Zur Feinstruktur und Taxonomie von Rhodopseudomonas gelatinosa

Zusammenfassung Die in einer früheren Arbeit postulierten Untergruppen innerhalb der Art Rps. gelatinosa werden näher charakterisiert. Die Gruppen unterscheiden sich in der Generationszeit, der Schleimproduktion, der Tendenz, sich in der Submerskultur abzusetzen, der Substratverwertung und den Eigenschaften des in vivo-Absorptionsspektrums. Artspezifisch und charakteristisch für alle Stämme ist die Fähigkeit zur Gelatineverflüssigung, die Nutzung von Asparagin als C-Quelle und das Unvermögen, autotroph zu wachsen.Die Thylakoide dieser Bakterien sink kleine, einzeln angeordnete Invaginationen der cytoplasmatischen Membran. Die geringe Entwicklung des intracellulären Membransystems gibt zu der Vermutung Anlaß, daß in den Photosynthese-apparat die cytoplasmatische Membran mit einbezogen ist. Die Verwendung von Merkmalen für die Artdiagnostik bei den Athiorhodaceae wird diskutiert.

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Substructure and Taxonomy of rhodopseudomonas gelatinosa

Summary The characterization of two sub-groups of the species Rhodopseudomonas gelatinosa was continued. Differences in relation to generation time, slime production, tendency to conglomerate, utilization of substrates and the characteristics of the in vivo-absorption spectra are described. All strains liquefy gelatin and grow very well on aspartic acid as substrate in anaerobic light cultures and with a lower rate in aerobic dark cultures, too.The thylakoids (chromatophores) of Rps. gelatinosa are small and single arranged invaginations of the cytoplasmic membrane. The low development of an intracellular membrane system in spite of a bacteriochlorophyll content of 10 to 25 g BChl.¹/mg protein suggests that the cytoplasmic membrane may be included in the system of the photosynthetic apparatus. All cells have a single polar flagellum. The use of data and methods for the identification of Athiorhodaceae is discussed.

     

Morphologie und Wirtskreis eines neu isolierten Rhodopseudomonas palustris-Phagen

Zusammenfassung Aus Teichwasser in der Nähe Freiburgs wurde ein Bacteriophage angereichert, der Rhodopseudomonas palustris, Stamm 1 e 5 befällt. Dieser, als Rp 1 bezeichnete Phage kann in anaeroben Lichtkulturen von Rps. palustris bis zu einem Titer von 10⁹ vermehrt werden. Der Infektionscyclus findet auch in aerober Dunkelkultur statt. Von 17 untersuchten Rps. palustris-Stämmen wird nur der Stamm 1 e 5 befallen und bei ihm die Entwicklung von Plaques beobachtet. Das Capsid des Phagen ist icosaedrisch. Die Capsomeren erscheinen pentagonal. Das Capsid hat einen Durchmesser von 380–390 . Die Festheftung des Phagen an der Oberfläche der Wirtszelle oder an den Thylakoiden erfolgt mit einer röhrenförmigen Struktur, die 200 lang und 55–60 dick ist.

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Morphology and host range of a new isolated Rhodopseudomonas palustris-phage

Summary A Phage active against Rhodopseudomonas palustris was isolated from freshwater ponds near Freiburg. The Phage is called Rp 1. Rp 1 could be propagated to titres of 10⁹ plaque-forming units/ml in anaerobic light cultures. It is also synthesized in aerobic dark cultures of Rps. palustris strain 1 e 5. The host range of this phage is very narrow. Only one of seventeen strains of Rps. palustris is able to propagate the phage. The other tested Athiorhodaceae do not give rise to plaque development. The Rp 1 virus consists of an icosahedral head (380–390 in diameter) and a tail like structure. After degradation of the capsid pentagonal capsomeres become visible. The phages are attached by a hollow tube (length 200 , diameter 55–60 ) to the host cell. In negative stained preparations of phages many Rp 1 are seen attached to the thylakoid of the bacteria.

     

Die Fraktionierung des Membransystems von Rhodopseudomonas capsulata und seine Morphogenese

Zusammenfassung Das Membransystem von Rps. capsulata setzt sich aus Cytoplasmamembran und intracytoplasmatischen Membranen zusammen. In anaeroben Lichtzellen und in Dunkelzellen unter geringen Sauerstoffpartialdrucken bestehen die intracytoplasmatischen Membranen aus Vesikeln, bei Anzucht unter hohen Sauerstoffspannungen sind sie tubulär. Auch nach einer 8stündigen Kultur bei 400 mm (Hg) Sauerstoffpartialdruck, d.h. unter Bedingungen, die eine BChl-Synthese vollständig hemmen, enthalten die Zellen noch intracytoplasmatische Tubuli an einem Zellpol.Nach Zellaufschluß mit der French pressure cell gelang es durch anschließende fraktionierte Zentrifugation und Reinigung der Partialfraktionen über Ficoll-Gradienten 3 membranhaltige Banden zu isolieren. Die leichte Bande besteht vorwiegend aus Membranfragmenten der Cytoplasmamembran. Die mittlere Bande enthält die intracytoplasmatischen Tubuli aerob angezogener Zellen. Die schwere Bande, die den höchsten Reinheitsgrad aufweist, setzt sich aus den intracytoplasmatischen Vesikeln der Licht-bzw. der semiaeroben Dunkelzellen zusammen. ¹⁴C-Markierungsexperimente und elektronenmikroskopische Beobachtungen sprechen für morphologische und morphogenetische Zusammenhänge zwischen den Membranfraktionen und stützen damit die Hypothese, daß alle Membrantypen in einer Zelle Teile eines zusammenhängenden Membransystems sind. Die einzelnen Membrantypen können reversibel ineinander überführt werden.

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The fractionation and morphogenesis of the membrane system of Rhodopseudomonas capsulata

Summary The membrane system of Rhodopseudomonas capsulata, strain 37b4 is investigated in cells cultivated anaerobically in the light and in the dark, respectively, at different oxygen partial pressures (pO₂). The intracytoplasmic membrane vesicles of anaerobically light grown and semiaerobically [5 mm (Hg) pO₂] dark grown cells show similar diameters (30–50 nm). Growing aerobically in darkness the cells contain tubular intracytoplasmic membranes with comparable diameters. An increase of the pO₂ up to 400 mm (Hg) results in a slightly decreased growth rate and in a complete inhibition of bacteriochlorophyll synthesis and intracytoplasmic membrane formation. After 8 h of cultivation under these conditions tubular membranes are still found. However, they are restricted to one cell pole only.In order to isolate the membranes, cells were broken by means of a French pressure cell. The crude, membrane fractions (sedimented at 104000-314000xg, 60 min) are purified by centrifugation on a Ficoll density gradient. This results in the formation of three membrane fractions. The light fraction consists of small vesicular particles derived from the cytoplasmic membrane. Crude membrane fractions of all cells sedimented at 314 000xg contain a relative high percentage of these particles. Intracytoplasmic membranes of aerobically grown cells are found in a middle band. Their tubular structure remains unaffected if the cells are treated by lower pressures during homogenization. The heavy band contains the intracytoplasmic membrane vesicles from light grown and from semiaerobically dark grown cells in a highly purified form.After treatment with butanol or NaCl and subsequently with lysozyme plus EDTA cells release flat membrane fragments which still exhibit invaginations. This shows once more that the membranes are connected to each other.Pulse chase experiments with (2-¹⁴C)-acetate support the hypothesis that the cytoplasmic membrane and intracytoplasmic membranes are transformable into each other. So they should be looked at as a morphogenetical unit.

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Abkürzungen BChl Bacteriochlorophyll - CM Cytoplasmamembran - ICM intracytoplasmatische Membran - pO₂ Sauerstoffpartialdruck     

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     

Die Fettsäuren in ganzen Zellen,Thylakoiden und Lipopolysacchariden von Rhodospirillum rubrum und Rhodopseudomonas capsulata

Zusammenfassung Die photosynthetischen Bakterien Rhodospirillum rubrum und Rhodopseudomonas capsulata wurden auf ihre Fettsäurezusammensetzung untersucht. Die Hauptfettsäuren von R. rubrum waren C_16:0 (11%), C_16:1 (30%) und C_18:1 (52%). Vaccensäure (C_18:1) bildete 94% der Fettsäuren von Rps. capsulata. Anaerobe Lichtzellen (thylakoidhaltig) unterschieden sich nicht in ihrem Fettsäuremuster von aeroben Dunkelzellen (thylakoidfrei). Gereinigte Thylakoide aus Lichtzellen zeigten das gleiche Fettsäuremuster wie die ganzen Zellen.Nach Phenol/Wasser-Extraktion der ganzen Zellen bei 68° C war bei beiden Organismen sowohl aus Licht- als auch aus Dunkelzellen eine Substanz aus der gäßrigen Phase isolierbar, welche in den Sedimentationseigenschaften mit den Lipopolysacchariden der Enterobacteriaceae übereinstimmte und nach orientierenden Untersuchungen Zucker enthält. Aus ihr wurde ein Fettsäuregemisch gewonnen, dessen Zusammensetzung von dem aus ganzen Zellen erheblich abwich. In Rps. capsulata enthielt es C_12:1 (40%) und C_16:0 (50%), während in R. rubrum sich das Fettsäuremuster über den Bereich von C₁₀ bis C₂₀ erstreckte. Licht- und Dunkelzellen wiesen in dieser Substanz Unterschiede in der Fettsäurezusammensetzung auf. Der quantitative Anteil der Fettsäuren in dieser Substanz, bezogen auf die Gesamtfettsäuren der Zelle, betrug in Licht- und Dunkelzellen 5–7%. Hydroxy-myristinsäure ließ sich in beiden Organismen nicht nachweisen.

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Fatty acid composition of whole cells, thylakoids and lipopolysaccharides of Rhodospirillum rubrum and Rhodopseudomonas capsulata

Summary The fatty acid composition of the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata was investigated. The bulk of fatty acids of R. rubrum consisted of C_16:0 (11%), C_16:1 (30%), and C_18:1 (52%). The major fatty acid of Rps. capsulata was vaccenic acid (C_18:1), which accounted for 94% of the total fatty acids. Cells of both organisms, which were grown anaerobically in the light and fitted out with thylakoids had the same fatty acid composition as cells grown aerobically in the dark, which have no thylakoids.Purified thylakoids had the same fatty acid pattern as whole cells. Whole cells of light and dark cultures were extracted with phenol/water at 68° C. An opalizing fraction in the aqueous phase was sedimentable in the ultracentrifuge like the lipopolysaccharides of the Enterobacteriaceae. The pattern of fatty acids in this compound differed considerably from that of whole cells. The major fatty acids in this macromolecular fraction were C_12:1 (40%) and C_16:0 (50%) in Rps. capsulata, whereas in R. rubrum the whole range of fatty acids from C₁₀ to C₂₀ was demonstrable. Light and dark grown cells differed in the fatty acid composition of that compound. The fatty acid content of the extracted fraction accounted for 5–7% of the total fatty acids of whole cells. No hydroxymyristic acid could be identified in either R. rubrum or Rps. capsulata.

     

Die Differenzierung des Membransystems von Rhodopseudomonas capsulata hinsichtlich seiner photosynthetischen und respiratorischen Funktionen

Zusammenfassung Membranfunktionen aus anaerob im Licht gewachsenen Zellen von Rps. capsulata und aus Zellen nach Anzucht im Dunkeln unter verschiedenen konstanten Sauerstoffpartialdrucken (5, 150, 400 Torr) wurden untersucht. Aus dem Vergleich ihres Aufbaues (Proteinmuster) und ihrer photosynthetischen und respiratorischen Aktivität (Funktionsmuster) ließ sich eine Vorstellung über das Differenzierungsgeschehen des gesamten Membransystems von Rps. capsulata ableiten.Die intracytoplasmatischen Vesikel aus Lichtzellen mit einem hohen BChl-Gehalt (bis 195 g BChl/mg Protein) und hohen Photophosphorylierungsraten (bis 19,5 mole PO₄ ^3-/mg Protein) können weitgehend als eine Struktur für den photosynthetischen Energieerwerb angesehen werden. Die entsprechenden Vesikel aus semiaerob im Dunkeln angezogenen Zellen zeigen mit über 50% des BChl-Gehaltes und der Photophosphorylierungsaktivität der Vesikel aus Lichtzellen ebenfalls den Charakter einer photosynthetischen Membran. Daneben tragen diese Strukturen jedoch noch weitgehend Funktionen des respiratorischen Apparates. Für die NADH-Oxydase-Aktivität wurden über 10mal höhere Werte und für die oxydative Phosphorylierung etwa doppelt so hohe Raten wie in den Vesikeln aus Lichtzellen gemessen.In den intracytoplasmatischen Tubuli sind die Aktivitäten von NADH-Oxydase und oxydativer Phosphorylierung gegenüber denen der Vesikel aus semiaerob angezogenen Zellen etwa 5 bzw. 2mal so hoch. Der Photosyntheseapparat ist in diesen Strukturen mit weniger als 5% des BChl-Gehaltes und etwa 10% der Photophosphorylierungsaktivität gegenüber den Vesikeln aus Lichtzellen nur sehr gering ausgebildet. Das Verhältnis von Photophosphory lierung zu oxydativer Phosphorylierung in intracytoplasmatischen Membranfraktionen (Phosphorylierungsquotient) kann als Maß für die Differenzierung der Membranen angesehen werden. Der Wert beträgt für Lichtzellen 25, für semiaerobe Dunkelzellen 5 und für aerobe Dunkelzellen 1. In der Cytoplasmamembran wird das Aktivitätsmuster abweichend von dem der intracytoplasmatischen Membran variiert. Die verschiedenen Membranfraktionen zeigen charakteristische Proteinmuster. Einzelne Banden ändern ihre Aktivität parallel mit bestimmten Funktionen der Membranen.

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Differentiation of membranes from Rps. capsulata with respect to their photosynthetic and respiratory functions

Summary Cultures of Rps. capsulata were grown anaerobically in the light and at different oxygen partial pressures (5, 150, and 400 Torr) in the dark. The respiratory and photosynthetic activities as well as the protein patterns of the membranes are influenced significantly by the oxygen partial pressure. These variations give an idea of the process of membrane differentiation in the membrane system of Rps. capsulata in vivo.The bacteriochlorophyll content of intracytoplasmic membranes is found to vary between 5 g bacteriochlorophyll per mg membrane protein (tubules from cells grown aerobically in the dark) to 195 g bacteriochlorophyll per mg membrane protein (vesicles from cells grown anaerobically in the light). Vesicles from cells grown semiaerobically in the dark exhibit a bacteriochlorophyll content of about 110 g per mg membrane protein. The bacteriochlorophyll values for light grown cells decrease to values below 1% (1.5 g per mg membrane protein) by cultivation at 400 Torr oxygen partial pressure in the dark. In the cytoplasmic membrane of semiaerobically grown cells the bacteriochlorophyll content increases up to 10 g per mg membrane protein. The bacteriochlorophyll content of the intracytoplasmic membranes varies in proportion to the bacteriochlorophyll content of the whole cells.Photophosphorylation of intracytoplasmic membranes is found to be highest in vesicles from light grown cells (19.5 moles PO₄ ^3- per mg membrane protein per 30 min). According to the bacteriochlorophyll content the rates decrease to 10% in tubular membrane fractions from aerobically grown cells. The activity of the NADH oxidase were determined as (moles NADH per mg protein per min): 0.83 in tubules from dark grown cells (150 mm pO₂), 0.17 in vesicles from dark grown cells (5 mm pO₂), and 0.011 in vesicles from cells grown anaerobically in the light. The activities of oxidative phosphorylation do not exactly run parallel to the respiratory values. They were determined as (moles PO₄ ^3- per 30 min per mg protein): 0.74 in vesicles from anaerobically light grown cells, 1.2 in vesicles from dark grown cells (5 mm pO₂) and 2.01 in tubules (150 mm pO₂).Variations of the photosynthetic and respiratory activities are also found in the cytoplasmic membrane, inferring specific processes of membrane differentiation in these structures, other than those in intracytoplasmic membranes. The state of membrane differentiation of intracytoplasmic membranes can be described by the ratio of photophosphorylation to oxidative phosphorylation. In vesicles isolated from light grown cells, the ratio is 25, from dark grown cells it is 5, and in tubules from aerobically grown cells it is 1.When treated with phenol, urea and acetic acid, membranes are split into 10 typical protein bands which can be obtained by polyacrylamide gel electrophoresis. According to the culture conditions, the pattern of specific protein bands is changed in relation to the variation of enzymatic activities and bacteriochlorophyll content of the membranes. During transient experiments, membrane proteins can be labelled specifically if cells are incubated with ¹⁴C (U) protein hydrolysate. In this way, membrane differentiation may be demonstrated by incorporation of specific newly synthesized proteins.

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Abkürzungen Bchl Bacteriochlorophyll - CM Cytoplasmamembran - ICM Intracytoplasmatische Membran - OP Oxydative Phosphorylierung - pO₂ Sauerstoffpartialdruck in mm Hg - PP Photophosphorylierung - GG Glycyl-Glycin-Puffer     

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.     

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     

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。     

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.     

Untersuchungen zum Cytochrom-Oxydase-System aus anaerob im Licht und aerob im Dunkeln gewachsenen Zellen von Rhodopseudomonas capsulata

Zusammenfassung Das aerobe Oxydase-System aus aerob im Dunkeln und anaerob im Licht gewachsenen Zellen von Rps. capsulata wurde untersucht. Die aus Ultraschallextrakten durch 140 000 g-Zentrifugation gewonnen Partikelfraktion katalysiert die Oxydation von NADH, Succinat und reduziertem Cytochrom c (aus Pferdeherz). Die Oxydase-Aktivitäten der Partikel aus aerob im Dunkeln gewachsenen Zellen variieren von 0,10–0,38 mole O₂/min · mg Protein und sind im Durchschnitt 10mal höher als die Oxydase-Aktivitäten der Partikel aus anaerob im Licht gewachsenen Zellen. Die Partikel enthalten Cytochrom vom b-Typ und c-Typ. Cytochrom a konnte weder in den Partikeln aus anaerob im Licht noch in den Partikeln aus aerob im Dunkeln gewachsenen Zellen nachgewiesen werden. In den Partikeln aus aerob gewachsenen Zellen ist das Verhältnis Cytochrom b:Cytochrom c größer als in den Partikeln aus anaerob im Licht gewachsenen Zellen. Die Cytochromoxydase reagiert mit Cytochrom c (aus Pferdeherz), DCPIP und TMPD. Die entsprechenden k _m -Werte betragen 5 · 10^-5 m, 1,5 · 10^-4 m und 2 · 10^-4 m. Die Cytochromoxydase hat ein breites pH-Optimum zwischen pH 8,5 und 9,5. Sättigung der Oxydase mit O₂ tritt erst bei einem Partialdruck von 15 bis 20% O₂ ein. Die Oxydase wird durch KCN und NaN₃ (50% Hemmung bei 10^-5 m), nicht aber durch CO gehemmt. Die Partikel aus aerob im Dunkeln und anaerob im Licht gewachsenen Zellen katalysieren eine mit der Succinat-Oxydation einhergehende Phosphorylierung von ADP mit P/O-Werten von maximal 0,3.

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Studies on the cytochrome oxidase system of ligh-anaerobically and dark-aerobically grown cells of Rhodopseudomonas capsulata

Summary The aerobic oxidase-system from dark-aerobically and light-anaerobically grown Rps. capsulata was investigated. The particulate fraction sedimented from ultrasonic extracts by 140,000 g-centrifugation, catalyzed the oxidation of NADH, succinate and reduced cytochrome c (horse heart). The oxidase activities of the particles from dark-aerobically grown cells were in the range of 0.10–0.38 moles O₂/min x mg protein and were usually ten times as high as the oxidase activities from light-grown cells. The particles contain cytochromes of b-type and c-type. Cytochrome of a-type could be detected neither in the particles from light-grown nor in the particles from dark-grown cells. The highest values of the relation between cytochrome b and cytochrome c were found in the particles from darkaerobically grown cells. The cytochrome oxidase reacts with cytochrome c (horse heart), DCPIP and TMPD. The k _m -values are 5×10^-5 m, 1.5×10^-4 m, or 2×10^-4 m, respectively. The cytochrome oxidase exhibits a broad pH-optimum in the range of pH 8.5–9.5. Saturation of the oxidase with O₂ is observed at a partial pressure of 15–20% O₂. The oxidase is inhibited by KCN and NaN₃ (half inhibition at 10^-5 m), but not by CO. The particles from dark-aerobically and light-anaerobically grown cells catalyze phosphorylation of ADP in the dark coupled to the oxidation of succinate with maximum P/O-values of 0.3.

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Abkürzungen ADP Adenosindiphosphat - ATP Adenosintriphosphat - BChl Bacteriochlorophyll - CCCP Carbonylcyanid-m-chlorphenylhydrazon - DCPIP 2,6-Dichlorphenolindophenol - DNP 2,4-Dinitrophenol - NAD(P) Nicotinamid-Adenin-Dinucleotid(phosphat) - NAD(P)H reduziertes NAD(P) - R. Rhodospirillum - Rps. Rhodopseudomonas - TMPD N-Tetramethyl-p-phenylendiamin     

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     

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.     

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.