Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum

The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur bacteria and green filamentous bacteria is also discussed.     

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.     

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.     

217 000-year-old DNA sequences of green sulfur bacteria in Mediterranean sapropels and their implications for the reconstruction of the paleoenvironment

Deep-sea sediments of the eastern Mediterranean harbour a series of dark, organic carbon-rich layers, so-called sapropels. Within these layers, the carotenoid isorenieratene was detected. Since it is specific for the obligately anaerobic phototrophic green sulfur bacteria, the presence of isorenieratene may suggest that extended water column anoxia occurred in the ancient Mediterranean Sea during periods of sapropel formation. Only three carotenoids (isorenieratene, beta-isorenieratene and chlorobactene) are typical for green sulfur bacteria and thus do not permit to differentiate between the approximately 80 known phylotypes. In order to reconstruct the paleoecological conditions in more detail, we searched for fossil 16S rRNA gene sequences of green sulfur bacteria employing ancient DNA methodology. 540 bp-long fossil sequences could indeed be amplified from up to 217 000-year-old sapropels. In addition, such sequences were also recovered from carbon-lean intermediate sediment layers deposited during times of an entirely oxic water column. Unexpectedly, however, all the recovered 16S rRNA gene sequences grouped with freshwater or brackish, rather than truly marine, types of green sulfur bacteria. It is therefore feasible that the molecular remains of green sulfur bacteria originated from populations which thrived in adjacent freshwater or estuarine coastal environments rather than from an indigenous pelagic population.     

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.     

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.     

Biosynthesis of the biomarker okenone: χ-ring formation

Purple sulfur bacteria (PSB) mainly occur in anoxic aquatic and benthic environments, where they play important roles in cycling carbon and sulfur. Many PSB characteristically produce the unique keto-carotenoid, okenone, which is important not only for its light absorption and photoprotection properties but also because of its diagenesis product, okenane, which is a biomarker for ancient sediments derived from anoxic environments. The specific methylation pattern of the χ-ring of okenane is unlikely to be formed by diagenetic processes and should therefore reflect an enzymatic activity from okenone biosynthesis. This study describes two enzymes that produce the χ-ring of okenone, the only structural element of okenone preserved in okenane. Genes encoding enzymes of carotenogenesis were identified in the draft genome sequence of an okenone-producing PSB, Thiodictyon sp. strain CAD16. Two divergently transcribed genes encoded a CrtY-type lycopene cyclase and a CrtU/CruE-type γ-carotene desaturase/methyltransferase. Expression of crtY in Escherichia coli showed that this gene encoded a lycopene cyclase that produced γ-carotene as the only product. Although the sequence of the γ-carotene desaturase/methyltransferase was more similar to CrtU sequences of green sulfur bacteria than to CruE sequences of cyanobacteria, expression of the crtU gene in Chlorobaculum tepidum showed that the enzyme produced carotenoids with χ-rings rather than φ-rings. Phylogenetic analysis of the carotene desaturase/methyltransferases revealed that enzymes capable of converting β-rings to χ-rings have independently evolved at least two times. These results indicate that it probably will not be possible to deduce the activity of carotene desaturase/methyltransferases solely from sequence data.     

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.     

A comparative study of<Emphasis Type="Italic"> bchG</Emphasis> from green photosynthetic bacteria

The gene bchG, coding for bacteriochlorophyll a synthase from a variety of green sulfur bacteria and the filamentous anoxygenic phototrophic bacteria, Chloroflexus aurantiacus, Chloronema sp., and Roseiflexus castenholzii HL08, was partially sequenced and compared. The deduced amino acid consensus sequences for green sulfur bacteria and green filamentous anoxygenic phototrophic bacteria were found to belong to the UbiA enzyme family of polyprenyltransferases with the most similar sequences being those of photosynthetic organisms. All deduced amino acid sequences showed a highly conserved region, which includes the motif DRXXD, characteristic of polyprenyltransferases, which was extended to DREVDAINEP for green sulfur bacteria. Neighbor-joining analysis of a protein similitude matrix displayed a relatively high distance between green sulfur bacteria and the other groups. Sequences from green sulfur bacteria were more closely related to those of purple bacteria than to those of filamentous anoxygenic phototrophic bacteria. In addition, internal grouping within green sulfur bacteria was congruent regarding taxonomic features including cell shape, presence of gas vacuoles and NaCl requirement. In addition to bchlG, another gene encoding for a second chlorophyll synthetase, previously tentatively identified as chlG, was also found in Chlorobium tepidum, showing the highest similarities with polyprenyltransferases from chlorophyll- a-containing organisms.     

Isorenieratene biosynthesis in green sulfur bacteria requires the cooperative actions of two carotenoid cyclases

The cyclization of lycopene to γ- or β-carotene is a major branch point in the biosynthesis of carotenoids in photosynthetic bacteria. Four families of carotenoid cyclases are known, and each family includes both mono- and dicyclases, which catalyze the formation of γ- and β-carotene, respectively. Green sulfur bacteria (GSB) synthesize aromatic carotenoids, of which the most commonly occurring types are the monocyclic chlorobactene and the dicyclic isorenieratene. Recently, the cruA gene, encoding a conserved hypothetical protein found in the genomes of all GSB and some cyanobacteria, was identified as a lycopene cyclase. Further genomic analyses have found that all available fully sequenced genomes of GSB encode an ortholog of cruA. Additionally, the genomes of all isorenieratene-producing species of GSB encode a cruA paralog, now named cruB. The cruA gene from the chlorobactene-producing GSB species Chlorobaculum tepidum and both cruA and cruB from the brown-colored, isorenieratene-producing GSB species Chlorobium phaeobacteroides strain DSM 266^T were heterologously expressed in lycopene- and neurosporene-producing strains of Escherichia coli, and the cruB gene of Chlorobium clathratiforme strain DSM 5477^T was also heterologously expressed in C. tepidum by inserting the gene at the bchU locus. The results show that CruA is probably a lycopene monocyclase in all GSB and that CruB is a γ-carotene cyclase in isorenieratene-producing species. Consequently, the branch point for the synthesis of mono- and dicyclic carotenoids in GSB seems to be the modification of γ-carotene, rather than the cyclization of lycopene as occurs in cyanobacteria.     

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.     

Polyclonal antibodies to chlorosome proteins as probes for green sulfur bacteria.

We found that polyclonal antibodies raised against chlorosome polypeptides from green sulfur bacteria reacted to Chlorobium tepidum, Chlorobium limicola, and Chlorobium phaeobacteroides but not to Chloroflexus aurantiacus. These antibodies successfully labeled only green sulfur species in marine microbial mat samples. Our results suggest that these antibodies may be useful as immunohistochemical probes.     

Nitrogen fixation by the thermophilic green sulfur bacterium Chlorobium tepidum.

The thermophilic green sulfur bacterium Chlorobium tepidum grew with N2, NH4+, or glutamine as the sole nitrogen source under phototrophic (anaerobic-light) conditions. Growth on N2 required increased buffering capacity to stabilize uncharacterized pH changes that occurred during diazotrophic growth. Increased sulfide levels were stimulatory for growth on N2. Levels of nitrogenase activity (acetylene reduction) in N2-grown C. tepidum cells were very high, among the highest ever reported for anoxygenic phototrophic bacteria. Maximal acetylene reduction rates in C. tepidum cells were observed at 48 to 50 degrees C, which is about 15 degrees C higher than the optimum temperature for nitrogenase activity in mesophilic chlorobia, and nitrogenase activity in C. tepidum responded to addition of ammonia by a "switch-off/switch-on" mechanism like that in phototrophic purple bacteria. C. tepidum cells assimilated ammonia mainly via the glutamine synthetase-glutamate synthase pathway, elevated levels of both of these enzymes being present in cells grown on N2. These results show that N2 fixation can occur in green sulfur bacteria up to at least 60 degrees C and that regulatory mechanisms important in control of nitrogenase activity in mesophilic anoxygenic phototrophs also appear to regulate thermally active forms of the enzyme.     

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.     

Physical map of the genome of the green phototrophic bacterium Chlorobium tepidum.

A physical restriction map of the chromosome of the green sulfur bacterium Chlorobium tepidum was generated by determining the order of the fragments obtained after digestion with the restriction endonucleases XbaI and PacI and subsequent separation of the fragments by pulsed-field gel electrophoresis. The size of the chromosome is estimated to be 2.1 Mb. Fifteen genes and operons, mainly encoding proteins involved in photosynthesis, have been placed on this map by hybridization to fragments obtained after single- and double-restriction digestions.     

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.     

Metabolic engineering for synthesis of aryl carotenoids in Rhodococcus

Rhodococcus erythropolis naturally synthesizes monocyclic carotenoids: 4-keto-γ-carotene and γ-carotene. The genes and the pathway for carotenoid synthesis in R. erythropolis were previously described. We heterologously expressed a β-carotene desaturase gene (crtU) from Brevibacterium in Rhodococcus to produce aryl carotenoids such as chlorobactene. Expression of the crtU downstream of a chloramphenicol resistance gene on pRhBR171 vector showed higher activity than expression downstream of a native 1-deoxyxylulose-5-phosphate synthase gene (dxs) on pDA71 vector. Expression of the crtU in the β-carotene ketolase (crtO) knockout Rhodococcus host produced higher purity chlorobactene than expression in the wild-type Rhodococcus host. Growth of the engineered Rhodococcus strain in eight different media showed that nutrient broth yeast extract medium supplemented with fructose gave the highest total yield of chlorobactene. This medium was used for growing the engineered Rhodococcus strain in a 10-l fermentor, and ∼18 mg of chlorobactene was produced as the almost exclusive carotenoid by fermentation.     

The reaction center of green sulfur bacteria(1).

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.     

The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum.

Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum. The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000. Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase. In addition to apparent structural similarities, the catalytic properties of C. tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C. tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.