Structural Variability in Wild-Type and bchQ bchR Mutant Chlorosomes of the Green Sulfur Bacterium Chlorobaculum tepidum

The self-aggregated state of bacteriochlorophyll (BChl) c molecules in chlorosomes belonging to a bchQ bchR mutant of the green sulfur bacteria Chlorobaculum tepidum, which mostly produces a single 17(2)-farnesyl-(R)-[8-ethyl,12-methyl]BChl c homologue, was characterized by solid-state nuclear magnetic resonance (NMR) spectroscopy and high-resolution electron microscopy. A nearly complete (1)H and (13)C chemical shift assignment was obtained from well-resolved homonuclear (13)C-(13)C and heteronuclear (1)H-(13)C NMR data sets collected from (13)C-enriched chlorosome preparations. Pronounced doubling (1:1) of specific (13)C and (1)H resonances revealed the presence of two distinct and nonequivalent BChl c components, attributed to all syn- and all anti-coordinated parallel stacks, depending on the rotation of the macrocycle with respect to the 3(1)-methyl group. Steric hindrance from the 20-methyl functionality induces structural differences between the syn and anti forms. A weak but significant and reproducible reflection at 1/0.69 nm(-1) in the direction perpendicular to the curvature of cylindrical segments observed with electron microscopy also suggests parallel stacking of BChl c molecules, though the observed lamellar spacing of 2.4 nm suggests weaker packing than for wild-type chlorosomes. We propose that relaxation of the pseudosymmetry observed for the wild type and a related BChl d mutant leads to extended domains of alternating syn and anti stacks in the bchQ bchR chlorosomes. Domains can be joined to form cylinders by helical syn-anti transition trajectories. The phase separation in domains on the cylindrical surface represents a basic mechanism for establishing suprastructural heterogeneity in an otherwise uniform supramolecular scaffolding framework that is well-ordered at the molecular level.     

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.     

Characterization of a plant-like protochlorophyllide a divinyl reductase in green sulfur bacteria

The green sulfur bacterium Chlorobium tepidum synthesizes three types of (bacterio)chlorophyll ((B)Chl): BChl a(P), Chl a(PD), and BChl c(F). During the synthesis of all three molecules, a C-8 vinyl substituent is reduced to an ethyl group, and in the case of BChl c(F), the C-8(2) carbon of this ethyl group is subsequently methylated once or twice by the radical S-adenosylmethionine enzyme BchQ. The C. tepidum genome contains homologs of two genes, bchJ (CT2014) and CT1063, that are highly homologous to genes, bchJ and AT5G18660, and that have been reported to encode C-8 vinyl reductases in Rhodobacter capsulatus and Arabidopsis thaliana, respectively. To determine which gene product actually encodes a C-8 vinyl reductase activity, the bchJ and CT1063 genes were insertionally inactivated in C. tepidum. All three Chls synthesized by the CT1063 mutant of C. tepidum have a C-8 vinyl group. Using NADPH but not NADH as reductant, recombinant BciA reduces the C-8 vinyl group of 3,8-divinyl-protochlorophyllide in vitro. These data demonstrate that CT1063, renamed bciA, encodes a C-8 divinyl reductase in C. tepidum. The bchJ mutant produces detectable amounts of Chl a(PD), BChl a(P), and BChl c(F), all of which have reduced C-8 substituents, but the mutant cells secrete large amounts of 3,8-divinyl-protochlorophyllide a into the growth medium and have a greatly reduced BChl c(F) content. The results suggest that BchJ may play an important role in substrate channeling and/or regulation of Chl biosynthesis but show that it is not a vinyl reductase. Because only some Chl-synthesizing organisms possess homologs of bciA, at least two types of C-8 vinyl reductases must occur.     

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.     

Chlorobium tepidum mutant lacking bacteriochlorophyll c made by inactivation of the bchK gene,encoding bacteriochlorophyll c synthase

The gene encoding bacteriochlorophyll (BChl) c synthase was identified by insertional inactivation in the photosynthetic green sulfur bacterium Chlorobium tepidum and was named bchK. The bchK mutant of C. tepidum was rusty-orange in color and completely lacked BChl c. Because of the absence of the BChl c antenna, the mutant grew about seven times slower than the wild type at light intensities that were limiting to the wild type (< 90 micromol m(-2) s(-1)). Various pheophorbides, which probably represent precursors of BChl c which had lost magnesium, accumulated in the mutant cells. A small fraction of these pheophorbides were apparently esterified by the remaining chlorophyll (Chl) a and BChl a synthases in cells. The amounts of BChl a, Chl a, isoprenoid quinones, carotenoids, Fenna-Matthews-Olson protein, and chlorosome envelope protein CsmA were not significantly altered on a cellular basis in the mutant compared to in the wild type. This suggests that the BChl a antennae, photosynthetic reaction centers, and remaining chlorosome components were essentially unaffected in the mutant. Electron microscopy of thin sections revealed that the mutant lacked normal chlorosomes. However, a fraction containing vestigial chlorosomes, denoted "carotenosomes," was partly purified by density centrifugation; these structures contained carotenoids, isoprenoid quinones, and a 798-nm-absorbing BChl a species that is probably protein associated. Because of the absence of the strong BChl c absorption found in the wild type, the bchK mutant should prove valuable for future analyses of the photosynthetic reaction center and of the roles of BChl a in photosynthesis in green bacteria. An evolutionary implication of our findings is that the photosynthetic ancestor of green sulfur bacteria could have evolved without chlorosomes and BChl c and instead used only BChl a-containing proteins as the major light-harvesting antennae.     

In vitro activity of C-20 methyltransferase, BchU, involved in bacteriochlorophyll c biosynthetic pathway in green sulfur bacteria

The activity of a methyltransferase, BchU, which catalyzes methylation at the C-20 position of chlorin ring in the biosynthetic pathway of bacteriochlorophyll c, was investigated in vitro. The bchU gene derived from the photosynthetic green sulfur bacterium, Chlorobium tepidum, was overexpressed in Escherichia coli as a His-tagged protein (His(6)-BchU), and the enzyme was purified. In the presence of S-adenosylmethionine, His(6)-BchU methylated zinc bacteriopheophorbide d at the C-20 position to give zinc bacteriopheophorbide c. Metal-free bacteriopheophorbide d could not be methylated by the BchU, indicating that the central metal in the chlorin should be required for the recognition by the BchU.     

Accumulation of chlorophyllous pigments esterified with the geranylgeranyl group and photosynthetic competence in the CT2256-deleted mutant of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria contain chlorophyllous pigments, chlorophyll (Chl) aPD and bacteriochlorophyll (BChl) aP, esterified with Delta2,6-phytadienol and phytol, respectively, which would be produced by reduction of the geranylgeranyl group at the C-17 propionate residue. In the genome of Chlorobium tepidum, two paralogous genes presumably encoding geranylgeranyl reductase, CT1232 and CT2256, are found. The deletion mutants of the CT1232 and CT2256 genes were constructed using an insertional inactivation method in order to clarify the biosynthetic process of the Delta2,6-phytadienyl and phytyl groups in green sulfur bacteria. The compositions of chlorophyllous pigments in the two mutants were determined by LC-MS analysis. The CT2256-deleted mutant accumulated Chl aGG and BChl aGG esterified with geranylgeraniol, indicating that CT2256 was involved in the production of both Delta2,6-phytadienyl and phytyl groups. The relatively high fluorescence emission from chlorosomes in the mutant also suggested some hindrance of the energy transfer from chlorosomes to the reaction center complex. However, the CT1232-deleted mutant almost showed no apparent phenotype compared to the wild type. Furthermore, the purple bacterium Rhodobacter capsulatus mutant defective in the bchP gene was partially complemented with the CT2256 gene; BChl aP was synthesized in the mutant in addition to accumulating other intermediates.     

Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes

Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope. These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes. Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known. Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA. All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within approximately 90% (CsmC(-) and CsmJ(-)) of those of the wild-type strain. Chlorosomes isolated from the mutants lacked only the protein whose gene had been inactivated and were generally similar to those from the wild-type strain with respect to size, shape, and BChl c, BChl a, and carotenoid contents. However, chlorosomes from the csmC mutant were about 25% shorter than those from the wild-type strain, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered. The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential for the biogenesis, light harvesting, or structural organization of BChl c and BChl a within the chlorosome. These results demonstrate that chlorosomes are remarkably robust structures that can tolerate considerable changes in protein composition.     

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.     

Spectroscopic properties and bacteriochlorophyll c isomer composition of extramembranous light-harvesting complexes in the green sulfur photosynthetic bacterium Chlorobium tepidum and its CT0388-deleted mutant under vitamin B12-limited conditions

The effects of exogenous vitamin B12 on the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum were examined. Wild-type cells and mutant cells lacking a gene CT0388 (denoted as VB0388) of Chl.tepidum were grown in liquid cultures containing different concentrations of vitamin B12. The VB0388 cells hardly grew in vitamin B12-limited media, indicating that the product of CT0388 actually played an important role in vitamin B12 biosynthesis in Chl. tepidum. Both wild-type and VB0388 cells in vitamin B12-limited media exhibited absorption bands and CD signals at the Qy region that were shifted to a shorter wavelength than those of cells grown in normal media. BChl c isomers that had S-stereochemistry at the 3(1)-position tended to increase in Chl. tepidum grown in vitamin B12-limited media.     

Studies on the biological role of DNA methylation: V. The pattern of E.coli DNA methylation.

The distribution of the methylatable sites GATC and CCATGG was studied by analyzing the molecular average size of restriction fragments of E. coli DNA. Both sites were found to be randomly distributed, reflecting a random pattern of methylation. The methylation pattern of specific sequences such as the origin of replication and rRNA genes has been studied in wild type E. coli and a methylation deficient (dam- dcm-) mutant. These sequences were found to be methylated in wild type cells and unmethylated in the mutant indicating that there is no effect of the state of methylation of these sequences on their expression. Analysis of the state of methylation of GATC sites in newly replicating DNA using the restriction enzyme Dpn I (cleaves only when both strands are methylated) revealed no detectable hemimethylated DNA suggesting that methylation occurs at the replication fork. Taking together the results presented here and previously published data (5), we arrive at the conclusion that the most likely function of E. coli DNA methylations is probably in preventing nuclease activity.     

Chlorobium Tepidum: Insights into the Structure,Physiology, and Metabolism of a Green Sulfur Bacterium Derived from the Complete Genome Sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.     

Redox effects on the excited-state lifetime in chlorosomes and bacteriochlorophyll c oligomers.

Oligomers of [E,E] BChl CF (8, 12-diethyl bacteriochlorophyll c esterified with farnesol (F)) and [Pr,E] BChl CF (analogously, M methyl, Pr propyl) in hexane and aqueous detergent or lipid micelles were studied by means of steady-state absorption, time-resolved fluorescence, and electron spin resonance spectroscopy. The maximum absorption wavelength, excited-state dynamics, and electron spin resonance (EPR) linewidths are similar to those of native and reconstituted chlorosomes of Chlorobium tepidum. The maximum absorption wavelength of oligomers of [E,E] BChl CF was consistently blue-shifted as compared to that of [Pr,E] BChl CF oligomers, which is ascribed to the formation of smaller oligomers with [E,E] BChl CF than [Pr,E] BChl CF. Time-resolved fluorescence measurements show an excited-state lifetime of 10 ps or less in nonreduced samples of native and reconstituted chlorosomes of Chlorobium tepidum. Under reduced conditions the excited-state lifetime increased to tens of picoseconds, and energy transfer to BChl a or long-wavelength absorbing BChl c was observed. Oligomers of [E,E] BChl CF and [Pr,E] BChl CF in aqueous detergent or lipid micelles show a similar short excited-state lifetime under nonreduced conditions and an increase up to several tens of picoseconds upon reduction. These results indicate rapid quenching of excitation energy in nonreduced samples of chlorosomes and aqueous BChl c oligomers. EPR spectroscopy shows that traces of oxidized BChl c radicals are present in nonreduced and absent in reduced samples of chlorosomes and BChl c oligomers. This suggests that the observed short excited-state lifetimes in nonreduced samples of chlorosomes and BChl c oligomers may be ascribed to excited-state quenching by BChl c radicals. The narrow EPR linewidth suggests that the BChl c are arranged in clusters of 16 and 6 molecules in chlorosomes of Chlorobium tepidum and Chloroflexus aurantiacus, respectively.     

Biosynthesis of unnatural bacteriochlorophyll c derivatives esterified with α,ω-diols in the green sulfur photosynthetic bacterium Chlorobaculum tepidum

Unnatural bacteriochlorophyll (BChl) c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain at the 17-propionate were biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculum tepidum. Addition of exogenous 1,8-octanediol, 1,12-dodecanediol, and 1,16-hexadecanediol in acetone to liquid cultures resulted in accumulation of BChl c monoesterified with the corresponding diols. The relative ratios of the novel BChl c derivatives esterified with 1,8-, 1,12-, and 1,16-diols to totally producing BChl c were 8.2, 50.2, and 57.6% in the cells grown with additive α,ω-diols at concentrations of 1.5, 0.06, and 0.06 mM, respectively, at the final concentration. The homologue composition of BChl c derivatives esterified with these α,ω-diols was similar to that of original, coexisting BChl c esterified with farnesol (BChl c(F)), suggesting that esterification of α,ω-diols occurred at the last step of the BChl c biosynthetic pathway by BChl c synthase, BchK, in the same manner as in BChl c(F). Chlorosomes, which were isolated from cells grown in the presence of exogenous α,ω-diols, contained a ratio and a composition of BChl c derivatives esterified with the diols similar to those in the whole cells, indicating that these BChl c derivatives were actually present in chlorosomes. Q(y) absorption bands of C. tepidum cells containing the novel BChl c derivatives were shifted to a shorter wavelength, although their bandwidths were analogous to those of cells obtained by normal cultivation. Circular dichroism spectra of cells that had BChl c derivatives esterified with α,ω-diols exhibited S-shaped signals in the Q(y) region, whose polarities were the reverse of those of cells grown in the normal medium and by supplementation with neat acetone as a control experiment. These spectral features of C. tepidum possessing BChl c derivatives esterified with α,ω-diols imply that the novel BChl c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain affect their self-assembly in chlorosomes.     

The photosystem of Rhodobacter sphaeroides assembles with zinc bacteriochlorophyll in a bchD (magnesium chelatase) mutant

A Rhodobacter sphaeroides bchD (magnesium chelatase) mutant was studied to determine the properties of its photosystem in the absence of bacteriochlorophyll (BChl). Western blots of reaction center H, M, and L (RC H/M/L) proteins from mutant membranes showed levels of 12% RC H, 32% RC L, and 46% RC M relative to those of the wild type. Tricine-SDS-PAGE revealed 52% light-harvesting complex alpha chain and 14% beta chain proteins compared to those of the wild type. Pigment analysis of bchD cells showed the absence of BChl and bacteriopheophytin (BPhe), but zinc bacteriochlorophyll (Zn-BChl) was discovered. Zn-BChl binds to light-harvesting 1 (LH1) and 2 (LH2) complexes in place of BChl in bchD membranes, with a LH2:LH1 ratio resembling that of wild-type cells under BChl-limiting conditions. Furthermore, the RC from the bchD mutant contained Zn-BChl in the special pair and accessory BChl binding sites, as well as carotenoid and quinone, but BPhe was absent. Comparison of the bchD mutant RC absorption spectrum to that of Acidiphilium rubrum, which contains Zn-BChl in the RC, suggests the RC protein environment at L168 contributes to A. rubrum special pair absorption characteristics rather than solely Zn-BChl. We speculate that Zn-BChl is synthesized via the normal BChl biosynthetic pathway, but with ferrochelatase supplying zinc protoporphyrin IX for enzymatic steps following the nonfunctional magnesium chelatase. The absence of BPhe in bchD cells is likely related to Zn2+ stability in the chlorin macrocycle and consequently high resistance of Zn-BChl to pheophytinization (dechelation). Possible agents prevented from dechelating Zn-BChl include the RC itself, a hypothetical dechelatase enzyme, and spontaneous processes.     

Internal structure of chlorosomes from brown-colored chlorobium species and the role of carotenoids in their assembly

Chlorosomes are the main light harvesting complexes of green photosynthetic bacteria. Recently, a lamellar model was proposed for the arrangement of pigment aggregates in Chlorobium tepidum chlorosomes, which contain bacteriochlorophyll (BChl) c as the main pigment. Here we demonstrate that the lamellar organization is also found in chlorosomes from two brown-colored species (Chl. phaeovibrioides and Chl. phaeobacteroides) containing BChl e as the main pigment. This suggests that the lamellar model is universal among green sulfur bacteria. In contrast to green-colored Chl. tepidum, chlorosomes from the brown-colored species often contain domains of lamellar aggregates that may help them to survive in extremely low light conditions. We suggest that carotenoids are localized between the lamellar planes and drive lamellar assembly by augmenting hydrophobic interactions. A model for chlorosome assembly, which accounts for the role of carotenoids and secondary BChl homologs, is presented.     

Factors controlling the biosynthesis of chlorosome antenna bacteriochlorophylls in green filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae

We determined the concentrations of bacteriochlorophylls (BChl) in the light-harvesting antennae of Oscillochloris trichoides (of the family Oscillochloridaceae belonging to green filamentous mesophilic bacteria) cultivated either with gabaculine, an inhibitor of the C-5 pathway of BChl biosynthesis in a number of bacteria, or at various illumination intensities. We determined the BChl c: BChl a molar ratios in intact cells, in chlorosome-membrane complexes, and in isolated chlorosomes. We revealed that BChl c synthesis in Osc. trichoides was more gabaculine-sensitive than BChl a synthesis. Accordingly, an increase in gabaculine concentrations in the medium resulted in a decrease in the BChl c: BChl a ratio in the tested samples. We suggest that BChl synthesis in Osc. trichoides proceeds via the C-5 pathway, similar to representatives of other families of green bacteria (Chlorobium limicola and Chloroflexus aurantiacus). We demonstrated that the BChl c: BChl a ratio in the chlorosomes varied from 55 : 1 to 110 : 1, depending on light intensity. This ratio is, therefore, closer to that of Chlorobiaceae, and it significantly exceeds the BChl c: BChl a ratio in Chloroflexaceae.     

Isolation and pigment composition of the antenna system of four species of green sulfur bacteria

New and rapid procedures were developed for the isolation of chlorosomes and FMO-protein from the green sulfur bacteria Prosthecochloris (P.) aestuarii, Chlorobium (Cb.) phaeovibrioides, Cb. tepidum and Cb. vibrioforme. The resulting preparations were free from contaminating pigments and proteins as was shown by absorption spectroscopy, pigment analysis and SDS-PAGE. Two spectrally different types of FMO-protein were found. The first type, present in P. aestuarii and Cb. vibrioforme, has a main absorption band at 6 K at 815 nm, whereas the second type, isolated from Cb. tepidum and Cb. phaeovibrioides, has a strong band at 806 nm. In contrast to what was recently suggested (Tronrud DE and Matthews BW (1993) In: Deisenhofer J and Norris J (eds) The Photosynthetic Reaction Center, Vol 1, pp 13–21. Academic Press, San Diego, CA) the FMO-proteins contained no polar BChl a homologue. The isolated chlorosomes showed a small blue-shift of the QY absorption maximum with respect to intact cells. For the different species, grown under the same light conditions, the homologue composition of BChls c and d was approximately identical whereas for the BChl e in Cb. phaeovibrioides the relative amounts of homologues with larger alkyl substituents at position 8 were considerably larger. Baseplate BChl a was present in all chlorosomes and comprised 1–2% of the chlorosomal BChl. Its QY absorption band was located at about 802 nm and was clearly separated from the major QY absorption band at 6 K. The predominant esterifying alcohol of BChl a in the chlorosomes as well as in the FMO-proteins was phytol, but both antenna complexes also contained small amounts of BChl a esterified with the metabolic intermediates geranylgeraniol, dihydrogeranylgeraniol and tetrahydrogeranylgeraniol, like most purple bacteria. Since the esterifying alcohols of the chlorosomal BChl a and of the main chlorosomal pigments (BChls c, d and e) are different, esterification, and perhaps also the synthesis, of the BChls in the interior of the chlorosome and of the BChls in the baseplate must be spatially and genetically separated processes.     

Studies of the location and function of isoprenoid quinones in chlorosomes from green sulfur bacteria

The chlorosome antenna of the green sulfur bacterium Chlorobium tepidum essentially consists of aggregated bacteriochlorophyll (BChl) c enveloped in a glycolipid monolayer. Small amounts of protein and the isoprenoid quinones chlorobiumquinone (CK) and menaquinone-7 (MK-7) are also present. Treatment of isolated chlorosomes from Cb. tepidum with sodium dodecyl sulfate (SDS) did not affect the quinones, demonstrating that these are located in a site which is inaccessible to SDS, probably in the interior of the chlorosomes. About half of the quinones were removed by Triton X-100. The non-ionic character of Triton probably allowed it to extract components from within the chlorosomes. MK-10 in chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus was likewise found to be located in the chlorosome interior. The excitation transfer in isolated chlorosomes from Cb. tepidum is redox-regulated. We found a ratio of BChl c fluorescenceintensity under reducing conditions (Fred) to that under oxidizing conditions (Fox) of approximately 40. The chlorosomal BChl a fluorescence was also redox-regulated. When the chlorosomal BChl c–BChl c interactions were disrupted by 1-hexanol, the BChl c Fred/Fox ratiodecreased to approximately 3. When CK and MK-7 were extracted from isolated chlorosomes with hexane, the BChl c Fred/Fox ratio also decreased to approximately 3. A BChl c Fred/Fox ratio of 3–5 was furthermore observed in aggregates of pure BChl c and in chlorosomes from Cfx. aurantiacus which do not contain CK. We therefore suggest that BChl c aggregates inherently exhibit a small redox-dependent fluorescence (Fred/Fox 3) and that the large redox-dependent fluorescence observed in chlorobial chlorosomes (Fred/Fox 40) is CK-dependent.     

Assembly of a mixture of isomeric BChl c from Chlorobium limicola as determined by intermolecular 13C-13C dipolar correlations: coexistence of dimer-based and pseudo-monomer-based stackings

A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate.