Chromatium Vinosum Strain Research Articles

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.

Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductase-deficient mutants of Chromatium vinosum.

In purple sulphur bacteria of the family Chromatiaceae sulphite oxidation via intermediary formation of adenylylsulphate is an enzymologically well characterized process. In contrast, the role of an alternative direct oxidation pathway via the enzyme sulphite:acceptor oxidoreductase has not been resolved. This paper reports the cloning of the genes encoding the adenylylsulphate-forming enzyme adenosine-5'-phosphosulphate (APS) reductase from Chromatium vinosum strain D (DSM 180'), a representative of the purple sulphur bacteria, and the construction of mutations in these genes by insertion of a kanamycin omega cartridge. The mutated genes were transferred to C. vinosum on suicide vectors of the pSUP series by conjugation and delivered to the chromosome by double homologous recombination. Southern hybridization and PCR analyses of the recombinants obtained verified the first insertional gene inactivation in purple sulphur bacteria. Enzymological studies demonstrated the absence of APS reductase from the mutants. Further phenotypic characterization showed no significant effect of APS reductase deficiency on the sulphite-oxidizing ability of the cells under photolithoautotrophic growth conditions. In the wild-type as well as in mutant strains, tungstate, the specific antagonist of molybdate, led to the intermediary accumulation of sulphite in the medium during sulphide oxidation and strongly inhibited growth with sulphite as photosynthetic electron donor; this indicates that a molybdoenzyme, probably sulphite:acceptor oxidoreductase, is the main sulphite-oxidizing enzyme in C. vinosum. Specific inactivation of selected genes as developed for C. vinosum in this study provides a powerful genetic tool for further analysis of sulphur metabolism and other metabolic pathways in phototrophic sulphur bacteria.

Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D.

From a genomic library of Chromatium vinosum strain D in lambda L47, a 16.5-kbp EcoRI-restriction fragment was identified by hybridization with a DNA fragment harboring the operon for Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthesis. This fragment and subfragments thereof restored the ability to synthesize and accumulate PHA in PHA-negative mutants of A. eutrophus. A region of 6977 bp was sequenced; seven open reading frames (ORFs) were identified which probably represent coding regions; six of these are most probably relevant for PHA biosynthesis in C. vinosum. The structural genes for biosynthetic acetyl-CoA acyltransferase (beta-ketothiolase; phbACv, 1188 bp) and NADH-dependent acetoacetyl-CoA reductase (phbBCv, 741 bp) were separated by ORF4 (462 bp) and ORF5 (369 bp). Downstream of phbBCv ORF7 (471 pb) was identified which was not completed at the 3' terminus. The functions of ORF4, ORF5, and ORF7 are not known. The amino acid sequences of beta-ketothiolase and acetoacetyl-CoA reductase deduced from phbACv and phbBCv, exhibited a similarity of 68.2% and 56.4% identical amino acids, respectively, to the corresponding enzymes of A. eutrophus. Antilinear to and upstream of the genes mentioned above, two genes were identified which were transcribed from a sigma 70-dependent promoter. This promoter overlapped with and was divergent to the phbACv promoter; the transcriptional start sites were mapped by S1 nuclease protection assays. These genes were ORF2 (1074 bp), whose function is not known but whose presence in Escherichia coli is essential for expression of PHA synthase activity, and the structural gene for a PHA synthase of low M(r) (phbCCv, 1068 bp). The gene products of ORF2 and phbCCv, with M(r) of 40,525 and 39,730, respectively, were expressed in E. coli applying the T7 RNA polymerase/promoter system. Although the amino acid sequence of PHA synthase deduced from phbCCv exhibited only 24.7% overall similarity with the PHA synthase of A. eutrophus, highly conserved regions were identified.

Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria

The formation of poly(3-hydroxyalkanoic acid), PHA, by various strains of chemolithotrophic and phototrophic bacteria has been examined. Chemolithotrophic bacteria were grown aerobically under nitrogen-limiting conditions on various aliphatic organic acids. Phototrophic bacteria were grown anaerobically in the light on a nitrogen-rich medium and were subsequently transferred to a nitrogen-free medium containing acetate, propionate, valerate, heptanoate or octanoate as carbon source. All 41 strains investigated in this study were able to synthesize and accumulate PHA. All 11 strains of chemolithotrophic bacteria and all 15 strains belonging to the non-sulfur purple bacteria synthesized a polymer, which contained 3-hydroxy-valerate (3HV) beside 3-hydroxybutyrate (3HB), if the cells were cultivated in the presence of propionate, valerate or heptanoate. Many non-sulfur purple bacteria synthesized copolyesters of 3HB and 3HV even with acetate as carbon source. In contrast, most sulfur purple bacteria did not incorporate 3HV at all. Among 15 strains tested, only Chromatium vinosum strain 1611, C. purpuratum strain BN5500 and Lamprocystis roseopersicina strain 3112 were able to synthesize polyesters containing 3HV with propionate, valerate or heptanoate as carbon source.

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N2 or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested. In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N2, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen. In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated “feeding” with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.

13C-NMR evidence of bacteriochlorophyll a formation by the C 5 pathway in Chromatium

The 13C-NMR spectra of bacteriochlorophyll a formed in the presence of l-[1 13C]glutamate and [2- 13C]glycine in Chromatium vinosum strain D were analyzed. The isotope in the glutamate was specifically incorporated into eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid (ALA), and the 13C in glycine was incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of bacteriochlorophyll a. These labeling patterns provide evidence for the exclusive operation of the C 5 pathway in ALA biosynthesis in the bacterium. The 13C chemical shifts of two quaternary carbons (C-9 and C-16) of bacteriochlorophyll a were reassigned in the present study.

Siroheme as an Active Catalyst in Sulfite Reduction

Siroheme extracted by acetone/HCl treatment of sulfite reductase from yeast and purified by column chromatography catalyzed the reduction of sulfite to thiosulfate and sulfide when coupled with a hydrogen-hydrogenase-methyl viologen system. The activity increased with decrease in pH from 7 to 4, and an apparent Km value of 50 mM for sulfite was obtained. In contrast to sirohydrochlorin plus Fe2+, addition of inorganic iron or 2,2'-bipyridine prior to the reduction reaction had scarcely any effect on the sulfite-reducing activity of siroheme. Hydroxylamine was reduced by siroheme at a much faster rate than sulfite, and the rate increased with increase in pH from 6 to 9. Siroheme extracted from Chromatium vinosum strain D sulfite reductase also reduced sulfite to thiosulfate and sulfide.

Effect of oxygen on acetylene reduction by photosynthetic bacteria

The effect of dissolved oxygen concentration on nitrogenase activity was studied in three species of photosynthetic bacteria. The O2 concentration in the cell suspension was measured with an O2 electrode inserted into the reaction vessel. Acetylene reduction by whole cells of Rhodopseudomonas capsulata, Rhodospirillum rubrum, and Chromatium vinosum strain D was inhibited 50% by 0.73, 0.32, and 0.26 microM O2, respectively. The inhibition of the activity by O2 in R. capsulata usually was reversed completely by reestablishing anaerobic conditions. In R. rubrum and C. vinosum the inhibition was only partially reversible. The respiration rate of R. capsulata was the highest of the three, that of R. rubrum was intermediate, and that of C. vinosum was lowest. R. capsulata and R. rubrum cells were broken after their acetylene reduction activity in vivo had been completely inhibited by O2, and nitrogenase was found to be active in vitro. A concentration of cyanide that did not affect acetylene reduction activity, but which inhibited 75 to 90% of the O2 uptake by whole cells of R. capsulata, shifted the O2 concentration causing 50% inhibition of nitrogenase activity from 0.73 microM to 2.03 microM. These results are in accordance with the assumption that within a limited range of O2 concentrations, the respiratory activity of the cells is enough to scavenge the O2 and to keep the interior of the cells essentially anaerobic. It is suggested that O2 inhibits nitrogenase activity by competing for a limited supply of electrons. When cyanide is present, respiration is slower but is adequate to keep the nitrogenase environment in the cell anaerobic. The lower respiration rate may allow a greater proportion of the electrons to be used for acetylene reduction.

Malic enzyme of Chromatium vinosum

Malic enzyme of the phototropic bacterium Chromatium vinosum strain D that lacks malate dehydrogenase was partially purified yielding a specific activity of 55 units/mg protein. The constitutive enzyme with a molecular weight of 110,000 and a pH optimum of 8.0 was absolutely dependent on the presence of a monovalent cation (NH4+, K+, Cs+, or Rb+) as well as a divalent cation (Mn2+, or Mg2+). The enzyme was inhibited by oxaloacetate, glyoxyate, and NADPH. The K0.5 value for L-malate and the inhibition constants for oxaloacetate and glyoxylate are dependent on the concentration of the monovalent cation, whereas the Km value for NADP (18 microM) and the KI value for NADPH (42 microM) are independent. Throughout all kinetic measurements hyperbolic saturation curves and linear double reciprocal plots were obtained.

The amino acid sequence of cytochrome c′ from the purple sulphur bacterium Chromatium vinosum

An amino acid sequence is proposed for the cytochrome c' from the photosynthetic purple sulphur bacterium Chromatium vinosum strain D. It is single polypeptide chain of 131 residues, with haem-attachment cysteine residues at positions 121 and 124. The results discredit an earlier report [Dus, Bartsch & Kamen (1962) J. Biol. Chem 237, 3083--3093] of a di-haem peptide sequence from this protein. The sequence belongs to the same class as the published Alcaligenes and Rhodospirillum rubrum cytochrome c' squences, but the resemblance is not close. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50,093 (15 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.

Siroheme sulfite reductase isolated from Chromatium vinosum. Purification and investigation of some of its molecular and catalytic properties

Cells of the phototrophic bacterium Chromatium vinosum strain D were shown to contain a siroheme sulfite reductase after autotrophic growth in a sulfide/bicarbonate medium. The enzyme could not be detected in cells grown heterotrophically in a malate/sulfate medium. Siroheme sulfite reductase was isolated from autotrophic cells and obtained in an about 80% pure preparation which was used to investigate some molecular and catalytic properties of the enzyme. It was shown to consist of two different types of subunits with molecular weights of 37,000 and 42,000, most probably arranged in an α4β4-structure. The molecular weight of the native enzyme was determined to 280,000, 51 atoms of iron and 47 atoms of acid-labile sulfur were found per enzyme molecule. The absorption spectrum indicated siroheme as prosthetic group; it had maxima at 280 nm, 392 nm, 595 nm, and 724 nm. The molar extinction coefficients were determined as 302×103 cm2xmmol-1 at 392 nm, 98×103 cm2 xmmol-1 at 595 nm and 22×103 cm2x-mmol-1 at 724 nm. With reduced viologen dyes as electron donor the enzyme reduced sulfite to sulfide, thiosulfate, and trithionate. The turnover number with 59 (2 e-/enzyme moleculexmin) was low. The pH-optimum was at 6.0. C. vinosum sulfite reductase closely resembled the corresponding enzyme from Thiobacillus denitrificans and also desulfoviridin, the dismilatory sulfite reductase from Desulfovibrio species. It is proposed that C. vinosum catalyses anaerobic oxidation of sulfide and/or elemental sulfur to sulfite in the course of dissimilatory oxidation of reduced sulfur compounds to sulfate.

Notizen: Formation of Erythroascorbic Acid and 2,3-Enediol Pentonic Acid from ʟ-Ascorbic Acid by Purple Sulfur Bacteria

Abstract Phototrophic growth of Chromatium vinosum strain D on pyruvate or malate, and of Thiocapsa roseopersicina strain 6311 on malate or sulfide as sole electron donors was promotedby 5 mᴍ ʟ-ascorbate, while growth of T. roseopersicina on fructose was inhibited. Slow growth was obtained also with ascorbate alone. In ascorbate-containing cultures the medium developed a yellow to red-brown colour which showed an absorption maximum at 310 -320 nm. As colourless products of ʟ-ascorbic acid degradation by the two strains examined erythroascorbic acid and 2,3-enediol pentonic acid were found in cell-free culture filtrates by gas chromatographic analysis of the silylated residues and identified by their mass, IR and UV spectra.

Utilization of nitrogen compounds and ammonia assimilation by Chromatiaceae.

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH4Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina. Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrated in the above organisms grown on NH4Cl.

Vergärung von Pyruvat durch sieben Arten phototropher Purpurbakterien

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.

Vergärung von Pyruvat durch sieben Arten phototropher Purpurbakterien

AbstractThe dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments.Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2–8 μmoles pyruvate/mg protein · h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4–2.0 μmoles pyruvate/mg protein · h), while the other strains tested fermented at quite low rates (0.2–0.4 μmoles pyruvate/mg protein · h). The extremes of fermentation times were from 30–380 hours.Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. R. acidophila and R. capsulata additionally form much 2,3‐butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate.The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2–6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.

The Complete Amino Acid Sequence of Chromatium High Potential Iron Sulfur Protein

Abstract High potential iron-sulfur protein isolated from both autotrophically and heterotrophically grown cells of Chromatium vinosum strain D was found to contain a single 85-amino acid residue polypeptide chain which is covalently linked to the iron-sulfur cluster via 4 residues of cysteine. The molecular weight of the protein, including the 4 atoms of iron and acid labile sulfur, is 9,254. The iron-sulfur complex must be removed from the protein before complete digestion by proteolytic enzymes like trypsin, chymotrypsin, or thermolysin can be attained. Digestion of the holoprotein by these enzymes leads to large peptides which contain the central portion of the sequence and comprise 35 to 45 residues, depending on the enzyme used, as well as residual iron presumably attached to the cysteines in positions 43, 45, 63, and 77. Both ends of the protein are readily accessible to exopeptidases and can be degraded substantially. Cysteines 43 and 45 and the single histidyl residue in position 42 are in a configuration reminiscent of that found at the heme attachment site of c-type cytochromes. The complete amino acid sequence of Chromatium high potential iron-sulfur protein was deduced from analyses of peptides obtained by proteolytic digestion of the apoprotein, the tryptic and chymotryptic core peptides, and of the two large peptides produced by chemical fragmentation of the apoprotein with BrCN. The first 18 residues of the protein were determined by stepwise Edman degradation. This approach was also applied to the core peptides and to both BrCN fragments.

Iron protein content of Thiocapsa pfennigii, a purple sulfur bacterium of atypical chlorophyll composition

Four out of five soluble electron transport iron proteins of Thiocapsa pfennigii plus the particulate cytochromes have been found to be analogous to those of Chromatium vinosum strain D. In addition to ferredoxin, high potential iron-sulfur protein. cytochrome c′, and cytochrome c-552(550), T. pfennigii contains a cytochrome c-552(545) not previously isolated from photosynthetic bacteria. It is concluded that T. pfennigii is more closely related to C. vinosum than to Rhodopseudomonas viridis, the only other known bacterial species having bacteriochlorophyll b.

Redox properties of the “P-836” pigment complex of Chromatium

The redox potential of the component responsible for the photobleaching at 836 nm in chromatophores of Chromatium vinosum strain D was determined to be 320 mV. This result may be of significance for suggestions regarding the existence of a low potential, non-cyclic photosystem in Chromatium.

Composition of the sulfur particle of Chromatium vinosum strain D.

Sulfur particles were isolated from the purple sulfur photosynthetic bacterium, Chromatium vinosum strain D. The composition of these particles was determined to be 93% sulfur, 5% protein, and 0.6% lipid. Gel electrophoresis indicated the presence of a single protein species with a molecular weight of 13,500 daltons. From these results, the sulfur particle is postulated to be bounded by a membrane consisting entirely of protein.