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.     

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.     

Spheroplast formation and partial purification of microbodies from hydrocarbon-grown cells ofCladosporium resinae

Summary Cells ofCladosporium resinae form greater numbers of microbodies when grown onn-alkanes than when grown on glucose. To facilitate isolation of microbodies, hydrocarbon-grown cells were spheroplasted. Of four spheroplasting agents and five osmotic supports examined, best results were obtained after a 4-h incubation with Novozym 234 plus chitinase and with 0.8 M sorbitol as osmotic support. Equal numbers of spheroplasts were obtained at pH 5.8 and at pH 7.0. Catalase was used as a marker for microbodies and cytochrome-c oxidase as a marker for mitochondria. Urate oxidase, a second marker for microbodies, was not detected in cell extracts. Microbodies were extremely fragile; of eight spheroplast disruption techniques attempted, the best yield of microbodies was obtained using a Teflon homogenizer for 5 min. Microbodies were partially purified by differential and density gradient centrifugation. Best results were obtained with discontinuous Percoll gradients which yielded a fraction enriched in microbodies and one enriched in mitochondria.     

Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity.

Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.     

Cytochemical demonstration of malate synthase and glycolate oxidase in microbodies of cucumber cotyledons

The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.     

WHAT ARE THE CHROMIDIA OF POLYPHAGUS EUGLENAE?

Zoospores, prosporangia, and asexual sporangia were studied with electron microscopy to determine the ultrastructural identification of “chromidia,” granular masses surrounding nuclei that classical mycologists believed to be extruded chromatin used for lipid synthesis. In the zoospore the nucleus was enclosed by an aggregation of ribosomes. In other developmental stages the behavior of microbodies was identical to that described for “chromidia.” A microbody network with interspersed ER surrounded nuclei in young prosporangia. As the prosporangium matured, lipid globules became associated with the microbodies. When the single, large nucleus migrated into the elongate asexual sporangium, microbodies still surrounded the nucleus; but after the nucleus divided and a multinucleate sporangium formed, microbodies were scattered throughout the cytoplasm. When incubated in the diaminobenzidine medium for the cytochemical detection of catalase, reaction product was found in these microbodylike structures, confirming that “chromidia” described in prosporangia and asexual sporangia by classical mycologists are really microbodies. Rather than giving rise to lipid, these microbodies are probably involved in the metabolism of the lipid globules with which they are associated. The “chromidia” in zoospores are not extruded chromatin as suggested earlier, but correspond in their location around the nucleus to an aggregation of ribosomes.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.     

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.     

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.     

OBSERVATIONS ON THE MICROBODIES IN THE GENUS TILLANDSIA (BROMELIACEAE)

Organelles morphologically similar to microbodies have been found in several tissues of atmospheric species of Tillandsia from different habitats. The presence of catalase was demonstrated by the DAB reaction thus confirming the microbody nature of these organelles. They are a feature of the Tillandsia species with normal photosynthetic carbon fixation and with CAM. Their size is consistently small. The nucleoid observed in the microbodies shows a characteristic morphology which has not been reported before within other plant microbodies. This nucleoid is composed of minute tubular structures, for which the authors here propose a three-dimensional arrangement.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

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.     

Changes in Parenchyma Cells of Poplar Xylem during Transition from Growing to Wintering Stages

Ultrastructural changes in xylem parenchyma cells during transitionfrom growing to wintering stages were investigated. Changesin the fine structures of parenchyma cells in differentiatedxylem started in mid-August. At this time, dictyosomes werefrequently found and vesicles were abundant in the cells inwhich active formation of cellular structures took place. Thecytosol was filled with polysomes. In mid-September, the cytoplasmof the parenchyma cells gradually began to fill up with microbodies,and the endoplasmic reticulum cisternae had already startedto decrease. Dictyosomes and vesicles were still abundant atthis stage and microbody-associated vesicles could be seen,indicating participation of the vesicles in the formation ofmicrobodies at this stage. A direct structural connection withrespect to the biogenesis of the microbodies remained unclear.In the early stage, the microbodies showed a dense center surroundedby a less dense outer surface, and in the latter stage in November,the outer surface exhibited an increased accumulation of osmiophilicmaterials. There were few further changes in the overall patternof the fine structures until mid-winter. (Received July 2, 1987; Accepted November 30, 1987)     

Mitochondrial morphology in Basidiobolus haptosporus

Young hyphal cells of the potentially zoopathogenic fungus Basidiobolus haptosporus characteristically exhibit unusual proportions of annulate views of mitochondria in the two-dimensional perspective of thin sections. Such views exhibit a central space containing cytoplasmic ground substance and often profiles of other cytoplasmic organelles (lipid bodies, other mitochondrial forms, and especially crystalloid-containing microbodies). Three-dimensional projections are presented to suggest that these mitochondria have assumed the form of a goblet-shaped enclosure, and that the various annulate views are the consequence of plane of section viewed by electron microscopy. Their frequent occurrence and consistent morphology argues against their being random expressions of mitochondrial plasticity, but rather for close spatial associations amongst cytoplasmic organelles of young hyphae. When the fungus is grown on xanthine or its catabolites as sole sources of nitrogen, there is a proliferation of crystalloid-containing microbodies, double-membraned vesicles, and ovate to ellipsoidal mitochondria. Annulate views of mitochondria then are no longer observed, but microbodies again frequently appear in close association with mitochondria and at times in intimate contact with the mitochondrial outer membrane.     

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.     

MICROBODIES IN EXPERIMENTALLY ALTERED CELLS

A rapid and sustained increase in the number of microbodies in liver and kidney cells can be induced in male rats by ethyl chlorophenoxyisobutyrate (CPIB), a hypolipidemic drug. This phenomenon permits investigation of several aspects of microbody behavior in experimental conditions. Reversal experiments demonstrate that liver cells revert to normal between 2 and 3 weeks after withdrawal of CPIB and that one of the mechanisms for removal of excess microbodies is their incorporation into structures indistinguishable from lysosomes. In a state of rapid cell division, such as that present during liver regeneration, microbody proliferation apparently occupies a high biological priority. In necrotic or degenerating cells microbody structure remains relatively normal. The increase in microbodies induced by CPIB is inhibited by chloramphenicol. No increase in microbodies occurred in female rats or in chickens, guinea pigs, or rabbits at the dosage used (0.25% in diet). No changes in microbodies were seen in monkey liver. Catalase activity was generally parallel to the numerical response in microbodies. Additional observations suggest that the microbody response to CPIB is not related to hepatomegaly induced by this agent but may be related to the hypolipidemic effect of CPIB, though hypolipidemia per se is not a specific or sufficient cause of microbody proliferation.     

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     

Microbodies in methanol-assimilating yeasts

Cells of 3 yeast species capable of assimilating methanol have been examined by electron microscopy. When grown on methanol as the sole source of carbon and energy they contained many microbodies. Cells grown on glucose or ethanol either did not contain such bodies at all, or only to a limited extent.     

Ultrastructural changes in Cladosporium cucumerinum during pathogenesis.

Three ultrastructural patterns, each one associated with a different stage of pathogenesis, were observed in Cladosporium cucumerinum infecting cucumber cytoledons. (i) Fungal prepenetration structures (conidia and germ tubes) contained numberous lipid bodies. Microbodies were abundant and often associated with lipid bodies. Mitochondria were also abundant. (ii) During the invasion stage, no lipid bodies and relatively few microbodies were observed. Mitochondria were abundant, but differed markedly morphologically from those observed in the prepenetration structures. (iii) In the colonizing hyphae, microbodies were the most abundant organelle, lipid bodies were seldom found, and mitochondria were as observed during invasion. These changes in numbers of organelles may be associated with differences in the metabolic activities of the pathogen at various stages of pathogenesis.