Identification of the bacteriochlorophylls, carotenoids, quinones, lipids, and hopanoids of "Candidatus Chloracidobacterium thermophilum"

"Candidatus Chloracidobacterium thermophilum" is a recently discovered chlorophototroph from the bacterial phylum Acidobacteria, which synthesizes bacteriochlorophyll (BChl) c and chlorosomes like members of the green sulfur bacteria (GSB) and the green filamentous anoxygenic phototrophs (FAPs). The pigments (BChl c homologs and carotenoids), quinones, lipids, and hopanoids of cells and chlorosomes of this new chlorophototroph were characterized in this study. "Ca. Chloracidobacterium thermophilum" methylates its antenna BChls at the C-8(2) and C-12(1) positions like GSB, but these BChls were esterified with a variety of isoprenoid and straight-chain alkyl alcohols as in FAPs. Unlike the chlorosomes of other green bacteria, "Ca. Chloracidobacterium thermophilum" chlorosomes contained two major xanthophyll carotenoids, echinenone and canthaxanthin. These carotenoids may confer enhanced protection against reactive oxygen species and could represent a specific adaptation to the highly oxic natural environment in which "Ca. Chloracidobacterium thermophilum" occurs. Dihydrogenated menaquinone-8 [menaquinone-8(H(2))], which probably acts as a quencher of energy transfer under oxic conditions, was an abundant component of both cells and chlorosomes of "Ca. Chloracidobacterium thermophilum." The betaine lipid diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine, esterified with 13-methyl-tetradecanoic (isopentadecanoic) acid, was a prominent polar lipid in the membranes of both "Ca. Chloracidobacterium thermophilum" cells and chlorosomes. This lipid may represent a specific adaptive response to chronic phosphorus limitation in the mats. Finally, three hopanoids, diploptene, bacteriohopanetetrol, and bacteriohopanetetrol cyclitol ether, which may help to stabilize membranes during diel shifts in pH and other physicochemical conditions in the mats, were detected in the membranes of "Ca. Chloracidobacterium thermophilum."     

Identification of a gene essential for the first committed step in the biosynthesis of bacteriochlorophyll c

Bacteriochlorophylls (BChls) c, d, and e are the major chlorophylls in chlorosomes, which are the largest and one of the most efficient antennae produced by chlorophototrophic organisms. In the biosynthesis of these three BChls, a C-13(2)-methylcarboxyl group found in all other chlorophylls (Chls) must be removed. This reaction is postulated to be the first committed step in the synthesis of these BChls. Analyses of gene neighborhoods of (B)Chl biosynthesis genes and distribution patterns in organisms producing chlorosomes helped to identify a gene (bciC) that appeared to be a good candidate to produce the enzyme involved in this biochemical reaction. To confirm that this was the case, a deletion mutant of an open reading frame orthologous to bciC, CT1077, was constructed in Chlorobaculum tepidum, a genetically tractible green sulfur bacterium. The CT1077 deletion mutant was unable to synthesize BChl c but still synthesized BChl a and Chl a. The deletion mutant accumulated large amounts of various (bacterio)pheophorbides, all of which still had C-13(2)-methylcarboxyl groups. A C. tepidum strain in which CT1077 was replaced by an orthologous gene, Cabther_B0081 [corrected] from "Candidatus Chloracidobacterium thermophilum" was constructed. Although the product of Cabther_B0081 [corrected] was only 28% identical to the product of CT1077, this strain synthesized BChl c, BChl a, and Chl a in amounts similar to wild-type C. tepidum cells. To indicate their roles in the first committed step of BChl c, d, and e biosynthesis, open reading frames CT1077 and Cabther_B0081 [corrected] have been redesignated bciC. The potential mechanism by which BciC removes the C-13(2)-methylcarboxyl moiety of chlorophyllide a is discussed.     

Comparison of the physical characteristics of chlorosomes from three different phyla of green phototrophic bacteria

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered “Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum” were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133 × 57 × 36 nm; 141,000 nm³ volume), compared with chlorosomes from Cfx. aurantiacus (120 × 44 × 30 nm; 84,000 nm³) and Ca. Cab. thermophilum (99 × 40 × 31 nm; 65,000 nm³). Reflecting the contributions of thousands of pigment–pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000–2000 pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30 nm wide and 6 nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.     

Triplet exciton formation as a novel photoprotection mechanism in chlorosomes of Chlorobium tepidum

Chlorosomes comprise thousands of bacteriochlorophylls (BChl c, d, or e) in a closely packed structure surrounded by a lipid-protein envelope and additionally contain considerable amounts of carotenoids, quinones, and BChl a. It has been suggested that carotenoids in chlorosomes provide photoprotection by rapidly quenching triplet excited states of BChl via a triplet-triplet energy transfer mechanism that prevents energy transfer to oxygen and the formation of harmful singlet oxygen. In this work we studied triplet energy transfer kinetics and photodegradation of chlorosomes isolated from wild-type Chlorobium tepidum and from genetically modified species with different types of carotenoids and from a carotenoid-free mutant. Supporting a photoprotective function of carotenoids, carotenoid-free chlorosomes photodegrade approximately 3 times faster than wild-type chlorosomes. However, a significant fraction of the BChls forms a long-lived, triplet-like state that does not interact with carotenoids or with oxygen. We propose that these states are triplet excitons that form due to triplet-triplet interaction between the closely packed BChls. Numerical exciton simulations predict that the energy of these triplet excitons may fall below that of singlet oxygen and triplet carotenoids; this would prevent energy transfer from triplet BChl. Thus, the formation of triplet excitons in chlorosomes serves as an alternative photoprotection mechanism.     

Characterization of the FMO protein from the aerobic chlorophototroph, Candidatus Chloracidobacterium thermophilum

Candidatus Chloracidobacterium (Cab.) thermophilum is a recently discovered aerobic chlorophototroph belonging to the phylum Acidobacteria. From analyses of genomic sequence data, this organism was inferred to have type-1 homodimeric reaction centers, chlorosomes, and the bacteriochlorophyll (BChl) a-binding Fenna–Matthews–Olson protein (FMO). Here, we report the purification and characterization of Cab. thermophilum FMO. Absorption, fluorescence emission, and CD spectra of the FMO protein were measured at room temperature and at 77 K. The spectroscopic features of this FMO protein were different from those of the FMO protein of green sulfur bacteria (GSB) and suggested that exciton coupling of the BChls in the FMO protein is weaker than in FMO of GSB especially at room temperature. HPLC analysis of the pigments extracted from the FMO protein only revealed the presence of BChl a esterified with phytol. Despite the distinctive spectroscopic properties, the residues known to bind BChl a molecules in the FMO of GSB are well conserved in the primary structure of the Cab. thermophilum FMO protein. This suggests that the FMO of Cab. thermophilum probably also binds seven or possibly eight BChl a(P) molecules. The results imply that, without changing pigment composition or structure dramatically, the FMO protein has acquired properties that allow it to perform light harvesting efficiently under aerobic conditions.     

Isolation and pigment composition of the antenna system of four species of green sulfur bacteria

New and rapid procedures were developed for the isolation of chlorosomes and FMO-protein from the green sulfur bacteria Prosthecochloris (P.) aestuarii, Chlorobium (Cb.) phaeovibrioides, Cb. tepidum and Cb. vibrioforme. The resulting preparations were free from contaminating pigments and proteins as was shown by absorption spectroscopy, pigment analysis and SDS-PAGE. Two spectrally different types of FMO-protein were found. The first type, present in P. aestuarii and Cb. vibrioforme, has a main absorption band at 6 K at 815 nm, whereas the second type, isolated from Cb. tepidum and Cb. phaeovibrioides, has a strong band at 806 nm. In contrast to what was recently suggested (Tronrud DE and Matthews BW (1993) In: Deisenhofer J and Norris J (eds) The Photosynthetic Reaction Center, Vol 1, pp 13–21. Academic Press, San Diego, CA) the FMO-proteins contained no polar BChl a homologue. The isolated chlorosomes showed a small blue-shift of the QY absorption maximum with respect to intact cells. For the different species, grown under the same light conditions, the homologue composition of BChls c and d was approximately identical whereas for the BChl e in Cb. phaeovibrioides the relative amounts of homologues with larger alkyl substituents at position 8 were considerably larger. Baseplate BChl a was present in all chlorosomes and comprised 1–2% of the chlorosomal BChl. Its QY absorption band was located at about 802 nm and was clearly separated from the major QY absorption band at 6 K. The predominant esterifying alcohol of BChl a in the chlorosomes as well as in the FMO-proteins was phytol, but both antenna complexes also contained small amounts of BChl a esterified with the metabolic intermediates geranylgeraniol, dihydrogeranylgeraniol and tetrahydrogeranylgeraniol, like most purple bacteria. Since the esterifying alcohols of the chlorosomal BChl a and of the main chlorosomal pigments (BChls c, d and e) are different, esterification, and perhaps also the synthesis, of the BChls in the interior of the chlorosome and of the BChls in the baseplate must be spatially and genetically separated processes.     

Selective protein extraction from Chlorobium tepidum chlorosomes using detergents. Evidence that CsmA forms multimers and binds bacteriochlorophyll a

Chlorosomes of the photosynthetic green sulfur bacterium Chlorobium tepidum consist of bacteriochlorophyll (BChl) c aggregates that are surrounded by a lipid-protein monolayer envelope that contains ten different proteins. Chlorosomes also contain a small amount of BChl a, but the organization and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters. When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes. Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape of these extracted chlorosomes did not initially differ significantly from untreated chlorosomes, the extracted chlorosomes gradually disintegrated, and rod-shaped BChl c aggregates were sometimes observed. These results strongly suggest that CsmA binds the BChl a in Chlorobium-type chlorosomes and further indicate that none of the nine other chlorosome envelope proteins are absolutely required for maintaining the shape and integrity of chlorosomes. Quantitative estimates suggest that chlorosomes contain approximately equimolar amounts of CsmA and BChl a and that roughly one-third of the surface of the chlorosome is covered by CsmA.     

The chlorosome: a prototype for efficient light harvesting in photosynthesis

Three phyla of bacteria include phototrophs that contain unique antenna systems, chlorosomes, as the principal light-harvesting apparatus. Chlorosomes are the largest known supramolecular antenna systems and contain hundreds of thousands of BChl c/d/e molecules enclosed by a single membrane leaflet and a baseplate. The BChl pigments are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Their excitation energy flows via a small protein, CsmA embedded in the baseplate to the photosynthetic reaction centres. Chlorosomes allow for photosynthesis at very low light intensities by ultra-rapid transfer of excitations to reaction centres and enable organisms with chlorosomes to live at extraordinarily low light intensities under which no other phototrophic organisms can grow. This article reviews several aspects of chlorosomes: the supramolecular and molecular organizations and the light-harvesting and spectroscopic properties. In addition, it provides some novel information about the organization of the baseplate.     

Internal structure of chlorosomes from brown-colored chlorobium species and the role of carotenoids in their assembly

Chlorosomes are the main light harvesting complexes of green photosynthetic bacteria. Recently, a lamellar model was proposed for the arrangement of pigment aggregates in Chlorobium tepidum chlorosomes, which contain bacteriochlorophyll (BChl) c as the main pigment. Here we demonstrate that the lamellar organization is also found in chlorosomes from two brown-colored species (Chl. phaeovibrioides and Chl. phaeobacteroides) containing BChl e as the main pigment. This suggests that the lamellar model is universal among green sulfur bacteria. In contrast to green-colored Chl. tepidum, chlorosomes from the brown-colored species often contain domains of lamellar aggregates that may help them to survive in extremely low light conditions. We suggest that carotenoids are localized between the lamellar planes and drive lamellar assembly by augmenting hydrophobic interactions. A model for chlorosome assembly, which accounts for the role of carotenoids and secondary BChl homologs, is presented.     

Chlorobium Tepidum: Insights into the Structure,Physiology, and Metabolism of a Green Sulfur Bacterium Derived from the Complete Genome Sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.     

Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes

Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope. These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes. Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known. Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA. All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within approximately 90% (CsmC(-) and CsmJ(-)) of those of the wild-type strain. Chlorosomes isolated from the mutants lacked only the protein whose gene had been inactivated and were generally similar to those from the wild-type strain with respect to size, shape, and BChl c, BChl a, and carotenoid contents. However, chlorosomes from the csmC mutant were about 25% shorter than those from the wild-type strain, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered. The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential for the biogenesis, light harvesting, or structural organization of BChl c and BChl a within the chlorosome. These results demonstrate that chlorosomes are remarkably robust structures that can tolerate considerable changes in protein composition.     

Studies of the location and function of isoprenoid quinones in chlorosomes from green sulfur bacteria

The chlorosome antenna of the green sulfur bacterium Chlorobium tepidum essentially consists of aggregated bacteriochlorophyll (BChl) c enveloped in a glycolipid monolayer. Small amounts of protein and the isoprenoid quinones chlorobiumquinone (CK) and menaquinone-7 (MK-7) are also present. Treatment of isolated chlorosomes from Cb. tepidum with sodium dodecyl sulfate (SDS) did not affect the quinones, demonstrating that these are located in a site which is inaccessible to SDS, probably in the interior of the chlorosomes. About half of the quinones were removed by Triton X-100. The non-ionic character of Triton probably allowed it to extract components from within the chlorosomes. MK-10 in chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus was likewise found to be located in the chlorosome interior. The excitation transfer in isolated chlorosomes from Cb. tepidum is redox-regulated. We found a ratio of BChl c fluorescenceintensity under reducing conditions (Fred) to that under oxidizing conditions (Fox) of approximately 40. The chlorosomal BChl a fluorescence was also redox-regulated. When the chlorosomal BChl c–BChl c interactions were disrupted by 1-hexanol, the BChl c Fred/Fox ratiodecreased to approximately 3. When CK and MK-7 were extracted from isolated chlorosomes with hexane, the BChl c Fred/Fox ratio also decreased to approximately 3. A BChl c Fred/Fox ratio of 3–5 was furthermore observed in aggregates of pure BChl c and in chlorosomes from Cfx. aurantiacus which do not contain CK. We therefore suggest that BChl c aggregates inherently exhibit a small redox-dependent fluorescence (Fred/Fox 3) and that the large redox-dependent fluorescence observed in chlorobial chlorosomes (Fred/Fox 40) is CK-dependent.     

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.     

Lamellar organization of pigments in chlorosomes, the light harvesting complexes of green photosynthetic bacteria

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations approximately 20 A apart. X-ray scattering from chlorosomes exhibited a feature with the same approximately 20 A spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.     

Complete genome of Candidatus Chloracidobacterium thermophilum, a chlorophyll-based photoheterotroph belonging to the phylum Acidobacteria

Candidatus Chloracidobacterium thermophilum, which naturally inhabits microbial mats of alkaline siliceous hot springs in Yellowstone National Park, is the only known chlorophototroph in the phylum Acidobacteria. The Ca. C. thermophilum genome was composed of two chromosomes (2,683,362 bp and 1,012,010 bp), and both encoded essential genes. The genome included genes to produce chlorosomes, the Fenna-Matthews-Olson protein, bacteriochlorophylls a and c as principal pigments, and type-1, homodimeric reaction centres. Ca. C. thermophilum is an aerobic photoheterotroph that lacks the ability to synthesize several essential nutrients. Key genes of all known carbon fixation pathways were absent, as were genes for assimilatory nitrate and sulfate reduction and vitamin B(12) synthesis. Genes for the synthesis of branched-chain amino acids (valine, isoleucine and leucine) were also absent, but genes for catabolism of these compounds were present. This observation suggested that Ca. C. thermophilum may synthesize branched-chain amino acids from an intermediate(s) of the catabolic pathway by reversing these reactions. The genome encoded an aerobic respiratory electron transport chain that included NADH dehydrogenase, alternative complex III and cytochrome oxidase. The chromosomes of the laboratory isolate were compared with assembled, metagenomic scaffolds from the major Ca. C. thermophilum population in hot-spring mats. The larger chromosomes of the two populations were highly syntenous but significantly divergent (~13%) in sequence. In contrast, the smaller chromosomes have undergone numerous rearrangements, contained many transposases, and might be less constrained by purifying selection than the large chromosomes. Some transposases were homologous to those of mat community members from other phyla.     

Isolation and characterization of homodimeric type-I reaction center complex from Candidatus Chloracidobacterium thermophilum, an aerobic chlorophototroph

The recently discovered thermophilic acidobacterium Candidatus Chloracidobacterium thermophilum is the first aerobic chlorophototroph that has a type-I, homodimeric reaction center (RC). This organism and its type-I RCs were initially detected by the occurrence of pscA gene sequences, which encode the core subunit of the RC complex, in metagenomic sequence data derived from hot spring microbial mats. Here, we report the isolation and initial biochemical characterization of the type-I RC from Ca. C. thermophilum. After removal of chlorosomes, crude membranes were solubilized with 0.1% (w/v) n-dodecyl β-D-maltoside, and the RC complex was purified by ion-exchange chromatography. The RC complex comprised only two polypeptides: the reaction center core protein PscA and a 22-kDa carotenoid-binding protein denoted CbpC. The absorption spectrum showed a large, broad absorbance band centered at ～483 nm from carotenoids as well as smaller Q(y) absorption bands at 672 and 812 nm from chlorophyll a and bacteriochlorophyll a, respectively. The light-induced difference spectra of whole cells, membranes, and the isolated RC showed maximal bleaching at 840 nm, which is attributed to the special pair and which we denote as P840. Making it unique among homodimeric type-I RCs, the isolated RC was photoactive in the presence of oxygen. Analyses by optical spectroscopy, chromatography, and mass spectrometry revealed that the RC complex contained 10.3 bacteriochlorophyll a(P), 6.4 chlorophyll a(PD), and 1.6 Zn-bacteriochlorophyll a(P)' molecules per P840 (12.8:8.0:2.0). The possible functions of the Zn-bacteriochlorophyll a(P)' molecules and the carotenoid-binding protein are discussed.     

The chlorosome of Chlorobaculum tepidum: size, mass and protein composition revealed by electron microscopy, dynamic light scattering and mass spectrometry-driven proteomics

Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.     

Redox effects on the excited-state lifetime in chlorosomes and bacteriochlorophyll c oligomers.

Oligomers of [E,E] BChl CF (8, 12-diethyl bacteriochlorophyll c esterified with farnesol (F)) and [Pr,E] BChl CF (analogously, M methyl, Pr propyl) in hexane and aqueous detergent or lipid micelles were studied by means of steady-state absorption, time-resolved fluorescence, and electron spin resonance spectroscopy. The maximum absorption wavelength, excited-state dynamics, and electron spin resonance (EPR) linewidths are similar to those of native and reconstituted chlorosomes of Chlorobium tepidum. The maximum absorption wavelength of oligomers of [E,E] BChl CF was consistently blue-shifted as compared to that of [Pr,E] BChl CF oligomers, which is ascribed to the formation of smaller oligomers with [E,E] BChl CF than [Pr,E] BChl CF. Time-resolved fluorescence measurements show an excited-state lifetime of 10 ps or less in nonreduced samples of native and reconstituted chlorosomes of Chlorobium tepidum. Under reduced conditions the excited-state lifetime increased to tens of picoseconds, and energy transfer to BChl a or long-wavelength absorbing BChl c was observed. Oligomers of [E,E] BChl CF and [Pr,E] BChl CF in aqueous detergent or lipid micelles show a similar short excited-state lifetime under nonreduced conditions and an increase up to several tens of picoseconds upon reduction. These results indicate rapid quenching of excitation energy in nonreduced samples of chlorosomes and aqueous BChl c oligomers. EPR spectroscopy shows that traces of oxidized BChl c radicals are present in nonreduced and absent in reduced samples of chlorosomes and BChl c oligomers. This suggests that the observed short excited-state lifetimes in nonreduced samples of chlorosomes and BChl c oligomers may be ascribed to excited-state quenching by BChl c radicals. The narrow EPR linewidth suggests that the BChl c are arranged in clusters of 16 and 6 molecules in chlorosomes of Chlorobium tepidum and Chloroflexus aurantiacus, respectively.     

The organization of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus and the structural role of carotenoids and protein

The organization of bacteriochlorophyll c (BChl c) molecules was studied in normal and carotenoid-deficient chlorosomes isolated from the green phototrophic bacterium Chloroflexus aurantiacus. Carotenoid-deficient chlorosomes were obtained from cells grown in the presence of 60 µg of 2-hydroxybiphenyl per ml. At this concentration, BChl c synthesis was not affected while the formation of the 5.7 kDa chlorosome polypeptide was inhibited by about 50% (M. Foidl et al., submitted). Absorption, linear dichroism and circular dichroism spectroscopy showed that the organization of BChl c molecules with respect to each other as well as to the long axis of the chlorosomes was similar for both types of chlorosomes. Therefore, it is concluded that the organization of BChl c molecules is largely independent on the presence of the bulk of carotenoids as well as of at least half of the normal amount of the 5.7 kDa polypeptide. The Stark spectra of the chlorosomes, as characterized by a large difference polarizability for the ground- and excited states of the interacting BChl c molecules, were much more intense than those of individual pigments. It is proposed that this is caused by the strong overlap of BChl c molecules in the chlorosomes. In contrast to individual chlorophylls, BChl c in chlorosomes did not give rise to a significant difference permanent dipole moment for the ground- and excited states. This observation favors models for the BChl c organization which invoke the anti-parallel stacking of linear BChl c aggregates above those models in which linear BChl c aggregates are stacked in a parallel fashion. The difference between the Stark spectrum of carotenoid-deficient and WT chlorosomes indicates that the carotenoids are in the vicinity of the BChls.     

A new bacteriochlorophyll a-protein complex associated with chlorosomes of green sulfur bacteria

Chlorosomes were prepared from Chlorobium limicola f. thiosulfatophilum by sucrose density gradient centrifugation. Cells broken in the presence of 2 M NaSCN yielded three chlorosome fractions in the gradient: low density (no sucrose), medium density (approx. 18% sucrose), and high density (approx. 26% sucrose). All fractions were stable at any chlorosome concentration. Cells broken in the absence of 2 M NaSCN also yielded three fractions, but only the high-density fraction contained stable chlorosomes. The medium-density chlorosomes were stable only when highly concentrated. Upon dilution, bacteriochlorophyll (BChl) c was degraded to bacteriopheophytin c and concomitantly a band at 794 nm (BChl a) was revealed. Two 794-nm fractions were observed with the same densities as low- and medium-density chlorosomes. The protein composition of the 794-nm fractions was similar to that of the stable chlorosome fractions. All showed a 4-5 kDa (Mr) protein as a major component, but no trace of the 40-kDa protein characteristic of the water-soluble BChl a-protein of green sulfur bacteria. BChl a was present in all types of chlorosomes, in stable chlorosomes the BChl c/BChl a ratio was approx. 90. A special BChl a-protein (794 nm) inside the chlorosome is postulated to mediate energy transfer from BChl c to the water-soluble BChl a-protein in the baseplate.