The lamellar spacing in self-assembling bacteriochlorophyll aggregates is proportional to the length of the esterifying alcohol

Chlorosomes from green photosynthetic bacteria are large photosynthetic antennae containing self-assembling aggregates of bacteriochlorophyll c, d, or e. The pigments within chlorosomes are organized in curved lamellar structures. Aggregates with similar optical properties can be prepared in vitro, both in polar as well as non-polar solvents. In order to gain insight into their structure we examined hexane-induced aggregates of purified bacteriochlorophyll c by X-ray scattering. The bacteriochlorophyll c aggregates exhibit scattering features that are virtually identical to those of native chlorosomes demonstrating that the self-assembly of these pigments is fully encoded in their chemical structure. Thus, the hexane-induced aggregates constitute an excellent model to study the effects of chemical structure on assembly. Using bacteriochlorophyllides transesterified with different alcohols we have established a linear relationship between the esterifying alcohol length and the lamellar spacing. The results provide a structural basis for lamellar spacing variability observed for native chlorosomes from different species. A plausible physiological role of this variability is discussed. The X-ray scattering also confirmed the assignments of peaks, which arise from the crystalline baseplate in the native chlorosomes.     

Effect of carotenoid deficiency on cells and chlorosomes of Chlorobium phaeobacteroides

The effects of inhibition of carotenoid biosynthesis by 2-hydroxybiphenyl on the photosynthetic growth, pigment composition and chlorosome structure of Chlorobium phaeobacteroides strain CL1401 were examined. At a concentration of 20 micrograms 2-hydroxybiphenyl .ml-1, carotenoid synthesis was largely inhibited (85%), but the photosynthetic growth rate was almost unaffected (mu control = 0.00525 +/- 0.00007 h-1 and mu HBP-treated = 0.00505 +/- 0.0005 h-1). Cells grown in the presence of the inhibitor were 5 microns-70 microns long, while control cells were between 2-5 microns long. Moreover, 2-hydroxybiphenyl-treated cells contained fewer, unevenly distributed chlorosomes per micron of cytoplasmic membrane with an irregular arrangement (2.5 +/- 1.5 vs of 9.1 +/- 1.9). This was concomitant to the 83% decrease in the content of bacteriochlorophyll (BChl) e in 2-hydroxybiphenyl-treated cells. Electron microscopy revealed that the shape of carotenoid-depleted chlorosomes changed from ellipsoidal to spherical, although the mean volume was similar to that of control chlorosomes. SDS-PAGE analysis of the chlorosome polypeptide composition showed that the amount of CsmA protein decreased by 60% in carotenoid-depleted chlorosomes. This was paralleled by a decrease in the baseplate BChl a content. The data suggest that carotenoids are close to the chlorosomal baseplate, where they carry out both structural and photoprotective functions.     

Impact of esterified bacteriochlorophylls on the biogenesis of chlorosomes in Chloroflexus aurantiacus

A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52 °C (an optimal growth temperature) and 44 °C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44 °C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44 °C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.     

Antenna organization and evidence for the function of a new antenna pigment species in the green photosynthetic bacterium Chloroflexus aurantiacus

Whole cells and isolated chlorosomes (antenna complex) of the green photosynthetic bacterium Chloroflexus aurantiacus have been studied by absorption spectroscopy (77 K and room temperature), fluorescence spectroscopy, circular dichroism, linear dichroism and electron spin resonance spectroscopy. The chlorosome absorption spectrum has maxima at 450 (contributed by carotenoids and bacteriochlorophyll (BChl) a Soret), 742 (BChl c) and 792 nm (BChl a) with intensity ratios of 20:25. The fluorescence emission spectrum has peaks at 748 and 802 nm when excitation is into either the 742 or 450 nm absorption bands, respectively. Whole cells have fluorescence peaks identical to those in chlorosomes with the addition of a major peak observed at 867 nm. The CD spectrum of isolated chlorosomes has an asymmetric-derivative-shaped CD centered at 739 nm suggestive of exciton interaction at least on the level of dimers. Linear dichroism of oriented chlorosomes shows preferential absorption at 742 nm of light polarized parallel to the long axis of the chlorosome. This implies that the transition dipoles are also oriented more or less parallel to the long axis of the chlorosome. Treatment with ferricyanide results in the appearance of a 2.3 G wide ESR spectrum at g 2.002. Whole cells grown under different light conditions exhibit different fluorescence behavior when absorption is normalized at 742 nm. Cells grown under low light conditions have higher fluorescence intensity at 748 nm and lower intensity at 802 nm than cells grown under high light conditions. These results indicate that the BChl c in chlorosomes is highly organized, and transfers energy from BChl c (742 nm) to a connector of baseplate BChl B792 (BChl a) presumably located in the chlorosome baseplate adjacent to the cytoplasmic membrane.     

Structure of Chlorosomes from the Green Filamentous Bacterium Chloroflexus aurantiacus

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.Chlorosomes (5, 13) are light-harvesting complexes found in three different phyla of photosynthetic bacteria. Chloroflexus aurantiacus belongs to the filamentous anoxygenic phototrophs (green nonsulfur bacteria) comprising members of the phylum Chloroflexi. All members of the green sulfur bacteria (phylum Chlorobi) contain chlorosomes. Very recently, a phototropic chlorosome-containing organism was found in the phylum Acidobacteria (9).Chlorosomes are oblong bodies attached to the inner side of the cytoplasmic membrane. A unique property of chlorosomes is that their main pigment, bacteriochlorophyll (BChl) c, d, or e, is organized in the form of an aggregate. A similar self-assembled aggregate can form in the absence of proteins and exhibits spectral and excitonic properties similar to those of pigments in the native chlorosomes (for a review, see reference 3). The BChl aggregates were suggested to form lamellar structures in chlorosomes of green sulfur bacteria with lamellar spacing between 2 and 3 nm, depending on the main BChl (BChl c or e) and the prevailing esterifying alcohol (38, 39). In this model, the lamellar layers are maintained by nonspecific hydrophobic interactions of the interdigitated esterifying alcohols, while the in-layer arrangement is mediated through specific interactions between the stacked chlorin rings. In BChl c-containing chlorosomes of Chlorobaculum tepidum (formerly Chlorobium tepidum), the lamellar system (spacing, ∼2 nm) often remains parallel for the whole length of the chlorosome (33, 38). In Chlorobaculum tepidum the lamellae exhibit considerable curvature, which was initially attributed to undulation (38), but recent end-on micrographs revealed a variety of curved lamellar structures, such as lamellar tubules or multilayered wraps, as well as undulations (33). Recently, when chlorosomes from a Chlorobaculum tepidum mutant with well-ordered BChl aggregates were used as a model for electron microscopy (EM) and nuclear magnetic resonance experiments, it was proposed that BChl aggregates form concentric nanotubes with the pigments arranged in helical spirals (14).In contrast, chlorosomes from BChl e-containing bacteria (e.g., Chlorobium phaeovibrioides) contain lamellar pigments that are organized into small domains with random orientations. It has been proposed that this arrangement improves the absorption of photons with different polarizations (39). This, together with aggregation-induced enlargement of the oscillator strength, enables the bacteria to survive under extremely low-light conditions. At this point it is unclear whether these domains also exhibit a multilayer tubular arrangement. The data suggest that while the lamellar nature of BChl aggregates seems to be conserved, the higher-order structure of chlorosomes may be different in different species.Chlorosomes attach to the cytoplasmic membrane via a crystalline baseplate that contains BChl a and carotenoids and acts as an intermediary in energy transfer from the chlorosome to the reaction centers in the membrane. The baseplate consists of multiple CsmA protein subunits (5.7 kDa in C. aurantiacus and 6.2 kDa in Chlorobaculum tepidum [8, 27, 34, 40]). In addition to its role in energy transfer, it has been proposed that the baseplate is essential for the long-range order of lamellar BChl aggregates (2, 19). In addition to CsmA, chlorosomes of C. aurantiacus contain a number of other proteins, all of which are located in the chlorosome envelope (for a review, see reference 13).Recent progress in understanding chlorosome structure has been limited to the Chlorobi, and it is unclear whether there is similar organization in chlorosomes from bacteria belonging to different phyla, such as the Chloroflexi. While Chloroflexi also employ chlorosomes as the main light-harvesting complex, genetically they are only distantly related to the Chlorobi. Chlorobi and Chloroflexi also exhibit substantial differences in the photosynthetic apparatus. The average size of chlorosomes from C. aurantiacus, the model organism of the Chloroflexi, has been reported to be smaller (100 by 30 by 15 nm) than the average size of chlorosomes from the Chlorobi (150 to 200 by 50 by 20 nm) (30, 32). C. aurantiacus chlorosomes contain a single homologue of BChl c (8-ethyl,12-methyl) (16) and several secondary homologues that harbor different esterifying alcohols. The main esterifying alcohol (stearol) and the minor secondary homologues have longer chains than the prevailing alcohol in Chlorobaculum tepidum (farnesol) (11, 16, 22).Carotenoids are thought to play important light-harvesting and protective roles in chlorosomes (10, 13, 26, 36, 37). These hydrophobic molecules were shown to partition into the apolar space between the chlorin planes together with the aliphatic chains of the esterifying alcohols (39), and they also contribute to the hydrophobic driving force during assembly (1, 20). C. aurantiacus exhibits much greater variability of the carotenoid/BChl molar ratio than the Chlorobi. This ratio was observed to increase at most 1.4-fold in the Chlorobi species studied, even if the light intensity was increased more than 2 orders of magnitude (from 0.1 to 50 microeinsteins m⁻² s⁻¹) (6, 7). However, when there was a moderate change in the light intensity (from 400 to 2,000 lx [41] or from 44 to 127 microeinsteins m⁻² s⁻¹ [22]), C. aurantiacus exhibited a robust increase (fivefold) in the carotenoid content. As a result, the carotenoid content can reach levels of approximately one carotenoid molecule per two BChl molecules (41). Thus, a C. aurantiacus chlorosome seems to be able to accumulate significantly more carotenoids than the average Chlorobaculum tepidum chlorosome, which exhibits about one carotenoid molecule per 10 BChl molecules (7, 39).In the present work we examined the overall structure, pigment arrangement, and composition of C. aurantiacus chlorosomes using cryo-electron tomography, X-ray scattering, and quantitative pigment analysis. C. aurantiacus chlorosomes appear to be thin with a distinct two-dimensional baseplate protein array. Our results also demonstrate that BChl c aggregates are lamellar, suggesting that this is a universal feature of chlorosome structure. The greater lamellar spacing is due to the longer esterifying alcohols and allows accommodation of more carotenoids.     

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.     

Isolation and Characterization of Carotenosomes from a Bacteriochlorophyll c-less Mutant ofChlorobium 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 × 66 ± 13 nm × 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.     

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.     

A model of the protein–pigment baseplate complex in chlorosomes of photosynthetic green bacteria

In contrast to photosynthetic reaction centers, which share the same structural architecture, more variety is found in the light-harvesting antenna systems of phototrophic organisms. The largest antenna system described, so far, is the chlorosome found in anoxygenic green bacteria, as well as in a recently discovered aerobic phototroph. Chlorosomes are the only antenna system, in which the major light-harvesting pigments are organized in self-assembled supramolecular aggregates rather than on protein scaffolds. This unique feature is believed to explain why some green bacteria are able to carry out photosynthesis at very low light intensities. Encasing the chlorosome pigments is a protein-lipid monolayer including an additional antenna complex: the baseplate, a two-dimensional paracrystalline structure containing the chlorosome protein CsmA and bacteriochlorophyll a (BChl a). In this article, we review current knowledge of the baseplate antenna complex, which physically and functionally connects the chlorosome pigments to the reaction centers via the Fenna–Matthews–Olson protein, with special emphasis on the well-studied green sulfur bacterium Chlorobaculum tepidum (previously Chlorobium tepidum). A possible role for the baseplate in the biogenesis of chlorosomes is discussed. In the final part, we present a structural model of the baseplate through combination of a recent NMR structure of CsmA and simulation of circular dichroism and optical spectra for the CsmA–BChl a complex.     

Hypothesis on chlorosome biogenesis in green photosynthetic bacteria

Chlorosomes are specialized compartments that constitute the main light harvesting system of green sulfur bacteria (GSB) and some filamentous anoxygenic phototrophs (FAP). Chlorosome biogenesis promises to be a complex process requiring the generation of a unilayer membrane and the targeting of bacteriochlorophyll, carotenoids, quinones, and proteins to the chlorosome. The biogenesis of chlorosomes as well as their presence in two distinct bacterial groups, GSB and FAP, remains enigmatic. The photosynthetic machinery and overall metabolic characteristics of these two bacterial groups are very different, and horizontal gene transfer has been proposed to explain chlorosome distribution. Chlorosomes have been considered to be unique structures that require a specific assembly machinery. We propose that no special machinery is required for chlorosome assembly. Instead, it is suggested that chlorosomes are a special form of lipid body. We present a model for chlorosome biogenesis that combines aspects of lipid body biogenesis with established chlorosome characteristics and may help explain the presence of chlorosomes in two metabolically diverse organism groups.     

Chlorosome antenna complexes from green photosynthetic bacteria

Chlorosomes are the distinguishing light-harvesting antenna complexes that are found in green photosynthetic bacteria. They contain bacteriochlorophyll (BChl) c, d, e in natural organisms, and recently through mutation, BChl f, as their principal light-harvesting pigments. In chlorosomes, these pigments self-assemble into large supramolecular structures that are enclosed inside a lipid monolayer to form an ellipsoid. The pigment assembly is dictated mostly by pigment–pigment interactions as opposed to protein–pigment interactions. On the bottom face of the chlorosome, the CsmA protein aggregates into a paracrystalline baseplate with BChl a, and serves as the interface to the next energy acceptor in the system. The exceptional light-harvesting ability at very low light conditions of chlorosomes has made them an attractive subject of study for both basic and applied science. This review, incorporating recent advancements, considers several important aspects of chlorosomes: pigment biosynthesis, organization of pigments and proteins, spectroscopic properties, and applications to bio-hybrid and bio-inspired devices.     

Utilization of blue-green light by chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus: Ultrafast excitation energy conversion and transfer

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 10⁴–10⁵ bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S₂ state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400–900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret → Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100–270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S₁ state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret → Q_y internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.     

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m^–2 s^–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the Q_Y band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - DAS decay associated spectrum - PMSF phenylmethylsulfonyl fluoride     

Optical and structural properties of chlorosomes of the photosynthetic green sulfur bacterium Chlorobium limicola

Isolated chlorosomes of the photosynthetic green sulfur bacterium Chorobium limicola upon cooling to 4 K showed, in addition to the near-infrared absorption band at 753 nm due to bacteriochlorophyll c, a weak band near 800 nm that could be attributed to bacteriochlorophyll a. The emission spectrum showed bands of bacteriochlorophyll c and a at 788 and 828 nm, respectively. The fluorescence excitation spectrum indicated a high efficiency of energy transfer from bacteriochlorophyll c to bacteriochlorophyll a. When all bacteriochlorophyll c absorption had been lost upon storage, no appreciable change in the optical properties of the bacteriochlorophyll a contained in these ‘depleted chlorosomes’ was observed. The fluorescence and absorption spectra of the chlorosomal bacteriochlorophyll a were clearly different from those of the soluble bacteriochlorophyll a protein present in these bacteria. The results provide strong evidence that bacteriochlorophyll a, although present in a small amount, is an integral constituent of the chlorosome. It presumably functions in the transfer of energy from the chlorosome to the photosynthetic membrane; its spectral properties and the orientation of its near-infrared optical transitions as determined by linear dichroism are such as to favor this energy transfer.     

Temperature and Carbon Assimilation Regulate the Chlorosome Biogenesis in Green Sulfur Bacteria

Green photosynthetic bacteria adjust the structure and functionality of the chlorosome—the light-absorbing antenna complex—in response to environmental stress factors. The chlorosome is a natural self-assembled aggregate of bacteriochlorophyll (BChl) molecules. In this study, we report the regulation of the biogenesis of the Chlorobaculum tepidum chlorosome by carbon assimilation in conjunction with temperature changes. Our studies indicate that the carbon source and thermal stress culture of C. tepidum grows slower and incorporates fewer BChl c in the chlorosome. Compared with the chlorosome from other cultural conditions we investigated, the chlorosome from the carbon source and thermal stress culture displays (a) smaller cross-sectional radius and overall size, (b) simplified BChl c homologs with smaller side chains, (c) blue-shifted Q_y absorption maxima, and (d) a sigmoid-shaped circular dichroism spectra. Using a theoretical model, we analyze how the observed spectral modifications can be associated with structural changes of BChl aggregates inside the chlorosome. Our report suggests a mechanism of metabolic regulation for chlorosome biogenesis.     

Temperature and Carbon Assimilation Regulate the Chlorosome Biogenesis in Green Sulfur Bacteria

Green photosynthetic bacteria adjust the structure and functionality of the chlorosome—the light-absorbing antenna complex—in response to environmental stress factors. The chlorosome is a natural self-assembled aggregate of bacteriochlorophyll (BChl) molecules. In this study, we report the regulation of the biogenesis of the Chlorobaculum tepidum chlorosome by carbon assimilation in conjunction with temperature changes. Our studies indicate that the carbon source and thermal stress culture of C. tepidum grows slower and incorporates fewer BChl c in the chlorosome. Compared with the chlorosome from other cultural conditions we investigated, the chlorosome from the carbon source and thermal stress culture displays (a) smaller cross-sectional radius and overall size, (b) simplified BChl c homologs with smaller side chains, (c) blue-shifted Q_y absorption maxima, and (d) a sigmoid-shaped circular dichroism spectra. Using a theoretical model, we analyze how the observed spectral modifications can be associated with structural changes of BChl aggregates inside the chlorosome. Our report suggests a mechanism of metabolic regulation for chlorosome biogenesis.     

Initial characterization of the photosynthetic apparatus of "Candidatus Chlorothrix halophila," a filamentous, anoxygenic photoautotroph

“Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of “Ca. Chlorothrix halophila,” that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ~6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “Ca. Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus.     

Supramolecular organization of chlorosomes (chlorobium vesicles) and of their membrane attachment sites in Chlorobium Limicola

The photosynthetic green bacterium Chlorobium limicola 6230 has been examined by freeze-fracture electron microscopy to investigate the size, form, distribution and supramolecular architecture of its chlorosomes (chlorobium vesicles) as well as the chlorosome attachment sites on the cytoplasmic membrane. The oblong chlorosomes that underlie the cytoplasmic membrane show a considerable variation in size from about 40 × 70 nm to 100 × 260 nm and exhibit no particular orientation. The chlorosome core, which appears to be hydrophobic in nature, contains between 10 and 30 rod-shaped elements (approx. 10 nm in diameter) surrounded by an unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. Separating the chlorosome core from the cytoplasm is a approx. 3 nm thick lipid-like envelope layer, which exhibits no substructure. A 5–6 nm thick, crystalline baseplate connects the chlorosome to the cytoplasmic membrane. The ridges of the baseplate lattice make an angle of between 40° and 60° with the longitudinal axis of the chlorosome and have a repeating distance of approx. 6 nm. In addition, each ridge exhibits a granular substructure with a periodicity of approx. 3.3 nm. The cytoplasmic membrane regions adjacent to the baseplates are enriched in large (greater than 9 nm) intramembrane particles, most of which belong to approx. 10 nm and approx. 12.5 nm particle size categories. Each chlorosome attachment site contains between 20 and 30 very large (greater than 12.0 nm diameter) intramembrane particles.The following interpretive model of a chlorosome is discussed in terms of biophysical, biochemical and structural information reported by others: it is proposed that the bacteriochlorophyll c (BChl c; chlorobium chlorophyll) is located in the rod elements of the core and that it is complexed with specific proteins. The cytoplasm-associated envelope layer is depicted as consisting of a monolayer of galactosyl diacylglycerol molecules. BChl a-protein complexes in a planar lattice configuration most likely make up the crystalline baseplate. The greater than 12-nm particles in the chlorosome attachment sites of the cytoplasmic membrane, finally, may correspond to complexes containing a reaction center and non-crystalline light-harvesting BChl a. The crystalline nature of the baseplate is consistent with the notion that it serves two functions: besides transferring excitation energy to the reaction centers it could also function as a distributor of this energy amongst the reaction centers.     

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.     

Phototrophic growth and chlorosome formation in Chloroflexus aurantiacus under conditions of carotenoid deficiency

Chloroflexus aurantiacus was grown phototrophically in the presence of 60 µg of 2-hydroxybiphenyl (HBP) per ml, which inhibited the formation of colored carotenoids up to about 90%, while bacteriochlorophyll (BChl) c formation and phototrophic growth were not significantly influenced. When the HBP concentration was increased, the latter two processes became inhibited, yet, no further inhibition of carotenoid formation was possible. The typical shape of chlorosomes was not affected after growth in the presence of 60 µg of HBP per ml. However, the chlorosome dimensions were slightly decreased. Chlorosome preparations from carotenoid-deficient cultures lacked significant amounts of colored carotenoids and exhibited lower buoyant densities than chlorosomes from uninhibited control cultures. As compared on a BChl c basis, the relative contents of the two larger chlorosomal polypeptides of 10.8 and 15.5 kDa (apparent M_r= 11000 and 18000) were not affected by HBP, while formation of the 5.7 kDa (M_r= 3700) polypeptide was inhibited by 50%. The data suggest that, in C. aurantiacus, colored carotenoids are largely dispensable for phototrophic growth and chlorosome formation. Moreover, chlorosome formation and BChl c incorporation into chlorosomes in the absence of about half of the regular amount of the 5.7 kDa polypeptide excludes a constant relationship between these parameters.