The lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum

The cell wall lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum was obtained by the phenol-chloroform-petroleum ether and the hot phenol-water methods, respectively. It contained mannose, glucose, galacturonic acid, glucosamine, glycine, and small amounts of rhamnose, galactose and glucuronic acid. In addition to d-glycero-d-mannoheptose, the corespecific constituents 2-keto-3-deoxyoctonate and l-glycero-d-mannoheptose were found. Polyacrylamide gel-electrophoresis in the presence of sodium deoxycholate gave no indication for the presence of O-specific repeating units. Degradation of the lipopolysaccharide required 10% acetic acid (100° C, 2 h). The lipid A moiety contained the total of glucosamine of the lipopolysaccharide as well as small amounts of 2,3-diamino-2,3-dideoxy-glucose. It was phosphate-free. The fatty acid spectrum comprised 3-OH-14:0, 3-OH-16:0, and iso-3-OH-18:0 besides little 12:0, 14:0 and 16:0. Hydroxylaminolysis and sodium methylate treatment revealed all of the three hydroxy fatty acids to be amidebound.Abbreviations DOC sodium deoxycholate - PAGE polyacrylamide gel-electrophoresis     

An isolated reaction center complex from the green sulfur bacterium Chlorobium vibrioforme can photoreduce ferredoxin at high rates

Chlorosome-depleted membranes and a reaction center complex with well-defined subunit composition were prepared from the green sulfur bacterium Chlorobium vibrioforme under anaerobic conditions. The reaction center complex contains a 15-kDa polypeptide with the N-terminal amino acid sequence MEPQLSRPETASNQVR/. This sequence is nearly identical to the N-terminus of the pscD gene product from Chlorobium limicola (Hager-Braun et al. (1995) Biochemistry 34: 9617–9624). In the presence of ferredoxin and ferredoxin:NADP⁺ oxidoreductase, the membranes and the isolated reaction center complex photoreduced NADP⁺ at rates of 333 and 110 mol (mg bacteriochlorophyll a)^–1 h^–1, respectively. This shows that the isolated reaction center complex contains all the components essential for steady state electron transport. Midpoint potentials at pH 7.0 of 160 mV for cytochrome c ₅₅₁ and of 245 mV for P840 were determined by redox titration. Antibodies against cytochrome c ₅₅₁ inhibit NADP⁺ reduction while antibodies against the bacteriochlorophyll a-binding Fenna-Matthews-Olson protein do not.Abbreviations FMO protein Fenna-Matthews-Olson protein - TMBZ 3,3,5,5-tetramethylbenzidine     

Cytochromes of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Purification,characterization and sulfur metabolism

Three cytochromes of the thiosulfate-utilizing green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum were highly purified by ion exchange column chromatography and ammonium sulfate fractionation. All three cytochromes are located in the soluble fraction. Cytochrome c-551 (highest purity index obtained: A₂₈₀/A₄₁₆=0.39) shows maxima at 551 nm (-band), 521 nm (-band), and 416 nm (-band) for the reduced form. This cytochrome is an acidic protein with a molecular weight of 32,000, a redox potential of 150 mV, and an isoelectric point at pH 6.0. Cytochrome c-553 (highest purity index obtained: A₂₈₀/A₄₁₇=0.8) is also an acidic protein with maxima at 553,5 nm, 523,5 nm and 417 nm for the reduced form, a molecular weight of 63,000, a redox potential of 90 mV, an isoelectric point at pH 6.3, and it contains FAD as flavin component. It is autoxidizable and participates in sulfide oxidation, but cannot catalyze the reverse reaction. The cytochrome c-555 (highest purity index obtained: A₂₈₀/A₄₁₈=0.16) is a small basic protein with maxima at 555 nm, 523 nm and 418 nm (reduced form), a molecular weight of 12,500, an isoelectric point between pH 10 and 10.5, and a redox potential of 155 mV. The ratio of the cytochrome contents to each other is constant and does not change when the organism has only thiosulfate or sulfide as the main electron donor in the medium.The soluble fraction further contains the non-heme ironcontaining proteins rubredoxin and ferredoxin. The anaerobic sulfide oxidation in a growing culture of Chlorobium vibrioforme f. thiosulfatophilum is accompanied by a rapid formation of thiosulfate, which is only utilized when sulfide is no longer available, while the elemental sulfur concentration increases constantly until thiosulfate is consumed.Non-common abbreviations C Chlorobium - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein     

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

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

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.     

Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Four ferredoxin (Fd) fractions, namely, FdA-D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A(385)/A(280) ratios of the purified Fds were 0.76-0.80. Analysis of the N-terminal amino acid sequences of these Fds (15-25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A(385) values of these Fds were unchanged when they were stored for a month at -80 degrees C under aerobic conditions and decreased by 10-15% when they were stored for 6 days at 4 degrees C under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP(+) reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP(+) at room temperature with the following K(m) and V(max) (in micromol NADP(+) micromol BChl a(-1) h(-1)): FdA, 2.0 microm and 258; FdB, 0.49 microM and 304; FdC, 1.13 microM and 226; FdD, 0.5 microM and 242; spinach Fd, 0.54 microM and 183. The V(max) value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria.     

Time-resolved step-scan FTIR investigation on the primary donor of the reaction center from the green sulfur bacterium Chlorobium tepidum

The vibrational properties of the primary donor P₈₄₀ in the reaction center (RC) of the green sulfur bacterium Chlorobium tepidum and its interactions with the surrounding protein environment have been investigated by Fourier transform infrared (FTIR) difference spectroscopy at cryogenic temperatures. By using the step-scan technique with a time resolution of 5 μs on RCs that had been depleted of the iron–sulfur electron acceptors, the formation and decay of the triplet state ³P₈₄₀ have been followed in infrared for the first time. The ³P₈₄₀/P₈₄₀ FTIR difference spectrum is compared to the P₈₄₀ ⁺/P₈₄₀ FTIR difference spectrum measured under identical conditions on untreated RCs and recorded with the same step-scan set-up. The latter P₈₄₀ ⁺/P₈₄₀ difference spectrum is essentially the same as those measured under steady-state conditions using the more conventional continuous illumination method. Comparison of the ³P₈₄₀/P₈₄₀ and P₈₄₀ ⁺/P₈₄₀ spectra provides unambiguous assignment of the vibration of the 9-keto C=O group(s) of P₈₄₀ at 1684 cm⁻¹ as the only common negative band in the two spectra. This frequency corresponds to carbonyl group(s) free from hydrogen bonding interactions. The obtained results are discussed in the framework of the structure and photochemistry of the primary donor P₈₄₀. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 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.     

Iron-sulfur centers in the photosynthetic reaction center complex fromChlorobium vibrioforme. Differences from and similarities to the iron-sulfur centers in Photosystem I

The photosynthetic reaction center complex from the green sulfur bacteriumChlorobium vibrioforme has been isolated under anaerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 30, 18, 15, and 9 kDa. The 80- and 18-kDa polypeptides are identified as the reaction center polypeptide and the secondary donor cytochromec ₅₅₁ encoded by thepscA andpscC genes, respectively. N-terminal amino acid sequences identify the 40-kDa polypeptide as the bacteriochlorophylla-protein of the baseplate (the Fenna-Matthews-Olson protein) and the 30-kDa polypeptide as the putative 2[4Fe-4S] protein encoded bypscB. Electron paramagnetic resonance (EPR) analysis shows the presence of an iron-sulfur cluster which is irreversibly photoreduced at 9K. Photoaccumulation at higher temperature shows the presence of an additional photoreduced cluster. The EPR spectra of the two iron-sulfur clusters resemble those of F_A and F_B of Photosystem I, but also show significantly differentg-values, lineshapes, and temperature and power dependencies. We suggest that the two centers are designated Center I (with calculatedg-values of 2.085, 1.898, 1.841), and Center II (with calculatedg-values of 2.083, 1.941, 1.878). The data suggest that Centers I and II are bound to thepscB polypeptide.     

Characterization of an improved reaction center preparation from the photosynthetic green sulfur bacterium Chlorobium containing the FeS centers FA and FB and a bound cytochrome subunit.

A photosynthetic reaction center complex was prepared from the green sulfur bacterium Chlorobium by solubilization of chlorosome-depleted membranes with lauryl maltoside, followed by anion-exchange chromatography and molecular sieve chromatography. The purified complex was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, optical spectroscopy, and EPR spectroscopy. The major bands migrated at apparent molecular masses of 50, 42, and 32 kDa (heme-staining) and additional weaker bands at 22, 15, and 12 kDa. The isolated reaction center complex contained about 40 bacteriochlorophyll alpha molecules per primary electron donor, P840, assayed by photooxidation. It was competent in stable low-temperature photoreduction of the FeS centers FA and FB. The spectra of these acceptors and their low-temperature photochemistry in the purified complex were the same as found in intact Chlorobium membranes and similar to what had been described for photosystem I from plants. Membrane-bound cytochrome c553 copurified with the reaction center complex. A ratio of about four hemes per P840 was determined. This result indicates that cytochrome c553 that is closely associated with the reaction center is a tetraheme cytochrome, as described for some purple bacteria.     

15-Cis-carotenoids found in the reaction center of a green sulfur bacterium Chlorobium tepidum and in the Photosystem I reaction center of a cyanobacterium Synechococcus vulcanus

Carotenoids were extracted, at 4 °C in complete darkness and under nitrogen atmosphere, from the reaction center (RC) of a green-sulfur bacterium and the Photosystem (PS) I RC of a cyanobacterium; each extract was subjected to high-performance liquid chromatography (HPLC) using an apparatus equipped with a two-dimensional diode-array detector in order to spectroscopically identify cis–trans carotenoids while performing HPLC analysis. In the extract from the RC of Chlorobium tepidum, 15-cis and all-trans--carotenes as well as 13-cis-, 15-cis- and all- trans-chlorobactenes (in the order of elution) were identified, whereas in the extract from the PS I RC of Synechococcus vulcanus, 15-cis-, all-trans- and 9-cis--carotenes were found. Thus, the universal presence of 15- cis carotenoids in the 'iron sulfur-type' RCs has been shown in addition to the previous cases of the 'quinone-type' RCs.     

Highly efficient integration of foreign DNA into the genome of the green sulfur bacterium,Chlorobium vibrioforme by homologous recombination

Highly efficient and reproducible transformation ofChlorobium vibrioforme with plasmid DNA has been achieved by electroporation. Specific parameters have been optimized for the electrotransformation procedure. The method was developed using a construct containing a full copy of thepscC gene encoding the cytochromec ₅₅₁ subunit of the photosynthetic reaction center complex and theaadA gene encoding streptomycin resistance as selectable marker. Southern blotting analysis showed that the tested colonies were true transformants with the plasmid integrated into the genome by single homologous recombination. No transformants were obtained using the vector without thepscC gene showing that this vector does not replicate inC. vibrioforme. Thus transformation is possible only by homologous recombination. When using constructs designed to inactivate thepscC gene by insertion no transformants were obtained, indicating that the gene is indispensable for growth. The vector pVS2 carrying genes for erythromycin and chloramphenicol resistance was shown to replicate inC. vibrioforme. The two transformations shown here, provide an important genetical tool in the further analysis of structure and function of the photosynthetic apparatus in green sulfur bacteria.     

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.     

Kinetics of electron transfer between soluble cytochrome c-554 and purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Soluble cytochrome c-554 (M _r 10 kDa) is purified from the green sulfur bacterium Chlorobium tepidum. Its midpoint redox potential is determined to be +148 mV from redox titration at pH 7.0. The kinetics of cytochrome c-554 oxidation by a purified reaction center complex from the same organism were studied by flash absorption spectroscopy at room temperature, and the results indicate that the reaction partner of cytochrome c-554 is cytochrome c-551 bound to the reaction center rather than the primary donor P840. The second-order rate constant for the electron donation from cytochrome c-554 to cytochrome c-551 was estimated to be 1.7×10⁷ M^–1 s^–1. The reaction rate was not significantly influenced by the ionic strength of the reaction medium.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

Photoreduction and reoxidation of the three iron-sulfur clusters of reaction centers of green sulfur bacteria

Iron-sulfur clusters are the terminal electron acceptors of the photosynthetic reaction centers of green sulfur bacteria and photosystem I. We have studied electron-transfer reactions involving these clusters in the green sulfur bacterium Chlorobium tepidum, using flash-absorption spectroscopic measurements. We show for the first time that three different clusters, named F(X), F(1), and F(2), can be photoreduced at room temperature during a series of consecutive flashes. The rates of electron escape to exogenous acceptors depend strongly upon the number of reduced clusters. When two or three clusters are reduced, the escape is biphasic, with the fastest phase being 12-14-fold faster than the slowest phase, which is similar to that observed after single reduction. This is explained by assuming that escape involves mostly the second reducible cluster. Evidence is thus provided for a functional asymmetry between the two terminal acceptors F(1) and F(2). From multiple-flash experiments, it was possible to derive the intrinsic recombination rates between P840(+) and reduced iron-sulfur clusters: values of 7, 14, and 59 s(-1) were found after one, two and three electron reduction of the clusters, respectively. The implications of our results for the relative redox potentials of the three clusters are discussed.