Haemoproteins and haem synthesis in facultative photosynthetic and denitrifying bacteria

1. A simple spectrophotometric method is described for the measurement of various haemoproteins in extracts of photosynthetic and non-photosynthetic bacteria. The method is based on measurements of difference spectra at the Soret maxima. 2. In photosynthetic bacteria of the Athiorhodaceae group the concentration of carbon monoxide-binding haemoprotein and of cytochromes of the b and c types is two to three times as high in anaerobically grown cells as in those grown aerobically. 3. During the adaptation of Rhodopseudomonas spheroides 8253 to form photosynthetic pigments the concentration of each of these haemoproteins increases in parallel with that of the bacteriochlorophyll. 4. The carbon monoxide-binding haemoprotein in aerobically grown Rps. spheroides 8253, in contrast with anaerobically grown cells, is predominantly in the particulate fraction of extracts prepared by ultrasonic vibration. The b- and c-type cytochromes are approximately equally distributed between each fraction in extracts from both types of cell. 5. Extracts of Micrococcus denitrificans grown anaerobically on nitrate contain more cytochromes of the b and c types, as well as of the carbon monoxide-binding pigment, than do those from aerobically grown cells. 6. The activity of ferrochelatase in both Rps. spheroides 8253 and M. denitrificans was similar in extracts from cells grown aerobically and anaerobically, though the haemoprotein content was higher under the latter conditions. Coproporphyrinogen oxidative decarboxylase could not be demonstrated in cell-free extracts of either organism.     

Specificity of the transhydrogenase factor for chromatophores of Rhodopseudomonas spheroides and Rhodospirillum rubrum

Extensive washing of chromatophores of Rhodospirillum rubrum and Rhodopseudomonas spheroides with dilute buffer results in a complete loss of the energylinked transhydrogenase activities of Rsp. rubrum but only a partial loss of the light-driven reaction in chromatophores of Rps. spheroides. It was not possible to reactivate the Rps. spheroides transhydrogenation with the Rsp. rubrum transhydrogenase factor nor with a protein fraction of Rps. spheroides isolated by procedures identical to that used for the isolation of the Rsp. rubrum transhydrogenase factor. The Rsp. rubrum factor is highly specific and cannot be replaced by a number of sulfhydryl compounds tested for reconstitution of Rsp. rubrum transhydrogenation. A published procedure for the isolation of a “transhydrogenase factor” from Rps. spheroides chromatophores yields a preparation having energy-dependent transhydrogenation when supplemented with dithiothreitol in the absence of added chromatophores.     

Differences in the control of bacteriochlorophyll formation by light and oxygen

Control of bacteriochlorophyll formation was studied with continuous cultures of Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas capsulata. Oxygen controlled specific bacteriochlorophyll contents of the three species in a hyperbolical fashion irrespective of the presence of light. In Rps. sphaeroides, this applied to oxygen concentrations above 16% air saturation of the medium while at lower oxygen concentrations control followed a kinetics with negative cooperativity. Cell protein formation of R. rubrum and Rsp. sphaeroides was independent of oxygen concentrations while protein formation of Rps. capsulata increased at lower concentrations. Light controlled bacteriochlorphyll contents of R. rubrum and Rps. sphaeroides in a sigmoidal fashion. When growing at a constant low oxygen concentration cell protein formation increased with light energy flux in Rps. sphaeroides but remained unaffected in R. rubrum. Protein formation of R. rubrum increased with light energy flux only under anaerobic conditions. Two factor analyses were performed with R. rubrum and Rps. sphaeroides to study the combined effects of light and oxygen on bacteriochlorophyll formation. The results showed that both factors act independent of each other.Abbreviations ALA 5-aminolevulinic acid - R Rhodospirillum - Rsp. Rhodopseudomonas     

Detection of two further b-type cytochromes in Rhodopseudomonas spheroides

The photosynthetically-incompetent mutant V-2 of Rhodopseudomonas spheroides which is incapable of synthesising bacteriochlorophyll was grown aerobically under conditions of both high and low aeration. Potentiometric titration at 560 nm minus 570 nm revealed the presence of several different components tentatively identified as b-type cytochromes. Two such components of oxidation-reduction midpoint potentials of +390 mV ± 10 mV and +255 mV ± 7 mV have not previously been detected in membranes of Rps. spheroides. These components have also been resolved by difference spectra at controlled oxidation-reduction potentials and fourth derivative spectra. Neither component appeared to react with CO. With increasing aeration of the culture medium the relative concentration of these two b-type cytochromes diminished, whilst that of the a-type oxidase increased.     

Preferential inhibition by ethidium bromide of photosynthetic apparatus formation in Rhodopseudomonas spheroides cells

Ethidium bromide (EB) inhibited growth of cells of the non-sulfurpurple photosynthetic bacterium Rhodopseudomonas spheroides.The inhibitory action of EB on the light-anaerobic culturedcells was stronger than on dark-aerobic cultured ones. EB alsosuppressed the induced synthesis of bacteriochlorophyll (Bchl)and carotenoids in the dark-aerobically grown cells incubatedwith gentle aeration under no-growth conditions, suggestingthat the target of the inhibitory action of EB on the photosyntheticgrowth of R. spheroides cells is chromatophore formation. EBdepressed the incorporation of ³H- or ¹⁴C-uracil into both RNAand DNA fractions from cells incubated with gentle aeration.In contrast, inhibition by EB of ³H-uracil incorporation intothe DNA fraction was not observed under vigorous aeration. Ourfindings seemed to favor the hypothesis described previously(10) that lowering the intracellular oxidation-reduction potentialmight bring about a unique synthesis or turnover of DNA responsiblefor chromatophore formation. (Received December 22, 1977; )     

Bacteriochlorophyll Synthesis and the Ultrastructure of Wild Type and Mutant Strains of Rhodopseudomonas spheroides

The ultrastructure of sectioned cells of mutant and wild type Rhodopseudomonas spheroides has been examined by electron microscopy. The characteristic vesicles associated with the presence of bacteriochlorophyll were found in wild type cells grown with low aeration. These were also found in mutant TA-R which forms bacteriochlorophyll under high aeration. None of the mutants with blocks in bacteriochlorophyll synthesis contained intracytoplasmic membrane. These included mutant 8-17 which accumulates bacteriochlorophyllide but fails at the phytolation step. We conclude that the intact bacteriochlorophyll molecule, or some particular membrane protein associated with it, is needed for the development of the characteristic intracytoplasmic membrane system in R. spheroides.     

Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeastSchizosaccharomyces pombe

Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grownSchizosaccharomyces pombe cells was assessed, and compared with that ofSaccharomyces cerevisiae in order to determine the catalase activity regulation inS. pombe. In contrast toS. cerevisiae, the total catalase activity of permeabilizedS. pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e. it is not induced by oxygen and/or substrates), and only a negligible part (3–5%) of it is contributed byde novo protein synthesis during aeration with or without substrates. The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7–8-fold in the presence of glucose or ethanol. The increase is not inhibited by cycloheximide and is thus not due tode novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.     

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     

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 Ausscheidung von partikelgebundenen Bacteriochlorophyllvorstufen durch die Mutante F9 von Rhodospirillum rubrum

Zusammenfassung Eine bacteriochlorophyllfreie Mutante von Rhodospirillum rubrum wird beschrieben. Diese Mutante scheidet die Bacteriochlorophyllvorstufen Phaeophorbid a und 2-Devinyl-2--Hydroxyäthyl-Phaeophorbid a unter semiaeroben Bedingungen in das Kulturmedium aus. Beide Pigmente sind mit einem Trägermolekül zu einem Komplex verbunden. Die Analyse des Komplexes ergab folgende Zusammensetzung: 49% Protein, 30% Pigment, 11% Lipide, 3% Zucker, sowie eine geringe Menge Phosphor (0,2%). Die Aminosäurezusammensetzung des Proteinanteils wird angegeben. Die Fettsäuren werden gaschromatographisch bestimmt. Die Zuckerkomponente ist anders zusammengesetzt als das Lipopolysaccharid aus Rhodospirillum rubrum. Das Molekulargewicht der kleinsten Proteinuntereinheit beträgt 16500, das Molekulargewicht des Pigmentkomplexes ergibt sich daraus mit 34000. Typische Thylakoide werden in der Mutante nicht ausgebildet. Die Notwendigkeit einer ungestört ablaufenden Bacteriochlorophyllsynthese für die normale Thylakoidmorphogenese wird diskutiert.

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The production of particle bound bacteriochlorophyll precursors by the mutant F9 of Rhodospirillum rubrum

Summary A mutant strain of Rhodospirillum rubrum is described which does not form bacteriochlorophyll. This mutant strain excretes the bacteriochlorophyll precursors pheophorbide a and 2-devinyl-2-hydroxyethylpheophorbide a into the cultural medium, when it is cultured under low aeration in the dark. The pigments were identified spectroscopically. Both of the pigments are bound to a macromolecular compound. The complete macromolecule was shown to be composed of 49% protein, 30% pigments, 11% lipid, 3% sugar, and a trace of phosphorus (0.2%). The amino acid composition of the protein as well as the fatty acid pattern of the lipid are presented. The percent portions of fatty acids in whole cells are quite different from that of the pigment complex. The composition of the sugar moiety was demonstrated to be different from that of the lipopolysaccharide of Rhodospirillum rubrum. The molecular weight of the smallest subunit of the protein is 16,500. This suggests a molecular weight of 34,000 for the total pigment complex. Typical thylakoids were not observed in cells of the mutant strain. The necessity of an unblocked bacteriochlorophyll synthesis for the normal thylakoid morphogenesis is discussed.

     

Mechanism of catalase induction in Rhodopseudomonas spheroides: Role of porphyrin excretion

Summary Catalase induction in Rhodopseudomonas spheroides and three locally isolated strains of Rhodopseudomonas (TL-1, TL-4 and Rps. D) was studied. A correlation between the rate of excretion of porphyrin and the inducibility of the culture was observed. 8-hydroxyquinoline at 4x10^-5 M had no effect on Bchl synthesis but it inhibited porphyrin excretion and catalase induction in Rhodopseudomonas spheroides. 8-OH Q at this concentration partially inhibited Bchl synthesis and catalase induction in strain TL-1. The data indicate that the amount of catalase produced is dependent on the pool size of porphyrins.Abbreviations used ALA -amino laevulinic acid - Bchl Bacteriochlorophyll - EDTA Ethylene diamine tetra acetate - DMSO Dimethyl sulfoxide - 8-OH Q 8-hydroxyquinoline - nm nanometer - Rh. Rhodopseudomonas This work is part of a thesis submitted by K. T. Shanmugam in partial fulfillment of the requirements for the Ph. D. degree.     

Ultrastructure of the Acidophilic Aerobic Photosynthetic Bacterium Acidiphilium rubrum

The ultrastructure of cells of Acidiphilium rubrum, which is an acidophilic aerobic photosynthetic bacterium containing zinc-complexed bacteriochlorophyll a, was studied by electron microscopy with the rapid substitution technique. Thin-section electron microscopy indicated that any type of internal photosynthetic membranes was not present in this organism despite a relatively high content of the photopigment. The majority of cells had poly-β-hydroxybutyrate granules and electron-dense spherical bodies identified as being polyphosphate granules. When the organism was grown chemotrophically with 0.1% FeSO₄, it produced another group of electron-dense granules that were associated with the inner part of the cytoplasmic membrane. An energy-dispersive X-ray analysis showed that these membrane-bound, electron-dense granules contained iron. Received: 24 November 1999 / Accepted: 5 January 2000     

Phytol and Bacteriochlorophyll Synthesis in Rhodopseudomonas spheroides

Phytol has been separated and identified in the unsaponifiable lipid fraction from wild type Rhodopseudomonas spheroides, but it was not detected in mutant strains blocked at various stages of bacteriochlorophyll synthesis. Incorporation of ¹⁴C-acetate into phytol paralleled bacteriochlorophyll synthesis in suspensions of the wild type incubated anaerobically in the light. The addition of chloramphenicol inhibited both processes. It is concluded that phytol formation is tightly coupled to the synthesis of the pyrrole component of bacteriochlorophyll.     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

The distribution of NADH oxidase in the membrane system of Rhodospirillum rubrum

Summary The activity of NADH oxidase in cell fractions of R. rubrum was measured. In cells grown photosynthetically (anaerobic in the light) and semiaerobically in the dark, the highest activity was found to be in the 19,000xg pellet. This consisted preponderantly of cytoplasmic membrane and cell wall material. Bchl was more enriched in the purified thylakoids than the NADH oxidase activity.In extracts of cells grown aerobically the oxidase activity was higher than in cells grown anaerobically or semiaerobically. The highest activity was recovered in the 220,000xg pellet. The data suggest that NADH oxidase as well as bacteri ochlorophyll can be localized in the total intracellular membrane system. However, the distribution is inhomogeneous. Most of the NADH oxidase activity is localized in the c. m. region and most of bchl in the thylakoids.Abbreviations c. m. cytoplasmic membrane - th. Thylakoid - bchl bacteriochlorophyll - Tris Tris (hydroxymethyl) amino-methane     

Effects of ionophores and TPMP+ on light-induced responses in Dictyostelium discoideum

In spite of previous reports, the activities of respiratory oxygen uptake by whole cells are higher with chemotrophically than with phototrophically grown cells of Rhodospirillum rubrum and Rhodospirillum tenue. The same applies to NADH dependent respiratory reactions as determined with isolated crede membrane preparations. This is largely, but not only, due to an outstandingly high increase in activity of cytochrome c-oxidase measurable upon adaptation of phototrophically grown cells to chemotrophic conditions. In R. rubrum the dependency of the total respiratory chain on the activities of different sections of this chain becomes confused by the presence of differently composed membranes (i.e. cytoplasmic and intracytoplasmic membranes) which under the experimental conditions become functionally differentiated to different extents. But in R. tenue, which does not produce intracytoplasmic membranes, respiration at low activities parallels clearly cytochrome c oxidase activities while high respiratory activities parallel the activities of NADH dehydrogenase. The data are interpreted to indicate that, in cells of facultative phototrophic bacteria, the formation of the respiratory chain, up to certain stages, depends on the formation of the terminal oxidase. At least in R. tenue this is comparable to the role of bacteriochlorophyll in the formation of the photosynthetic apparatus.Abbreviation Bchl bacteriochlorophyll     

Cytochromes ofAcetobacter pasteurianus NCIB 6428: Reaction with oxygen of cytochromeo in cells,membranes, and nonsedimentable subcellular fractions

Cytochromeo ofAcetobacter pasteurianus NCIB 6428, grown with either vigorous or limiting aeration, was located in both membranes and a fraction nonsedimentable by centrifugation (13.5×10⁶ g min) of a French press extract. In oxygen-limited cells, the reaction with oxygen of the oxidase at low temperatures resembled that inEscherichia coli with respect to the spectrum and formation kinetics of an oxygenated intermediate. Both membranous and nonsedimentable cytochromeso formed this intermediate, but faster and at lower temperatures in the latter. The role of this cytochrome, particularly in the light of other bacterial, soluble, ligand-binding hemoproteins, and the kinetic constraints on its reaction are discussed.     

Microbial oxidation of protoporhydrinogen, an intermediate in heme and chlorphyll biosynthesis.

The oxidation of protoporphyrinogen to protoporphyrin, a late step in heme and chlorophyll synthesis, is catalyzed aerobically by a particulate fraction of Escherichia coli at a rate significantly higher than the rate of autooxidation. This activity is heat labile and is markedly inhibited by addition of respiratory substrates such as NADH. NADH is oxidized at a rate 100-fold higher than protoporphyrinogen. Particles from a cytochrome-less mutant of E. coli were markedly deficient in protoporphyrinogen oxidizing activity. Particles from a quinone-deficient mutant were also deficient. These findings suggest a possible role for the electron transport system in aerobic protoporphyrinogen oxidation. This activity was also examined in a variety of other bacteria. Particles from Streptococcus faecalis, which does not synthesize heme, were unable to oxidize protoporphyrinogen, confirming the specificity of this activity. Particles from aerobically grown Staphylococcus aureus exhibited protoporphyrinogen oxidizing activity, but particles from anaerobically grown cells had no activity above that of the nonenzymatic control. This indicates the repressible nature of this activity, and may also explain why Staphylococci synthesize cytochromes during aerobic, but not during anaerobic growth. Particles from photosynthetically grown Rhodopseudomonas spheroides, which contain both chlorophyll and heme, oxidized protoporphyrinogen at a rate no higher than the nonenzymic control. However, particles from cells grown aerobically, when bacteriochlorophyll synthesis is markedly repressed, readily exhibited protoporphyrinogen oxidizing activity. These initial findings suggest that this activity is detectable in cells primarily synthesizing heme, but not in cells primarily synthesizing bacteriochlorophyll, and could have implications both for the mechanism and regulation of the heme and bacteriochlorophyll pathways.     

Effect of iron and aeration on superoxide dismutase and catalase activity of PHB-producing Azotobacter chroococcum

The effect of aeration level and iron concentration on Azotobacter chroococcum 23 growth, PHB accumulation and antioxidative enzyme activities was investigated in shake flask experiments. Biomass yield and carbon source conversation coefficients increased in the presence of iron in the growth medium and under decreased aeration. The highest biomass production was observed for the culture grown in a medium with 36 μM of initial iron concentration and moderate aeration level. The highest PHB accumulation level (70–72% from cell dry weight) under our experimental conditions was observed at decreased aeration in the growth medium with 180 μM of initial iron concentration. Results obtained prove that both aeration level and iron supply have a marked influence on the activity of SOD and catalase. Bearing in mind the necessity of iron for the synthesis of both enzymes, only catalase showed a specific dependence on the intracellular iron accumulation level.     

Vitamin B(12) in photosynthetic bacteria and methionine synthesis by Rhodopseudomonas spheroides

1. Assay of some photosynthetic bacteria for vitamin B₁₂ showed them to be relatively rich in this factor. Rhodopseudomonas spheroides, grown photosynthetically in Co²⁺-supplemented medium, contained about 100μg./g. dry wt. 2. Extracts of wild-type Rps. spheroides methylated homocysteine by a mechanism similar to the cobalamin-dependent pathway present in Escherichia coli. However, no mechanism similar to the cobalamin-independent N⁵-methyltetrahydrofolate–homocysteine transmethylase of E. coli could be detected in Rps. spheroides. 3. N⁵N¹⁰-Methylenetetrahydrofolate-reductase activity was found in Rps. spheroides. 4. A methionine-requiring mutant strain of Rps. spheroides (strain 2/33), which does not respond to homocysteine, made the same amount of vitamin B₁₂ as the parent organism. Extracts did not form methionine from N⁵-methyltetrahydrofolate and homocysteine even in the presence of cofactors shown to be necessary with the parent strain, and it is concluded that the mutant is blocked in the formation of the apoenzyme of a homocysteine-methylating system similar to the vitamin B₁₂-dependent one in E. coli.