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.     

Genetic transfer by conjugation in the thermophilic green sulfur bacterium Chlorobium tepidum.

The broad-host-range IncQ group plasmids pDSK519 and pGSS33 were transferred by conjugation from Escherichia coli into the thermophilic green sulfur bacterium Chlorobium tepidum. C. tepidum exconjugants expressed the kanamycin and ampicillin-chloramphenicol resistances encoded by pDSK519 and pGSS33, respectively. Ampicillin resistance was a particularly good marker for selection in C. tepidum. Both pDSK519 and pGSS33 were stably maintained in C. tepidum at temperatures below 42 degrees C and could be transferred between C. tepidum and E. coli without modifications. Conjugation frequencies ranged from 10(-1) to 10(-4) exconjugants per donor cell, and frequencies of 10(-2) to 10(-3) were consistently obtained when ampicillin resistance was used as a selectable marker. Methods for growth of C. tepidum on agar, isolation of plating strains and antibiotic-resistant mutants of wild-type C. tepidum cells, and optimum conditions for conjugation were also investigated.     

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.     

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.     

Crystal structure of a RuBisCO-like protein from the green sulfur bacterium Chlorobium tepidum

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the incorporation of atmospheric CO(2) into ribulose 1,5-bisphosphate (RuBP). RuBisCOs are classified into four forms based on sequence similarity: forms I, II and III are bona fide RuBisCOs; form IV, also called the RuBisCO-like protein (RLP), lacks several of the substrate binding and catalytic residues and does not catalyze RuBP-dependent CO(2) fixation in vitro. To contribute to understanding the function of RLPs, we determined the crystal structure of the RLP from Chlorobium tepidum. The overall structure of the RLP is similar to the structures of the three other forms of RuBisCO; however, the active site is distinct from those of bona fide RuBisCOs and suggests that the RLP is possibly capable of catalyzing enolization but not carboxylation. Bioinformatic analysis of the protein functional linkages suggests that this RLP coevolved with enzymes of the bacteriochlorophyll biosynthesis pathway and may be involved in processes related to photosynthesis.     

Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.     

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     

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.     

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     

Membrane proteome of the green sulfur bacterium Chlorobium tepidum (syn. Chlorobaculum tepidum) analyzed by gel-based and gel-free methods

Chlorobium tepidum is a Gram-negative bacterium of the green sulfur phylum (Chlorobia). Chlorobia are obligate anaerobic photolithoautotrophs that are widely distributed in aquatic environments where anoxic layers containing reduced sulfur compounds are exposed to light. The envelope of C. tepidum is a complex organelle composed of the outer membrane, the periplasm–peptidoglycan layer, and the cytoplasmic membrane. In addition to the outer and plasma membranes, C. tepidum contains chlorosomes attached to the cytoplasmic side of the plasma membrane. Each cellular compartment has a unique set of proteins, called sub-proteome. An important aim of proteome analysis is to study the level of the expressed genes and their response to environmental changes. Membrane protein studies are of primary importance to understand how nutrients are transported inside the cell, how toxic molecules are exported, and the mechanisms of photosynthesis and energy metabolism.     

Identification of a thiosulfate utilization gene cluster from the green phototrophic bacterium Chlorobium limicola

Chlorobium is an autotrophic, green phototrophic bacterium which uses reduced sulfur compounds to fix carbon dioxide in the light. The pathways for the oxidation of sulfide, sulfur, and thiosulfate have not been characterized with certainty for any species of bacteria. However, soluble cytochrome c-551 and flavocytochrome c (FCSD) have previously been implicated in the oxidation of thiosulfate and sulfide on the basis of enzyme assays in Chlorobium. We have now made a number of observations relating to the oxidation of reduced sulfur compounds. (1) Western analysis shows that soluble cytochrome c-551 in Chlorobium limicola is regulated by thiosulfate, consistent with a role in the utilization of thiosulfate. (2) A membrane-bound flavocytochrome c-sulfide dehydrogenase (which is normally a soluble protein in other species) is constitutive and not regulated by sulfide as expected for an obligately autotrophic species dependent upon sulfide. (3) We have cloned the cytochrome c-551 gene from C. limicola and have found seven other genes, which are also presumably involved in sulfur metabolism and located near that for cytochrome c-551 (SoxA). These include genes for a flavocytochrome c flavoprotein homologue (SoxF2), a nucleotidase homologue (SoxB), four small proteins (including SoxX, SoxY, and SoxZ), and a thiol-disulfide interchange protein homologue (SoxW). (4) We have established that the constitutively expressed FCSD genes (soxEF1) are located elsewhere in the genome. (5) Through a database search, we have found that the eight thiosulfate utilization genes are clustered in the same order in the Chlorobium tepidum genome (www.tigr.org). Similar thiosulfate utilization gene clusters occur in at least six other bacterial species but may additionally include genes for rhodanese and sulfite dehydrogenase.     

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

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

A thermophilic green sulfur bacterium from New Zealand hot springs,Chlorobium tepidum sp. nov.

Thermophilic green sulfur bacteria of the genus Chlorobium were isolated from certain acidic high sulfide New Zealand hot springs. Cells were Gram-negative nonmotile rods of variable length and contained bacteriochlorophyll c and chlorosomes. Cultures of thermophilic chlorobia grew only under anaerobic, phototrophic conditions, either photoautotrophically or photoheterotrophically. The optimum growth temperature for the strains of thermophilic green sulfur bacteria isolated was 47–48°C with generation times of about 2 h being observed. The upper temperature limit for growth was about 52°C. Thiosulfate was a major electron donor for photoautotrophic growth while sulfide alone was only poorly used. N₂ fixation was observed at 48°C and cell suspensions readily reduced acetylene to ethylene. The G+C content of DNA from strains of thermophilic chlorobia was 56.5–58.2 mol% and the organisms positioned phylogenetically within the green sulfur bacterial branch of the domain Bacteria. The new phototrophs are described as a new species of the genus Chlorobium, Chlorobium tepidum.This paper is dedicated to Professor Norbert Pfennig on the occasion of his 65th birthday     

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.     

Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.     

Rubredoxin from the green sulfur bacterium Chlorobium tepidum functions as an electron acceptor for pyruvate ferredoxin oxidoreductase.

Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of pyruvate to acetyl-CoA and CO(2), exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the pyruvate synthase or CO(2) fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOR/pyruvate synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mM(-1) cm(-1) with an A(492)/A(280) ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be -87 mV, which is the most electronegative value reported for Rd from any source.     

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.     

A reconstituted light-harvesting complex from the green sulfur bacterium Chlorobium tepidum containing CsmA and bacteriochlorophyll a

Green sulfur bacteria possess two light-harvesting antenna systems, the chlorosome and the Fenna-Matthews-Olson (FMO) protein. In addition to self-aggregated bacteriochlorophyll (BChl) c, chlorosomes of Chlorobium tepidum contain a small amount of BChl a (ratio 100:1). The chlorosomal BChl a is associated with CsmA, a 6.2 kDa protein that accounts for more than 50% of the protein content of chlorosomes. This CsmA-BChl a complex is located in the chlorosome baseplate with the hydrophilic C-terminal part of CsmA in contact with the FMO protein. CsmA was purified from Chl. tepidum. Isolated chlorosomes were lyophilized and extracted with chloroform/methanol (1:1, v/v). The extract was further purified using gel filtration and reverse-phase HPLC and the purity of the preparation confirmed by SDS-PAGE. Mass spectrometric analysis showed an m/z of 6154.8, in agreement with the calculated mass of the csmA gene product after C-terminal processing. CD spectroscopy of the isolated protein showed that the main structural motif was an alpha-helix. We have reconstituted the isolated CsmA protein with BChl a in micelles of n-octyl beta-d-glucopyranoside. The resulting preparation reproduced the spectral characteristics of the CsmA-BChl a complex present in the chlorosome baseplate.     

Electron transfer kinetics in purified reaction centers from the green sulfur bacterium Chlorobium tepidum studied by multiple-flash excitation.

Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash-absorption spectroscopy in the near-UV, visible, and near-infrared regions. The decay kinetics of the photooxidized primary donor P840(+), together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840(+) was reduced by cytochrome c with a t(1/2) of approximately 65 micros after the first flash, 80% with a t(1/2) of approximately 100 micros after the second flash, and 23% with a t(1/2) of approximately 100 micros after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred. The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840. The data are well fitted assuming a standard free energy change DeltaG degrees of -53 meV for electron transfer from one cytochrome c to P840(+), DeltaG degrees being independent of the oxidation state of the other cytochrome c. These observations support a model with two monoheme cytochromes c which are symmetrically arranged around the reaction center core. From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center. However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 micros. No recombination reaction between the primary partners P840(+) and A(0)(-) could be detected under normal conditions. Such a recombination was observed (t(1/2) approximately 19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons. From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria.