Isolation and structural determination of C8-vinyl-bacteriochlorophyll d from the bciA and bchU double mutant of the green sulfur bacterium Chlorobaculum tepidum

The mutant lacking enzymes BciA and BchU, that catalyzed reduction of the C8-vinyl group and methylation at the C20 position of bacteriochlorophyll (BChl) c, respectively, in the green sulfur bacterium Chlorobaculum tepidum, were constructed. This mutant accumulated C8-vinyl-BChl d derivatives, and a molecular structure of the major pigment was fully characterized by its NMR, mass, and circular dichroism spectra, as well as by chemical modification: (3¹ R)-8-vinyl-12-ethyl-(R[V,E])BChl d was confirmed as a new BChl d species in the cells. In vitro chlorosome-like self-aggregates of this pigment were prepared in an aqueous micellar solution, and formed more rapidly than those of (3¹ R)-8,12-diethyl-(R[E,E])BChl d isolated from the green sulfur bacterium Chlorobaculum parvum NCIB8327d synthesizing BChl d homologs. Their red-shifted Q _y absorption bands were almost the same at 761 nm, and the value was larger than those of in vitro self-aggregates of R[E,E]BChl c (737 nm) and R[V,E]BChl c (726 nm), while the monomeric states of the former gave Q _y bands at shorter wavelengths than those of the latter. Red shifts by self-aggregation of the two BChl d species were estimated to be 110 nm and much larger than those by BChls c (75 nm for [E,E] and 64 nm for [V,E]).     

Isolation and characterization of a new bacteriochlorophyll-c bearing a neopentyl substituent at the 8-position from the bciD-deletion mutant of the brown-colored green sulfur bacterium Chlorobaculum limnaeum

We recently constructed the mutant of the brown-colored green sulfur bacterium Chlorobaculum limnaeum lacking BciD which was responsible for formation of a formyl group at the 7-position in bacteriochlorophyll(BChl)-e biosynthesis. This mutant exclusively gave BChl-c, but not BChl-e, as the chlorosome pigments (Harada et al. in PLoS One 8(4):e60026, 2013). By the mutation, the homolog and epimer composition of the pigment was drastically altered. The methylation at the 8²-position in the mutant cells proceeded to create BChl-c carrying large alkyl substituents at this position. Correspondingly, the content of BChls-c having the (S)-configuration at the chiral 3¹-position remarkably increased and accounted for 80.6 % of the total BChl-c. Based on the alteration of the pigment composition in the mutant cells, a new BChl-c bearing the bulkiest, triple 8²-methylated neopentyl substituent at the 8-position ([N,E]BChl-c) was identified. The molecular structure of [N,E]BChl-c was fully determined by its NMR, mass, and circular dichroism spectra. The newly identified [N,E]BChl-c was epimerically pure at the chiral 3¹-position and its stereochemistry was determined to be an (S)-configuration by modified Mosher’s method. Further, the effects of the C8²-methylation on the optical absorption properties of monomeric BChls-c were investigated. The Soret but not Qy absorption bands shifted to longer wavelengths by the extra methylation (at most 1.4 nm). The C8²-methylation induced a slight but apparent effect on absorption properties of BChls-c in their monomeric states.     

Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer

The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investigated by means of normal-phase high-performance liquid chromatography. From both species a number of homologues was isolated, which were identified by absorption and ²⁵²Cf-plasma desorption mass spectroscopy. Besides BChl d, C. vibrioforme contained a significant amount of BChl c, which may provide an explanation for the previous observation of at least two spectrally different pools of BChl in the chlorosomes of green sulfur bacteria (Otte et al. 1991). C. phaeovibrioides contained various homologues of BChl e only. Absorption spectra in acetone of BChl c, d and e, as well as bacteriopheophytin e are presented. No systematic differences were found for the various homologues of each pigment. In addition to farnesol, the mass spectra revealed the presence of various minor esterifying alcohols in both species, including phytol, oleol, cetol and 4-undecyl-2-furanmethanol, as well as an alcohol of low molecular mass, which is tentatively assumed to be decenol.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin (used as a general name for the Mg-free compound, irrespective of the esterifying alcohol) - HPLC high-performance liquid chromatography     

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.     

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.     

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.     

<Emphasis Type="Italic">Chlorobaculum tepidum</Emphasis> regulates chlorosome structure and function in response to temperature and electron donor availability

Green sulfur bacteria (GSB) rely on the chlorosome, a light-harvesting apparatus comprised almost entirely of self-organizing arrays of bacteriochlorophyll (BChl) molecules, to harvest light energy and pass it to the reaction center. In Chlorobaculum tepidum, over 97% of the total BChl is made up of a mixture of four BChl c homologs in the chlorosome that differ in the number and identity of alkyl side chains attached to the chlorin ring. C. tepidum has been reported to vary the distribution of BChl c homologs with growth light intensity, with the highest degree of BChl c alkylation observed under low-light conditions. Here, we provide evidence that this functional response at the level of the chlorosome can be induced not only by light intensity, but also by temperature and a mutation that prevents phototrophic thiosulfate oxidation. Furthermore, we show that in conjunction with these functional adjustments, the fraction of cellular volume occupied by chlorosomes was altered in response to environmental conditions that perturb the balance between energy absorbed by the light-harvesting apparatus and energy utilized by downstream metabolic reactions.     

In vitro demethoxycarbonylation of various chlorophyll analogs by a BciC enzyme

Unique light-harvesting antennas in the green sulfur bacterium Chlorobaculum tepidum, called chlorosomes, consist of self-aggregates of bacteriochlorophyll (BChl) c. In the biosynthesis of BChl c, BciC demethoxycarbonylase removes the C13²-methoxycarbonyl group to facilitate the self-aggregation of BChl c. We previously reported the in vitro BciC-enzymatic reactions and discussed the function of this enzyme in the biosynthesis of BChl c. This study aims to examine the substrate specificity of BciC in detail using several semi-synthetic (bacterio)chlorophyll derivatives. The results indicate that the substrate specificity of BciC is measurably affected by structural changes on the A/B rings including the bacteriochlorin π-systems. Moreover, BciC showed its activity on a Zn-chelated chlorophyll derivative. On the contrary, BciC recognized structural modifications on the D/E rings, including porphyrin pigments, which resulted in the significant decrease in the enzymatic activity. The utilization of BciC provides mild conditions that may be useful for the in vitro preparation of various chemically (un)stable chlorophyllous pigments.

     

Tubular exciton models for BChl c antennae in chlorosomes from green photosynthetic bacteria

Exciton calculations on tubular pigment aggregates similar to recently proposed models for BChl c/d/e antennae in light-harvesting chlorosomes from green photosynthetic bacteria yield electronic absorption spectra that are super-impositions of linear J-aggregate spectra. While the electronic spectroscopy of such antennae differs considerably from that of linear J-aggregates, tubular exciton models (which may be viewed as cross-coupled J-aggregates) may be constructed to yield spectra that resemble that of the BChl c antenna in the green bacterium Chloroflexus aurantiacus. Highly symmetric tubular models yield absorption spectra with dipole strength distributions essentially identical to that of a J-aggregate; strong symmetry-breaking is needed to simulate the absorption spectrum of the BChl c antenna.Abbreviations BChl bacteriochlorophyll - [E,M] BChl c _S bacteriochlorophyll c with ethyl and methyl substituents in the 8- and 12-positions, and with stearol as the esterifying alcohol     

Mutational analysis of three <Emphasis Type="Italic">bchH</Emphasis> paralogs in (bacterio-)chlorophyll biosynthesis in <Emphasis Type="Italic">Chlorobaculum tepidum</Emphasis>

The first committed step in the biosynthesis of (bacterio-)chlorophyll is the insertion of Mg²⁺ into protoporphyrin IX by Mg-chelatase. In all known (B)Chl-synthesizing organisms, Mg-chelatase is encoded by three genes that are homologous to bchH, bchD, and bchI of Rhodobacter spp. The genomes of all sequenced strains of green sulfur bacteria (Chlorobi) encode multiple bchH paralogs, and in the genome of Chlorobaculum tepidum, there are three bchH paralogs, denoted CT1295 (bchT), CT1955 (bchS), and CT1957 (bchH). Cba. tepidum mutants lacking one or two of these paralogs were constructed and characterized. All of the mutants lacking only one of these BchH homologs, as well as bchS bchT and bchH bchT double mutants, which can only produce BchH or BchS, respectively, were viable. However, attempts to construct a bchH bchS double mutant, in which only BchT was functional, were consistently unsuccessful. This result suggested that BchT alone is unable to support the minimal (B)Chl synthesis requirements of cells required for viability. The pigment compositions of the various mutant strains varied significantly. The BChl c content of the bchS mutant was only ~10% of that of the wild type, and this mutant excreted large amounts of protoporphyrin IX into the growth medium. The observed differences in BChl c production of the mutant strains were consistent with the hypothesis that the three BchH homologs function in end product regulation and/or substrate channeling of intermediates in the BChl c biosynthetic pathway. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physiology and Phylogeny of Green Sulfur Bacteria Forming a Monospecific Phototrophic Assemblage at a Depth of 100 Meters in the Black Sea

The biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophyll e (BChl e) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter⁻¹. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChl e. Only traces of BChl e (8 ng liter⁻¹) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m⁻² s⁻¹ at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of the Chlorobiaceae and was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m⁻² s⁻¹ for photosynthetic ¹⁴CO₂ fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.     

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.     

Characterization and In Situ Carbon Metabolism of Phototrophic Consortia

A dense population of the phototrophic consortium “Pelochromatium roseum” was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany). Fluorescence in situ hybridization revealed that the brown epibionts of “P. roseum” constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline. Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low. The 465-bp 16S rRNA gene sequence of the epibiont of “P. roseum” was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR. The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria. Maximum light-dependent H¹⁴CO₃⁻ fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria. The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (δ¹³C) of their specific biomarkers. Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl) e and smaller amounts of BChl a and d and chlorophyll a in the chemocline. Unexpectedly, isorenieratene and β-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent. Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their δ¹³C values were determined. Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in ¹³C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria. The difference between the δ¹³C values of farnesol, the major esterifying alcohol of BChl e, and CO₂ was −7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of “P. roseum” in situ is photoautotrophic.     

Excitation Energy Transfer Dynamics and Excited-State Structure in Chlorosomes of Chlorobium phaeobacteroides

The excited-state relaxation within bacteriochlorophyll (BChl) e and a in chlorosomes of Chlorobium phaeobacteroides has been studied by femtosecond transient absorption spectroscopy at room temperature. Singlet-singlet annihilation was observed to strongly influence both the isotropic and anisotropic decays. Pump intensities in the order of 10¹¹ photons × pulse⁻¹ × cm⁻² were required to obtain annihilation-free conditions. The most important consequence of applied very low excitation doses is an observation of a subpicosecond process within the BChl e manifold (~200–500 fs), manifesting itself as a rise in the red part of the Q_y absorption band of the BChl e aggregates. The subsequent decay of the kinetics measured in the BChl e region and the corresponding rise in the baseplate BChl a is not single-exponential, and at least two components are necessary to fit the data, corresponding to several BChl e→BChl a transfer steps. Under annihilation-free conditions, the anisotropic kinetics show a generally slow decay within the BChl e band (10–20 ps) whereas it decays more rapidly in the BChl a region (~1 ps). Analysis of the experimental data gives a detailed picture of the overall time evolution of the energy relaxation and energy transfer processes within the chlorosome. The results are interpreted within an exciton model based on the proposed structure.     

Temperature shift effect on the Chlorobaculum tepidum chlorosomes

Chlorobaculum [Cba.] tepidum is known to grow optimally at 48–52 °C and can also be cultured at ambient temperatures. In this paper, we prepared constant temperature, temperature shift, and temperature shift followed by backshift cultures and investigated the intrinsic properties and spectral features of chlorosomes from those cultures using various approaches, including temperature-dependent measurements on circular dichroism (CD), UV–visible, and dynamic light scattering. Our studies indicate that (1) chlorosomes from constant temperature cultures at 50 and 30 °C exhibited more resistance to heat relative to temperature shift cultures; (2) as temperature increases bacteriochlorophyll c (BChl c) in chlorosomes is prone to demetalation, which forms bacteriopheophytin c, and degradation under aerobic conditions. Some BChl c aggregates inside reduced chlorosomes prepared in low-oxygen environments can reform after heat treatments; (3) temperature shift cultures synthesize and incorporate more BChl c homologs with a smaller substituent at C-8 on the chlorin ring and less BChl c homologs with a larger long-chain alcohol at C-17³ versus constant-temperature cultures. We hypothesize that the long-chain alcohol at C-17³ (and perhaps together with the substituent at C-8) may account for thermal stability of chlorosomes and the substituent at C-8 may assist self-assembling BChls; and (4) while almost identical absorption spectra are detected, chlorosomes from different growth conditions exhibited differences in the rotational length of the CD signal, and aerobic and reduced chlorosomes also display different Q_y CD intensities. Further, chlorosomes exhibited changes of CD features in response to temperature increases. Additionally, we compare temperature-dependent studies for the Cba. tepidum chlorosomes and previous studies for the Chloroflexus aurantiacus chlorosomes. Together, our work provides useful and novel insights on the properties and organization of chlorosomes.     

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.     

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     

Pigment composition of the photosynthetic membrane and reaction center of the green bacterium Prosthecochloris aestuarii

We have performed a quantitative analysis of the pigment composition of different pigment-protein complexes present in the membrane of the green sulfur bacterium Prosthecochloris aestuarii, using the resolving power of reversed-phase high-performance liquid chromatography. The most purified photochemically active complexes contained only carotenoids (OH-chlorobactene and rhodopin), bacteriochlorophyll a and a chlorophyllous pigment with absorption maxima at 663 and 433 nm, like bacteriochlorophyll c. However, the lipophilicity of this pigment, labeled BChl 663, is quite high and indicates that it contains 5–6 additional methylene groups compared to the BChl c homologue known as most lipophilic. Comparison of the BChl 663 content of various pigment-protein complexes indicates that BChl 663 is present in an amount of 10–15 molecules per reaction center. BChl 663 absorbs at 670 nm in vivo, with a specific extinction coefficient of 85 (±10) mM⁻¹ · cm⁻¹. In view of the evidence that the primary electron acceptor in P. aestuarii is a pigment with absorption maximum at 670 nm (Nuijs, A.M., Vasmel, H., Joppe, H.L.P., Duysens, L.N.M. and Amesz, J. (1985) Biochim. Biophys. Acta 807, 24–34) a direct consequence of these experiments is the fact that only BChl 663 can be a likely candidate for the role of primary electron acceptor as no other pigments absorbing around 670 nm (e.g., bacteriopheophytin c) are present in a photochemically active pigment-protein complex derived from the membrane of this green bacterium.     

Atomic structure of the bacteriochlorophyll c assembly in intact chlorosomes from Chlorobium limicola determined by solid-state NMR

Green sulfur photosynthetic bacteria optimize their antennas, chlorosomes, especially for harvesting weak light by organizing bacteriochlorophyll (BChl) assembly without any support of proteins. As it is difficult to crystallize the organelles, a high-resolution structure of the light-harvesting devices in the chlorosomes has not been clarified. We have determined the structure of BChl c assembly in the intact chlorosomes from Chlorobium limicola on the basis of ¹³C dipolar spin-diffusion solid-state NMR analysis of uniformly ¹³C-labeled chlorosomes. About 90 intermolecular C–C distances were obtained by the simultaneous assignment of distance correlations and the structure optimization preceded by the polarization-transfer matrix analysis. An atomic structure was obtained, using these distance constraints. The determined structure of the chlorosomal BChl c assembly is built with the parallel layers of piggyback-dimers. This supramolecular structure would provide insights into the mechanism of weak-light capturing.     

Fluorescence detected magnetic resonance (FDMR) of green sulfur photosynthetic bacteria Chlorobium sp.

Fluorescence Detected Magnetic Resonance (FDMR) spectra have been measured for whole cells and isolated chlorosomal fractions for the green photosyntheic bacteria Chlorobium phaeobacteroides (containing bacteriochlorophyll e, and isorenieratene as major carotenoid) and Chlorobium limicola (containing bacteriochlorophyll c, and chlorobactene as major carotenoid). The observed transition at 237 MHz (identical in both bacteria) and > 1100 MHz can be assigned, by analogy with published data on other carotenoids, to the 2E and D + E transitions, respectively, of Chlorobium carotenoids. Their zero field splitting (ZFS) parameters are estimated to be: |D|=0.0332 cm^–1 and |E|=0.0039 cm^–1 (chlorobactene), and |D|=0.0355 cm^–1 and |E|=0.0039 cm^–1 (isorenieratene). In the intermediate frequency range 300–1000 MHz the observed transitions can be assigned to chlorosomal bacteriochlorophylls c and e, and to bacteriochlorophyll a located in the chlorosome envelope and water-soluble protein. The bacteriochlorophyll e triplet state measured in 750 nm fluorescence (aggregated chlorosomal BChl e) is characterised by the ZFS parameters: |D|=0.0251 cm^–1 and |E|=0.0050 cm^–1.Abbreviations BChl - bacteriochlorophyll - BPh - bacteriopheophytin - Chl. - Chlorobium - F(A)(O)DMR - fluorescence (absorption) (optical) detected magnetic resonance - FF - fluorescence fading - ISC - intramolecular intersystem crossing - RC - reaction center - ZFS - zero field splitting