Long-range organization of bacteriochlorophyll in chlorosomes of Chlorobium tepidum investigated by cryo-electron microscopy

Intact chlorosomes of Chlorobium tepidum were embedded in amorphous ice layers and examined by cryo-electron microscopy to study the long-range organization of bacteriochlorophyll (BChl) layers. End-on views reveal that chlorosomes are composed of several multi-layer tubules of variable diameter (20-30 nm) with some locally undulating non-tubular lamellae in between. The multi-layered tubular structures are more regular and larger in a C. tepidum mutant that only synthesizes [8-ethyl, 12-methyl]-BChl d. Our data show that wild-type C. tepidum chlorosomes do not have a highly regular, long-range BChl c layer organization and that they contain several multi-layered tubules rather than single-layer tubules or exclusively undulating lamellae as previously proposed.     

Characterization of csmB genes,encoding a 7.5-kDa protein of the chlorosome envelope,from the green sulfur bacteria Chlorobium vibrioforme 8327D and Chlorobium tepidum

The csmB gene, encoding the 7.5-kDa “Gerola-Olson” protein of chlorosomes, has been cloned and sequenced from the green sulfur bacteria Chlorobium vibrioforme strain 8327D and Chlorobium tepidum. Two potential start codons were identified, and the csmB gene may be translated into a preprotein with an amino-terminal extension. Two forms of the mature CsmB protein (74 or 75 amino acids in length) were identified that differ by the presence or absence of a methionine residue at the amino terminus. The csmB gene of Chl. tepidum is transcribed as an abundant monocistronic mRNA of approximately 350 nucleotides; primer extension mapping of the 5′ endpoint of the csmB mRNA suggests there is strong similarity between the csmB promoter and the σ⁷⁰ promoters of Escherichia coli. The CsmB protein of Chl. tepidum was overproduced as a histidine-tagged fusion protein in E. coli, purified to homogeneity by Ni²⁺ chelation affinity chromatography, and used to raise polyclonal antibodies in rabbits. Protease susceptibility mapping and agglutination experiments with isolated chlorosomes using anti-CsmB antibodies indicate that the CsmB protein is a component of the chlorosome envelope. Received: 28 May 1996 / Accepted: 17 July 1996     

Quinones in chlorosomes of green sulfur bacteria and their role in the redox-dependent fluorescence studied in chlorosome-like bacteriochlorophyll c aggregates

The light-harvesting chlorosome antennae of anaerobic, photosynthetic green sulfur bacteria exhibit a highly redox-dependent fluorescence such that the fluorescence intensity decreases under oxidizing conditions. We found that chlorosomes from Chlorobium tepidum contain three isoprenoid quinone species (chlorobiumquinone, menaquinone-7, and an unidentified quinone that probably is a chlorobiumquinone derivative) at a total concentration of approximately 0.1 mol per mol bacteriochlorophyll c. Most of the cellular chlorobiumquinone was found in the chlorosomes and constituted about 70% of the total chlorosome quinone pool. When the quinones were added to artificial, chlorosome-like bacteriochlorophyll c aggregates in an aqueous solution, a high redox dependency of the fluorescence was observed. Chlorobiumquinones were most effective in this respect. A lesser redox dependency of the fluorescence was still observed in the absence of quinones, probably due to another unidentified redox-active component. These results suggest that quinones play a significant, but not exclusive role in controlling the fluorescence and in inhibiting energy transfer in chlorosomes under oxic conditions. Chlorosomes from Chloroflexus aurantiacus contained menaquinone in an amount similar to that of total quinone in Chlorobium tepdium chlorosomes, but did not contain chlorobiumquinones. This may explain the much lower redox-dependent fluorescence observed in Chloroflexus chlorosomes. Received: 4 November 1996 / Accepted: 18 February 1997     

Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria are obligate anaerobic phototrophs, which in addition to outer and plasma membranes contain chlorosomes. The analysis of the membrane proteome of Chlorobium tepidum from chlorosome-depleted membranes is described in this study. The membranes were purified by sucrose density centrifugation and characterized by 1-DE and 2-DE coupled with MS, absorption spectroscopy, and electron microscopy. 1-DE and 2-DE were employed to analyze the membrane proteins and to characterize the capabilities of the methods. Solubilization of the membrane proteins prior to 2-DE was improved by using a series of zwitterionic detergents. Based on the resolved spots after 2-DE, the combination of amidosulfobetaine 14 with Triton X-100 is more efficient than the combination of CHAPS, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, and Triton X-100. From the application of 1-DE and 2-DE, 167 and 202 unique proteins were identified, respectively, using PMF by MALDI-TOF MS. Both methods resulted in the detection of 291 different proteins of which only 88 were predicted membrane proteins, indicating the limitation of membrane protein detection after separation with electrophoresis methods. In addition, 53 of these proteins were identified as outer membrane proteins.     

Growth-rate-dependent bacteriochlorophyll c/d ratio in the antenna of Chlorobium limicola strain UdG6040

The green sulfur bacterium Chlorobium limicola UdG6040 exhibited a significant change in the spectral properties of its antenna when transferred from batch culture to a sulfide-limited chemostat. In steady-state continuous cultures, the in vivo absorption maximum of the culture changed to shorter wavelengths according to the dilution rate. The maximum difference observed was of 15 nm when cells were growing at 0.087 h^–1. HPLC analyses revealed that the observed spectral change was caused by a partial enrichment of the original BChl c-containing antenna with BChl d molecules together with a change in the homolog composition of both pigments. The relative amount of BChl d reached a maximum value of 50% when cells were growing at 0.087 h^–1. The content of BChl d decreased to less than the 22% when the dilution rate was diminished to 0.015 h^–1. An unbalance of pigment synthesis at high dilution rates is suspected to be responsible of the changes observed in the antenna composition. Chlorosomes isolated from Chl. limicola UdG6040 growing at 0.070 h^–1 contain organised pools of BChl c and BChl d in equal amounts. Received: 2 December 1998 / Accepted: 25 February 1999     

Role of Phe-99 and Trp-196 of sepiapterin reductase from Chlorobium tepidum in the production of L-threo-tetrahydrobiopterin

Sepiapterin reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct tetrahydrobiopterin (BH4), L -threo-BH4, different from the mammalian enzyme product. The 3-D crystal structure of cSR has revealed that the product configuration is determined solely by the substrate binding mode within the well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180° around the active site from the position in mouse sepiapterin reductase. To confirm their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine (F99A, W196A) by site-directed mutagenesis and comparatively examined substrate binding of the purified proteins by kinetics analysis and differential scanning calorimetry. These mutants had higher K _m values than the wild type. Remarkably, the W196A mutation resulted in a higher K _m increase compared with the F99A mutation. Consistent with the results, the melting temperature ( T _m) in the presence of sepiapterin was lower in the mutant proteins and the worst was W196A. These findings indicate that the two residues are indispensable for substrate binding in cSR, and Trp-196 is more important than Phe-99 for different stereoisomer production.     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.     

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t_1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t_1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

Incorporation of exogenous long-chain alcohols into bacteriochlorophyll c homologs by Chloroflexus aurantiacus

Chloroflexus aurantiacus grown in batch culture took up exogenous alcohols and incorporated these into bacteriochlorophyll c as the esterifying alcohol. It was possible to change the distribution of the naturally occurring homologs of bacteriochlorophyll c esterified with phytol, hexadecanol, and octadecanol by adding the appropriate alcohol. The corresponding homolog then made up at least 60% of the cellular bacteriochlorophyll c. It was also possible to obtain novel bacteriochlorophyll homologs not found in detectable amounts in control cells by adding fatty alcohols with short chains (C10, C12) or long chains (C20). These changes in bacteriochlorophyll composition had no detectable effects on the spectral properties of the chlorosomes.Abbreviation BChl Bacteriochlorophyll     

Quantitative relationship between bacteriochlorophyll content,cytoplasmic membrane structure and chlorosome size in Chloroflexus aurantiacus

Continuous cultures of Chloroflexus aurantiacus were cultivated in a chemostat in the light with varying bacteriochlorophyll (BChl) a/c ratios by changing the growth rate. Under these culture conditions all cells were homogeneously and reproducibly equipped with chlorosomes. In order to determine the number and size of chlorosomes in relation to different BChl contents morphometric measurements were performed on electron micrographs. The linear increase of BChl a contents coincided with an increasing number of chlorosomes per membrane area and per bacterium rather than with an enlargement of the average size of chlorosomes. The numbers of chlorosomes and therefore the percentage of chlorosome-covered cytoplasmic membrane increased linearly with increasing BChl a contents. The average size of the baseplates was largely constant in all cultures (mean 3,222±836 nm²). However, within individual cells the size of baseplates varied by a factor of 3.0, especially by the variation of the length. The exponential increase in BChl c contents coincided with an increasing number of chlorosomes (up to a factor of 2.3) and an enlargement of the average chlorosome volume (up to a factor of 1.9). The number of BChl a molecules per chlorosome was about 1,484±165, thus the number of reaction centers per chlorosome was 58±12. The data suggest, firstly, that BChl a is confined to areas (cytoplasmic membrane plus baseplate) as represented by the chlorosome attachment sites; secondly, that the degree of packing of BChl c molecules within chlorosomes increases with increasing BChl c contents.     

The three-dimensional structure of CsmA: a small antenna protein from the green sulfur bacterium Chlorobium tepidum

The structure of the chlorosome baseplate protein CsmA from Chlorobium tepidum in a 1:1 chloroform:methanol solution was determined using liquid-state NMR spectroscopy. The data reveal that the 59-residue protein is predominantly alpha-helical with a long helical domain extending from residues V6 to L36, containing a putative bacteriochlorophyll a binding domain, and a short helix in the C-terminal part extending from residues M41 to G49. These elements are compatible with a model of CsmA having the long N-terminal alpha-helical stretch immersed into the lipid monolayer confining the chlorosome and the short C-terminal helix protruding outwards, thus available for interaction with the Fenna-Matthews-Olson antenna protein.     

Femtosecond energy transfer and spectral equilibration in bacteriochlorophyll a--protein antenna trimers from the green bacterium Chlorobium tepidum.

Femtosecond energy transfer processes in a bacteriochlorophyll a-protein antenna complex from the green sulfur bacterium Chlorobium tepidum have been studied by one-color, two-color, and broadband absorption difference spectroscopy. Much of the spectral excitation equilibration in this antenna occurs with 350 to 450 fs kinetics. The anisotropy decay functions r(t) exhibit two major lifetime components, 100 to 130 fs and 1.7 to 2.0 ps. The short component lifetimes may represent single-step energy transfer kinetics in this antenna; the long component is similar to the anisotropy decay observed in earlier picosecond pump-probe experiments.     

Manipulation of the bacteriochlorophyll c homolog distribution in the green sulfur bacterium Chlorobium tepidum

We have shown that the green sulfur bacterium Chlorobium tepidum can be grown in batch culture supplemented with potentially toxic fatty alcohols without a major effect on the growth rate if the concentration of the alcohols is kept low either by programmed addition or by adding the alcohol as an inclusion complex with -cyclodextrin. HPLC and GC analysis of pigment extracts from the supplemented cells showed that the fatty alcohols were incorporated into bacteriochlorophyll c as the esterifying alcohol. It was possible to change up to 43% of the naturally occurring farnesyl ester of bacteriochlorophyll c with the added alcohol. This change in the homolog composition had no effect on the spectral properties of the cells when farnesol was partially replaced by stearol, phytol or geranylgeraniol. However, with dodecanol we obtained a blue-shift of 6 nm of the Q_y band of the bacteriochlorophyll c and a concomitant change in the fluorescence emission was observed. The possible significance of these findings is discussed in the light of current ideas about bacteriochlorophyll organization in the chlorosomes.Abbreviations -CD -cyclodextrin - BChl bacteriochlorophyll - BChl c _H bacteriochlorophyllide c - [E,M] BChl c _F 8-ethyl, 12-methyl, farnesyl BChl c - [E,E] BChl c _F 8-ethyl, 12-ethyl, farnesyl BChl c - [P,E] BChl c _F 8-propyl, 12-ethyl, farnesyl BChl c - [I,E] BChl c _F 8-isobutyl, 12-ethyl, farnesyl BChl c - Car carotenoids     

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

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

Low-temperature energy transfer in FMO trimers from the green photosynthetic bacterium Chlorobium tepidum

The pump-probe kinetics of the slowest spectral equilibrations between inequivalent BChl a Q_y states in FMO trimers from Chlorobium tepidum are decelerated by nearly two orders of magnitude when the temperature is lowered from 300 K to 19 K. The pump-probe anisotropy decays are also markedly slower at 19 K than at 300 K. Singlet-singlet annihilation in FMO trimers is negligible at the laser powers used here. However, reduced temperatures greatly accentuate the probability of singlet-triplet annihilation, due to accumulation of metastable BChl a states under high laser repetition rates.Abbreviations BChl bacteriochlorophyll - FMO Fenna-Matthews-Olson - fwhm full width at half maximum - PB photobleaching - SE stimulated emission     

The bchU gene of Chlorobium tepidum encodes the c-20 methyltransferase in bacteriochlorophyll c biosynthesis

Bacteriochlorophylls (BChls) c and d, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl c. A gene potentially encoding the C-20 methyltransferase, bchU, was identified by comparative analysis of the Chlorobium tepidum and Chloroflexus aurantiacus genome sequences. Homologs of this gene were amplified and sequenced from Chlorobium phaeobacteroides strain 1549, Chlorobium vibrioforme strain 8327d, and C. vibrioforme strain 8327c, which produce BChls e, d, and c, respectively. A single nucleotide insertion in the bchU gene of C. vibrioforme strain 8327d was found to cause a premature, in-frame stop codon and thus the formation of a truncated, nonfunctional gene product. The spontaneous mutant of this strain that produces BChl c (strain 8327c) has a second frameshift mutation that restores the correct reading frame in bchU. The bchU gene was inactivated in C. tepidum, a BChl c-producing species, and the resulting mutant produced only BChl d. Growth rate measurements showed that BChl c- and d-producing strains of the same organism (C. tepidum or C. vibrioforme) have similar growth rates at high and intermediate light intensities but that strains producing BChl c grow faster than those with BChl d at low light intensities. Thus, the bchU gene encodes the C-20 methyltransferase for BChl c biosynthesis in Chlorobium species, and methylation at the C-20 position to produce BChl c rather than BChl d confers a significant competitive advantage to green sulfur bacteria living at limiting red and near-infrared light intensities.     

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl₄, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at 747 nm.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGGipm0dc9vqaqpepu0xbbG8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeq4Xdm2aaW% baaSqabeaacaaIYaaaaaaa!3777!\[\chi ^2 \] chi-square - FWHM full-width at half-maximum     

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