Purple Sulfur Bacterium Chromatium Vinosum Research Articles

The role of high-potential iron protein and cytochrome c(8) as alternative electron donors to the reaction center of Chromatium vinosum.

Under anaerobic conditions, intact cells of the purple sulfur bacterium Chromatium vinosum exhibit rapid photooxidation of the two low-potential hemes of the c-type cytochrome associated with the reaction center, after exposure to two short light flashes separated by a dark interval. Reduction of the photooxidized low-potential hemes is very slow under these conditions. On subsequent flashes, rapid photooxidation of a high-potential reaction center heme occurs and is followed by its rereduction on the millisecond time scale. Cells maintained under aerobic conditions exhibit the millisecond time scale reduction of the photooxidized high-potential heme after each flash. Cells grown autotrophically in the presence of Na(2)S and Na(2)S(2)O(3) appear to use the soluble [4Fe-4S]-containing protein, HiPIP, as the only direct electron donor to the reaction center heme under aerobic conditions. In contrast, cells grown in the presence of organic compounds, but in the absence of Na(2)S and Na(2)S(2)O(3), appear to use a soluble c-type cytochrome (most likely cytochrome c(8)) as the only electron donor to the reaction center heme under aerobic conditions. Cells grown autotrophically, in the presence of Na(2)S and Na(2)S(2)O(3), have a slightly higher ratio of HiPIP to cytochrome c(8) and a ratio of Rieske iron-sulfur protein to reaction center that is approximately one-half that of cells grown in the absence of Na(2)S and Na(2)S(2)O(3) but in the presence of organic compounds.

Genes encoding light-harvesting and reaction center proteins from Chromatium vinosum

Sequencing of a 3.4 kb DNA fragment isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum and of PCR products has resulted in identification of the Chr. vinosum pufL, pufM, and pufC genes, reading from the 5′ to the 3′ direction, and coding, respectively, for the L, M and cytochrome c subunits of the reaction center of this bacterium. Other PCR products have been used to obtain complete sequences for the pufB and pufA genes, located immediately upstream from pufL and encoding the apoproteins of two Chr. vinosum light- harvesting proteins. The 3′-portion of the bchZ gene, a gene that codes for a protein involved in the biosynthesis of bacteriochlorophyll, has been located immediately upstream from pufB. A second pufB gene, pufB2, has been located downstream from pufC, as has the 5′-portion of a second pufA gene, pufA2. The location of a second set of pufB and pufA genes, encoding light- harvesting proteins, downstream from pufC has not previously been reported for any photosynthetic bacterium. Translation of the gene sequences encoding these Chr. vinosum light-harvesting proteins reveals both similarities to and differences from the amino acid sequences, obtained from direct sequencing of the apoproteins, previously reported for Chr. vinosum light-harvesting proteins. Translation of these gene sequences, and of those for pufL, pufM and pufC, revealed significant homology, at the amino acid level, to the corresponding peptides of photosynthetic purple non-sulfur bacteria.

Acclimation of the photosynthetic response of Chromatium vinosum to light-limiting conditions.

The photosynthetic response of the purple sulfur bacterium Chromatium vinosum DSM 185 to different degrees of illumination was analyzed. The microorganism was grown in continuous culture, and samples were taken from the effluent of the culture and incubated at different irradiances to determine the specific rate of sulfur oxidation as a measure of the photosynthetic activity of the organism. The activities obtained were plotted as a function of the specific rate of light uptake, and for each set of data a photosynthesis equation was fitted, which allowed the estimation of Pmax (photosynthetic capacity), qk (the threshold irradiance for light limitation), and m (maintenance coefficient). The results indicated that cells grown under light limitation are able to achieve higher photosynthetic activities than cells grown under light saturation. The photosynthetic capacity (Pmax) remained constant under all the conditions of illumination tested, while the maintenance expenses (m) were higher under light limitation. The parameter qk, on the contrary, decreased considerably at limiting irradiances.

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum.

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180(T)) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum.

The pet operon, encoding the prosthetic group-containing subunits of the cytochrome bc1 complex, of the purple sulfur bacterium Chromatium vinosum

The pet operon, encoding the prosthetic group-containing subunits of the cytochrome bc 1 complex of the purple sulfur bacterium Chromatium vinosum, has been cloned and sequenced. The 5′ to 3′ order of the C. vinosum genes is: petA, encoding the Rieske iron-sulfur protein; petB, encoding cytochrome b; and petC, encoding cytochrome c1. Cytochrome b is the best conserved subunit of the C. vinosum complex, when compared to the corresponding proteins from four photosynthetic purple non-sulfur bacteria (70 to 74% identity). Identities for the C. vinosum Rieske protein and those from purple non-sulfur bacteria range from 60 to 64%. The C-terminal region of the C. vinosum Rieske protein is quite similar to those of purple non-sulfur bacteria, while the N-terminal region is more closely related to mitochondrial Rieske proteins of organisms such as Neurospora crassa. Cytochrome c1 is the least well-conserved protein of the C. vinosum cytochrome bc1 complex, with identities ranging from 49 to 51% when compared to the corresponding proteins from purple non-sulfur bacteria. A well-conserved negatively-charged region of the cytochromes c1 of the purple non-sulfur bacteria, thought to be involved in binding the electron acceptor for the complex, cytochrome c2, is absent in C. vinosum cytochrome c1. A positive Southern hybridization using a probe constructed from the Rhodobacter sphaeroides fbcQ gene, which codes for a fourth subunit of the cytochrome bc1 complex in that bacterium, suggests the presence of a homologous gene in C. vinosum.

Cloning and sequencing of the gene encoding the high potential iron-sulfur protein (HiPIP) from the purple sulfur bacterium Chromatium vinosum

The gene encoding the high potential iron-sulfur protein (HiPIP) of Chromatium vinosum strain D (DSM 180 T) was cloned from an EcoRI- HindIII digest of genomic DNA. A nucleotide sequence of 648 bp length was determined which contained the coding region and putative promoter and termination sites. The gene codes for a 122 residue 12761 Da protein. The C-terminal 85 residues are those of the previously biochemically determined sequence, whereas the N-terminal 37 residues constitute a leader peptide which shows characteristics of the double arginine signal sequences of complex cofactor containing periplasmic proteins.

Molecular cloning and sequencing of cytochrome c′ from the phototrophic purple sulfur bacterium Chromatium vinosum

The gene for cytochrome c′ from Chromatium vinosum was cloned from a HindIII- SalI digest of genomic DNA. A 1.4 kbp fragment containing the gene was sequenced in both directions using the Sanger dideoxy method. The cytochrome c′ gene codes for a 154-residue peptide, of which the last 131 amino acids match the previously determined sequence of the protein. The remaining 23 residues represent a signal sequence that is cleaved from the polypeptide upon translocation to the periplasmic space. An additional open reading frame on the other strand of the fragment codes for a peptide that contains four regions that are homologous to corresponding regions of the cytochrome b-type subunit of several Ni-Fe hydrogenases.

Spectroscopic characterization of flavocytochrome c-552 from the photosynthetic purple sulfur bacterium Chromatium vinosum

The identities of the axial ligands to the two hemes of the flavocytochrome c-552 isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum have been investigated by visible / near-infrared absorption and magnetic circular dichroism (MCD) spectroscopies, with parallel electron paramagnetic resonance (EPR) studies. One of the hemes has histidine and methionine as axial ligands and has a local environment that is relatively insensitive to the composition of the bulk medium. The second heme, the local environment of which is sensitive to changes in the composition of the bulk medium, exists as a mixture of two forms, only one of which has histidine / methionine axial ligation. One the basis of its EPR characteristics, the other form most likely has histidine / lysine axial ligation. In aqueous solution near neutral pH, more than half of the second heme is present as the histidine / lysine form, while in 50:50 water / ethylene glycol the histidine / methionine form is the dominant one.

The ubiquinol:cytochrome [formula omitted] oxidoreductase of Chromatium vinosum

Chromatophore membranes isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum exhibit a quinol:cytochrome c 2( c) oxidoreductase activity that is sensitive to two specific inhibitors of cytochrome bc 1 complexes, antimycin A and myxothiazol. Digests of C. vinosum DNA hybridize to probes constructed from portions of the pet(fbc) operons that code for the cytochrome bc 1 complexes of the photosynthetic purple non-sulfur bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodospirillum rubrum. Despite the fact that it has not yet proven possible to isolate a detergent-solubilized, purified cytochrome bc 1 complex from C. vinosum, these new results, when combined with spectroscopic and kinetic data available from the literature, indicate that this purple sulfur bacterium contains a cytochrome bc 1 complex similar in structure to the well-characterized complexes found in purple non-sulfur bacteria. Equine cytochrome c and cytochromes c 2 isolated from the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodopseudomonas viridis all function as effective electron acceptors from the C. vinosum cytochrome bc 1 complex. In contrast, HiPIP (high-potential iron protein) isolated from two purple sulfur bacteria, C. vinosum and Chromatium tepidum, are reduced only at low rates by quinol in the presence of C. vinosum membranes and their reduction is not inhibited by either antimycin A or myxothiazol. These observations support the hypothesis that a protein structurally related to cytochrome c 2, rather than HiPIP, is the physiological electron acceptor for the C. vinosum cytochrome bc 1 complex. Although it has not yet proven possible to purify the putative C. vinosum cytochrome c 2 to homogeneity, we report here that digests of C. vinosum DNA hybridize with probes constructed from portions of the cycA genes coding for cytochromes c 2 from Rb. capsulatus, Rps. viridis and Rb. sphaeroides. These DNA hybridization results provide further support for the hypothesis that a cytochrome c 2-like protein is present in C. vinosum.

Sequence and expression of genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chromatium vinosum

A DNA fragment bearing genes for the large ( rbcL) and small ( rbcS) subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was cloned from the photosynthetic purple sulfur bacterium Chromatium vinosum. Enzymatically fully active RuBisCO was synthesized in Escherichia coli cells when the cloned DNA was placed downstream of tac promoter. Nucleotide (nt) sequences of rbcL-rbcS were more homologous to cyanobacterial counterparts than to those from Alcaligenes eutrophus or higher plants. However, the amino acid (aa) sequence in a domain responsible for CO 2 activation in the C. vinosum rbcL product resembled the corresponding aa sequence in higher plant RuBisCOs, but not in the cyanobacterial enzymes. Chemically determined aa sequences at the N terminals of both subunits of RuBisCO purified from C. vinosum were not identical to those deduced from the nt sequences, although they were completely the same as aa sequences deduced from rbcA-rbcB, another locus encoding RuBisCO in C. vinosum. Therefore, the rbcL-rbcS locus seems to be barely expressed under a standard condition for photoautotrophic growth. The homology of the nt sequences between rbcL-and rbcA was 82%, and that between rbcS and rbcB was 63 %, whereas the codon usages of these genes were basically identical. The rbcL-rbcS and rbcA-rbcB loci therefore must have evolved from a common ancestral set of genes after duplication, instead of lateral gene transfer.

Distinct properties of Escherichia coli products of plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase directed by two sets of genes from the photosynthetic bacterium Chromatium vinosum.

We have recently described the existence of two sets of genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase), rbcA-rbcB and rbcL-rbcS, in the photosynthetic purple sulfur bacterium Chromatium vinosum (Viale, A.M., Kobayashi, H., and Akazawa, T. (1989) J. Bacteriol. 171, 2391-2400). These genes were cloned in plasmid vectors, and their expression was studied in Escherichia coli. Expression of rbcA-rbcB in E. coli was obtained under the control of its own promoter. On the other hand, expression of rbcL-rbcS in this host was not observed unless these genes were cloned under the control of the tac promoter. Purified rbcA-rbcB and rbcL-rbcS products from E. coli consisted of large and small subunits in equimolar ratios. They also showed very close elution profiles to Rbu-P2 carboxylase isolated from C. vinosum in size-exclusion chromatography columns, thus suggesting hexadecameric (L8S8) structures. Vmax of Rbu-P2 carboxylase were very similar for both enzymes, but the Km values for CO2 and ribulose 1,5-bisphosphate showed some differences. Immunochemical and N-terminal amino acid sequence analyses of the large and small subunits encoded by rbcA-rbcB and rbcL-rbcS also differed, especially at the level of the small subunits. The comparisons described above as well as the analysis of C. vinosum crude extracts by anion-exchange chromatography indicated that Rbu-P2 carboxylase encoded by rbcA-rbcB was the only species detected in the photosynthetic bacterium.

Lysine and arginine transport in the photosynthetic bacterium Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum can take up both arginine and lysine in the light and, to a lesser extent, in the dark. Competitive inhibition experiments suggest the likely presence of two transport systems in this bacterium: One capable of transporting either lysine or arginine and a second capable of transporting arginine but not lysine. Uptake of both amino acids is electrogenic and appears to involve the cotransport of neither protons nor sodium ions. It is suggested that the transport occurs via an electrogenic uniport.

Spectroscopic and kinetic properties of an oxygen-binding heme protein from Chromatium vinosum.

Resonance Raman and electron paramagnetic resonance spectroscopy have been utilized to identify histidine as an axial heme ligand in a high spin, heme c-containing protein isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum. Resonance Raman spectroscopy has also been used to characterize the CO adduct of the C. vinosum hemoprotein. Resonance Raman spectra of the heme site obtained within 10 ns of CO photolysis from the ferrous hemoprotein are virtually identical to those of the unligated protein, indicating that there is little or no rearrangement of the heme pocket in response to ligand photolysis. The equilibrium constant for CO binding to the ferrous hemeprotein was measured to be 1.7 X 10(-5) M-1 and the CO association rate constant determined to be 5.4 X 10(3) M-1 S-1. The quantum efficiency for photodissociation of the hemoprotein X CO complex was greater than or equal to 0.9.

Complex formation between Chlorobium limicola f. thiosulfatophilumc-type cytochromes

It has been possible to demonstrate, using affinity chromatography, that Chlorobium flavocytochrome c-553 forms an electrostatically stabilized complex with Chlorobium cytochrome c-555. The binding site for cytochrome c-555 appears to be located on the heme-containing subunit of flavocytochrome c-553. This complex appears to be involved in the flavocytochrome c-553-catalyzed transfer of electrons from sulfide to cytochrome c-555. Complex formation has also been demonstrated between Chlorobium cytochromes c-555 and c-551, two components involved in the oxidation of thiosulfate by this green sulfur bacterium. Affinity chromatography data also suggest the possibility that the cytochrome binding sites on the Chlorobium flavocytochrome c-553 and on flavocytochrome c-552 from the purple sulfur bacterium Chromatium vinosum may be similar.

Evidence for two calcium transport systems in the photosynthetic bacterium Chromatium vinosum

Light-dependent Ca 2+ efflux via the Ca 2+/H + antiport in the photosynthetic purple sulfur bacterium Chromatium vinosum was inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca 2+ uptake by C. vinosum in the dark nor any effect on the light-dependent efflux of either Na + or Tl + catalyzed, respectively, by the C. vinosum Na +/H + or K +/H + antiports. Ruthenium red and LaCl 3, neither of which inhibited light-dependent Ca 2+ efflux in C. vinosum, markedly inhibited Ca 2+ uptake in the dark by C. vinosum cells. Ruthenium red had no effect on the uptake of either Na +or the K + analog T1 + by C. vinosum cells in the dark. These results have been interpreted in terms of two separate Ca 2+ transport systems in C. vinosum: (i) a phenothiazine-sensitive and ruthenium red, La 3+-insensitive Ca 2+/H + antiport responsible for Ca 2+ efflux in the light; and (ii) a ruthenium red and La 3+-sensitive but phenothiazine-insensitive Ca 2+ uptake system.

Interaction of Chromatium vinosum flavocytochrome c-552 with cytochromes c studied by affinity chromatography

Flavocytochrome c-552, isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum, after linkage to an Affigel 10-affinity matrix, will bind equine and yeast mitochondrial cytochromes c, C. vinosum cytochrome c-550 and cytochrome c 2 from the photosynthetic purple non-sulfur bacterium Rhodo-pseudomonas viridis. Similarly, an equine cytochrome c-Sepharose 4B affinity column will bind the C. vinosum flavocytochrome c-552 and its separated heme-containing subunit. These results and the correlation between the effect of ionic strength on binding of the flavocytochrome c-552 to cytochrome c and on the sulfide:cytochrome c oxidoreductase activity catalyzed by C. vinosum flavocytochrome c-552 support the idea of the involvement of an electrostatic complex between the two proteins during sulfide oxidation.

The pathway of cyclic electron transport in chromatophores of Chromatium vinosum. Evidence for a Q-cycle mechanism

Chromatophores of the purple sulfur bacterium Chromatium vinosum were shown to contain a cytochrome similar to cytochrome c 1 and two b cytochromes. Cytochrome b can be accumulated in the reduced form upon illumination at an ambient redox potential of +415 mV in the presence of the electron transport inhibitors antimycin A or HOQNO. The reductions of cytochrome b, of the high-potential cytochrome c 555 and of the primary electron donor P-870 are all inhibited by myxothiazol. Dark-adapted C. vinosum chromatophores show little cytochrome b reduction on the first flash. Considerable cytochrome b reduction (1 cytochrome b:8 P-870 present) is observed on the second flash. This observation and the 1:1 stoichiometry observed between cytochrome b reduction and P-870 + reduction after the second flash support a Q-cycle model for cyclic electron flow in C. vinosum.

Inhibition of photosynthetic sulfide oxidation by organic cations

Photosynthetic sulfide oxidation by the purple sulfur bacterium Chromatium vinosum is strongly inhibited by the organic cations benzyl viologen (BV 2+) and tetraphenylphosphonium (TPP +) at micromolar concentrations. Both are much more inhibitory at pH 8 than at pH 7. Inhibition probably results from uptake of benzyl viologen and tetraphenylphosphonium in response to an electrical potential gradient across the plasma membrane which increases in magnitude with increasing pH. Although both compounds appear to have the same mode of action, the biochemical mechanism of their inhibition remains to be determined.

L-Aspartate transport in the photosynthetic bacterium Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum appears to contain two active transport systems for l-aspartate. The higher affinity system ( S 0.5 = 60 μM) appears to be an electrogenic aspartate/H + symport and the lower affinity system ( S 0.5 = 220 μM appears to involve an aspartate/Na + symport. In addition to a possible role in providing the driving force for aspartate uptake, transmembrane Na + gradients may also have allosteric effects on aspartate transport in C. vinosum.

Isolation and characterization of two soluble heme c-containing proteins from Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum has been shown to possess two previously undetected heme c-containing, soluble proteins. One is an acidic, c-type cytochrome with a molecular weight of 12 300 and an oxidation-reduction midpoint potential (at pH 8.0) of −82 mV. The other protein is a basic protein with a molecular weight of 11 900 and an oxidation-reduction midpoint potential (at pH 8.0) of −110 mV. The basic protein, in both oxidized and reduced forms, has optical spectra similar to those of myoglobin and the oxidized C. vinosum protein exhibits a high-spin heme EPR spectrum similar to that of metmyoglobin. Furthermore, the basic C. vinosum protein binds CO and O 2. The spectra of the CO and O 2 complexes show significant similarities with the respective myoglobin complexes. Possible functions for an O 2-binding protein in C. vinosum are discussed.