Chlorobium Tepidum Research Articles

Light intensity effects on pigment composition and organisation in the green sulfur bacterium Chlorobium tepidum

We have investigated the changes in the pigment composition and organisation of the light-harvesting apparatus of the green sulfur bacterium Chlorobium tepidum growing under different light intensities. Cells grown at lower light intensities had lower exponential growth rates and increased amounts of the main light-harvesting pigments, bacteriochlorophyll c and carotenoids, on a cell protein basis. Absorption spectra of chlorosomes isolated from cells grown at low light intensities revealed a red-shift of up to 8 nm in the Qy band of bacteriochlorophyll c compared to chlorosomes from high light grown cells. A similar red-shift of up to 4 nm was also observed in the corresponding fluorescence emission peaks. HPLC analysis of pigment extracts showed a correlation between the red-shift and the content of the more alkylated BChl c homologs, which increased as light intensity for growth was lower. Furthermore, analysis of the carotenoid composition in chlorosomes re vealed a conspicuous change in the ratio between chlorobactene and 1′, 2′-dihydrochlorobactene, which dramatically decreased from 5 to 0.7 in light-limited cultures.

Excited-State Structure and Dynamics in FMO Antenna Complexes from Photosynthetic Green Sulfur Bacteria

Absorption difference spectra for the singlet excited states of the Fenna−Matthews−Olson (FMO) complex from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum were simulated by exciton theory. The same assumptions and parameters were used as applied earlier (Louwe, R. J. W.; Vrieze, J.; Hoff, A. J.; Aartsma, T. J. J. Phys. Chem. B 1997, 101, 11280. Vulto, S. I. E.; de Baat, M. A.; Louwe, R. J. W.; Permentier, H. P.; Neef, T.; Miller, M.; van Amerongen, H.; Aartsma, T. J. J. Phys. Chem. B 1998, 102, 9577). The difference spectra show a bleaching near the wavelength of excitation, due to ground-state bleaching and stimulated emission. Additional negative and positive bands reflect changes in interaction with other bacteriochlorophylls than the one that is mainly excited at the transition frequency. Simulated spectra were compared with experimental difference spectra obtained by pump−probe experiments in the femto- and picosecond time region with excitation pulses in the spectral ran...

Theory of Ultrafast Exciton Motion in Photosynthetic Antenna Systems

To investigate ultrafast exciton motion, exciton relaxation and related nonlinear optical spectra of photosynthetic antenna systems the dissipative multi-exciton theory is utilized. This type of approach accounts for optically induced transitions to the band of delocaliced single-exciton and two-exciton states as well as takes notice of the coupling to the vibrations of the protein matrix. Different applications are mentioned with emphasis on the Fenna Matthews Olson (FRO) photosynthetic antenna complex of Chlorobium Tepidum.

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies

Spin polarized transient EPR spectra taken at X-band (9 GHz) and K-band (24 GHz) of membrane fragments of Chlorobium tepidum and Heliobacillus mobilis are presented along with the spectra of two fractions obtained in the purification of reaction centers (RC) from C. tepidum. The lifetime of P +· is determined by measuring the decay of the EPR signals following relaxation of the initial spin polarization. All samples except one of the RC fractions show evidence of light induced charge separation and formation of chlorophyll triplet states. The lifetime of P +· is found to be biexponential with components of 1.5 ms and 30 ms for C. tepidum and 1.0 and 4.5 ms for Hc. mobilis at 100 K. In both cases, the rates are assigned to recombination from F − X. The spin polarized radical pair spectra for both species are similar and those from Hc. mobilis at room temperature and 100 K are identical. In all cases, an emission/absorption polarization pattern with a net absorption is observed. A slight narrowing of the spectra and a larger absorptive net polarization is found at K-band. No out-of-phase echo modulation is observed. Taken together, the recombination kinetics, the frequency dependence of the spin polarization and the absence of an out-of-phase echo signal lead to the assignment of the spectra to the contribution from P +· to the state P +·F − X. The origin of the net polarization and its frequency dependence are discussed in terms of singlet–triplet mixing in the precursor. It is shown that the field-dependent polarization expected to develop during the 600–700 ps lifetime of P +·A −· 0 is in qualitative agreement with the observed spectra. The identity that the acceptor preceding F X and the conflicting evidence from EPR, optical methods and chemical analyses of the samples are discussed.

Exciton Simulations of Optical Spectra of the FMO Complex from the Green Sulfur Bacterium Chlorobium tepidum at 6 K

Optical spectra (absorption, circular dichroism, linear dichroism, and triplet-minus-singlet), measured at 6 K, are presented for the Fenna−Matthews−Olson (FMO) complex from the green sulfur bacterium Chlorobium (C.) tepidum. Significant differences are observed in comparison with the corresponding spectra of Prosthecochloris (P.) aestuarii. The spectra of C. tepidum FMO were simulated using exciton calculations, based on the recently resolved structure of this complex [Li et al. J. Mol. Biol. 1997, 271 456]. These calculations apply the same basic assumptions as were earlier used for the FMO complex from P. aestuarii [Louwe et al. J. Phys. Chem. B 1997, 101, 11280], except that the site energies of all but one of the bacteriochlorophylls had to be changed in order to optimize the simulations. The good agreement that was obtained supports the assumptions in these model calculations, and the assignment of the site energies makes it possible to consider the spectra in relation to the structural differences between the two types of FMO complex.

Characterization of the group 1 and group 2 sigma factors of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus.

The group 1 and group 2 sigma70-type sigma factors of the green sulfur bacterium Chlorobium tepidum and of the green nonsulfur bacterium Chloroflexus aurantiacus were cloned and characterized. Cb. tepidum was found to contain one sigma70-type sigma factor; the expression of the gene was analyzed by Northern blot hybridization and primer-extension mapping. Cf. aurantiacus has genes encoding four sigma factors of groups 1 and 2. The expression of these genes was examined in cells grown aerobically and anaerobically. The sigC gene was expressed at approximately equal levels under both conditions, resulting in its designation as the group 1 sigma factor of this organism. The only other detectable transcripts arose from the sigB gene, which was expressed at higher levels during aerobic growth. A phylogenetic tree was obtained using the group 1 sigma factors of Cb. tepidum, Cf. aurantiacus, and diverse eubacteria as the molecular marker. The resulting phylogenetic tree shows that Cb. tepidum and Cf. aurantiacus are related to each other and to the cyanobacteria. The relationship of the group 2 sigma factors of Cf. aurantiacus and the cyanobacteria was more specifically examined phylogenetically. The group 2 sigma factors of Cf. aurantiacus probably arose by gene duplication events after the split of the green nonsulfur bacteria from other photosynthetic eubacteria.

Membrane-bound cytochrome cz couples quinol oxidoreductase to the P840 reaction center complex in isolated membranes of the green sulfur bacterium Chlorobium tepidum.

The reaction of quinol oxidoreductase and membrane-bound c-type cytochromes was studied in chlorosome-depleted membranes isolated from Chlorobium tepidum. Rapid oxidations of c-type cytochromes were detected after flash excitation. Their re-reductions occurred in parallel with the reduction of cytochrome b, especially in the presence of antimycin A, whereas reductions of both cytochromes c and b were suppressed by added stigmatellin. These results indicate the tight coupling between the photosynthetic reaction center and quinol oxidoreductase. Turnovers of two types of cytochromes c were detected. One was assigned to the monoheme-type cytochrome c (designated cytochrome cz), which is known to be tightly bound to the reaction center complex. The other was a new c-type cytochrome, cytochrome c-556, which functions the same as cytochrome c1. The steps of electron-transfer scheme, menaquinol --> Rieske FeS center --> cytochrom c-556 --> cytochrome cz --> P840, are estimated to have reaction times of 20 ms and 560, 150, and 40 microseconds, respectively. We conclude that quinol oxidoreductase and the reaction center complex in Chlorobium tepidum are linked by two distinct membrane-bound cytochromes, cz and c-556, with no involvement of water-soluble cytochromes.

Optical dephasing in photosynthetic pigment–protein complexes

We have studied optical dephasing in a number of pigment–protein complexes. Remarkably we find that the energies of the modes involved in the dephasing of the optical transition of the 825 nm band of the FMO-complex of Chlorobium tepidum, the blue-shifted B800 band of two mutants of Rhodobacter sphaeroides and the B777 subparticle of the LH1-complex of Rhodospirillum rubrum are the same, 9 and 42 cm −1. Except for the B777 subparticle, we find that the data can be described using identical coupling strengths for these modes. For the B777 particle the coupling strengths appeared to be slightly larger. The signals from the blue-shifted B800 band of the mutants we ascribe to semi-detached bacteriochlorophyll molecules. These results suggest that bacteriochlorophyll a molecular modes are involved in the dephasing process.

Multidimensional CP-MAS 13C NMR of uniformly enriched chlorophyll

The progress toward structure refinement of solid-type uniformly 13C enriched ([U- 13C]) chlorophyll-containing biological preparations is summarised. Solid state carbon chemical shifts of aggregated [U- 13C] bacteriochlorophyll (BChl) c in intact chlorosomes of Chlorobium tepidum and in [U- 13C] BChl c aggregates were determined by the application of homonuclear ( 13C 13C) magic angle spinning (MAS) NMR dipolar correlation spectroscopy. It was found that the arrangement of BChl c molecules in the chlorosomes and in the aggregates is highly similar, which provides convincing evidence that self-organisation of the BChl c is the main mechanism to support the structure of the chlorosomes. Additionally, high field 2-D ( 1H 13C) and 3-D ( 1H 13C 13C) dipolar correlation spectroscopy was applied to determine solid state proton chemical shifts of aggregated [U- 13C] BChl c in intact chlorosomes. From the high-field assignments, evidence is found for the existence of at least two well-defined interstack arrangements.

Insertional inactivation studies of the csmA and csmC genes of the green sulfur bacterium Chlorobium vibrioforme 8327: the chlorosome protein CsmA is required for viability but CsmC is dispensable

Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327. Under the photoautotrophic growth conditions employed, CsmA is required for the viability of the cells but CsmC is dispensable. The absence of CsmC caused a small red shift in the near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled in and could be isolated from the csmC mutant. The doubling time of the csmC mutant was approximately twice that of the wild-type strain. Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to another pigment, perhaps bacteriochlorophyll a, emitting at 800–804 nm, was less efficient in the csmC mutant cells than in wild-type cells. These studies establish that transformation and homologous recombination can be employed in targeted mutagenesis of Chlorobium sp. and further demonstrate that chlorosome proteins play important roles in the structure and function of these light-harvesting organelles.

Insertional inactivation studies of the csmA and csmC genes of the green sulfur bacterium Chlorobium vibrioforme 8327: the chlorosome protein CsmA is required for viability but CsmC is dispensable.

Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327. Under the photoautotrophic growth conditions employed, CsmA is required for the viability of the cells but CsmC is dispensable. The absence of CsmC caused a small red shift in the near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled in and could be isolated from the csmC mutant. The doubling time of the csmC mutant was approximately twice that of the wild-type strain. Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to another pigment, perhaps bacteriochlorophyll a, emitting at 800-804 nm, was less efficient in the csmC mutant cells than in wild-type cells. These studies establish that transformation and homologous recombination can be employed in targeted mutagenesis of Chlorobium sp. and further demonstrate that chlorosome proteins play important roles in the structure and function of these light-harvesting organelles.

Toward Level-to-Level Energy Transfers in Photosynthesis: The Fenna−Matthews−Olson Protein

The trimeric Fenna−Matthews−Olson (FMO) protein is a bacteriochlorophyll (BChl) a antenna complex, whose X-ray structure is now known at the atomic level for two green sulfur bacterial species (Prosthecochloris aestuarii and Chlorobium tepidum). Its steady-state Qy absorption spectrum at low temperature exhibits considerable structure, with at least eight bands attributable to well-defined (groups of) BChl a exciton levels. The low-temperature absorption difference spectra of Cb. tepidum trimers excited at 789 nm are correspondingly multifeatured, and they show rich spectral evolution due to femtosecond and picosecond electronic energy transfers. Global analyses of these time-dependent ΔA spectra lead to a phenomenological scenario for cascading and branching among exciton level groups responsible for specific steady-state absorption bands. The missing link in our understanding of structure−function causality in this protein stems from a lack of an ab initio theory for the effects of known protein environments on BChl a transition energies; this problem still limits our insights into the workings of spectrally heterogeneous antennas with multiple nonequivalent pigment sites. Optical anisotropy studies strongly suggest that FMO excitations are typically localized to the 7 BChl a pigments within one protein subunit, rather than delocalized over the whole trimer. This localization occurs because the resonance couplings between BChls belonging to different subunits (<20 cm-1) are several times smaller than the diagonal energy disorder (∼70 cm-1). Strong oscillations appear in the anisotropies (∼220 fs period) for pump−probe wavelengths that simultaneously overlap both of the exciton level groups responsible for the 825 and 815 nm bands in the Qy spectrum. These are not vibational coherences, but arise from quantum beating between levels with nearly perpendicular transition moments.

Transient Absorption Spectroscopy of Energy-Transfer and Trapping Processes in the Reaction Center Complex of Chlorobium tepidum

Reaction center complexes from the green sulfur bacterium Chlorobium tepidum were examined using picosecond absorption difference spectroscopy at room temperature. The complexes include the core proteins containing the primary electron donor P840 and associated core antenna pigments and also include some of the Fenna−Matthews−Olson (FMO) antenna protein. Upon excitation at 590 nm, long-lived absorption difference changes were observed that are interpreted to arise from a combination of the excited FMO protein and the P840-containing complex. Most of the FMO protein does not transfer energy to the core in this preparation. Excitation at 840 nm directly into the core complex gave an overall excited-state decay of 35 ps, forming the long-lived charge-separated state P840+A0- and no excited FMO complex. No antenna components were observed with absorption at wavelengths longer than the primary donor, in contrast to the situation in the related reaction centers from heliobacteria and photosystem I.

Ultrafast Exciton Motion in Photosynthetic Antenna Systems: The FMO-Complex

Excitation energy transfer in the Fenna−Matthews−Olson (FMO) photosynthetic antenna complex of chlorobium tepidum is investigated theoretically. On the basis of a dissipative multiexciton theory, the numerical simulations reproduce the cw-absorption and, in using the same parameters, the differential absorption of ultrafast pump−probe experiments. Exciton relaxation processes are included via a coupling to the vibrations of the protein matrix. In order take into account a delocalized protein−pigment interaction a correlation radius of the protein vibrations is introduced. The model allows for the study of the temperature dependence of optical spectra and enables one to utilize exciton relaxation data as a probe for a global-shape estimation of the spectral density of low-frequency protein vibrations. In fitting the cw-absorption measured at 5 and 107 K, the strength of the exciton-vibrational coupling, the related correlation radius and spectral density, and the inhomogeneous broadening of the exciton lev...

Fast Energy Transfer and Exciton Dynamics in Chlorosomes of the Green Sulfur Bacterium Chlorobium tepidum

The excited-state structure and energy-transfer dynamics, including their dependence on temperature and redox conditions, were studied in chlorosomes of the green sulfur bacterium Chlorobium tepidum at low temperatures by two independent methods: spectral hole burning in absorption and fluorescence spectra and isotropic one-color pump−probe spectroscopy with ∼100 fs resolution. Hole-burning experiments show that the lowest excited state (LES) of BChl c aggregates is distributed within approximately 760−800 nm, while higher excitonic states of BChl c (with absorption maximum at 750 nm) possess the main oscillator strength. The excited-state lifetime determined from hole-burning experiments at anaerobic conditions was 5.75 ps and most likely reflects energy transfer between BChl c clusters. Isotropic one-color absorption difference signals were measured from 720 to 790 nm at temperatures ranging from 5 to 65 K, revealing BChl c photobleaching and stimulated emission kinetics with four major components, with lifetimes of 200−300 fs, 1.7−1.8 ps, 5.4−5.9 ps, and 30−40 ps at anaerobic conditions. The lifetimes are attributed to different relaxation processes of BChl c, taking into account their different spectral distributions as well as limitations arising from results of hole burning. Evidence for at least two spectral forms of BChl c in chlorosome is reported. There is a striking similarity between the spectrum and kinetics of the 5.4−5.9 ps component with those of the LES determined from hole burning. A pronounced change of isotropic decays was observed at around 50 K. The temperature dependence of the isotropic decays is correlated with temperature-dependent changes of BChl c fluorescence emission. Further, the temperature decrease leads to an increase in the relative amplitude of the 200−300 fs component. At aerobic conditions, both hole burning and pump−probe spectroscopy show that the lifetime of the LES shortens to ∼2.6 ps, as a result of excitation quenching by a mechanism presumably protecting the cells against superoxide-induced damage. This mechanism operates on at least two levels, the second one being characterized by a 14−16 ps lifetime.

The phylogenetic relationships of Chlorobium tepidum and Chloroflexus aurantiacus based upon their RecA sequences

Using RecA as the phylogenetic marker, the relationships of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus to other eubacteria were investigated. The recA genes of the two organisms were cloned, and the resulting protein sequences aligned with 86 other eubacterial RecA sequences. Cb. tepidum was placed as the nearest relative to the Cytophaga/ Flexibacter/ Bacteroides group, a relationship supported by results obtained with several phylogenetic markers. Cf. aurantiacus was placed near Chlamydia trachomatis and the high-GC Gram-positives; however, this branching pattern was not strongly supported statistically by bootstrap analyses. Possible reasons for this ambiguity are discussed.

The phylogenetic relationships of Chlorobium tepidum and Chloroflexus aurantiacus based upon their RecA sequences.

Using RecA as the phylogenetic marker, the relationships of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus to other eubacteria were investigated. The recA genes of the two organisms were cloned, and the resulting protein sequences aligned with 86 other eubacterial RecA sequences. Cb. tepidum was placed as the nearest relative to the Cytophaga/Flexibacter/Bacteriodes group, a relationship supported by results obtained with several phylogenetic markers. Cf. aurantiacus was placed near Chlamydia trachomatis and the high-GC Gram-positives; however, this branching pattern was not strongly supported statistically by bootstrap analyses. Possible reasons for this ambiguity are discussed.

Stark Hole-Burning Studies of Three Photosynthetic Complexes

Stark hole-burning spectroscopy at 1.8 K was used to determine the dipole moment changes fΔμ (f, the local field correction factor) for the B800 absorption band of the light harvesting 2 (LH2) complex of Rhodobacter sphaeroides, Rhodopseudomonas acidophila (strain 10050), and Rhodospirillum molischianum. Hole-burning values of fΔμ for the lowest energy exciton level (B870) associated with LH2's B850 band have recently been reported (Rätsep et al. Spectrochim. Acta, in press). Values for the lowest energy exciton level (B896) associated with the B875 band of the LH1 complex of Rb. sphaeroides (wild-type chromatophores and an LH1-only mutant) and the 825 nm band of the bacteriochlorophyll a (FMO) antenna complex of Chlorobium tepidum are also reported. For each band, fΔμ was determined for burn laser polarization parallel and perpendicular to the Stark field ES and several burn frequencies. The dependencies on laser polarization and burn frequency are typically quite weak. Importantly, fΔμ values for the above bands are small, falling in the range ∼0.5−1.2 D, with the lowest and highest values associated with the 825 nm band of the FMO complex and B800 band of the LH2 complex, respectively. For the B896 band of the LH1 complex, fΔμ ≈ 0.8 D. Such small values are consistent with the very weak linear electron−phonon coupling of antenna protein complexes as determined by hole-burning spectroscopy. Overall, the values for fΔμ from classical Stark modulation (CSM) studies (Gottfried et al. Biochim. Biophys. Acta 1991, 1059, 63; Beekman et al. J. Phys. Chem. B 1997, 101, 7293) are larger, in the cases of B850 and B875, by a factor of 3−4. (In CSM spectroscopy, one analyzes the response of the entire absorption band to the external field.) Discussion of the discrepancies between the two Stark techniques is given. It appears that difficulties inherent to the analysis procedure of CSM spectroscopy can lead to unreliable values for dipole moment and polarizability changes associated with absorption bands of photosynthetic complexes, especially when several excitonic levels contribute to the band, e.g., B850 and B875. An explanation for the small hole-burning values of fΔμ for the B870 and B896 levels associated with Cn cyclic arrays of strongly coupled BChl a dimers is given based on structural, symmetry, and energy disorder considerations. A key point is that, in the absence of energy disorder, the component of Δμj (j labeling the exciton level) perpendicular to the Cn axis is zero. Energy disorder, which destroys the Cn symmetry and leads to localization effects results in nonzero values which may depend on j when the protein-induced contribution to Δμj is taken into account.

Tepidopterin, 1- O-( l-threo-biopterin-2′-yl)-β- N-acetylglucosamine from Chlorobium tepidum

A novel pterin compound, designated as tepidopterin, was detected from a thermophilic photosynthetic green sulphur bacterium, Chlorobium tepidum. The amount of tepidopterin inside the cell was estimated to be 2–5 μmol g −1 dry weight of cell. This compound was purified through a high performance liquid chromatography using preparative DeltaPak C 18 column. This compound was characterized by chromatographic behavior and by absorption and fluorescence properties. Its structure was determined to be 1- O-( l-threo-biopterin-2′-yl)-β- N-acetylglucosamine by 1D- and 2D-NMR spectroscopy, mass spectrometry and CD. The relative amount of tetrahydrotepidopterin was estimated to be 96.7% inside the cell, that of dihydrotepidopterin 2.9%, and that of fully oxidized tepidopterin 0.4%. The amount of tepidopterin within the cell increased continuously until the beginning of the stationary phase of the cell growth.

15-Cis-carotenoids found in the reaction center of a green sulfur bacterium Chlorobium tepidum and in the Photosystem I reaction center of a cyanobacterium Synechococcus vulcanus

Carotenoids were extracted, at ∼4 °C in complete darkness and under nitrogen atmosphere, from the reaction center (RC) of a green-sulfur bacterium and the Photosystem (PS) I RC of a cyanobacterium; each extract was subjected to high-performance liquid chromatography (HPLC) using an apparatus equipped with a two-dimensional diode-array detector in order to spectroscopically identify cis–trans carotenoids while performing HPLC analysis. In the extract from the RC of Chlorobium tepidum, 15-cis and all-trans-γ-carotenes as well as 13-cis-, 15-cis- and all- trans-chlorobactenes (in the order of elution) were identified, whereas in the extract from the PS I RC of Synechococcus vulcanus, 15-cis-, all-trans- and 9-cis-β-carotenes were found. Thus, the universal presence of 15- cis carotenoids in the 'iron sulfur-type' RCs has been shown in addition to the previous cases of the 'quinone-type' RCs.