenna complexes arise from vibrational wave packets on the ground state or, alternatively, coherences arise from mixed electronic and vibrational states. Understanding origin of coherences is important for designing molecules for efficient light harvesting. Here, we give a direct experimental observation from a mutant of LH2, which does not have B800 chromophores, to distinguish between electronic, vibrational, and vibronic coherence. We also present a minimal theoretical model to characterize the coherences both in the two limiting cases of purely vibrational and purely electronic coherence as well as in the intermediate, vibronic regime.

Structural factors which control the position of the Q(y) absorption band of bacteriochlorophyll a in purple bacterial antenna complexes.

This paper presents a concise review of the structural factors which control the energy of the Q(y) absorption band of bacteriochlorophyll a in purple bacterial antenna complexes. The energy of these Q(y) absorption bands is important for excitation energy transfer within the bacterial photosynthetic unit.

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].

The purple photosynthetic bacterium Rhodopseudomonas acidophila contains multiple puc peripheral antenna complex (LH2) genes: Cloning and initial characterisation of four ß/α pairs.

The genome of the purple non-sulphur photosynthetic bacterium Rhodopseudomonas acidophila has been found to contain multiple copies of puc light-harvesting (LH2) peripheral antenna complex genes. Three wild-type isolates each exhibiting dissimilar peripheral antenna complex phenotypes in response to growth at reduced light intensity, were found to contain different numbers of these genes. Twenty-three puc cross-hybridising clones were isolated from a genomic library constructed from Rhodopseudomonas acidophila strain 7050; two of which were examined further; 2.6kb from one clone was sequenced and found to contain three ß/α gene pairs designated puc (1)BA, puc (2)BA and puc (3)BA. The putative translated polypeptides are very like, but not identical to those from B800-820 complexes and upstream sequence homologies suggests that this treble gene cluster has arisen through a relatively recent gene duplication event. From the other clone 0.6kb was sequenced and found to contain a further gene pair, puc (4)BA, which is capable of encoding apoproteins for a B800-850-like complex. When the cells are grown at 'high' or 'low' light intensity Northern analyses showed that only puc (4)BA is expressed under 'high' light conditions. Furthermore, pucBA mRNA transcripts were detected in all three species in the range 500-780 nt. In Rhodopseudomonas acidophila post-transcriptional regulatory mechanisms also play a role in determining the amount of peripheral antenna present in the intra-cytoplasmic membrane.

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. sphaeroides mutants; DBC omega/G5 and DD13 (DD13/G1); the resulting transconjugant strains synthesized LH2 complexes that were examined using absorption and fluorescence spectroscopy, and Western blotting. Thus, we have created a heterologous expression system which supports the assembly of a functional "foreign" light-harvesting complex. This work opens up the possibility of creating site-directed LH2 mutants from bacteria for which no genetic system is available; this is particularly significant in the case of Rps. acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic resolution.

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.N. Hunter, Biochem. J. 298:197-205, 1994). The phytoene synthase and desaturase enzymes mediate the first steps specific for carotenoid biosynthesis up to and including the synthesis of the colored carotenoid neurosporene. In this report, we present the DNA and deduced amino acid sequences of the genes encoding these proteins, namely, crtB and crtI, from R. sphaeroides and present evidence for the existence of a crtIB operon. Both genes have been shown to possess putative puc and puf operon-like promoter sequences, and oxygen regulation and the point of initiation of the crtI transcript have been demonstrated. The complete crtI gene has been overexpressed in Escherichia coli and R. sphaeroides and shown to catalyze three desaturations of phytoene to give neurosporene. This activity was shown to be ATP dependent, and the cofactor requirement was investigated by using a spectroscopic assay for in vitro carotenogenic activity. Although the crtI and crtB genes have been sequenced from a number of different organisms, the transcriptional organization and regulation of these genes have not been analyzed in detail. In this report, we have located the transcription initiation point and have shown that R. sphaeroides possesses an oxygen-regulated CrtI-type phytoene desaturase gene that forms a transcriptional operon with crtB.

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 12.8-kb region and have been mapped and partially sequenced. In this paper, part of the E. herbicola crt cluster has been excised and expressed in various crt strains of Rhodobacter sphaeroides. This has produced light-harvesting complexes with a novel carotenoid composition, in which the foreign carotenoids such as beta-carotene function successfully in light harvesting. The outcome of the combination of the crt genes in R. sphaeroides with those from E. herbicola has, in some cases, resulted in an interesting rerouting of the expected biosynthetic sequence, which has also provided insights into how the various enzymes of the carotenoid biosynthetic pathway might interact. Clearly this approach has considerable potential for studies on the control and organization of carotenoid biosynthesis, as well as providing novel pigment-protein complexes for functional studies.

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. However, at no light-intensities studied is this changeover complete when the cells are grown at 30°C. If however, the light-intensity is lowered at temperatures below 25°C with strain 7750 there is a complete replacement of the B800-850 complex by the B800-820 complex. At all light-intensities and temperatures tested, strain 10050 only synthesised the B800-850 complex. Strain 7050 also responded to changes in light-intensity by altering its carotenoid composition. At high light-intensity the major carotenoids were rhodopin and rhodopin-glucoside, while at low light-intensities the major ones were rhodopinal and rhodopinal-glucoside. This change in carotenoid content started to occur at rather higher light-intensities than the switchover from B800-850 to B800-820.