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 demonstrate in the above organisms grown on NH₄Cl.     

Capacity of chromatiaceae for chemotrophic growth. Specific respiration rates of Thiocystis violacea and Chromatium vinosum

The capacity for chemoautotrophic, mixotrophic and organotrophic growth in the dark was tested with 45 strains of 17 species (11 genera) of the Chromatiaceae. The auxanographic deep agar shake culture method was used; the gas phase contained 5% O₂ and 1% CO₂ in N₂. All strains tested of Chromatium vinosum, C. minus, C. violascens, C. gracile, Thiocystis violacea, Amoebobacter roseus, Thiocapsa roseopersicina gave positive growth responses under chemoautotrophic and mixotrophic conditions (extra carbon source acetate); one strain of Thiocapsa roseopersicina grew also organotrophically on acetate alone. No growth was obtained with the remaining 17 strains of ten species. None of the five type species (three genera) of the Chlorobiaceae grew under chemotrophic conditions. With Thiocystis violacea 2311 a growth yield of 11.3g dry weight per mol thiosulfate consumed was obtained under chemoautotrophic conditions; under mixotrophic conditions with acetate the yield increased to 69g dry weight per mol thiosulfate consumed. With Thiocystis violacea 2311 maximal specific respiration rates were obtained with thiosulfate as electron donor irrespective of the presence or absence of sulfur globules in the cells; organic substrates served as carbon sources only and did not support respiration. With Chromatium vinosum D utilization of thiosulfate was not constitutive; maximal respiration rates on thiosulfate were obtained only with thiosulfate grown cells containing sulfur globules. Respiration rates were further increased by malate, fumarate or propionate; these substrates also served as sole electron donors for respiration. Acetate and pyruvate were used as carbon sources only. The ecological significance of the chemotrophic metabolism is discussed.     

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.     

Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180^T). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon. All three genes are preceded by sequences resembling σ⁷⁰-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules. All three sgp genes encode translation products which – when compared to the isolated proteins – carry amino-terminal extensions. These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Δp that drives ATP synthesis and reverse electron flow in Chr. vinosum. Received: 1 October 1997 / Accepted: 17 December 1997     

Buoyant density changes due to intracellular content of sulfur in Chromatium warmingii and Chromatium vinosum

Average specific density of individual cells of pure cultures of Chromatium warmingii and Chromatium vinosum were measured by isopicnic gradient centrifugation with Percoll during growth at constant illumination as a function of the increasing content of intracellular sulfur. Cell number and volume, bacteriochlorophyll a, sulfide, and sulfur were followed in the cultures along with cellular buoyant density. Poly--hydroxybutyrate was monitored at several points during growth of the cultures. The density of C. warmingii changed from 1.071 to 1.108 g cm^-3 (sulfur content per cell varied from 0 to 1.71pg). C. vinosum changed its density from 1.096 to 1.160 g cm^-3 (sulfur content per cell varied from 0 to 0.43 pg). Maximum sulfur content in pg of sulfur per m³ of cell volume were 0.178 for C. warmingii and 0.294 for C. vinosum. Measurement of the differences in buoyant density, volume and sulfur content before and after ethanol extraction of cells with and without intracellular sulfur, allowed tentatively to estimate the density of sulfur inside the cells as 1.219 g cm^-3. Isolation of sulfur globules and centrifugation in density gradients gave a density higher than 1.143 g cm^-3 for these intracellular inclusions.Non-common abbreviations Bchl Bacteriochlorophyll - DMB Density Marker Beads - PHB poly--hydroxybutyrate     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Phylogenetic characterization of the purple sulfur bacterium Thiocapsa sp. BBS by analysis of the 16S rRNA, cbbL, and nifH genes and its description as Thiocapsa bogorovii sp. nov., a new species

Strain BBS, the purple sulfur bacterium assigned initially to the species Thiocapsa roseopersicina, is the best studied representative of this species. However, no molecular phylogenetic analysis has been performed to confirm its systematic position. Based on the results of analysis of the sequences of 16S rRNA, cbbL, and nifH genes, DNA-DNA hybridization with the T. roseopersicina type strain, and comparative analysis of the phenotypic characteristics of various species belonging to the genus Thiocapsa, we suggest that strain BBS should be assigned to a new species of the genus Thiocapsa, Thiocapsa bogorovii sp. nov. Original Russian Text ? T.P. Tourova, O.I. Keppen, O.L. Kovaleva, N.V. Slobodova, I.A. Berg, R.N. Ivanovsky, 2009, published in Mikrobiologiya, 2009, Vol. 78, No. 3, pp. 381–392.     

The role of the sulfur globule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT-PCR

During oxidation of reduced sulfur compounds, the purple sulfur bacterium Allochromatium vinosum stores sulfur in the periplasm in the form of intracellular sulfur globules. The sulfur in the globules is enclosed by a protein envelope that consists of the homologous 10.5-kDa proteins SgpA and SgpB and the smaller 8.5-kDa SgpC. Reporter gene fusions of sgpA and alkaline phosphatase showed the constitutive expression of sgpA in A. vinosum and yielded additional evidence for the periplasmic localization of the sulfur globules. Expression analysis of the wild-type sgp genes by quantitative RT-PCR using the LightCycler system showed the constitutive expression of all three sgp genes. The expression of sgpB and sgpC is significantly enhanced under photolithotrophic conditions. Interestingly, sgpB is expressed ten times less than sgpA and sgpC implying that SgpA and SgpC are the main proteins of the sulfur globule envelope. Mutants with inactivated sgpA or sgpB did not show any differences in comparison with the wild-type, i.e., the encoded proteins can replace each other, whereas inactivation of sgpC leads to the formation of considerably smaller sulfur globules. This indicates a role of SgpC for globule expansion. A sgpBC double mutant was unable to grow on sulfide and could not form sulfur globules, showing that the protein envelope is indispensible for the formation and deposition of intracellular sulfur.The paper is dedicated to Prof. Dr. Dr. h.c. mult. Hans Günter Schlegel, Göttingen, on the occasion of his 80th birthday on October 24th, 2004, with great gratitude, as our interest in microbial sulfur metabolism goes back to the early 1960s, when HGT worked in Prof. Schlegels laboratory and in 1972 established this field in Bonn.     

Towards a phylogeny of phototrophic purple sulfur bacteria—16S rRNA oligonucleotide cataloguing of 11 species of Chromatiaceae

11 species from 7 genera of Chromatiaceae, depositing sulfur globules inside their cells, were analyzed by comparative oligonucleotide cataloguing of their 16S ribosomal RNA in order to determine the genealogical relationships to each other and to other Gram-negative phototrophic purple and non-phototrophic eubacteria. The rRNA data reveal that members of Chromatium, Amoebobacter, Lamprocystis, Thiocapsa, Thiocystis, Thiodictyon and Thiospirillum form a phylogenetically coherent grouping. The species investigated do not in each case cluster according to their present classification. Organisms storing sulfur inside their cells are moderately related to but clearly separated from members of Ectothiorhodospira, with which they form one of several sublines of group III of phototrophic purple bacteria as defined by Gibson et al. (1979).Dedicated to Professor H. G. Schlegel on the occasion of his 60th birthday.     

Localization of ribulose-bisphosphate carboxylase-oxygenase and its putative binding protein in the cell envelope of Chromatium vinosum

Antibodies to the large and small subunits of ribulose-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1-1.39) and a putative binding protein (PBP) for RuBisCO from Chromatium vinosum have been used to localize these proteins in thin sections. Immunogold techniques employing single and double antibodies establish that RuBisCO and the RuBisCO PBP are concentrated in the cell envelope of C. vinosum.Abbreviations kDa kilodalton - L large subunit of RuBisCO - PBP putative binding protein of RuBisCO - RuBisCO ribulose-bisphosphate carboxylase-oxygenase - S small subunit of RuBisCO To whom correspondence should be addressed.     

pH-Dependent metabolism of thiosulfate and sulfur globules in the chemolithotrophic marine bacterium Thiomicrospira crunogena

Respiring cells of the chemolithotrophic bacterium Thiomicrospira crunogena produced sulfur globules from the sulfane sulfur of thiosulfate below pH 7, and consumed the globules above pH 7. The switch in metabolism was immediate and reversible upon titration of the culture. The consumed sulfur globules remained in a membrane-bound form and were not oxidized unless the medium was depleted of thiosulfate. Sulfur globule production but not uptake was blocked by azide. Anoxia, thiol-binding agents, and inhibitors of protein synthesis blocked globule uptake. Transitory accumulations of sulfite and polythionates appeared to be reaction products of thiosulfate and sulfur globules. A model depicting the pH sensitivity and biochemistry of sulfur globule production and consumption is proposed.     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

The capacity of phototrophic sulfur bacterium Thiocapsa roseopersicina for chemosynthesis

Purple sulfur bacterium Thiocapsa roseopersicina strain BBS requiring vitamin B₁₂ may grow in the dark in media containing no other organic compounds. Under such conditions the cells oxidize sulfide and thiosulfate with the use of O₂ and assimilate carbon dioxide. After 10–30 s assimilation of NaH¹⁴CO₃ about 60% of radioactivity is found in phosphorylated compounds characteristic for the reductive pentose phosphate cycle. The possibility of the function of this cycle in the dark in the presence of O₂ is confirmed by the capacity of cells grown under such conditions to synthesize ribulose-1,5-diphosphate carboxylase. All this evidence suggests the ability of T. roseopersicina to change from phototrophy to aerobic chemolithoautotrophy.     

Ammonia inhibition of nitrogenase activity in purple and green bacteria

Ammonia reversibly inhibits the nitrogenase activity not only in purple nonsulfur bacteria but in purple (Thiocapsa roseopersicina) and green (Chlorobium limicola forma thiosulfatophilum) sulfur bacteria as well.The complete inhibition of nitrogenase activity (acetylene reduction) is observed about 30 s after addition of NH ₄ ⁺ (2.5×10^-6 M) to cell suspensions. The pattern of ammonia inhibition of acetylene reduction in T. roseopersicina does not differ from the action of tetrabutylammonium and tetraphenylphosphonium (3 · 10^-6-5·10^-5 M) on nitrogenase activity of this bacterium.Simultaneously with the switch-off effect of NH ₄ ⁺ a considerable increase of ATP in cells of Rhodobacter sphaeroides and C. limicola f. thiosulphatophilum was observed.     

Influence of sulfur accumulation and composition of sulfur globule on cell volume and buoyant density of Chromatium vinosum

Average cell volume and cell buoyant density of Chromatium vinosum DSM 185 growing in sulfide limited continuous cultures, were found to increase with increasing dilution rate. It was found that the increase in buoyant density was mainly a consequence of the accumulation of elemental sulfur. The contribution of other compounds such as protein, bacteriochlorophyll a and glycogen, was almost negligible. It was concluded that the sulfur globule is constituted by at least two fractions, sulfur and an unidentified moiety with a density lower than that of sulfur, probably water.A model was developed to explain the relation between the specific content of sulfur and cell buoyant density. The model also predicts the impact of elemental sulfur on the volume of the cell. It was found that in addition to the accumulation of sulfur the average cell volume also changes with the specific growth rate.In shift-up experiments (sulfur accumulation) the actual phenomena agreed with those predicted by the model, however, this was not so during shift-down (sulfur depletion). It is suggested that this difference is due to the fact that during the shift-down, elemental sulfur and the unidentified moiety are being depleted at different rates.Non-standard abbreviations BChl bacteriochlorophyll - PHB poly--hydroxybutyric acid - D dilution rate - specific growth rate - S _R reservoir concentration of limiting substrate     

Antenna organization in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum

Structural aspects of the core antenna in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum were studied by means of fluorescence emission and singlet-singlet annihilation measurements. In both species the number of bacteriochlorophylls of the core antenna between which energy transfer can occur corresponds to one core-reaction center complex only. From measurements of variable fluorescence we conclude that in C. tepidum excitation energy can be transferred back from the core antenna (B920) to the peripheral B800–850 complex in spite of the relatively large energy gap, and on basis of annihilation measurements a model of separate core-reaction center units accompanied by their own peripheral antenna is suggested. C. vinosum contains besides a core antenna, B890, two peripheral antennae, B800–820 and B800–850. Energy transfer was found to occur from the core to B800–850, but not to B800–820, and it was concluded that in C. vinosum each core-reaction center complex has its own complement of B800–850. The results reported here are compared to those obtained earlier with various strains and species of purple non-sulfur bacteria.Abbreviations BChl- bacteriochlorophyll - B800–820 and B800–850- antenna complexes with Q_y-band absorption maxima near 800 nm and 820 or 850 nm, respectively - B890 and B920- antenna complexes with Q_y-band absorption maxima near 890 and 920 nm, respectively - LH1- light harvesting 1 or core antenna - LH2- light harvesting 2 or peripheral antenna     

Localization of hydrogenase in the purple sulphur bacterium Thiocapsa roseopersicina

The localization of hydrogenase in the phototrophic bacterium Thiocapsa roseopersicina was investigated by subcellular fractionations, and transmission electron microscopic immunocytochemistry. By using sonicated cells and measuring in vitro hydrogenase activities in soluble and membrane fractions, respectively, a weak hydrophobic interaction between the hydrogenase enzyme and the T. roseopersicina membranes was observed. Polyclonal antisera directed against the purified hydrogenase were raised in rabbits and exhibited one band in native-PAGE/Western immunoblot analysis. Native-PAGE/activity stain confirmed the identity of this band as being hydrogenase. Immunocytolocalization experiments using ultrathin sections showed an internal localization of the hydrogenase enzyme. A higher specific labeling was associated with chromatophores, indicating a possible coupling of hydrogenase with the photosynthetic membranes in the T. roseopersicina cells.     

Viable phototrophic sulfur bacteria from the Black-Sea bottom

Phototrophic sulfur bacteria were isolated from Black-Sea mud at depths of 660 and 2,240 m. The species obtained in pure cultures were identified asChromatium warmingii andThiocapsa roseopersicina. In addition,Chlorobium phaeovibrioides occurred in enrichment cultures. The findings prove that Chromatiaceae and Chlorobiaceae are capable of survival in 2000 m depth, in the dark, in the presence of H₂S and organic materials. There is, however, no evidence for growth or reproduction of these organisms in this environment.     

酒色着色菌利用产酸克雷伯氏菌发酵废液产氢

研究了酒色着色菌(Chromatium vinosum DSM185)利用产酸克雷伯氏菌（Klebsiella oxytoca HP1）发酵产氢废液进行光发酵和暗发酵产氢的可行性,以达到对产氢底物的充分利用和对产氢废液的进一步处理。研究结果表明C.vinosum可以利用K.oxytoca的发酵废液进行光发酵产氢和暗发酵产氢。C.vinosum发酵产氢后废液中残余还原糖和主要有机酸（丁酸）的含量明显降低,发酵产氢的最佳pH为6.5,添加0.1%(W/W)NH₄Cl能促进产氢。在光照条件下丁酸利用率可达54.38%,产氢量达36.97 mL/mg;在黑暗条件下丁酸利用率可达36.01%,产氢量达37.50mL/mg。     

Coexistence of Chlorobium and Chromatium in a sulfide-limited continuous culture

Competition experiments between Chromatium vinosum and Chlorobium limicola in sulfide-limited continuous culture under photolithoautotrophic conditions resulted in the coexistence of both organisms. The ratio between the two bacteria was dilution-rate as well as pH dependent. The observed coexistence can be explained as a hitherto not reported form of dual substrate limitation. The two substrates involved are the electron donors sulfide (growth-limiting substrate in the reservoir vessel) and extracellular elemental sulfur (formed by Chlorobium as a result of sulfide oxidation). It is argued that, although Chlorobium may have the better affinity for both substrates involved, Chromatium can compete successfully on the basis of its intracellular storage of sulfur. Ecological implication of the observed coexistence with respect to natural blooms are discussed.