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PubMed2005

Expression, purification, crystallization and preliminary X-ray analysis of sepiapterin reductase from Chlorobium tepidum.

Sepiapterin reductase from Chlorobium tepidum (CT-SR) produces L-threo-tetrahydrobiopterin, an isomer of tetrahydrobiopterin, in the last step of de novo synthesis initiating from GTP. Native CT-SR and a selenomethionine (SeMet) derivative of CT-SR have been crystallized by the hanging-drop vapour-diffusion method using PEG 400 as precipitant. CT-SR crystals belong to space group R32, with unit-cell parameters a = b = 201.142, c = 210.184 A, and contain four molecules in the asymmetric unit. Diffraction data were collected to 2.1 A resolution using synchrotron radiation. The structure of CT-SR has been determined using MAD phasing. There is one CT-SR tetramer in the asymmetric unit formed by two closely interacting CT-SR dimers. The solvent content is calculated to be about 67.2%.

Alcohol Oxidoreductases↗
PubMed2006

Crystallization, preliminary crystallographic analysis and phasing of the thiosulfate-binding protein SoxY from Chlorobium limicola f. thiosulfatophilum.

The 22 kDa SoxYZ protein complex from the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum is a central player in the sulfur-oxidizing (Sox) enzyme system of the organism by activating thiosulfate for oxidation by SoxXA and SoxB. It has been proposed that SoxYZ exists as a heterodimer or heterotetramer, but the properties and role of the individual components of the complex thus far remain unknown. Here, the heterologous expression, purification, and the crystallization of stable tetrameric SoxY are reported. Crystals of SoxY diffract to 2.15 A resolution and belong to space group C222(1), with unit-cell parameters a = 41.22, b = 120.11, c = 95.30 A. MIRAS data from Pt(2+)- and Hg(2+)-derivatized SoxY crystals resulted in an interpretable electron-density map at 3 A resolution after density modification.

Bacterial Proteins↗
PubMed2006

Both subunits of ATP-citrate lyase from Chlorobium tepidum contribute to catalytic activity.

ATP-citrate lyase (ACL) is an essential enzyme of the reductive tricarboxylic acid (RTCA) pathway of CO(2) assimilation. The RTCA pathway occurs in several groups of autotrophic prokaryotes, including the green sulfur bacteria. ACL catalyzes the coenzyme A (CoA)-dependent and MgATP-dependent cleavage of citrate into oxaloacetate and acetyl-CoA, representing a key step in the RTCA pathway. To characterize this enzyme from the green sulfur bacterium Chlorobium tepidum and determine the role of its two distinct polypeptide chains, recombinant holo-ACL as well as its two individual subunit polypeptides were synthesized in Escherichia coli. The recombinant holoenzyme, prepared from coexpressed large and small ACL genes, and the individual large and small subunit polypeptides, prepared from singly expressed genes, were all purified to homogeneity to high yield. Purified recombinant holo-ACL was isolated at high specific activity, and its k(cat) was comparable to that of previously prepared native C. tepidum ACL. Moreover, the purified recombinant large and small subunit polypeptides were able to reconstitute the holo-ACL in vitro, with activity levels approaching that of recombinant holo-ACL prepared from coexpressed genes. Stoichiometric amounts of each subunit protein were required to maximize the activity and form the most stable structure of reconstituted holo-ACL. These results suggested that this reconstitution system could be used to discern the catalytic role of specific amino acid residues on each subunit. Reconstitution and mutagenesis studies together indicated that residues of each subunit contributed to different aspects of the catalytic mechanism, suggesting that both subunit proteins contribute to the active site of C. tepidum ACL.

ATP Citrate (pro-S)-Lyase↗
PubMed2006

Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum.

The green sulfur bacterium Chlorobium tepidum produces chlorobactene as its primary carotenoid. Small amounts of chlorobactene are hydroxylated by the enzyme CrtC and then glucosylated and acylated to produce chlorobactene glucoside laurate. The genes encoding the enzymes responsible for these modifications of chlorobactene, CT1987, and CT0967, have been identified by comparative genomics, and these genes were insertionally inactivated in C. tepidum to verify their predicted function. The gene encoding chlorobactene glucosyltransferase (CT1987) has been named cruC, and the gene encoding chlorobactene lauroyltransferase (CT0967) has been named cruD. Homologs of these genes are found in the genomes of all sequenced green sulfur bacteria and filamentous anoxygenic phototrophs as well as in the genomes of several nonphotosynthetic bacteria that produce similarly modified carotenoids. The other bacteria in which these genes are found are not closely related to green sulfur bacteria or to one another. This suggests that the ability to synthesize modified carotenoids has been a frequently transferred trait.

Acyltransferases↗
PubMed2004

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.

Alkyl and Aryl Transferases↗
PubMed2004

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.

Bacterial Proteins↗
PubMed2005

Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit.

delta-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His(6) tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His(6)-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNA(Glu) and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of beta-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.

Aldehyde Oxidoreductases↗
PubMed2004

Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation.

Inactivation of a chromosomal gene is a useful approach to study the function of the gene in question and can be used to produce a desired phenotype in the organism. This chapter describes how to generate such mutants of the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum by natural transformation with synthetic DNA constructs. Two alternative methods to generate the DNA constructs, both performed entirely in vitro and based on the polymerase chain reaction (PCR), are also presented. These methods are ligation of DNA fragments with T4 DNA ligase, and megaprimer PCR.

Chlorobium↗
PubMed2006

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.

Bacteriochlorophylls↗
PubMed2004

The role of carotenoids in the photoadaptation of the brown-colored sulfur bacterium Chlorobium phaeobacteroides.

The brown-colored sulfur bacterium Chlorobium (Cb.) phaeobacteroides 1549 (new name, Chlorobaculum limnaeum 1549) contains many kinds of carotenoids as well as bacteriochlorophyll (BChl) e. These carotenoids were identified with C18-high-performance liquid chromatography, absorption, mass and proton nuclear magnetic resonance spectroscopies and were divided into two groups: the first is carotenoid with one or two phi-end groups such as isorenieratene and beta-isorenieratene and the second is carotenoid with one or two beta-end groups such as p-zeacarotene, beta-carotene and 7,8-dihydro-beta-carotene. The latter 7,8-dihydro-beta-carotene was found to be a novel carotenoid in nature. OH-gamma-Carotene glucoside laurate and OH-chlorobactene glucoside laurate were also found as minor components. The distribution of BChl e homologs in Cb. phaeobacteroides cultivated under various light intensities did not change, but the carotenoid to BChl e ratio changed markedly: carotenoid with the phi-end group maintained the same ratio to BChl e, whereas that with the beta-end group increased with increasing light intensity. The cells cultured under low-light intensity contained more phi-end carotenoids than beta-end. In Cb. phaeobacteroides the wavelength of the Qy band of BChl e aggregates did not change. We suggested that Cb. phaeobacteroides photoadapts to light intensity by changing the carotenoid composition.

Bacteriochlorophylls↗
PubMed2003

Presence of exclusively bacteriochlorophyll-c containing substrain in the culture of green sulfur photosynthetic bacterium Chlorobium vibrioforme strain NCIB 8327 producing bacteriochlorophyll-d.

The light-dependent composition change of light harvesting bacteriochlorophyll(BChl)s in the present culture of a green sulfur photosynthetic bacterium Chlorobium (Chl.) vibrioforme f. sp. thiosulfatophilum strain NCIB 8327 was investigated by visible absorption spectroscopy and HPLC analyses. When the culture was repeatedly grown in liquid media under a low light condition, both the Soret and Qy absorption bands of the in vivo spectrum were shifted to longer wavelengths. Analysis of the extracted pigments by HPLC revealed that the ratio of the amount of BChl-c to that of BChl-d molecules gradually increased during repeated cultivation. In contrast, when the culture grown under a low light intensity was transferred to a high light condition and continued to be grown, the absorption bands were shifted to shorter wavelengths and the ratio of BChls-c/d decreased finally to the almost original value. Colonies were prepared on solid agar media from the liquid culture containing both BChls-c and d, which was grown under a low light intensity. Each colony obtained was found to contain either BChl-c or d, but not both of them. Two types of cells isolated in this study were derived from the same clone, judged from their genetic analyses. The variation of pigment composition in our liquid culture observed here could be ascribed to the difference of growth rates between two substrains containing BChl-c and BChl-d, respectively, depending on light conditions.

Bacterial Proteins↗
PubMed1997

Crystal structure of the bacteriochlorophyll a protein from Chlorobium tepidum.

The bacteriochlorophyll (BChl) a protein from Chlorobium tepidum, which participates in energy transfer in green photosynthetic bacteria, has been crystallized using the sitting drop method of vapor diffusion. X-ray diffraction data collected from these crystals indicate that the crystals belong to the cubic space group P4132 with cell dimensions of a=b=c=169.5 A. A native X-ray diffraction data set has been collected to a resolution of 2.2 A. The initial solution was determined by using the molecular replacement method using the structure of the previously solved BChl a protein from Prosthecochloris aestuarii. A unique rotation and translation solution was obtained for two monomers in the asymmetric unit giving a pseudo-body centered packing. After rebuilding and refinement the model yields an R factor of 19.0%, a free R-factor of 28.3%, and good geometry with root-mean-square deviations of 0.013 A and 2.1 degrees for the bond lengths and angles, respectively. The structure of the BChl a protein from C. tepidum consists of three identical subunits related by a 3-fold axis of crystallographic symmetry. In each subunit the polypeptide backbone forms large beta-sheets and encloses a central core of seven BChl a molecules. The distances between neighboring bacteriochlorin systems within a subunit range between 4 A to 11 A and that between two bacteriochlorins from different subunits is more than 20 A. The overall structure is comparable with that of P. aestuarii but significant differences are observed for the individual bacteriochlorophyll structures. The surface of the trimer has a hydrophobic region that is modeled as the complex being a peripheral membrane protein partially embedded in the membrane. A general model is presented for the membrane organization with two of the bacteriochlorophyll structures in the membrane and transferring energy to the reaction center complex. In this model these two bacteriochlorophyll structures serve a similar role to the cofactors of integral membrane light-harvesting complexes although the protein structure surrounding the cofactors is significantly different for the BChl a protein compared with the integral membrane complexes.

Bacterial Proteins↗
PubMed1999

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.

Bacterial Proteins↗
PubMed1991

Structure and expression of the Chlorobium vibrioforme hemA gene.

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Oxidoreductases↗
PubMed1979

Biosynthesis of aryl carotenoids: inhibitor studies of chlorobactene biosynthesis in Chlorobium limicola f. thiosulfatophilum.

The biosynthesis of the aryl carotenoid, chlorobactene, was examined in the green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum. Nicotine, which was used to inhibit carotenoid cyclization, caused the accumulation of the acyclic carotenoid, lycopene. Cells reincubated in fresh medium, after removal of nicotine, synthesized chlorobactene more readily from newly synthesized lycopene rather than from the pool of lycopene accumulated during nicotine inhibition. When the cells were reincubated in the presence of diphenylamine, which inhibited de novo carotenogenesis, a portion of the lycopene which had accumulated during nicotine inhibition was converted into chlorobactene. There was no evidence that neurosporene, rather than lycopene, was the precyclization intermediate. The involvement of gamma-carotene as the cyclic precursor of chlorobactene also was shown. The pathway for chlorobactene biosynthesis is discussed in terms of a possible arrangement of the enzymes involved in carotenoid biosynthesis.

Carotenoids↗
PubMed1975

Photoassimilation of acetate and metabolism of carbohydrate in Chlorobium thiosulfatophilum.

1. Washed cell suspensions of Chlorobium thiosulfatophilium form large amounts of a polyglucose in the light. Addition of acetate to the cells increases the formation of polysaccharide considerable. During incubation in the dark, polysaccharide decreases with time, and organic acids such as succinic and propionic acid are excreted into the medium. 2. Glucose isolated from cells which had photoassimilated 1-, 2-, and U-14C-acetate had a specific activity which lay between 1 and 2 times that of the acetate substrates. 3. To analyse the distribution of radioactivity in the glucose units formed during photoassimilation of 14C-acetate, 2 microbial degradations, with bakers' yeast and Zymomonas mobilis respectively, were used. The results show that acetate gives rise to carbon atoms 1 plus 2 and 5 plus 6 of glucose, wheras carbon atomes 3 plus 4 are not labelled. Further, the results indicate that glucose is not formed via the reductive pentose phosphate cycle when acetate is present.

Acetates↗
PubMed1977

Synthesis, storage and degradation of polyglucose in Chlorobium thiosulfatophilum.

Cultures of Chlorobium thiosulfatophilum form polyglucose during growth. The polyglucose is laid down within the cells as rosette-like granules, which are made up from smaller grains. The size of each granule appears to be limited to less than 30 nm, since an increase in polyglucose content leads to more granules being formed rather than an increase in granule size. The polyglucose in washed cells is fermented in the dark to acetate, propionate, caproate and succinate, of which acetate by far comprises the largest fraction (68%). During incubation of washed cells without hydrogen donor, the level of polyglucose decreases regardless of whether the cells are incubated in the dark or in the light. Since the products formed from polyglucose under the two different conditions are not the same, it is suggested that polyglucose in the dark serves as an energy source, whereas when in the light the role of polyglucose is mainly to provide the cell with reducing power.

Darkness↗
PubMed1994

Redox effects on the bacteriochlorophyll a-containing Fenna-Matthews-Olson protein from Chlorobium tepidum.

The BChl a-containing Fenna-Matthews-Olson (FMO) protein from the green sulfur bacterium Chlorobium tepidum was purified and characterized. Fluorescence spectra indicate that efficient excited state quenching occurs at neutral or oxidizing redox potentials. The major fluorescence lifetime at room temperature is approximately 60 ps in samples that are in neutral or oxidizing conditions, and approximately 2 ns in samples where the strong reductant sodium dithionite has been added. A similar change is observed in pump-probe picosecond absorbance difference experiments, where the long life time component increases after dithionite addition. A 16 Gauss wide EPR signal with g factor = 2.005 is observed in samples without dithionite. This signal largely disappears upon addition of dithionite. Dithionite induces large reversible changes in the 77 K absorbance spectra of the purified FMO protein and in whole cells. These results indicate that the FMO protein contains redox active groups, which may be involved in the regulation of energy transfer. Room temperature circular dichroism and low temperature absorption spectra show that dithionite also induces conformational or structural changes of the FMO protein complex.

Bacterial Proteins↗