Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha.

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorphia has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop growth on or methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H2O2 dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H2O2 dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H2O2 was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H2O2 for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.     

Separation of mitochondria from microbodies of Pisum sativum (L. cv. Alaska) cotyledons

A method is described for separating mitochondria from microbodies in cotyledon preparations of Pisum sativum L. cv. Alaska. Pure and intact mitochondria were obtained on a continuous: discontinuous sucrose density gradient as shown by marke-enzyme assay and electron microscopy. Manipulation of sucrose-gradient construction to widen the distance between organelles provided a quick method for the separation of the mitochondria from the microbodies. The shorter time of exposure of mitochondria to centrifugation and osmotic stress produces mitochondria free of contamination.     

Microbody of n-alkane-grown yeast

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and d-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, malate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 nor NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.     

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorpha has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop during growth on methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H₂O₂ dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H₂O₂ dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H₂O₂ was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H₂O₂ for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.     

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b₅ reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm⁻³, a membrane fraction at 1.17 g × cm⁻³ containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.     

Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively. Received: 21 September 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

The occurrence of microbodies and peroxisomal enzymes in achlorophyllous leaves

Summary Several types of leaves of leaf parts lacking chlorophyll were fixed and embedded according to conventional procedures and examined electron-microscopically for microbodies. Comparisons of relative abundance of microbodies, plastids and mitochondria were made by computing the average numbers of organelle profiles per cell section. Similar leaves were homogenized and assayed for three enzymes characteristic of leaf peroxisomes. The localization of these enzymes in microbodies was indicated for the achlorophyllous tissues by the positive result obtained when 3,3-diaminobenzidine was used as an electron cytochemical stain for catalase activity.Microbodies were present in all non-photosynthetic leaves or leaf parts examined, including yellowish-white segments of variegated leaves, albino leaves, and etiolated leaves of two species. In several cases, the numbers of microbody profiles per cell section were as great in the achlorophyllous leaves as in the chlorophyllous. The levels of peroxisomal enzyme activity in the yellowish-white leaves were substantial, although often not as high as in the green leaves. It was concluded that enzymatically these microbodies are probably similar to the peroxisomes characterized from chlorophyllous leaves. In the absence of the photosynthetic product, glycolate, however, it seems unlikely that the organelle is performing the same functions as in green leaves. It is also apparent that the initial formation of peroxisomes in leaves can occur when neither light nor a photosynthate such as glycolate is present as an inducer.     

The intramitochondrial location of cytochrome c peroxidase in wild-type and petite Saccharomyces cerevisiae

Cytochemical and ultrastructural analysis of wild-type cells of Saccharomyces cerevisiac, grown aerobically in a glucose-limited chemostat, shows that cytochrome c peroxidase is localized between the membranes of the cristae, that is, in the intracristal space. This enzyme is thus positioned appropriately within the organelle to act as an alternate terminal oxidase for the respiratory chain. The proximity of the peroxidase to major sites of generation of its two substrates may account for the small leakage of hydrogen peroxide from yeast mitochondria, as compared with the larger outflow from mammalian mitochondria.In the cytoplasmic petite mutant, gross distortion of promitochondrial membrane arrangement is evident. Nevertheless, cytochrome c peroxidase activity is present in the same amounts as is found in wildtype cell, and is localized predominantly within annuli of membrane which constitute the promitochondria in these cells.No unequivocal evidence was obtained for the localization of catalase in microbodies or other organelles in either wild-type or petite cells.     

Occurrence of microbodies in the green algaBracteacoccus cinnabarinus grown heterotrophically

Summary A comparative study was made of the ultrastructure and abundance of microbodies in the green algaBracteacoccus cinnabarinus grown photoautotrophically and heterotrophically on a conventional culture medium containing sodium acetate, potassium acetate and glucose. Several changes were observed in the cells maintained under these conditions. Most noticeably, cells grown on acetate in both light and dark were packed with lipid bodies. Microbodies were found to be closely appressed to the lipid bodies in cells grown heterotrophically in the dark on sodium acetate and potassium acetate. The average number of microbody profiles per cell was in general threefold greater in cells grown on sodium acetate than those grown on potassium acetate. No microbodies were observed in cells maintained photoautotrophically on the three carbon sources or in cells maintained photoautotrophically on Bristol's inorganic medium alone. Cytochemical staining with 3,3-diaminobenzidine indicated the presence of catalase in the microbodies. The presence of microbodies suggests that the organelle may be performing functions similar to glyoxysomes in higher plants, namely the net conversion to succinate of acetyl CoA derived from lipid degredation. It is also apparent thatBracteacoccus can grow well as a heterotroph in the dark when acetate is included in the culture medium as a source of carbon.     

Evidence for two classes of microbodies in meiocytes of the red algaPalmaria palmata

Summary Microbodies, usually spherical and about 0.2 m in diameter, were found to be associated with prophase nuclei in vegetative cells and meiocytes of the red algaPalmaria palmata. Nucleus-associated microbodies in meiocytes were numerous, but they did not react to the DAB cytochemical test for catalase and peroxidase activity. Microbodies not associated with nuclei in the same cells were intensely DAB-positive. Neither aminotriazole nor potassium cyanide inhibited the DAB reaction.     

Ultrastructure of methanotrophic yeasts.

The cellular structure of two yeast strains capable of growth on methane was investigated by electron microscopy. Microbodies were observed in cells of Sporobolomyces roseus strain Y and Rhodotorula glutinis strain CY when grown on methane but rarely when grown on glucose. The size of the microbodies and the number observed per cell in a thin section did not increase with culture age. No crystalline organization was observed within these organelles. Similar microbodies were also observed in cells of R. glutinis CY grown on hexadecane. The plasma membranes of both methane and hexadecane-grown cells exhibited increased invagination compared to that of glucose-grown cells. Catalase activity was detected in the microbodies of alkane-grown cells by using 3,3'-diaminobenzidine as a cytochemical stain. The data presented suggest that microbodies, and the catalase contained within them, play a role in eucaryotic methane metabolism.     

Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger

Summary The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both enzymes were located in microbodies. In addition, when the organism was grown on glucose with methylamine as a nitrogen source, amine oxidase activity was detected in the microbodies. These organelles can therefore be designated as peroxisomes.     

The structure of microbodies and their associations with other organelles in zoosporangia ofEntophlyctis variabilis

Summary The ultrastructure of microbodies in developing zoosporangia ofEntophlyctis variabilis was studied by three dimensional reconstructions from serial sections and by cytochemical localization of catalase activity. The morphology of microbodies and the spatial association of microbodies with other organelles varied during fungal development. In incipient zoo-sporangia, granular dilations resembling microbodies arose from rough ER. Young, enlarging zoosporangia contained elongate, contorted microbodies continuous with ER and aligned along bundles of microtubules. Oval, paired microbodies, lying on each side of an ER cisternae, were found in all zoosporangia, but in older zoosporangia this configuration of microbodies predominated. Analysis of serial sections revealed that these oval, paired microbodies were sometimes continuous with each other, with ER, and also apparently with the ER cisterna interposed between them. Other paired, oval microbodies were clearly discrete. Constrictions were found along the length of elongate microbodies and at junctions between oval microbodies. These constrictions may represent stages in fragmentation of microbodies from pre-existing microbodies. These observations suggest that microbodies originated in three ways: 1. as local dilations in tubular ER, 2. as lateral buds from opposite sides of ER cisternae, and 3. as fragments from elongate microbodies.Microbodies were consistently spatially associated with ER, nuclear envelopes, and mitochondria. The cisterna of ER passing between paired microbodies sometimes extended into a branching, tubular system of ER which curved around the side of one microbody and lay between this microbody and the forming face of a dictyosome. The cytochemical localization of thiamine pyrophosphatase activity in this cisterna when it is not associated with dictyosomes suggests a role in metabolic control. These spatial associations indicate that the microbody assemblage with other organelles represents functional units where propinquity to other organelles and intraluminal continuities insure a system for transport of substrates and products.     

Physiological role of microbodies in the yeast Trichosporon cutaneum during growth on ethylamine as the source of energy,carbon and nitrogen

Compartmentation of the metabolism of ethylamine in Trichosporon cutaneum X4 was studied in cells, grown on this compound as the sole source of energy, carbon, and nitrogen. Transfer experiments indicated that an amine oxidase is involved in the early metabolism of ethylamine. The synthesis of this enzyme was induced by primary amines and was subject to partial carbon catabolite repression. Repression by ammonium ions was not observed. Adaptation of glucose-grown cells to growth on ethylamine was associated with the development of many microbodies, which developed from already existing organelles present in the inoculum cells and multiplied by division. Cytochemical experiments indicated that the organelles contained amine oxidase and catalase. Therefore, they were considered to play a key role in the metabolism of ethylamine. The physiological significance of the microbodies was investigated by fractionation studies of homogenized protoplasts from ethylamine-grown cells by differential- and sucrose-gradient centrifugation of subcellular organelles. Intact microbodies were only obtained when the isolation procedure was performed at pH 5.8 in the absence of Mg²⁺-ions. Analysis of the different fractions indicated that the key enzymes of the glyoxylate cycle, namely isocitrate lyase and malate synthase, cosedimented together with catalase and amine oxidase. In addition, activities of malate dehydrogenase, glutamate:oxaloacetate aminotransferase (GOT) and (NAD-dependent) glutamate dehydrogenase were detected in these fractions. Electron microscopy revealed that they mainly contained microbodies. Cytochemical experiments indicated that the above enzymes were all present in the same organelle. These findings suggest that microbodies of ethylamine-grown T. cutaneum X4 produce aspartate, so allowing NADH generated in the oxidation of malate by malate dehydrogenase to be quantitatively reoxidized inside the organelles in a series of reactions involving GOT and glutamate dehydrogenase. Aspartase and fumarase were not detected in the microbodies; activities of these two enzymes were present in the cytoplasm.Abbreviations ABTS 2,2-Azino-di(3-ethylbenzthiazoline sulfonate [6]) - DTT dithiothreitol - GOT glutamate:oxaloacetate aminotransferase - DTNB 5,5-dithiobis-2-nitrobenzoate - DAB diaminobenzidine - BSPT 2-(2-benzothiazolyl)-3-(4-phthalhydrazidyl)-t-styryl-sH-tetrazolium chloride - PF convex fracture face - EF concave fracture face     

Occurrence of microbodies in chloromonadophycean algae

Microbody-like organelles occur in the cytoplasm of two chloromonadophycean algae,Vacuolaria virescens Cienkowsky andGonyostomum semen Diesing. Microbodies ofVacuolaria andGonyostomum have a granular matrix which lacks a crystalloid core; they are often present in close association with elements of the endoplasmic reticulum. The occurrence of microbodies in other algae is briefly reviewed.     

Isolation and Characterization of Microbodies from the Alga Chlorogonium elongatum

The alga Chlorogonium elongatum was grown autotrophically or heterotrophically on acetate. Cells harvested in the logarithmic phase of growth were disrupted, and the whole homogenates were fractionated on sucrose gradients. Protein and enzyme determinations carried out on the fractions led to the following conclusions. Chloroplast fragments which represent the major portion of particulate protein in autotrophic cells migrate to density 1.17 g/cm³. In heterotrophic cells, mitochondria comprise most of the particulate protein, and these particles accumulate at density 1.19 g/cm³, as shown by a peak of cytochrome oxidase in this region. Part of the catalase and uricase, two marker enzymes for microbodies, were found in the soluble fractions, but 60% or more of these activities were recovered at density 1.225 g/cm³ from autotrophic cells. Electron micrographs showed that in this region there were microbodies with a diameter of 0.4 micrometer. The isolated microbodies contained no isocitrate lyase, a marker enzyme of glyoxysomes. This enzyme was completely soluble and therefore seems not to be associated with organelles in this organism.     

Isolation of microbodies from, sweet potato root tissue and their development during aging of slices

Microbodies were isolated from, sweet potato root tissue bydifferential and linear sucrose density gradient centrifugation.When the tissue was homogenized in the presence of PolyclarAT, the microbodies sedimented together with the mitochondriathrough the sucrose gradients. The microbodies had a densityof 1.25 g/cm³, and contained catalase and urate oxidase, butnot malate dehydrogenase, isocitrate lyase, glycolate oxidase,hydroxypyruvate reductase and the cyanide-insensitive palmitoylCoA-oxidation system. A small amount of o-diphenol oxidase alsoseemed to be present. Catalase, but not urate oxidase, activity in the crude extractincreased during aging of the sliced tissue. A similar resultwas obtained with the microbody fraction after linear sucrosedensity gradient centrifugation. We propose that microbodiescontaining only catalase develop during aging of sliced sweetpotato root tissue. ¹ This work was supported in part by a Grant-in-Aid (No. 311908)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received June 20, 1979; )     

Tubuläre Einschlüsse in Microbodies vonSaccharomycopsis (Candida) lipolytica-Protoplasten

Zusammenfassung Auf Ultradünnschnitten Glutaraldehyd-OsO₄ oder KMnO₄ fixierterSaccharomycopsis lipolytica-Zellen, die in einem Medium mit Laktat als C-Quelle wuchsen, werden durchschnittlich 3–4 Anschnitte von Microbodies gefunden. Ihre Anzahl erhöht sich etwa um das Dreifache, wenn n-Hexadecan die C-Quelle ist.Nach der Umwandlung der Zellen zu Protoplasten mit Hilfe von Enzymgemischen vonHelix pomatia enthalten die Microbodies tubuläre Einschlüsse. Diese Tubuli, deren äußerer Durchmesser etwa 25 nm beträgt, haben eine zentrale Verdichtung und erreichen Längen bis zu 0,8 m. Sie sind in Bündeln oder Schichten angeordnet. Die Tubuli bilden sich möglicherweise durch Assemblierung von Enzymproteinen, wenn die Zellen während der Umwandlung zu Protoplasten mit hypertonischen Lösungen osmotisch geschockt werden.

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Tubular inclusions within microbodies ofSaccharomycopsis (Candida) lipolytica-Protoplasts

Summary Electron micrographs ofSaccharomycopsis lipolytica-cells grown in a medium with lactate as carbon source and fixed with glutaraldehyde-OsO₄ or KmnO₄ show 3–4 profiles of microbodies per section in average. The number of microbodies is about three times greater when cells are cultivated in a medium with n-hexadecane. The spherical to avoid microbodies (0.3–0,8 m in diameter) have a homogeneous matrix.After conversion of cells into protoplasts by the aid of snail gut juice tubular inclusions occur in microbodies. The single tubulus has an outer diameter of about 25 nm, a length up to 0.8 m and contains a central rod. Several tubules form bundles or layers. It is suggested that the tubules could be enzyme protein assembling by osmotic shock with hypertonic solutions during preparation of protoplasts.

     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.