[2Fe-2S] proteins in Chlorosomes: redox properties of CsmI, CsmJ, and CsmX of the Chlorosome envelope of Chlorobaculum tepidum.

The chlorosome envelope of Chlorobaculum tepidum contains 10 polypeptides, three of which, CsmI, CsmJ, and CsmX, have an adrenodoxin-like domain harboring a single [2Fe-2S] cluster. Mutants that produced chlorosomes containing two, one, or none of these Fe-S proteins were constructed [Li, H., et al. (2013) Biochemistry 52, preceding paper in this issue ( DOI: 10.1021/bi301454g )]. The electron paramagnetic resonance (EPR) spectra, g values, and line widths of the Fe-S clusters in individual CsmI, CsmJ, and CsmX proteins were obtained from studies with isolated chlorosomes. The Fe-S clusters in these proteins were characterized by EPR and could be differentiated on the basis of their g values and line widths. The EPR spectrum of wild-type chlorosomes could be simulated by a 1:1 admixture of the CsmI and CsmJ spectra. No contribution of CsmX to the EPR spectrum of chlorosomes was observed because of its low abundance. In chlorosomes that contained only CsmI or CsmJ, the midpoint potential of the [2Fe-2S] clusters was -205 or 8 mV, respectively; the midpoint potential of the [2Fe-2S] cluster in CsmX was estimated to be more oxidizing than -180 mV. In wild-type chlorosomes, the midpoint potentials of the [2Fe-2S] clusters were -348 mV for CsmI and 92 mV for CsmJ. The lower potential for CsmI in the presence of CsmJ, and the higher potential for CsmJ in the presence of CsmI, were attributed to interactions that occur when these proteins form complexes in the chlorosome envelope. The redox properties of CsmI and CsmJ are consistent with their proposed participation in the transfer of electrons to and from quenchers of energy transfer in chlorosomes.

[2Fe-2S] proteins in Chlorosomes: CsmI and CsmJ participate in light-dependent control of energy transfer in Chlorosomes of Chlorobaculum tepidum.

Chlorosomes of Chlorobaculum tepidum are formed from stacks of syn-anti coordinated bacteriochlorophyll c dimers, which form a suprastructure comprised of coaxial nanotubes and are surrounded by a glycolipid monolayer envelope containing 10 proteins. Three of these proteins, CsmI, CsmJ, and CsmX, have sequences very similar in their N-terminal domains to those of [2Fe-2S] ferredoxins of the adrenodoxin/putidaredoxin subfamily. The roles of these proteins in chlorosomes were studied in single-, double-, and triple-mutant strains. In each mutant, only the protein(s) corresponding to the mutated gene(s) was missing, and the amounts of other chlorosome proteins did not vary significantly. Electrophoretic analyses and immunoblotting showed that CsmX was much less abundant than CsmI or CsmJ. The growth rates and the pigment and isoprenoid quinone contents of isolated chlorosomes of the mutants were similar to wild-type values. Quenching and recovery of energy transfer in isolated chlorosomes and intact cells were studied by measuring fluorescence emission after exposure to or removal of oxygen. Oxygen-induced activation of the quencher in isolated chlorosomes or in intact cells was largely independent of CsmI and CsmJ. This may be because oxygen can diffuse across the chlorosome envelope easily and directly reacts with the quencher. However, CsmI and CsmJ were required to restore energy transfer fully after isolated chlorosomes were exposed to oxygen. Studies with intact cells suggested that cells contain both light-dependent and light-independent pathways for reducing the quenching species in chlorosomes and that CsmI and CsmJ are components of a light-dependent pathway.

Membrane proteome of the green sulfur bacterium Chlorobium tepidum (syn. Chlorobaculum tepidum) analyzed by gel-based and gel-free methods.

Chlorobium tepidum is a Gram-negative bacterium of the green sulfur phylum (Chlorobia). Chlorobia are obligate anaerobic photolithoautotrophs that are widely distributed in aquatic environments where anoxic layers containing reduced sulfur compounds are exposed to light. The envelope of C. tepidum is a complex organelle composed of the outer membrane, the periplasm-peptidoglycan layer, and the cytoplasmic membrane. In addition to the outer and plasma membranes, C. tepidum contains chlorosomes attached to the cytoplasmic side of the plasma membrane. Each cellular compartment has a unique set of proteins, called sub-proteome. An important aim of proteome analysis is to study the level of the expressed genes and their response to environmental changes. Membrane protein studies are of primary importance to understand how nutrients are transported inside the cell, how toxic molecules are exported, and the mechanisms of photosynthesis and energy metabolism.

Anisotropic distribution of emitting transition dipoles in chlorosome from Chlorobium tepidum: fluorescence polarization anisotropy study of single chlorosomes.

The polarization anisotropy of fluorescence spectra from single chlorosomes isolated from a green sulfur bacterium, Chlorobium (Cb.) tepidum, was observed at 13 K. As the polarizer was rotated, the intensities of the fluorescence bands of both bacteriochlorophyll (BChl)-c self-aggregates and BChl-a in baseplate proteins showed clear oscillations. From the oscillation, the values of the degree of polarization (DP) and the phase shift (PS) between the BChl-c and BChl-a bands were determined for each single chlorosome. The DP versus PS plot for Cb. tepidum chlorosomes showed linear correlations between the PS and the DP values for both BChl-c and BChl-a fluorescence bands. This tendency could be explained from a simulation assuming a random orientation of chlorosomes and a triaxial orientation distribution of emitting transition dipoles within a single chlorosome. The intensity ratios among the X-/Y-/Z-principal transition dipoles were estimated to be 0.3/0.5/1 and 1/0.6/0.1 for the BChl-c and BChl-a fluorescence bands, respectively. Here, the X-, Y-, and Z-axes are perpendicular, parallel to the cytoplasmic membrane, and parallel to the chlorosome long axis, respectively. A theoretical calculation based on the exciton theory was conducted to reproduce the observed triaxial orientation distribution of emitting transition dipoles. The simulation revealed that a deformation introduced to the circular cross section of the rod-shaped BChl-c self-aggregates could qualitatively reproduce results of this study.

Long-range organization of bacteriochlorophyll in chlorosomes of Chlorobium tepidum investigated by cryo-electron microscopy.

Intact chlorosomes of Chlorobium tepidum were embedded in amorphous ice layers and examined by cryo-electron microscopy to study the long-range organization of bacteriochlorophyll (BChl) layers. End-on views reveal that chlorosomes are composed of several multi-layer tubules of variable diameter (20-30 nm) with some locally undulating non-tubular lamellae in between. The multi-layered tubular structures are more regular and larger in a C. tepidum mutant that only synthesizes [8-ethyl, 12-methyl]-BChl d. Our data show that wild-type C. tepidum chlorosomes do not have a highly regular, long-range BChl c layer organization and that they contain several multi-layered tubules rather than single-layer tubules or exclusively undulating lamellae as previously proposed.

Isotopic replacement of pigments and a lipid in chlorosomes from Chlorobium limicola: characterization of the resultant chlorosomes.

Pigments including bacteriochlorophyll (BChl) c, carotenoids, and a trace of BChl a together with a lipid, monogalactosyl diglyceride (MGDG), were extracted with chloroform/methanol (1:1 v/v) from an aqueous suspension (50 mM Tris-HCl, pH 8.0) of chlorosomes from Chlorobium limicola; other lipids and proteins were left behind in the aqueous layer by funnel separation. The chloroform layer was dried by purging N2 gas, dissolved in methanol, and rapidly injected into the aqueous layer to reassemble chlorosomes. This technique has been developed to replace one-half of the inherent 12C-BChl c by 13C-BChl c to identify the intermolecular 13C...13C magnetic dipole correlation peaks (that are supposed to reduce their intensities to one-fourth by reducing the 13C-BChl c concentration into one-half) and to determine the structure of BChl c aggregates in the rod elements by means of solid-state NMR spectroscopy. The isotopically replaced chlorosomes were characterized (1) by sucrose density gradient centrifugation, zeta potential measurement, electron microscopy, and dynamic light scattering measurement to determine the morphology of chlorosomes, (2) by 13C NMR spectroscopy, electronic absorption and circular dichroism spectroscopies, and low-angle X-ray diffraction to determine the pigment assembly in the rod elements, and (3) by subpicosecond time-resolved absorption spectroscopy to determine the excited-state dynamics in the pigment assembly. The results characterized the reassembled chlorosomes to have (1) similar but longer morphological structures, (2) almost the same pigment assembly in the rod elements, and (3) basically the same excited-state dynamics in the pigment assembly.

Assembly of a mixture of isomeric BChl c from Chlorobium limicola as determined by intermolecular 13C-13C dipolar correlations: coexistence of dimer-based and pseudo-monomer-based stackings.

A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate.

Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium "Chlorochromatium aggregatum".

A symbiotic green sulfur bacterium, strain CaD, was isolated from an enrichment culture of the phototrophic consortium "Chlorochromatium aggregatum". The capability of the epibiont to grow in pure culture indicates that it is not obligately symbiotic. Cells are Gram-negative, nonmotile, rod-shaped and contain chlorosomes. Strain CaD is obligately anaerobic and photolithoautotrophic, using sulfide as electron donor. Acetate and peptone are photoassimilated in the presence of sulfide and hydrogencarbonate. Photosynthetic pigments contain bacteriochlorophylls a and c, and gamma-carotene and OH-gamma-carotene glucoside laurate as the dominant carotenoids. In cells from pure cultures, chlorosomes are equally distributed along the inner face of the cytoplasmic membrane. In contrast, the distribution of the chlorosomes in symbiotic epibiont cells is uneven, with chlorosomes being entirely absent at the site of attachment to the central bacterium. The symbiotic epibiont cells display a conspicuous additional layered structure at the attachment site. The G + C content of genomic DNA of strain CaD is 46.7 mol%. On the basis of 16S rRNA sequence comparison, the strain is distantly related to Chlorobium species within the green sulfur bacteria phylum (

The ultrastructure of Chlorobium tepidum chlorosomes revealed by electron microscopy.

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.

Isolation and characterization of carotenosomes from a bacteriochlorophyll c-less mutant of Chlorobium tepidum.

Chlorosomes are the light-harvesting organelles in photosynthetic green bacteria and typically contain large amounts of bacteriochlorophyll (BChl) c in addition to smaller amounts of BChl a, carotenoids, and several protein species. We have isolated vestigial chlorosomes, denoted carotenosomes, from a BChl c-less, bchK mutant of the green sulfur bacterium Chlorobium tepidum. The physical shape of the carotenosomes (86 +/- 17 nm x 66 +/- 13 nm x 4.3 +/- 0.8 nm on average) was reminiscent of a flattened chlorosome. The carotenosomes contained carotenoids, BChl a, and the proteins CsmA and CsmD in ratios to each other comparable to their ratios in wild-type chlorosomes, but all other chlorosome proteins normally found in wild-type chlorosomes were found only in trace amounts or were not detected. Similar to wild-type chlorosomes, the CsmA protein in the carotenosomes formed oligomers at least up to homo-octamers as shown by chemical cross-linking and immunoblotting. The absorption spectrum of BChl a in the carotenosomes was also indistinguishable from that in wild-type chlorosomes. Energy transfer from the bulk carotenoids to BChl a in carotenosomes was poor. The results indicate that the carotenosomes have an intact baseplate made of remarkably stable oligomeric CsmA-BChl a complexes but are flattened in structure due to the absence of BChl c. Carotenosomes thus provide a valuable material for studying the biogenesis, structure, and function of the photosynthetic antennae in green bacteria.