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1.
Biochemistry ; 60(44): 3302-3314, 2021 11 09.
Article in English | MEDLINE | ID: mdl-34699186

ABSTRACT

Light-harvesting 2 (LH2) antenna complexes augment the collection of solar energy in many phototrophic bacteria. Despite its frequent role as a model for such complexes, there has been no three-dimensional (3D) structure available for the LH2 from the purple phototroph Rhodobacter sphaeroides. We used cryo-electron microscopy (cryo-EM) to determine the 2.1 Å resolution structure of this LH2 antenna, which is a cylindrical assembly of nine αß heterodimer subunits, each of which binds three bacteriochlorophyll a (BChl) molecules and one carotenoid. The high resolution of this structure reveals all of the interpigment and pigment-protein interactions that promote the assembly and energy-transfer properties of this complex. Near the cytoplasmic face of the complex there is a ring of nine BChls, which absorb maximally at 800 nm and are designated as B800; each B800 is coordinated by the N-terminal carboxymethionine of LH2-α, part of a network of interactions with nearby residues on both LH2-α and LH2-ß and with the carotenoid. Nine carotenoids, which are spheroidene in the strain we analyzed, snake through the complex, traversing the membrane and interacting with a ring of 18 BChls situated toward the periplasmic side of the complex. Hydrogen bonds with C-terminal aromatic residues modify the absorption of these pigments, which are red-shifted to 850 nm. Overlaps between the macrocycles of the B850 BChls ensure rapid transfer of excitation energy around this ring of pigments, which act as the donors of energy to neighboring LH2 and reaction center light-harvesting 1 (RC-LH1) complexes.


Subject(s)
Bacterial Proteins/ultrastructure , Light-Harvesting Protein Complexes/metabolism , Light-Harvesting Protein Complexes/ultrastructure , Bacterial Proteins/metabolism , Bacteriochlorophyll A/metabolism , Carotenoids/chemistry , Carotenoids/metabolism , Cryoelectron Microscopy/methods , Energy Transfer , Rhodobacter sphaeroides/metabolism , Rhodobacter sphaeroides/ultrastructure
2.
Colloids Surf B Biointerfaces ; 172: 362-371, 2018 Dec 01.
Article in English | MEDLINE | ID: mdl-30189387

ABSTRACT

Biological processes using microorganisms for nanoparticle synthesis are appealing as eco-friendly nanofactories. The response of the photosynthetic bacterium Rhodobacter sphaeroides to gold exposure and its reducing capability of Au(III) to produce stable gold nanoparticles (AuNPs), using metabolically active bacteria and quiescent biomass, is reported in this study. In the former case, bacterial cells were grown in presence of gold chloride at physiological pH. Gold exposure was found to cause a significant increase of the lag-phase duration at concentrations higher than 10 µM, suggesting the involvement of a resistance mechanism activated by Au(III). Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM/EDS) analysis of bacterial cells confirmed the extracellular formation of AuNPs. Further studies were carried out on metabolically quiescent biomass incubated with gold chloride solution. The biosynthesized AuNPs were spherical in shape with an average size of 10 ±â€¯3 nm, as analysed by Transmission Electron Microscopy (TEM). The nanoparticles were hydrophilic and stable against aggregation for several months. In order to identify the functional groups responsible for the reduction and stabilization of nanoparticles, AuNPs were analysed by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, X-ray Photoelectron Spectroscopy (XPS), X-ray Fluorescence Spectrometry (XRF) and X-ray Absorption Spectroscopy (XAS) measurements. The obtained results indicate that gold ions bind to functional groups of cell membrane and are subsequently reduced by reducing sugars to gold nanoparticles and capped by a protein/peptide coat. Gold nanoparticles demonstrated to be efficient homogeneous catalysts in the degradation of nitroaromatic compounds.


Subject(s)
Gold/toxicity , Metal Nanoparticles/toxicity , Photosynthesis , Rhodobacter sphaeroides/metabolism , Anaerobiosis , Biomass , Catalysis , Metal Nanoparticles/ultrastructure , Photosynthesis/drug effects , Rhodobacter sphaeroides/drug effects , Rhodobacter sphaeroides/growth & development , Rhodobacter sphaeroides/ultrastructure
3.
Mol Microbiol ; 109(6): 812-825, 2018 09.
Article in English | MEDLINE | ID: mdl-29995992

ABSTRACT

The size of whole Rhodobacter sphaeroides prevents 3D visualization of centermost chromatophores in their native environment. This study combines cryo-focused ion beam milling with cryo-electron tomography to probe vesicle architecture both in situ and in 3D. Developing chromatophores are membrane-bound buds that remain in topological continuity with the cytoplasmic membrane and detach into vesicles when mature. Mature chromatophores closest to the cell wall are typically isolated vesicles, whereas centermost chromatophores are either linked to neighboring chromatophores or contain smaller, budding structures. Isolated chromatophores comprised a minority of centermost chromatophores. Connections between vesicles in growing bacteria are through ~10 nm-long, ~5 nm-wide linkers, and are thus physical rather than functional in terms of converting photons to ATP. In cells in the stationary phase, chromatophores fuse with neighboring vesicles, lose their spherical structure, and greatly increase in volume. The fusion and morphological changes seen in older bacteria are likely a consequence of the aging process, and are not representative of connectivity in healthy R. sphaeroides. Our results suggest that chromatophores can adopt either isolated or connected morphologies within a single bacterium. Revealing the organization of chromatophore vesicles throughout the cell is an important step in understanding the photosynthetic mechanisms in R. sphaeroides.


Subject(s)
Bacterial Chromatophores/ultrastructure , Rhodobacter sphaeroides/ultrastructure , Cell Membrane/metabolism , Cryoelectron Microscopy , Electron Microscope Tomography , Photosynthesis/physiology
4.
Enzyme Microb Technol ; 96: 103-110, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27871369

ABSTRACT

In Rhodobacter sphaeroides, carbonic anhydrase (CA; EC 4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3- while phosphoenolpyruvate carboxylase (PEPC; 4.1.1.31), an enzyme involved in the carbon metabolism that catalyzed the fixation of CO2 to PEP, is a key factor for biological fixation of CO2 and enhances the production of organic compounds. In this study, the recombinant R. sphaeroides with highly-expressed CA was developed based on a surface displayed system of CA (pJY-OmpCA) on the outer membrane of R. sphaeroides using outer membrane protein (Omp) in R. sphaeroides, Finally, two more different recombinant R. sphaeroides were developed, which transformed with a two-vector system harboring cytosolic expressed CA (pJY-OmpCA-CA)or PEPC (pJY-OMPCA-PEPC) in R. sphaeroides with surface displayed CA on the outer membrane. In case of recombinant R. sphaeroides with the pJY-OmpCA-PEPC, it has shown the highest CO2 reduction efficiency and the production of several organic compounds (carotenoids, polyhydroxybutyrate, malic acid, succinic acid). It means that the surface displayed CA on the R. sphaeroides would accelerate the CO2-bicabonate conversion on the bacterial outer membrane. Moreover, inducible over-expression of PEPC with surface-displayed CA was successfully used to facilitate a rapider CO2 reduction and quicker production of organic compounds.


Subject(s)
Carbon Dioxide/metabolism , Carbonic Anhydrases/metabolism , Phosphoenolpyruvate Carboxylase/metabolism , Rhodobacter sphaeroides/metabolism , Carbonic Anhydrases/genetics , Cell Membrane/metabolism , Cell Surface Display Techniques/methods , Hydroxybutyrates/metabolism , Phosphoenolpyruvate Carboxylase/genetics , Photosynthesis , Polyesters/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/ultrastructure
5.
J Bacteriol ; 197(17): 2859-66, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26124240

ABSTRACT

UNLABELLED: Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 µm and a diameter of 1.4 µm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. IMPORTANCE: R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1(-) mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism.


Subject(s)
Bacterial Proteins/metabolism , Flagella/ultrastructure , Flagellin/genetics , Gene Expression Regulation, Bacterial/physiology , Rhodobacter sphaeroides/ultrastructure , Bacterial Proteins/genetics , Flagellin/metabolism , Polymorphism, Genetic , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/metabolism
6.
Micron ; 72: 8-14, 2015 May.
Article in English | MEDLINE | ID: mdl-25725215

ABSTRACT

Bacteria have been studied using different microscopy methods for many years. Recently, the developments of high-speed atomic force microscopy have opened the doors to study bacteria in new ways due to the fact that it uses much less force on the sample while imaging. This makes the high-speed atomic force microscope an indispensable technique for imaging the surface of living bacterial cells because it allows for the high-resolution visualization of surface proteins in their natural condition without disrupting the cell or the activity of the proteins. Previous work examining living cells of Magnetospirillum magneticum AMB-1 demonstrated that the surface of these bacteria was covered with a net-like structure that is mainly composed of porin molecules. However, it was unclear whether or not this feature was unique to other living bacteria. In this study we used the high-speed atomic force microscope to examine the surface of living cells of Escherichia coli and Rhodobacter sphaeroides to compare their structure with that of M. magneticum. Our research clearly demonstrated that both of these types of cells have an outer surface that is covered in a network of nanometer-sized holes similar to M. magneticum. The diameter of the holes was 8.0±1.5 nm for E. coli and 6.6±1.1 nm for R. sphaeroides. The results in this paper confirm that this type of outer surface structure exists in other types of bacteria and it is not unique to Magnetospirillum.


Subject(s)
Escherichia coli/ultrastructure , Membrane Proteins/ultrastructure , Nanostructures/ultrastructure , Rhodobacter sphaeroides/ultrastructure , Magnetospirillum/ultrastructure , Microbial Viability , Microscopy, Atomic Force , Porins/ultrastructure
7.
Photosynth Res ; 119(3): 283-90, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24146256

ABSTRACT

Oxygen dictates the catabolic "lifestyle" of Rhodobacter sphaeroides. When it is present, the bacteria are fully equipped for aerobic respiration. When it is absent, the cells outfit themselves to make use of energy-gathering options that do not require oxygen. Thus, while respiring on alternate electron acceptors in the absence of oxygen even in the dark, the cells are fully enabled for phototrophy. PrrA, PpsR, and FnrL are global regulatory proteins mediating oxygen control of gene expression in this organism. For each of these, regulon members include a subset of a cluster of genes known as the photosynthesis genes, which encode the structural proteins and enzymes catalyzing biosynthesis of the pigments of the light-harvesting and reaction center complexes. The complexes are housed in a specialized structure called the intracytoplasmic membrane (ICM). Although details are emerging as to the differentiation process leading to fully formed ICM, little is known of necessary regulatory events beyond changes in photosynthesis gene transcription. This study used transmission electron microscopy toward gaining additional insights into potential roles of PrrA, PpsR, and FnrL in the formation of ICM. The major findings were (1) the absence of either PrrA or FnrL negatively affects ICM formation, (2) the lack of ICM in the absence of PrrA is partially, but not fully reversed by removing PpsR from the cell, (3) unlike R. sphaeroides, ICM formation in Rhodobacter capsulatus does not require FnrL. New avenues these findings provide toward identifying additional genes involved in ICM formation are discussed.


Subject(s)
Bacterial Proteins/metabolism , Intracellular Membranes/metabolism , Rhodobacter sphaeroides/metabolism , Bacterial Proteins/genetics , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Intracellular Membranes/ultrastructure , Microscopy, Electron, Transmission/methods , Mutation , Phototrophic Processes , Rhodobacter capsulatus/genetics , Rhodobacter capsulatus/ultrastructure , Rhodobacter sphaeroides/ultrastructure , Trans-Activators/genetics , Trans-Activators/metabolism
8.
Photosynth Res ; 116(2-3): 333-48, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23708977

ABSTRACT

Studies on membrane development in purple bacteria during adaptation to alterations in light intensity and oxygen tension are reviewed. Anoxygenic phototrophic such as the purple α-proteobacterium Rhodobacter sphaeroides have served as simple, dynamic, and experimentally accessible model organisms for studies of the photosynthetic apparatus. A major landmark in photosynthesis research, which dramatically illustrates this point, was provided by the determination of the X-ray structure of the reaction center (RC) in Blastochloris viridis (Deisenhofer and Michel, EMBO J 8:2149-2170, 1989), once it was realized that this represented the general structure for the photosystem II RC present in all oxygenic phototrophs. This seminal advance, together with a considerable body of subsequent research on the light-harvesting (LH) and electron transfer components of the photosynthetic apparatus has provided a firm basis for the current understanding of how phototrophs acclimate to alterations in light intensity and quality. Oxygenic phototrophs adapt to these changes by extensive thylakoid membrane remodeling, which results in a dramatic supramolecular reordering to assure that an appropriate flow of quinone redox species occurs within the membrane bilayer for efficient and rapid electron transfer. Despite the high level of photosynthetic unit organization in Rba. sphaeroides as observed by atomic force microscopy (AFM), fluorescence induction/relaxation measurements have demonstrated that the addition of the peripheral LH2 antenna complex in cells adapting to low-intensity illumination results in a slowing of the rate of electron transfer turnover by the RC of up to an order of magnitude. This is ascribed to constraints in quinone redox species diffusion between the RC and cytochrome bc1 complexes arising from the increased packing density as the intracytoplasmic membrane (ICM) bilayer becomes crowded with LH2 rings. In addition to downshifts in light intensity as a paradigm for membrane development studies in Rba. sphaeroides, the lowering of oxygen tension in chemoheterotropically growing cells results in a gratuitous formation of the ICM by an extensive membrane biogenesis process. These membrane alterations in response to lowered illumination and oxygen levels in purple bacteria are under the control of a number of interrelated two-component regulatory circuits reviewed here, which act at the transcriptional level to regulate the formation of both the pigment and apoprotein components of the LH, RC, and respiratory complexes. We have performed a proteomic examination of the ICM development process in which membrane proteins have been identified that are temporally expressed both during adaptation to low light intensity and ICM formation at low aeration and are spatially localized in both growing and mature ICM regions. For these proteomic analyses, membrane growth initiation sites and mature ICM vesicles were isolated as respective upper-pigmented band (UPB) and chromatophore fractions and subjected to clear native electrophoresis for isolation of bands containing the LH2 and RC-LH1 core complexes. In chromatophores, increasing levels of LH2 polypeptides relative to those of the RC-LH1 complex were observed as ICM membrane development proceeded during light-intensity downshifts, along with a large array of other associated proteins including high spectral counts for the F1FO-ATP synthase subunits and the cytochrome bc1 complex, as well as RSP6124, a protein of unknown function, that was correlated with increasing LH2 spectral counts. In contrast, the UPB was enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane protein assembly factors confirming the origin of the UPB from both peripheral respiratory membrane and sites of active CM invagination that give rise to the ICM. The changes in ICM vesicles were correlated to AFM mapping results (Adams and Hunter, Biochim Biophys Acta 1817:1616-1627, 2012), in which the increasing LH2 levels were shown to form densely packed LH2-only domains, representing the light-responsive antenna complement formed under low illumination. The advances described here could never have been envisioned when the author was first introduced in the mid-1960s to the intricacies of the photosynthetic apparatus during a lecture delivered in a graduate Biochemistry course at the University of Illinois by Govindjee, to whom this volume is dedicated on the occasion of his 80th birthday.


Subject(s)
Intracellular Membranes/metabolism , Light , Oxygen/pharmacology , Photosynthesis/radiation effects , Rhodobacter sphaeroides/metabolism , Rhodobacter sphaeroides/radiation effects , Intracellular Membranes/drug effects , Intracellular Membranes/radiation effects , Photosynthesis/drug effects , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Proteomics , Rhodobacter sphaeroides/drug effects , Rhodobacter sphaeroides/ultrastructure , Spectroscopy, Near-Infrared , Thylakoids/drug effects , Thylakoids/metabolism , Thylakoids/radiation effects
9.
J Mol Microbiol Biotechnol ; 23(1-2): 48-62, 2013.
Article in English | MEDLINE | ID: mdl-23615195

ABSTRACT

The results of a detailed structural and functional proteomic analysis of intracytoplasmic membrane (ICM) assembly in the model purple phototrophic bacterium Rhodobacter sphaeroides are reviewed in this report. Proteomics approaches have focused upon identification of membrane proteins temporally expressed during ICM development and spatially localized within the internal cell membranes, together with their structural and functional correlates. For the examination of temporal protein expression, procedures were established for the induction of ICM formation at low oxygen tension and for ICM remodeling in cells adapting to low intensity illumination, which permitted isolation by rate-zone sedimentation of ICM growth initiation sites (CM invaginations) in an upper-pigmented band (UPB), together with more mature ICM vesicles (chromatophores) as the main band. Nondenaturing clear native gel electrophoresis of the chromatophore fraction gave rise to four pigmented bands: the top and bottom bands contained the reaction center-light-harvesting 1 (RC-LH1) core complex and the LH2 peripheral antenna, respectively, while two bands of intermediate migration exhibited distinct associations of LH2 and core complexes. Proteomic analysis of the gel bands revealed developmental changes including increasing levels of LH2 polypeptides relative to those of core complexes as ICM development proceeded, as well as a large array of other associated proteins including high spectral counts for the F1FO-ATP synthase subunits, and the cytochrome bc1 complex. High counts were also observed for RSP6124, a protein of unknown function, that were correlated with increasing LH2 levels. RC-LH1-containing clear native electrophoresis gel bands from the UPB were enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane assembly factors (viz., preprotein translocases YidC, YajC and SecY, bacterial type 1 signal peptidase and twin arg translocation subunit TatA), thereby confirming the origin of the UPB from both peripheral respiratory membrane and sites of active CM invagination in which preferential assembly of the RC-LH1 complex occurs. Functional aspects of the photosynthetic unit assembly process were monitored by fluorescence induction/relaxation measurements of the variable fluorescence arising from LH-bacteriochlorophyll a. Slowing of the rate of RC electron transfer turnover (τQA), as assessed from the relaxation phase, was correlated with the growth of the functional absorption cross section (σ) and LH2/LH1 molar ratios. This is thought to arise from the imposition of constraints upon free diffusion of ubiquinone (UQ) redox species between the RC and cytochrome bc1 complex as the ICM bilayer becomes densely packed with LH2 rings. Such LH2 packing was confirmed in a comparison by high-resolution atomic force microscopy of ICM patches from cells grown at high and low light intensity [Adams and Hunter: Biochim Biophys Acta 2012;1817:1616-1627], in which the increasing LH2 levels form densely packed LH2-only domains, representing the light-responsive antenna complement arising under low illumination. In contrast, LH2 is initially dispersed in rows and small cluster-separating linear arrays of largely dimeric RC-LH1 core complexes, which become filled with LH2 during acclimation to reduced light intensity. In phototrophically grown cells that were transferred to oxic conditions in the dark, fluorescence induction/relaxation measurements showed that despite a growth burst independent of photosynthetic pathways, functional photosynthetic units were maintained for up to 24 h after the transition. The τQA was accelerated from ∼1 to 0.5 ms by 8 h, reflecting the decrease in LH2 levels, facilitating more rapid UQ redox species diffusion in the membrane bilayer as crowding by LH2 is overcome. Under these circumstances, UPB levels were elevated with significant increases in LH1/LH2 molar ratio. These changes indicate that vesiculation of CM growth initiation sites to form vesicular ICM was arrested under oxic conditions.


Subject(s)
Intracellular Membranes/metabolism , Intracellular Membranes/ultrastructure , Light-Harvesting Protein Complexes/metabolism , Rhodobacter sphaeroides/metabolism , Rhodobacter sphaeroides/ultrastructure , Bacterial Proteins/metabolism , Bacteriochlorophylls/metabolism , Electron Transport , Membrane Proteins/metabolism , Microscopy, Atomic Force , Photosynthesis , Proteomics
10.
Biofizika ; 57(1): 88-92, 2012.
Article in Russian | MEDLINE | ID: mdl-22567913

ABSTRACT

A comparative study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant was done by atomic force and electron microscopy. It was shown that transformation led to a decrease in the number or total disappearance of the flagella, as well as to changes in the structure of the outer membrane of the bacteria cell wall. On the wild-type cell surface phage-like structures were found, and in transformed cells at their places hollows were identified. This study significantly extends an understanding of the changes occurring in the ipt-transformants of phototrophic purple bacterium Rhodobacter sphaeroides. This investigation not only confirmed earlier obtained data about the differences in the wild-type and ipt-transformant phototrophic purple bacteria cell wall, but also showed fine changes in the structure of its outer membrane.


Subject(s)
Bacterial Proteins/genetics , Cell Wall/ultrastructure , Cytokinins/genetics , Flagella/ultrastructure , Rhodobacter sphaeroides/ultrastructure , Cell Wall/genetics , Flagella/genetics , Genotype , Microscopy, Atomic Force , Microscopy, Electron , Rhodobacter sphaeroides/genetics , Transformation, Bacterial
11.
Biochim Biophys Acta ; 1807(1): 95-107, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20937243

ABSTRACT

The PufX polypeptide is an integral component of some photosynthetic bacterial reaction center-light harvesting 1 (RC-LH1) core complexes. Many aspects of the structure of PufX are unresolved, including the conformation of its long membrane-spanning helix and whether C-terminal processing occurs. In the present report, NMR data recorded on the Rhodobacter sphaeroides PufX in a detergent micelle confirmed previous conclusions derived from equivalent data obtained in organic solvent, that the α-helix of PufX adopts a bent conformation that would allow the entire helix to reside in the membrane interior or at its surface. In support of this, it was found through the use of site-directed mutagenesis that increasing the size of a conserved glycine on the inside of the bend in the helix was not tolerated. Possible consequences of this bent helical structure were explored using a series of N-terminal deletions. The N-terminal sequence ADKTIFNDHLN on the cytoplasmic face of the membrane was found to be critical for the formation of dimers of the RC-LH1 complex. It was further shown that the C-terminus of PufX is processed at an early stage in the development of the photosynthetic membrane. A model in which two bent PufX polypeptides stabilise a dimeric RC-LH1 complex is presented, and it is proposed that the N-terminus of PufX from one half of the dimer engages in electrostatic interactions with charged residues on the cytoplasmic surface of the LH1α and ß polypeptides on the other half of the dimer.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Light-Harvesting Protein Complexes/chemistry , Light-Harvesting Protein Complexes/metabolism , Rhodobacter sphaeroides/enzymology , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Conserved Sequence , Dimerization , Intracellular Membranes/enzymology , Light-Harvesting Protein Complexes/isolation & purification , Magnetic Resonance Spectroscopy/methods , Micelles , Microscopy, Atomic Force/methods , Models, Molecular , Molecular Sequence Data , Photosynthesis , Protein Conformation , Rhodobacter sphaeroides/growth & development , Rhodobacter sphaeroides/ultrastructure , Sequence Alignment , Sequence Homology, Amino Acid
12.
Mol Microbiol ; 76(4): 833-47, 2010 May.
Article in English | MEDLINE | ID: mdl-20444085

ABSTRACT

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.


Subject(s)
Cell Membrane/ultrastructure , Rhodobacter sphaeroides/ultrastructure , Bacterial Proteins/chemistry , Cryoelectron Microscopy , Light-Harvesting Protein Complexes/chemistry , Microscopy, Atomic Force , Microscopy, Fluorescence
13.
Mol Microbiol ; 76(4): 803-5, 2010 May.
Article in English | MEDLINE | ID: mdl-20412442

ABSTRACT

A major feature that distinguishes prokaryotic organisms from eukaryotes is their less complex internal structure, in which all membrane-associated functions are thought to be present within a continuous lipid-protein bilayer, rather than with distinct organelles. Contrary to this notion, as described by Tucker and co-workers in this issue of Molecular Microbiology, the application of cryo-electron tomography to the purple bacterium Rhodobacter sphaeroides has demonstrated a heretofore unrecognized ultrastructural complexity within the intracytoplasmic membrane (ICM) housing the photosynthetic apparatus. In addition to distinguishing invaginations of the cytoplasmic membrane (CM) and interconnected vesicular structures still attached to the CM, a eukaryote-like ICM budding process was revealed, which results in the formation of fully detached vesicular structures. These bacterial organelles are able to carry out both the light-harvesting and light-driven energy transduction activities necessary for the cells to assume a photosynthetic lifestyle. Their formation is shown to represent the final stage in a membrane invagination and growth process, originating with small CM indentations, which after cell disruption give rise to a membrane fraction that can be separated from mature ICM vesicles by rate-zone sedimentation.


Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Cytoplasmic Vesicles/metabolism , Energy Metabolism , Light-Harvesting Protein Complexes/metabolism , Rhodobacter sphaeroides/metabolism , Biological Transport , Eukaryota/metabolism , Photosynthesis , Rhodobacter sphaeroides/ultrastructure
14.
Biophys J ; 97(1): 321-9, 2009 Jul 08.
Article in English | MEDLINE | ID: mdl-19580770

ABSTRACT

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 approximately 250-550 A, 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.


Subject(s)
Bacterial Proteins/chemistry , Cell Membrane/chemistry , Light-Harvesting Protein Complexes/chemistry , Light-Harvesting Protein Complexes/metabolism , Membrane Proteins/chemistry , Rhodobacter sphaeroides/chemistry , Bacterial Proteins/metabolism , Cell Membrane/ultrastructure , Chromatophores/chemistry , Computer Simulation , Membrane Proteins/metabolism , Microscopy, Electron , Models, Molecular , Models, Theoretical , Protein Multimerization , Rhodobacter sphaeroides/ultrastructure , Video Recording
15.
J Am Soc Mass Spectrom ; 20(2): 198-212, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18835523

ABSTRACT

The identification and structural characterization of a series of ornithine lipids extracted from the cell membranes of wild-type Rhodobacter sphaeroides, as well as from a glycerophosphocholine-deficient strain, have been achieved by multistage tandem mass spectrometry of their protonated and deprotonated precursor ions in a linear quadrupole ion trap. Systematic examination of the multistage gas-phase fragmentation reactions of these ions, combined with the use of hydrogen/deuterium exchange, has enabled the pathways responsible for sequential losses of the 3-hydroxy linked fatty acyl chain and the amide linked 3-OH fatty acyl chain from these lipids, as well as for formation of the previously reported ornithine specific positively charged "fingerprint" ion at m/z 115, to be determined. Additionally, the fragmentation pathways responsible for formation of a previously unreported ornithine lipid head group-specific product ion at m/z 131 in negative ion mode have been examined. Based on these results, and by comparison with the fragmentation behavior of model lipoamino acid standard compounds, a series of novel glutamine containing lipids have also been identified, with analogous structures but with masses 14 Da higher than those of several of the ornithine lipids observed in this study. Characteristic "fingerprint" ions indicative of these glutamine lipids were found at m/z 147, 130, and 129 in positive ion mode and at m/z 145 and 127 in negative ion mode. The results from this study establish an experimental basis for future efforts aimed at the sensitive identification, characterization, and quantitative analysis of ornithine and glutamine lipids in complex unfractionated cellular extracts.


Subject(s)
Cell Membrane/chemistry , Glutamine/chemistry , Lipids/chemistry , Ornithine/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Glycerylphosphorylcholine/deficiency , Ornithine/analogs & derivatives , Rhodobacter sphaeroides/chemistry , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/ultrastructure , Tandem Mass Spectrometry/methods
16.
J Biol Chem ; 283(45): 30772-9, 2008 Nov 07.
Article in English | MEDLINE | ID: mdl-18723509

ABSTRACT

The mapping of the photosynthetic membrane of Rhodobacter sphaeroides by atomic force microscopy (AFM) revealed a unique organization of arrays of dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) core complexes surrounded and interconnected by light-harvesting LH2 complexes (Bahatyrova, S., Frese, R. N., Siebert, C. A., Olsen, J. D., van der Werf, K. O., van Grondelle, R., Niederman, R. A., Bullough, P. A., Otto, C., and Hunter, C. N. (2004) Nature 430, 1058-1062). However, membrane regions consisting solely of LH2 complexes were under-represented in these images because these small, highly curved areas of membrane rendered them difficult to image even using gentle tapping mode AFM and impossible with contact mode AFM. We report AFM imaging of membranes prepared from a mutant of R. sphaeroides, DPF2G, that synthesizes only the LH2 complexes, which assembles spherical intracytoplasmic membrane vesicles of approximately 53 nm diameter in vivo. By opening these vesicles and adsorbing them onto mica to form small, < or =120 nm, largely flat sheets we have been able to visualize the organization of these LH2-only membranes for the first time. The transition from highly curved vesicle to the planar sheet is accompanied by a change in the packing of the LH2 complexes such that approximately half of the complexes are raised off the mica surface by approximately 1 nm relative to the rest. This vertical displacement produces a very regular corrugated appearance of the planar membrane sheets. Analysis of the topographs was used to measure the distances and angles between the complexes. These data are used to model the organization of LH2 complexes in the original, curved membrane. The implications of this architecture for the light harvesting function and diffusion of quinones in native membranes of R. sphaeroides are discussed.


Subject(s)
Cell Membrane/ultrastructure , Light-Harvesting Protein Complexes/ultrastructure , Microscopy, Atomic Force , Models, Molecular , Rhodobacter sphaeroides/ultrastructure , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Membrane/genetics , Cell Membrane/metabolism , Light-Harvesting Protein Complexes/genetics , Light-Harvesting Protein Complexes/metabolism , Mutation , Rhodobacter sphaeroides/enzymology , Rhodobacter sphaeroides/genetics
17.
FEBS J ; 275(12): 3157-66, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18479459

ABSTRACT

Microscopic and light spectroscopic investigations on the supramolecular architecture of bacterial photosynthetic membranes have revealed the photosynthetic protein complexes to be arranged in a densely packed energy-transducing network. Protein packing may play a determining role in the formation of functional photosynthetic domains and membrane curvature. To further investigate in detail the packing effects of like-protein photosynthetic complexes, we report an atomic force microscopy investigation on artificially created 2D crystals of the peripheral photosynthetic light-harvesting complexes 2 (LH2's) from the bacterium Rhodobacter sphaeroides. Instead of the usually observed one or two different crystallization lattices for one specific preparation protocol, we find seven different packing lattices. The most abundant crystal types all show a tilting of LH2. Most surprisingly, although LH2 is a monomeric protein complex in vivo, we find an LH2 dimer packing motif. We further characterize two different dimer configurations: in type 1, the LH2's are tilted inwards, and in type 2, they are tilted outwards. Closer inspection of the lattices surrounding the LH2 dimers indicates their close resemblance to those LH2's that constitute a lattice of zig-zagging LH2's. In addition, analyses of the tilt of the LH2's within the zig-zag lattice and that observed within the dimers corroborate their similar packing motifs. The type 2 dimer configuration exhibits a tilt that, in the absence of up-down packing, could bend the lipid bilayer, leading to the strong curvature of the LH2 domains as observed in Rhodobacter sphaeroides photosynthetic membranes in vivo.


Subject(s)
Bacterial Proteins/ultrastructure , Light-Harvesting Protein Complexes/ultrastructure , Rhodobacter sphaeroides/ultrastructure , Bacterial Proteins/chemistry , Crystallization , Dimerization , Light-Harvesting Protein Complexes/chemistry , Microscopy, Atomic Force
18.
Biol Chem ; 389(1): 69-78, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18095871

ABSTRACT

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and play crucial roles in many cellular and physiological processes. Functional production of recombinant GPCRs is one of the main bottlenecks to obtaining structural information. Here, we report the use of a novel bacterial expression system based on the photosynthetic bacterium Rhodobacter sphaeroides for the production of human recombinant GPCRs. The advantage of employing R. sphaeroides as a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. The system was tailored to overexpress recombinant receptors under the control of the moderately strong and highly regulated superoperonic photosynthetic promoter pufQ. We tested this system for the expression of some class A GPCRs, namely, the human adenosine A2a receptor (A2aR), the human angiotensin AT1a receptor (AT1aR) and the human bradykinin B2 receptor (B2R). Several different constructs were examined and functional production of the recombinant receptors was achieved. The best-expressed receptor, AT1aR, was solubilized and affinity-purified. To the best of our knowledge, this is the first report of successful use of a bacterial host--R. sphaeroides--to produce functional recombinant GPCRs under the control of a photosynthetic gene promoter.


Subject(s)
Receptors, G-Protein-Coupled/biosynthesis , Rhodobacter sphaeroides/metabolism , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Chromatography, Affinity , Chromatography, Gel , Cloning, Molecular , Gene Expression Regulation, Bacterial , Genetic Vectors , Humans , Indicators and Reagents , Microscopy, Electron , Photosynthesis , Radioligand Assay , Receptor, Adenosine A2A/biosynthesis , Receptor, Adenosine A2A/genetics , Receptor, Angiotensin, Type 1/biosynthesis , Receptor, Angiotensin, Type 1/genetics , Receptor, Bradykinin B2/biosynthesis , Receptor, Bradykinin B2/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Rhodobacter sphaeroides/ultrastructure
19.
Mol Microbiol ; 67(1): 63-77, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18034796

ABSTRACT

It is well established that assembly of the peripheral antenna complex, LH2, is required for proper photosynthetic membrane biogenesis in the purple bacterium Rhodobacter sphaeroides. The underlying interactions are, as yet, not understood. Here we examined the relationship between the morphology of the photosynthetic membrane and the lipid-protein interactions at the LH2-lipid interface. The non-bilayer lipid, phosphatidylethanolamine, is shown to be highly enriched in the boundary lipid phase of LH2. Sequence alignments indicate a putative lipid binding site, which includes beta-glutamate-20 and the adjacent carotenoid end group. Replacement of beta-glutamate-20 with alanine results in significant reduction of phosphatidylethanolamine and concomitant raise in phosphatidylcholine in the boundary lipid phase of LH2 without altering the lipid composition of the bulk phase. The morphology of the LH2 housing membrane is, however, unaffected by the amino acid replacement. In contrast, simultaneous modification of glutamate-20 and exchange of the carotenoid sphaeroidenone with neurosporene results in significant enlargement of the vesicular membrane invaginations. These findings suggest that the LH2 complex, specifically beta-glutamate-20 and the carotenoids' polar head group, contribute to the shaping of the photosynthetic membrane by specific interactions with surrounding lipid molecules.


Subject(s)
Cell Membrane/chemistry , Glutamates/metabolism , Light-Harvesting Protein Complexes/chemistry , Light-Harvesting Protein Complexes/genetics , Mutation, Missense , Phospholipids/metabolism , Rhodobacter sphaeroides/metabolism , Amino Acid Sequence , Binding Sites , Glutamates/genetics , Mass Spectrometry , Molecular Structure , Mutagenesis, Site-Directed , Protein Conformation , Rhodobacter sphaeroides/chemistry , Rhodobacter sphaeroides/ultrastructure , Spectrophotometry, Ultraviolet
20.
Biotechnol Bioeng ; 99(5): 1045-54, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-17969153

ABSTRACT

A Rhodobacter sphaeroides-supported dried, ground palladium catalyst ("Rs-Pd(0)") was compared with a Desulfovibrio desulfuricans-supported catalyst ("Dd-Pd(0)") and with unsupported palladium metal particles made by reduction under H2 ("Chem-Pd(0)"). Cell surface-located clusters of Pd(0) nanoparticles were detected on both D. desulfuricans and R. sphaeroides but the size and location of deposits differed among comparably loaded preparations. These differences may underlie the observation of different activities of Dd-Pd(0) and Rs-Pd(0) when compared with respect to their ability to promote hydrogen release from hypophosphite and to catalyze chloride release from chlorinated aromatic compounds. Dd-Pd(0) was more effective in the reductive dehalogenation of polychlorinated biphenyls (PCBs), whereas Rs-Pd(0) was more effective in the initial dehalogenation of pentachlorophenol (PCP) although the rate of chloride release from PCP was comparable with both preparations after 2 h.


Subject(s)
Desulfovibrio desulfuricans/metabolism , Hydrocarbons, Chlorinated/metabolism , Palladium/chemistry , Rhodobacter sphaeroides/metabolism , Biodegradation, Environmental , Biomass , Catalysis , Chlorides/chemistry , Desulfovibrio desulfuricans/ultrastructure , Hydrocarbons, Chlorinated/chemistry , Microscopy, Electron, Transmission , Oxidation-Reduction , Palladium/metabolism , Phosphites/chemistry , Rhodobacter sphaeroides/ultrastructure
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