DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

Purification and properties of the adenylate kinases from Rhodopseudomonas palustris,Rhodopseudomonas sphaeroides and Rhodopseudomonas rubrum

The adenylate kinases (EC 2.7.4.3) from photosynthetically grown Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum were purified to homogeneity by the same procedure. The purified enzymes showed optimal rates of activity with MgCl₂ at 25° C and pH 8.0. They were found to be heat labile and were characterized by pI-values of 4.5. Apparent molecular weights of 33 500 for R. palustris, 34 400 for R. sphaeroides and 32 100 for R. rubrum were determined by high performance liquid chromatography. No separation into subunits was observed by use of sodium dodecylsulfate polyacrylamide gel electrophoresis. The apparent K _m -values for ADP corresponded to 0.26 mM for R. palustris, 0.27 mM for R. sphaeroides and 0.24 mM for R. rubrum. ADP in excess had a strong inhibitory effect. Competitive product inhibition was found for AMP, with K _i-values of 0.017 mM for R. palustris, 0.018 mM for R. sphaeroides and 0.014 mM for R. rubrum. A competitive inhibitor likewise was P¹,P⁵-di(adenosine-5)pentaphosphate with K _i-values of 0.020 M for R. palustris and R. sphaeroides, and 0.017 M for R. rubrum. Sulfhydryl-reacting reagents like p-chloromercuribenzoate and iodoacetic acid were found to be non-inhibitory. All measurements of adenylate kinase activity were carried out with the stabilized and most sensitive luciferin-luciferase system.     

Characterization of Rhodopseudomonas capsulata

Thirty-three strains of Rhodopseudomonas capsulata have been studied in order to develop a more comprehensive characterization of the species. On the basis of morphological, nutritional, physiological and other properties, the characteristics of an ideal biotype have been defined, which can be used to distinguish Rps. capsulata from similar purple bacteria. In this connection, two properties of Rps. capsulata are of particular note: a) sensitivity to penicillin G is 10³–10⁵ times greater than that shown by closely related species, and b) all strains examined are susceptible to lysis by one or more strains of host species-specific virulent bacteriophages. It appears that members of the species Rps. capsulata form a stringent taxonomic grouping.     

沼泽红假单胞菌的研究进展

概述了沼泽红假单胞菌菌株的分离纯化,主要特性及应用研究方面的进展。     

Bacteriophages of Rhodopseudomonas spheroides: Isolation and Characterization of a Rhodopseudomonas spheroides Bacteriophage

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.     

Thylakoidmorphogenese bei Rhodopseudomonas palustris

Zusammenfassung Rhodopseudomonas palustris, Stamm 11/1, kann in Dunkelkulturen durch Absenken des Sauerstoffpartialdruckes in der Submerskultur von 100 auf etwa 5 mm Hg [pO₂] zur Bacteriochlorophyllsynthese und Thylakoidmorphogenese induziert werden.Die Thylakoidbildung beginnt meistens an einem Zellpol (Vorderende). Sie erfolgt durch Vorstülpung der cytoplasmatischen Membran in das Zellinnere. Diese Invaginationsbezirke wachsen dann flächenförmig, in der Regel parallel zur cytoplasmatischen Membran aus. Die Thylakoide ihrerseits können durch lokal begrenzte Wachstumsprozesse in der Fläche und an den Kanten sich ausdehnen, sich verzweigen, sich falten und wieder mit anderen Thylakoiden oder der cytoplasmatischen Membran verschmelzen. An Hand von Serienschnitten und daraus rekonstruierten räumlichen Modellen wird versucht, die Stapelbildung durch lokale Überschiebungsund Überwachsungsprozesse zu erklären.

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The morphogenesis of the thylakoids of Rhodopseudomonas palustris

Summary Rhodopseudomonas palustris, strain 11/1 is able to grow aerobically in the dark and likewise anaerobically in the light. The biosynthesis of bacteriochlorophyll and the morphogenesis of the thylakoids (chromatophores) are light independent processes. They are induced by lowering the oxygen partial pressure in the culture from 100 to 5 mm Hg [pO₂].The morphogenesis starts in a defined polar region of the cell. The cytoplasmic membrane forms channel-like invagination structures. The arising protuberances grow plain like and parallel to the cytoplasmic membrane producing the flat vesicels of the thylakoids. These thylakoids are able to branch, to fuse with another and with the cytoplasmic membrane.From serial sections three-dimensional models of the thylakoid pattern in the cell are constructed. The evaluation has suggested that the staples of thylakoids are caused by overlapping, overgrowing and thylakoid invaginations.

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Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.     

Porphobilinogenase from Rhodopseudomonas palustris

1. Porphobilinogenase (PBGase) from Rp. palustris has been isolated and some properties of a partially purified fraction were studied. 2. PBGase has an optimum pH of 7.4 when activity was expressed in terms of porphyrins formed and two pH maxima at 7.4 and 8.5 when activity was based on the amount of PBG consumed. 3. Cyclotetramerization rate and distribution of reaction products were not affected either by the presence or absence of oxygen. 4. Two PBGase active species of mol. wt 115,000 and 50,000 were found, by means of gel filtration through a calibrated Sephadex G-100 column. 5. Kinetic data show the existence of positive cooperative effects for porphyrin formation, while a hyperbolic behaviour for PBG consumption was observed.     

Ferrochelatase of Rhodopseudomonas spheroides

Extracts of Rhodopseudomonas spheroides contain two ferrochelatases: one is soluble and forms metalloporphyrins from deuteroporphyrin and haematoporphyrin; the other is particulate and forms metalloporphyrins from protoporphyrin, mesoporphyrin, deuteroporphyrin and haematoporphyrin. Neither enzyme incorporates Mg²⁺ into porphyrins or Fe²⁺ into porphyrin cytochrome c. By using the particulate enzyme, plots of 1/v versus 1/s when one substrate was varied and the other kept constant showed that neither substrate affected the K_m of the other. The suggested sequential mechanism for the reaction is supported by derivative plots of slopes and intercepts. The K_m for deuteroporphyrin was 21.3μm and that for Co²⁺ was 6.13μm. The enzyme incorporated Co²⁺, Fe²⁺, Zn²⁺, Ni²⁺ and Mn²⁺; Cd²⁺ was not incorporated and was an inhibitor, competitive with respect to Co²⁺, non-competitive with respect to deuteroporphyrin. The K_i for Cd²⁺ was 0.73μm. Ferrochelatase was inhibited by protohaem, non-competitively with respect to Co²⁺ or with respect to deuteroporphyrin. Inhibition by magnesium protoporphyrin was non-competitive with respect to deuteroporphyrin, uncompetitive with respect to Co²⁺. The inhibitory concentrations of the metalloporphyrins are lower than those required for the inhibition of δ-aminolaevulate synthetase by protohaem. Fe²⁺ is not incorporated aerobically into porphyrins unless an electron donor, succinate or NADH, is supplied; the low aerobic rate of metalloporphyrin synthesis obtained is insensitive to rotenone and antimycin. The rate of Fe³⁺ incorporation increases as anaerobic conditions are achieved.     

Porin from Rhodopseudomonas sphaeroides

A protein homooligomer was purified from both the cell envelope fractions and the saline extracts of Rhodopseudomonas sphaeroides cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with egg phosphatidylcholine. In the saline extracts of both chemotrophically and phototrophically grown cells, the porin oligomer was the most predominant polypeptide, which produced pores whose behavior toward various sugars could be approximated by hollow cylinders of 0.62 nm in radius. The oligomer was dissociated, in the presence of EDTA, into monomers that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as though their molecular weight was about 47,000. The monomer was active in the reconstitution assay and produced pores with sizes comparable to those produced by the oligomer. Circular dichroism spectra indicated the predominance of beta-sheet structure in both the oligomeric and EDTA-dissociated monomeric forms. Drastic conditions, for example, precipitation with 10% trichloroacetic acid or heating for a few hours at 100 degrees C in sodium dodecyl sulfate, were necessary to denature the protein into a form with a reduced content of beta-sheet structure.     

Phosphoribulokinase from Rhodopseudomonas acidophila

Phosphoribulokinase from the nonsulfur purple bacterium Rhodopseudomonas acidophila has been purified to apparent homogeneity, using affinity chromatography on Cibacron Blue-agarose and AMP-agarose. The relative molar mass of the enzyme was determined by sucrose density gradient centrifugation to be M _r=248,000 with a sedimentation coefficient of s _20,w=10.9 S. Dodecyl sulfate polyacrylamide gel electrophoresis revealed that the enzyme consists of identical size subunits of M _r=32,000, suggesting an octameric structure of the holoenzyme. The enzyme cross-reacted with heterologous antibodies raised against phosphoribulokinase from the hydrogen bacterium Alcaligenes eutrophus. The pH optimum of the enzyme was shifted from 8.4 in the absence of the activator NADH to 7.6 in the presence of the effector. Mg²⁺ ions were the most effective divalent cations required for activity. Specificity of the enzyme for the sugar phosphate substrate ribulose 5-phosphate was high whereas a variety of nucleoside triphosphates besides ATP could serve as phosphate donors. NADH was a strong activator of the enzyme (K _a=0.05 mM) that primarily affected the maximal reaction velocity in a pH-dependent manner. The only other effector identified was phosphoenolpyruvate. It moderately inhibited the enzyme (I _0.5=0.32 mM).Abbreviation PRK phosphoribulokinase Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen     

Substrate specificity of citrate lyase deacetylase of Rhodopseudomonas gelatinosa and Rhodopseudomonas palustris.

Citrate lyase (EC 4.1.3.6) isolated from Rhodopseudomonas palustris was investigated with regard to its kinetic properties and its subunit composition. This enzyme was inactivated by citrate lyase deacetylase (EC 3.1.2.-) of Rhodopseudomonas gelatinosa. A corresponding cross-reaction was measured with partially purified deacetylase of R. palustris and citrate lyase of R. gelatinosa. The three different subunit types (alpha, beta, and gamma) of citrate lyase from R. gelatinosa wee purified to homogeneity, and antibodies were prepared against each of the three subunits and against the native enzyme complex. In corresondence with the enzymatic interactions, immunological cross-reactions were found between anti-enzyme and anti-large subunit antibodies and citrate lyase from R. palustris. On the other hand, no immunological cross-reactions were detectable among each of the antibodies and citrate lyases from Enterobacter aerogenes, Streptococcus diacetilactis, and Clostridium sphenoides. Antibodies against the large subunit of citrate lyase inhibited the deacetylase, but antibodies against the middle and small subunits did not, indicating that the large subunits of citrate lyase are involved in binding the deacetylase.     

深层液体培养法生产沼泽红假单胞菌

根据沼泽红假单胞菌 (Rhodopseudomonaspalustris)在好氧黑暗条件下也可生长的特性 ,采用液体深层培养方法 ,在通气式发酵罐中进行大规模生产。对培养基中的碳源、氮源、生长因子和微量元素进行了优化 ,用正交试验确定了主要培养基成分的添加量和接种量。在 10 0 0mL三角瓶和 2 5L全自动通气式发酵罐上进行了放大试验 ,在适宜工艺条件下培养 2 4h ,菌体浓度可达 6 0亿 /mL。     

Nickel uptake in Rhodopseudomonas capsulata

Nickel uptake system was investigated with a wild-type cell of Rhodopseudomonas capsulata and two mutants lacking uptake hydrogenase (Hup^-). Wild type cells grown photoheterotrophically incorporated ⁶³Ni²⁺ by a high affinity system. The uptake system had a pH of 7.0 and a temperature optimum of 28°C. Both Mg²⁺ and Co²⁺ ions severely repressed the uptake of Ni²⁺. Nickel transport was also inhibited by metabolic inhibitors including cyanide, azide, 2,4-dinitrophenol and m-chlorophenyl carbonylcyanidehydrazone. These data imply that Ni²⁺ uptake system occurs by the energy-linked system for Mg²⁺ transport. The intracellular distribution of ⁶³Ni²⁺ in Hup^- cells showed the same pattern as that of wild-type cells, indicate that the Hup^- strains have no deficiency in Ni²⁺ transport.Abbreviations CCCP m-chlorophenyl carbonylcyanidehydrazone - HEPES N-2-hydroxylethylpiperazine-N-2-ethane-sulfuric acid - HOQNO 2-n-nonly-4-hydroxyquinoline-N-oxide - TMA tetramethylammonium hydroxide     

Zur Taxonomie von Rhodopseudomonas palustris

Zusammenfassung Eine Reihe von Rhodopseudomonas palustris-Stämmen aus verschiedenen Herkünften wurden vergleichend unter Verwendung folgender Merkmale untersucht: Substratverwertung, in vivo-Absorptionsspektrum und Serologie der O-Antigene. Die gegen 2 Stämme gerichteten Antiseren zeigen hohe Spezifität. Die Verwendbarkeit der serologischen Kreuzreaktion für taxonomische Untersuchungen bei photosynthetischen Bakterien wird diskutiert.

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On the taxonomy of Rhodopseudomonas palustris

Summary Strains of Rhodopseudomonas palustris isolated from different habitats were compared with respect to their taxonomic features. All strains grew very well on formiate, acetate, propionate, butyrate, aspartate, inositol, ethanol, fructose, and p-amino-benzoate, respectively, as single carbon source. Most of the strains were able to use benzoic acid or glucose, too. But alanine was not found to be a good substrate. The maxima of the bacteriochlorophyll in-vivo-absorption spectra were estimated to be 376, 589, 802–805, and 858–875 nm. The shift of the infrared peak in the different strains is loosely correlated with the change of the carotenoid in vivo spectrum, the maxima of which were measured to be 470–480 nm (shoulder) 495–505 nm, and 520–545 nm (shoulder). Antisera were prepared against the strains 1e5 and 11/1. It was demonstrated that these antisera were directed against the lipopolysaccharides (O-antigen) of these bacteria. The antigen of 1e5 does not cross react with the antigen of 11/1. Strain 1e5 is the only one of 17 strains tested which is sensitive to the bacteriophage Rp1. The antigen of this strain cross reacted only with the antigen of strain K1. In contrast, the antigen of strain 11/1 cross reacted in some degree with most of the tested strains of Rps. palustris. No or very weak cross reaction was observed between the antigens of Rps. palustris (1e5, 11/1) and Rps. capsulata, Rps. spheroides, or R. rubrum, respectively. In contrast to 11/1 only heat-killed cells of strain 1e5 were agglutinated by anti-1e5.

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Im Text verwendete Abkürzungen LPS Lipopolysaccharid - R Rhodospirillum - Rps. Rhodopseudomonas - i.m. intramuskulär - s.c. subcutan - i.v. intravenös