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     

Anisotropic distribution of emitting transition dipoles in chlorosome from <Emphasis Type="Italic">Chlorobium tepidum</Emphasis>: fluorescence polarization anisotropy study of single chlorosomes

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

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.     

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Q_y absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. One-color and two-color pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.     

Characterization of the chlorosome antenna of the filamentous anoxygenic phototrophic bacterium<Emphasis Type="Italic"> Chloronema</Emphasis> sp. strain UdG9001

The absorption and fluorescence properties of chlorosomes of the filamentous anoxygenic phototrophic bacterium Chloronema sp. strain UdG9001 were analyzed. The chlorosome antenna of Chloronema consists of bacteriochlorophyll (BChl) d and BChl c together with -carotene as the main carotenoid. HPLC analysis combined with APCI LC-MS/MS showed that the chlorosomal BChls comprise a highly diverse array of homologues that differ in both the degree of alkylation of the macrocycle at C-8 and/or C-12 and the alcohol moiety esterified to the propionic acid group at C-17. BChl c and BChl d from Chloronema were mainly esterified with geranylgeraniol (33% of the total), heptadecanol (24%), octadecenol (19%), octadecanol (14%), and hexadecenol (9%). Despite this pigment heterogeneity, fluorescence emission of the chlorosomes showed a single peak centered at 765 nm upon excitation at wavelengths ranging from 710 to 740 nm. This single emission, assigned to BChl c, indicates an energy transfer from BChl d to BChl c within the same chlorosome. Likewise, incubation of chlorosomes under reducing conditions caused a weak increase in fluorescence emission, which indicates a small redox-dependent fluorescence. Finally, protein analysis of Chloronema chlorosomes using SDS-PAGE and MALDI-TOF-MS revealed the presence of a chlorosomal polypeptide with a molecular mass of 5.7 kDa, resembling the CsmA protein found in Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. Several minor polypeptides were also detected but not identified. These results indicate that, compared with other members of filamentous anoxygenic phototrophic bacteria and green sulfur bacteria, Chloronema possesses an antenna system with novel features that may be of interest for further investigations.Abbreviations APCI LC-MS/MS Atmospheric pressure chemical ionization liquid chromatography mass spectrometry - BChl Bacteriochlorophyll - Chl. Chlorobium - Cfl. Chloroflexus - MALDI-TOF-MS Matrix assisted laser desorption/ionization time-of-flight mass spectrometry - [Et] Ethyl - [i-Bu] Isobutyl - [Me] Methyl - [neo-Pent] Neopentyl - [n-Pr] Propyl - t _R Retention time     

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.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Q_y transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Q_y transition of BChl c and the -carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Q_y transition is close to the magic angle (55°).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - LD linear dichroism Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

Self quenching of chlorosome chlorophylls in water and hexanol-saturated water

The optical properties of a methyl ester homolog of bacteriochlorophylld (BChld _M ) and bacteriochlorophyllc (BChlc) in H₂O, hexanol-saturated H₂O and methanol were studied by absorption, fluorescence emission, and circular dichroism (CD). In H₂O, BChld _M spontaneously forms an aggregate similar to that formed in hexane, with absorption maximum at 730 nm and fluorescence emission at 748 nm. For the pigment sample in hexanol-saturated H₂O, while the absorption peaks at 661 nm, only slightly red-shifted compared to the monomer, the fluorescence emission is highly quenched. When diluted 2–3 fold with H₂O, the absorption returns to around 720 nm, characteristic of an aggregate. The CD spectrum of the H₂O aggregate exhibits a derivative-shaped feature with positive and negative peaks, while the amplitude is lower than that of chlorosomes. The Fourier transform infrared spectra of BChld _M aggregates in H₂O and hexane were measured. A 1644 cm^–1 band, indicative of a bonded 13¹-keto group, is detected for both samples. A marker band for 5-coordinated Mg was observed at 1611 cm^–1 for the two samples as well. To study the kinetic behavior of the samples, both single-photon counting (SPC) fluorescence and transient absorption difference spectroscopic measurements were performed. For BChld _M in hexanol-saturated H₂O, a fast decay component with a lifetime of 10 to 14 ps was detected using the two different techniques. The fast decay could be explained by the concentration quenching phenomenon due to a high local pigment concentration. For the pigment sample in H₂O, SPC gave a 16 ps component, whereas global analysis of transient absorption data generated two fast components: 3.5 and 26 ps. The difference may arise from the different excitation intensities. With a much higher excitation in the latter measurements, other quenching processes, e.g. annihilation, might be introduced, giving the 3.5 ps component. Finally, atomic force microscopy was used to examine the ultrastructure of BChld _M in H₂O and hexanol-saturated H₂O. Pigment clusters with diameters ranging from 15 to 45 nm were observed in both samples.     

The formation and characterization of the in vitro polymeric aggregates of bacteriochlorophyllc homologs fromChlorobium limicola in aqueous suspension in the presence of monogalactosyl diglyceride

Artificial aggregates of bacteriochlorophyllc (BChlc) were formed in an aqueous medium in the presence of a lipid, monogalactosyl diglyceride (MGDG), and the optical properties of those aggregates were studied by absorption and circular dichroism (CD) mainly. Four BChlc homologs, ([E,E]BChlc _F, [P,E]BChlc _F, [E,M]BChlc _F and [I,E]BChlc _F), were isolated from the green photosynthetic bacteriumChlorobium limicola strain 6230. Above 0.0004%, MGDG induced a red-shift of the absorption maxima of BChlc aggregates. At 0.003% MGDG BChlc aggregates showed absorption maxima in the range of 724 to 745 (±3) nm with a shift of 12 to 24 (±3) nm depending on the homolog species. Four kinds of BChlc-MGDG aggregates showed characteristic CD spectra. [E,M]BChlc _F gave rise to a CD spectrum similar to that of chlorosomes, while the other three gave spectra of opposite sign. These aggregates are sensitive to 1-hexanol treatment; in a saturating amount (0.85%) of 1-hexanol, all the homologs gave a monomer-like absorption spectrum peaking at 670nm. At an intermediate concentration (0.5%), [E,M]BChlc _F showed an enhanced CD intensity, as observed in native chlorosomes. Resonance Raman spectra of the monomer-like BChlc samples indicated that the keto vibrational band at ca. 1640 cm^–1 was considerably weakened by the 0.85% 1-hexanol treatment, however the 1680 cm^–1 band characteristic of a free keto group did not appear. These results indicate that the artificial aggregates formed by purified BChlc homologs and MGDG are good models for studying chlorosomes structure.     

Polarized fluorescence of aggregated bacteriochlorophyll c and baseplate bacteriochlorophyll a in single chlorosomes isolated from Chloroflexus aurantiacus

The polarization anisotropy of fluorescence from single chlorosomes isolated from a green filamentous bacterium, Chloroflexus aurantiacus, was measured using a confocal laser microscope at 13 K. Each single chlorosome that is floating in a frozen solvent exhibited strong polarization anisotropy of fluorescence. We calculated the degrees of fluorescence polarization for 51 floating single chlorosomes. The value ranged from 0.1 to 0.76 for the BChl-c aggregate in the core chlorosomes and from 0 to 0.4 for the energy acceptor BChl-a in the baseplate protein in the outer membrane. The shifts in polarization angles between the two emission bands were distributed over all the possible values with a sharp peak around 90 degrees , suggesting the perpendicular orientation between the transition dipoles of the fluorescence emission from the BChl-c aggregate and that from BChl-a. A simulation assuming a random orientation of chlorosomes reproduced the experimental results exactly. The analysis further indicated the appreciable contribution of the transition dipole of BChl-c that has an orientation perpendicular to the major polarization axis in each chlorosome. Small values of the degrees of polarization implied the BChl-a transition dipole to be somewhat tilted with respect to the normal of the cytoplasmic membrane to which chlorosomes are attached. These conclusions can be obtained only by observing the fluorescence of single chlorosomes.     

Bacteriochlorophyllc formation and chlorosome development inChloroflexus aurantiacus

The dependence of chlorosome development on bacteriochlorophyll (BChl)c synthesis was studied with the phototrophic green bacteriumChloroflexus aurantiacus. By selecting defined culture conditions, three possibilities could be identified. Upon addition of 5-aminolevulinic acid, cells of resting cultures increased their specific BChlc contents as well as the volumes of already existing chlorosomes. The number of chlorosomes, however, remained constant. Serine-limited chemostat cultures grown under steady state conditions exhibited constant rates of synthesis of both BChlc as well as of chlorosomes. The volume of the latter remained constant, as well. Upon addition of ALA to chemostat cultures, chlorosomes were synthesized at the same rate as before but their volumes increased as a consequence of increased BChlc incorporation. In chlorosomes isolated from resting cultures supplied with ALA the amounts of all of the polypeptides increased only slightly, if at all. Moreover, the ratio of all of the chlorosomal polypeptides remained largely constant. These results show that chlorosomes may incorporate newly synthesized BChlc without concomitant formation of chlorosomal polypeptides. This means that there was no obvious coordination of polypeptide and BChlc synthesis. On this basis, it appears unlikely that one of the chlorosomal polypeptides functions as an apoprotein of a presumed BChlc holochrome complex.     

Study of the chlorosomal antenna of the green mesophilic filamentous bacterium Oscillochloris trichoides

Whole cells, chlorosome-membrane complexes and isolated chlorosomes of the green mesophilic filamentous bacterium Oscillochloris trichoides, representing a new family of the green bacteria Oscillochloridaceae, were studied by optical spectroscopy and electron microscopy. It was shown that the main light-harvesting pigment in the chlorosome is BChl c. The presence of BChl a in chlorosomes was visualized only by pigment extraction and fluorescence spectroscopy at 77 K. The molar ratio BChl c: BChl a in chlorosomes was found to vary from 70:1 to 110:1 depending on light intensity used for cell growth. Micrographs of negatively and positively stained chlorosomes as well as of ultrathin sections of the cells were obtained and used for morphometric measurements of chlorosomes. Our results indicated that Osc. trichoides chlorosomes resemble, in part, those from Chlorobiaceae species, namely, in some spectral features of their absorption, fluorescence, CD spectra, pigment content as well as the morphometric characteristics. Additionally, it was shown that similar to Chlorobiaceae species, the light-harvesting chlorosome antenna of Osc. trichoides exhibited a highly redox-dependent BChl c fluorescence. At the same time, the membrane B805–860 BChl a antenna of Osc. trichoides is close to the membrane B808–866 BChl a antenna of Chloroflexaceae species. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

The effect of detergent on the structure and composition of chlorosomes isolated from Chloroflexus aurantiacus

Isolated chlorosomes, treated with the detergent lithium dodecyl sulfate (LDS), can be separated into two green fractions by agarose gel electrophoresis. One fraction contains chlorosomes with a full complement of proteins and antenna BChl c absorbing at 740 nm, but with a more spherical form than the normal ellipsoid shape observed in control chlorosomes. The second fraction was completely devoid of proteins but had a similar absorption spectrum. Electron micrographs of the protein-free fraction indicated the presence of stain-excluding spheres with overall dimensions resembling those of intact chlorosomes (40–100 nm). These spheres are probably micelles of BChl c liberated from the chlorosomes during the detergent treatment, since similar structures could be produced when purified BChl c, dissolved in 1-hexanol, was dispersed in buffer, producing an aggregate absorbing at 742 nm. These results suggest that the chlorosome proteins are not required to produce an arrangement of BChl c chromophores which gives rise to a 740 nm absorption peak resembling that of intact chlorosomes. It seems probable, however, that proteins have a role in determining the overall shape of the chlorosome. Treatment with cross-linking reagents did not prevent the detergent-induced changes in chlorosome morphology.Abbreviations BChl bacteriochlorophyll - DSP dithiobis-succinimidyl-2-propionate - EM electron microscopy - LDS lithium dodecyl sulfate - MGDG monogalactosyl diacylglycerol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Giant circular dichroism of chlorosomes fromChloroflexus aurantiacus treated with 1-hexanol and proteolytic enzymes

The circular dichroism (CD) spectrum of isolated chlorosomes fromChloroflexus aurantiacus showed a conservative, S-shaped signal with a negative maximum at 723 nm, a positive maximum at 750 nm and a zero-crossing at 740 nm. Proteolytic treatment of chlorosomes with trypsin at 37°C did not change the CD signal or the absorption spectrum in contrast to treatment with proteinase K, where a twofold increase in rotational strength and a slight decrease of the absorption band at 740 nm were observed. Treatment with saturating 1-hexanol concentrations resulted in a blue shift of the absorption band at 740 nm as well as in changes of the CD spectrum. These changes reversed when the sample was diluted to half the saturating 1-hexanol concentration. In contrast to that, we observed an irreversible formation of a giant CD signal using the combination of 1-hexanol and proteinase K treatment. Electron micrographs of chlorosomes treated with both 1-hexanol and proteinase K showed large aggregates of multiple chlorosome size. By comparison of proteinase K induced effects with trypsin effects it appeared that the 5.7 kDa polypeptide has a structural role in the organisation of BChlc in the chlorosome.     

Development and pigmentation of chlorosomes in Chloroflexus aurantiacus strain Ok-70-fl

The development of chlorosomes and their pigmentation were studied by growing Chloroflexus aurantiacus strain Ok-7o-fl first under conditions under which BChl c-synthesis is low (50°C, 2000 lux and 30°C, 1500 lux) and subsequently under conditions promoting high BChl c-synthesis (50°C, 400 lux). Electron microscopic observations on and chemical analyses of isolated cell components showed that in BChl c-depleted cells chlorosome-like structures (chlorosome bags) are attached to fragments of cytoplasmic membranes. These chlorosome bags exhibit a periodic fine structure caused by the construction of the baseplates of the chlorosomes. The baseplates are closely attached to the cytoplasmic membrane, they are rich in phospholipids and apparently contain a 790 nm-BChl a-complex. Chlorosome bags of BChl c-depleted cells always contain a limited amount of light-harvesting pigment complexes (BChlc, - and -carotene). The light-harvesting system is restored (50°C, 400 lux) by first refilling the existing chlorosome bags before cell division takes place.Abbreviations BChl Bacteriochlorophyll - LH Light-harvesting complex - RC Reaction center     

Excitation energy transfer in chlorosomes of Chlorobium phaeobacteroides strain CL1401: the role of carotenoids

The role of carotenoids in chlorosomes of the green sulfur bacterium Chlorobium phaeobacteroides, containing bacteriochlorophyll (BChl) e and the carotenoid (Car) isorenieratene as main pigments, was studied by steady-state fluorescence excitation, picosecond single-photon timing and femtosecond transient absorption (TA) spectroscopy. In order to obtain information about energy transfer from Cars in this photosynthetic light-harvesting antenna with high spectral overlap between Cars and BChls, Car-depleted chlorosomes, obtained by inhibition of Car biosynthesis by 2-hydroxybiphenyl, were employed in a comparative study with control chlorosomes. Excitation spectra measured at room temperature give an efficiency of 60–70% for the excitation energy transfer from Cars to BChls in control chlorosomes. Femtosecond TA measurements enabled an identification of the excited state absorption band of Cars and the lifetime of their S₁ state was determined to be 10 ps. Based on this lifetime, we concluded that the involvement of this state in energy transfer is unlikely. Furthermore, evidence was obtained for the presence of an ultrafast (>100 fs) energy transfer process from the S₂ state of Cars to BChls in control chlorosomes. Using two time-resolved techniques, we further found that the absence of Cars leads to overall slower decay kinetics probed within the Q_y band of BChl e aggregates, and that two time constants are generally required to describe energy transfer from aggregated BChl e to baseplate BChl a.This revised version was published online in October 2005 with corrections to the Cover Date.     

Structures of chlorosomes and aggregated BChlc inChlorobium tepidum from solid state high resolution CP/MAS13C NMR

Cross polarization/magic angle spinning (CP/MAS)¹³C (solid state high resolution) NMR spectra were observed for chlorosomes and BChlc aggregates. Similarity of both kinds of spectra (except for some signals assignable to proteins and lipids in chlorosomes) indicates that BChlc's in chlorosomes are present just as in synthetic BChlc aggregates. Chemical shifts for C13¹ carbonyl and C3¹ hydroxylethyl carbons indicate hydrogen bonding between them. Comparison of solution and solid state¹³C NMR chemical shifts shows the five coordinated nature of BChlc aggregates. Some chemical shift differences were attributable to ring currents shifts. Their comparisons with calculated ring current shift values predicted structures for the aggregates. Cross polarization dynamics of the CP/MAS¹³C NMR signals explored dynamic and structural nature of the BChlc aggregates.     

Hole burning study of excited state structure and energy transfer dynamics of bacteriochlorophyll c in chlorosomes of green sulphur photosynthetic bacteria

Results of low temperature fluorescence and spectral hole burning experiments with whole cells and isolated chlorosomes of the green sulfur bacterium Chlorobium limicola containing BChl c are reported. At least two spectral forms of BChl c (short-wavelength and long-wavelength absorbing BChl c) were identified in the second derivative fluorescence spectra. The widths of persistent holes burned in the fluorescence spectrum of BChl c are determined by excited state lifetimes due to fast energy transfer. Different excited state lifetimes for both BChl c forms were observed. A site distribution function of the lowest excited state of chlorosomal BChl c was revealed. The excited state lifetimes are strongly influenced by redox conditions of the solution. At anaerobic conditions the lifetime of 5.3 ps corresponds to the rate of energy transfer between BChl c clusters. This time shortens to 2.6 ps at aerobic conditions. The shortening may be caused by introducing a quencher. Spectral bands observed in the fluorescence of isolated chlorosomes were attributed to monomeric and lower state aggregates of BChl c. These forms are not functionally connected with the chlorosome.Abbreviations BChl bacteriochlorophyll - EET electronic energy transfer - FWHM full width at half maximum - SDF site distribution function - RC reaction centre