Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC–LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

Selective expression of light-harvesting complexes alters phospholipid composition in the intracytoplasmic membrane and core complex of purple phototrophic bacteria

Phospholipid–protein interactions play important roles in regulating the function and morphology of photosynthetic membranes in purple phototrophic bacteria. Here, we characterize the phospholipid composition of intracytoplasmic membrane (ICM) from Rhodobacter (Rba.) sphaeroides that has been genetically altered to selectively express light-harvesting (LH) complexes. In the mutant strain (DP2) that lacks a peripheral light-harvesting (LH2) complex, the phospholipid composition was significantly different from that of the wild-type strain; strain DP2 showed a marked decrease in phosphatidylglycerol (PG) and large increases in cardiolipin (CL) and phosphatidylcholine (PC) indicating preferential interactions between the complexes and specific phospholipids. Substitution of the core light-harvesting (LH1) complex of Rba. sphaeroides strain DP2 with that from the purple sulfur bacterium Thermochromatium tepidum further altered the phospholipid composition, with substantial increases in PG and PE and decreases in CL and PC, indicating that the phospholipids incorporated into the ICM depend on the nature of the LH1 complex expressed. Purified LH1–reaction center core complexes (LH1–RC) from the selectively expressing strains also contained different phospholipid compositions than did core complexes from their corresponding wild-type strains, suggesting different patterns of phospholipid association between the selectively expressed LH1–RC complexes and those purified from native strains. Effects of carotenoids on the phospholipid composition were also investigated using carotenoid-suppressed cells and carotenoid-deficient species. The findings are discussed in relation to ICM morphology and specific LH complex–phospholipid interactions.     

Protein-Induced Membrane Curvature Investigated through Molecular Dynamics Flexible Fitting

In the photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides, light is absorbed by membrane-bound light-harvesting (LH) proteins LH1 and LH2. LH1 directly surrounds the reaction center (RC) and, together with PufX, forms a dimeric (RC-LH1-PufX)₂ protein complex. In LH2-deficient Rba. sphaeroides mutants, RC-LH1-PufX dimers aggregate into tubular vesicles with a radius of ∼250-550 Å, making RC-LH1-PufX one of the few integral membrane proteins known to actively induce membrane curvature. Recently, a three-dimensional electron microscopy density map showed that the Rba. sphaeroides RC-LH1-PufX dimer exhibits a prominent bend at its dimerizing interface. To investigate the curvature properties of this highly bent protein, we employed molecular dynamics simulations to fit an all-atom structural model of the RC-LH1-PufX dimer within the electron microscopy density map. The simulations reveal how the dimer produces a membrane with high local curvature, even though the location of PufX cannot yet be determined uniquely. The resulting membrane curvature agrees well with the size of RC-LH1-PufX tubular vesicles, and demonstrates how the local curvature properties of the RC-LH1-PufX dimer propagate to form the observed long-range organization of the Rba. sphaeroides tubular vesicles.     

Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC–LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC–LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC–LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC–LH1 complexes, as long-range order appears to be induced only through its role of configuring RC–LH1 complexes into dimers.     

Membrane invagination in Rhodobacter sphaeroides is initiated at curved regions of the cytoplasmic membrane,then forms both budded and fully detached spherical vesicles

The purple phototrophic bacteria synthesize an extensive system of intracytoplasmic membranes (ICM) in order to increase the surface area for absorbing and utilizing solar energy. Rhodobacter sphaeroides cells contain curved membrane invaginations. In order to study the biogenesis of ICM in this bacterium mature (ICM) and precursor (upper pigmented band – UPB) membranes were purified and compared at the single membrane level using electron, atomic force and fluorescence microscopy, revealing fundamental differences in their morphology, protein organization and function. Cryo‐electron tomography demonstrates the complexity of the ICM of Rba. sphaeroides. Some ICM vesicles have no connection with other structures, others are found nearer to the cytoplasmic membrane (CM), often forming interconnected structures that retain a connection to the CM, and possibly having access to the periplasmic space. Near‐spherical single invaginations are also observed, still attached to the CM by a ‘neck’. Small indents of the CM are also seen, which are proposed to give rise to the UPB precursor membranes upon cell disruption. ‘Free‐living’ ICM vesicles, which possess all the machinery for converting light energy into ATP, can be regarded as bacterial membrane organelles.     

Quinone transport in the closed light-harvesting 1 reaction center complex from the thermophilic purple bacterium Thermochromatium tepidum

Redox-active quinones play essential roles in efficient light energy conversion in type-II reaction centers of purple phototrophic bacteria. In the light-harvesting 1 reaction center (LH1-RC) complex of purple bacteria, Q_B is converted to Q_BH₂ upon light-induced reduction and Q_BH₂ is transported to the quinone pool in the membrane through the LH1 ring. In the purple bacterium Rhodobacter sphaeroides, the C-shaped LH1 ring contains a gap for quinone transport. In contrast, the thermophilic purple bacterium Thermochromatium (Tch.) tepidum has a closed O-shaped LH1 ring that lacks a gap, and hence the mechanism of photosynthetic quinone transport is unclear. Here we detected light-induced Fourier transform infrared (FTIR) signals responsible for changes of Q_B and its binding site that accompany photosynthetic quinone reduction in Tch. tepidum and characterized Q_B and Q_BH₂ marker bands based on their ¹⁵N- and ¹³C-isotopic shifts. Quinone exchanges were monitored using reconstituted photosynthetic membranes comprised of solubilized photosynthetic proteins, membrane lipids, and exogenous ubiquinone (UQ) molecules. In combination with ¹³C-labeling of the LH1-RC and replacement of native UQ₈ by ubiquinones of different tail lengths, we demonstrated that quinone exchanges occur efficiently within the hydrophobic environment of the lipid membrane and depend on the side chain length of UQ. These results strongly indicate that unlike the process in Rba. sphaeroides, quinone transport in Tch. tepidum occurs through the size-restricted hydrophobic channels in the closed LH1 ring and are consistent with structural studies that have revealed narrow hydrophobic channels in the Tch. tepidum LH1 transmembrane region.     

FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells

In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical ‘chromatophore’ vesicles. These bacterial ‘organelles’ are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc₁ complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c₂. Proximity between cytochrome bc₁ and RC-LH1-PufX minimises quinone/quinol/cytochrome c₂ diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc₁ with yellow or cyan fluorescent proteins (YFP/CFP) and record the lifetimes of YFP/CFP Förster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution atomic force microscopy (AFM) of intact, purified chromatophores verifies the close association of cytochrome bc₁ complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling between RC-LH1-PufX and cytochrome bc₁ observed by spectroscopy, and explain how quinols/quinones and cytochrome c₂ shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.     

Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: implications for energy transduction mechanisms and membrane modeling

Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc ₁ complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc ₁ complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.     

Native architecture of the photosynthetic membrane from Rhodobacter veldkampii

The photosynthetic membrane in purple bacteria contains several pigment–protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1–RC (light-harvesting complex 1 – reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1–RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb. veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC–LH1–PufX cores, as it has been proposed in Rb. sphaeroides.     

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.     

Intrinsic curvature properties of photosynthetic proteins in chromatophores

In purple bacteria, photosynthesis is carried out on large indentations of the bacterial plasma membrane termed chromatophores. Acting as primitive organelles, chromatophores are densely packed with the membrane proteins necessary for photosynthesis, including light harvesting complexes LH1 and LH2, reaction center (RC), and cytochrome bc₁. The shape of chromatophores is primarily dependent on species, and is typically spherical or flat. How these shapes arise from the protein-protein and protein-membrane interactions is still unknown. Now, using molecular dynamics simulations, we have observed the dynamic curvature of membranes caused by proteins in the chromatophore. A membrane-embedded array of LH2s was found to relax to a curved state, both for LH2 from Rps. acidophila and a homology-modeled LH2 from Rb. sphaeroides. A modeled LH1-RC-PufX dimer was found to develop a bend at the dimerizing interface resulting in a curved shape as well. In contrast, the bc₁ complex, which has not been imaged yet in native chromatophores, did not induce a preferred membrane curvature in simulation. Based on these results, a model for how the different photosynthetic proteins influence chromatophore shape is presented.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

Adaptation of intracytoplasmic membranes to altered light intensity in Rhodobacter sphaeroides

The model photosynthetic bacterium Rhodobacter sphaeroides uses a network of bacteriochlorophyll (BChl)-protein complexes embedded in spherical intracytoplasmic membranes (ICM) to collect and utilise solar energy. We studied the effects of high- and low-light growth conditions, where BChl levels increased approximately four-fold from 1.6×10(6) to 6.5×10(6) molecules per cell. Most of this extra pigment is accommodated in the proliferating ICM system, which increases from approximately 274 to 1468 vesicles per cell. Thus, 16×10(6)nm(2) of specialised membrane surface area is made available for harvesting and utilising solar energy compared to 3×10(6)nm(2) under high-light conditions. Membrane mapping using atomic force microscopy revealed closely packed dimeric and monomeric reaction centre-light harvesting 1-PufX (RC-LH1-PufX) complexes in high-light ICM with room only for small clusters of LH2, whereas extensive LH2-only domains form during adaptation to low light, with the LH2/RC ratio increasing three-fold. The number of upper pigmented band (UPB) sites where membrane invagination is initiated hardly varied; 704 (5.8×10(5) BChls/cell) and 829 (4.9×10(5) BChls/cell) UPB sites per cell were estimated under high- and low-light conditions, respectively. Thus, the lower ICM content in high-light cells is a consequence of fewer ICM invaginations reaching maturity. Taking into account the relatively poor LH2-to-LH1 energy transfer in UPB membranes it is likely that high-light cells are relatively inefficient at energy trapping, but can grow well enough without the need to fully develop their photosynthetic membranes from the relatively inefficient UPB to highly efficient mature ICM.     

Aerobic chemolithoautotrophic growth and RubisCO function in Rhodobacter capsulatus and a spontaneous gain of function mutant of Rhodobacter sphaeroides

Photosynthetic prokaryotes that assimilate CO₂ under anoxic conditions may also grow chemolithoautotrophically with O₂ as the electron acceptor. Among the nonsulfur purple bacteria, two species (Rhodobacter capsulatus and Rhodopseudomonas acidophilus), exhibit aerobic chemolithoautotrophic growth with hydrogen as the electron donor. Although wild-type strains of Rhodobacter sphaeroides grow poorly, if at all, with hydrogen plus oxygen in the dark, we report here the isolation of a spontaneous mutant (strain HR-CAC) of Rba. sphaeroides strain HR that is fully capable of this mode of growth. Rba. sphaeroides and Rba. capsulatus fix CO₂ via the reductive pentose phosphate pathway and synthesize two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO levels in the aerobic-chemolithoautotrophic-positive strain of Rba. sphaeroides were similar to those in wild-type strains of Rba. sphaeroides and Rba. capsulatus during photoheterotrophic and photolithoautotrophic growth. Moreover, RubisCO levels of Rba. sphaeroides strain HR-CAC approximated levels obtained in Rba. capsulatus when the organisms were grown as aerobic chemolithoautotrophs. Either form I or form II RubisCO was able to support aerobic chemolithoautotrophic growth of Rba. capsulatus strain SB 1003 and Rba. sphaeroides strain HR-CAC at a variety of CO₂ concentrations, although form II RubisCO began to lose the capacity to support aerobic CO₂ fixation at high O₂ to CO₂ ratios. The latter property and other facets of the physiology of this system suggest that Rba. sphaeroides and Rba. capsulatus strains may be effectively employed for the biological selection of RubisCO molecules of altered substrate specificity. Received: 8 August 1997 / Accepted: 26 December 1997     

Membrane Curvature Induced by Aggregates of LH2s and Monomeric LH1s

The photosynthetic apparatus of purple bacteria is contained within organelles called chromatophores, which form as extensions of the cytoplasmic membrane. The shape of these chromatophores can be spherical (as in Rhodobacter sphaeroides), lamellar (as in Rhodopseudomonas acidophila and Phaeospirillum molischianum), or tubular (as in certain Rb. sphaeroides mutants). Chromatophore shape is thought to be influenced by the integral membrane proteins Light Harvesting Complexes I and II (LH1 and LH2), which pack tightly together in the chromatophore. It has been suggested that the shape of LH2, together with its close packing in the membrane, induces membrane curvature. The mechanism of LH2-induced curvature is explored via molecular dynamics simulations of multiple LH2 complexes in a membrane patch. LH2s from three species—Rb. sphaeroides, Rps. acidophila, and Phsp. molischianum—were simulated in different packing arrangements. In each case, the LH2s pack together and tilt with respect to neighboring LH2s in a way that produces an overall curvature. This curvature appears to be driven by a combination of LH2's shape and electrostatic forces that are modulated by the presence of well-conserved cytoplasmic charged residues, the removal of which inhibits LH2 curvature. The interaction of LH2s and an LH1 monomer is also explored, and it suggests that curvature is diminished by the presence of LH1 monomers. The implications of our results for chromatophore shape are discussed.     

Structure and dynamics of photosynthetic membrane-bound proteins in Rhodobacter Sphaeroides,studied with solid-state NMR spectroscopy

The photosynthetic purple bacteria such as Rb. sphaeroides possesses an intracytoplasmic membrane (ICM) and a variety of pigment-binding membrane proteins located in the ICM, acting as photoreceptor. Such photosynthetic apparatus is concentrated in the ICM. It is composed of three multimeric membrane-bound proteins; light-harvesting complexes (LH 1, LH 2), a reaction center (RC) and a cytochrome b/c1 complex. We have purified these membranes, which are called chromatophores, and characterized the structure and dynamics of the photosynthetic membrane-bound proteins by means of multi-nuclear solid state NMR. First, the isotropic chemical shift of carbonyl carbons in natural abundance and [1-¹³C] Phe labeled chromatophores indicates that the membrane-bound proteins take mainly the helical conformation. Second, the chemical shifts of side-chain resonances of uniformly ¹⁵N-labeled chromatophores indicate the side-chain histidine residue is mainly hydrogen bonded, whereas structural heterogeneity of arginine and lysine side-chains are probed by those wide distribution of ¹⁵N shifts. Thirdly, the [β-²H₃]Ala and [ε-²H₂]Tyr labeling of the chromatophores are performed and dynamics of the [β-²H]Ala and the [ε-²H₂]Tyr labeled chromatophores are studied by means of ²H solid state NMR. The dynamics of [β-²H₃]Ala is found to be a 10⁸Hz three-site jump motion with 10° liberation along the Cα-Cβ bond axis. The ²H-NMR powder pattern spectrum of [ε-²H₂] Tyr labeled chromatophores was interpreted with an averaged correlation time of 5×10⁵ Hz with 180° two-fold flips, the result of the averaging of two kinds of split spectra in terms of motional time scale. This revised version was published online in June 2006 with corrections to the Cover Date.     

PucC and LhaA direct efficient assembly of the light‐harvesting complexes in Rhodobacter sphaeroides

The mature architecture of the photosynthetic membrane of the purple phototroph Rhodobacter sphaeroides has been characterised to a level where an atomic‐level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light‐harvesting LH2 and reaction centre‐light‐harvesting1‐PufX (RC‐LH1‐PufX) photosystem complexes using spectroscopy, pull‐downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lhaA and pucC mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in ΔlhaA mutants assemble to form normal RC‐LH1‐PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG‐SecDF‐YajC‐YidC assembly machinery, thereby co‐ordinating pigment delivery, the co‐translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.